WO2023155881A1

WO2023155881A1 - Methods for regulating secretion via migrasomes

Info

Publication number: WO2023155881A1
Application number: PCT/CN2023/076780
Authority: WO
Inventors: Li Yu; Haifeng JIAO
Original assignee: Tsinghua University
Priority date: 2022-02-18
Filing date: 2023-02-17
Publication date: 2023-08-24

Abstract

Provided is a method for regulating secretion via migrasomes, also providesd is a method for regulating the secretion of a substance by a migrasome, comprising regulating the number and/or function of an intraluminal vesicle in the migrasome.

Description

METHODS FOR REGULATING SECRETION VIA MIGRASOMES

BACKGROUND OF THE INVENTION

As migrating cells move, they leave long tubular strands, called retraction fibers, behind them. Large vesicles, which contain numerous smaller vesicles, grow on the tips and intersections of retraction fibers. These fibers, which connect the vesicles with the main cell body, eventually break, and the vesicles are released into the extracellular space or directly taken up by surrounding cells. Since the formation of these vesicles is migration-dependent, the vesicles are named as “migrasomes” . However, few possible roles have been identified.

SUMMARY OF THE INVENTION

The present disclosure provides a method for regulating secretion via migrasomes.

Regulating the formation and/or function of a migrasome and/or a migrasome mediated biological process

In one aspect, the present application provides a method for regulating the formation and/or function of a migrasome and/or a migrasome mediated biological process, comprising regulating transportation of an intraluminal vesicle into said migrasome.

In one embodiment, wherein regulating the transportation of said intraluminal vesicle comprises regulating the amount and/or function of a motor protein in a cell generating said migrasome.

In one embodiment, the method increases the transportation of said intraluminal vesicle into said migrasome and comprises increasing the amount and/or function of said motor protein in said cell.

In one embodiment, wherein increasing the amount and/or function of the motor protein comprises overexpressing said motor protein, a functional fragment thereof, a motor domain thereof, and/or a functional variant thereof in said cell.

In one embodiment, the method decreases the transportation of said intraluminal vesicle into said migrasome and comprises decreasing the amount and/or function of said motor protein in said cell.

In one embodiment, wherein decreasing the amount and/or function of said motor protein comprises knocking out or knocking down the expression of a gene encoding for said motor protein in said cell.

In one embodiment, wherein decreasing the amount and/or function of said motor protein comprises treating said cell with a motor protein inhibitor.

In one embodiment, wherein said motor protein is a Myosin family member.

In one embodiment, wherein said motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.

In one embodiment, the method is an in vivo method.

In one embodiment, the method is an in vitro method or an ex vivo method.

In one aspect, the present application provides an agent capable of regulating transportation of an intraluminal vesicle into a migrasome, for use in regulating migrasome formation, function, and/or a migrasome-mediated biological process.

In one embodiment, the agent is capable of regulating the amount and/or function of a motor protein in a cell generating said migrasome.

In one embodiment, the agent is capable of increasing the amount and/or function of the motor protein, for use in increasing the transportation of said intraluminal vesicle into said migrasome.

In one embodiment, the agent is capable of resulting in overexpression of said motor protein, a motor domain thereof, a functional fragment thereof, and/or a functional variant thereof in said cell.

In one embodiment, the agent comprises the motor protein, a motor domain thereof, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.

In one embodiment, the agent is capable of decreasing the amount and/or function of the motor protein, for use in decreasing the transportation of said intraluminal vesicle into said migrasome.

In one embodiment, the agent is capable of knocking out or knocking down the expression of a gene encoding for said motor protein in said cell.

In one embodiment, the agent comprises a motor protein inhibitor.

In one embodiment, wherein said motor protein is a Myosin family member.

In one aspect, the present application provides an engineered cell with altered ability for forming migrasomes containing intraluminal vesicles comparing to a corresponding unmodified cell, said engineered cell has been modified to alter transportation of the intraluminal vesicles into said migrasome.

In one embodiment, wherein the expression and/or function of a motor protein has been modified.

In one embodiment, wherein the expression and/or function of the motor protein has been increased.

In one embodiment, wherein said motor protein, a motor domain thereof, a functional fragment thereof, and/or a functional variant thereof has been overexpressed.

In one embodiment, wherein the expression and/or function of the motor protein has been decreased.

In one embodiment, wherein a gene encoding for said motor protein has been knocked out or knocked down.

In one embodiment, the engineered cell has been treated with a motor protein inhibitor.

In one embodiment, wherein said motor protein is a Myosin family member.

In one aspect, the present application provides a use of the agent capable of regulating transportation of an intraluminal vesicle into a migrasome of the present application and/or the engineered cell with altered ability for forming migrasomes containing intraluminal vesicles of the present application in the preparation of a regulator for: i) migrasome formation and/or function; and/or ii) a migrasome-mediated biological process.

In one aspect, the present application provides a composition, comprising the agent capable of regulating transportation of an intraluminal vesicle into a migrasome of the present application and/or the engineered cell with altered ability for forming migrasomes containing intraluminal vesicles of the present application.

In one embodiment, the composition is a pharmaceutical composition and optionally comprises a pharmaceutically acceptable excipient.

In one aspect, the present application provides a kit, comprising the agent capable of regulating transportation of an intraluminal vesicle into a migrasome of the present application, the engineered cell with altered ability for forming migrasomes containing intraluminal vesicles of the present application, and/or the composition of the present application.

Characterizing a migrasome and/or an intraluminal vesicle in a migrasome

In one aspect, the present application provides a method for characterizing a migrasome and/or an intraluminal vesicle in a migrasome, comprising determining the presence and/or amount of a secretory vesicle marker.

In one embodiment, comprising determining the presence and/or amount of the secretory vesicle marker in the migrasome.

In one embodiment, wherein said determining comprises using an agent capable of specifically identifying said secretory vesicle marker.

In one embodiment, wherein said agent capable of specifically identifying said secretory vesicle marker comprises an antibody against said marker or an antigen binding fragment thereof.

In one embodiment, wherein said antigen binding fragment comprises Fab, F (ab) 2, F (ab’) , F (ab’) 2, scFv, affibody and/or VHH.

In one embodiment, wherein said agent capable of specifically identifying said secretory vesicle marker further comprises a detectable label.

In one embodiment, wherein said determining comprises detecting the presence and/or amount of a modified secretory vesicle marker containing a detectable label.

In one embodiment, wherein said detectable label comprises a fluorescent label, a luminescent label, and/or a non-optically detectable label.

In one embodiment, wherein said secretory vesicle marker comprises a Rab protein and/or a SNAP Receptor (SNARE) .

In one embodiment, wherein said Rab protein comprises Rab8.

In one embodiment, wherein said SNARE comprises a t-SNARE and/or a v-SNARE.

In one embodiment, wherein said t-SNARE comprises SNAP23.

In one embodiment, wherein said v-SNARE comprises VAMP2 and/or VAMP7.

In one embodiment, further comprising determining the presence and/or amount of Tspan4, Integrin, Pleckstrin Homology (PH) domain, NDST1, PIGK, CPQ, and/or EOGT.

In one embodiment, further comprising staining with wheatgerm agglutinin (WGA) .

In one aspect, the present application provides a method for isolating and/or regulating a migrasome and/or an intraluminal vesicle in a migrasome, comprising:

i) characterizing the migrasome and/or the intraluminal vesicle in a migrasome according to the method for characterizing a migrasome and/or an intraluminal vesicle in a migrasome of the present application; and

ii) isolating the characterized migrasome and/or the characterized intraluminal vesicle, and/or administering a regulating agent to said characterized migrasome and/or said characterized intraluminal vesicle.

In one embodiment, the method is an in vitro or ex vivo method.

In one embodiment, the method is an in vivo method.

In one aspect, the present application provides an agent capable of determining the presence and/or amount of a secretory vesicle marker, for use in characterizing a migrasome and/or an intraluminal vesicle in a migrasome.

In one embodiment, the agent is capable of specifically identifying said secretory vesicle marker.

In one embodiment, the agent comprises an antibody against said marker or an antigen binding fragment thereof.

In one embodiment, the agent further comprises a detectable label.

In one embodiment, the agent comprises a modified secretory vesicle marker containing a detectable label.

In one embodiment, wherein said Rab protein comprises Rab8.

In one embodiment, wherein said SNARE comprises a t-SNARE and/or a v-SNARE.

In one embodiment, wherein said t-SNARE comprises SNAP23.

In one embodiment, wherein said v-SNARE comprises VAMP2 and/or VAMP7.

In one aspect, the present application provides a composition, comprising the agent capable of determining the presence and/or amount of a secretory vesicle marker of the present application.

In one embodiment, further comprising a second agent capable of determining the presence and/or amount of a migrasome.

In one embodiment, wherein said second agent is capable of determining the presence and/or amount of Tspan4, Integrin, Pleckstrin Homology (PH) domain, NDST1, PIGK, CPQ, and/or EOGT.

In one embodiment, wherein said second agent comprises WGA.

In one embodiment, wherein said agent of the present application is not mixed with said second agent.

In one aspect, the present application provides a kit, comprising the agent capable of determining the presence and/or amount of a secretory vesicle marker of the present application, and/or the composition of the present application.

In one aspect, the present application provides a use of the agent capable of determining the presence and/or amount of a secretory vesicle marker of the present application, the composition of the present application, and/or the kit of the present application, in the preparation of an indicator for a migrasome and/or an intraluminal vesicle in a migrasome.

Regulating the secretion of a substance by a migrasome

In one aspect, the present application provides a method for regulating the secretion of a substance by a migrasome, comprising regulating the number and/or function of an intraluminal vesicle in said migrasome.

In one embodiment, wherein said migrasome is generated by a cell.

In one embodiment, wherein said cell is a migrating cell.

In one embodiment, wherein said cell is a circulating cell.

In one embodiment, wherein said cell is a cell in or from the blood.

In one embodiment, wherein said cell is an immune cell.

In one embodiment, wherein said immune cell comprises a monocyte, a macrophage, a neutrophil and/or a T cell.

In one embodiment, wherein said substance to be secreted is comprised in said intraluminal vesicle.

In one embodiment, wherein said substance to be secreted comprises one or more signaling molecules.

In one embodiment, wherein said substance to be secreted comprises one or more protein or polypeptide containing a signal peptide.

In one embodiment, wherein said substance to be secreted comprises a cytokine and/or a flavonoid.

In one embodiment, the method increases the secretion of the substance, and comprises increasing the number and/or function of the intraluminal vesicles in said migrasome.

In one embodiment, wherein said increasing the number and/or function of the intraluminal vesicles in said migrasome comprises increasing the transportation of the intraluminal vesicles into said migrasome.

In one embodiment, wherein said increasing the number and/or function of the intraluminal vesicles in said migrasome comprises increasing the amount and/or function of a motor protein in said cell.

In one embodiment, wherein said increasing the number and/or function of the intraluminal vesicles in said migrasome comprises increasing fusion of the membrane of said migrasome with the intraluminal vesicle therein.

In one embodiment, wherein increasing said fusion comprises increasing the amount and/or function of a SNARE complex in said migrasome.

In one embodiment, wherein increasing said fusion comprises increasing calcium in said migrasome.

In one embodiment, the method inhibits the secretion of the substance, and comprises reducing the number and/or function of the intraluminal vesicles in said migrasome.

In one embodiment, wherein said reducing the number and/or function of the intraluminal vesicles in said migrasome comprises inhibiting the transportation of the intraluminal vesicles into said migrasome.

In one embodiment, wherein said reducing the number and/or function of the intraluminal vesicles in said migrasome comprises reducing the amount and/or function of a motor protein in said cell.

In one embodiment, wherein said reducing the number and/or function of the intraluminal vesicles in said migrasome comprises inhibiting fusion of the membrane of said migrasome with the intraluminal vesicle therein.

In one embodiment, wherein inhibiting said fusion comprises reducing the amount and/or function of a SNARE complex in said migrasome.

In one embodiment, wherein reducing the amount and/or function of a SNARE complex comprises inhibiting the expression of one or more components of the SNARE complex.

In one embodiment, wherein reducing the amount and/or function of a SNARE complex comprises knocking out or knocking down the expression of a gene encoding for one or more components of the SNARE complex.

In one embodiment, wherein inhibiting said fusion comprises reducing calcium in said migrasome.

In one embodiment, wherein reducing calcium in said migrasome comprises administering a calcium chelator.

In one embodiment, wherein said calcium chelator comprises BAPTA-AM.

In one embodiment, wherein said motor protein comprises a Myosin.

In one embodiment, wherein said SNARE comprises a v-SNARE and/or a t-SNARE.

In one embodiment, wherein said t-SNARE comprises SNAP23.

In one embodiment, wherein said v-SNARE comprises VAMP2 and/or VAMP7.

In one aspect, the present application provides an agent capable of regulating the number and/or function of an intraluminal vesicle in a migrasome, for use in regulating the secretion of a substance by said migrasome.

In one embodiment, wherein said migrasome is generated by a cell.

In one embodiment, wherein said cell is a migrating cell.

In one embodiment, wherein said cell is a circulating cell.

In one embodiment, wherein said cell is a cell in or from the blood.

In one embodiment, wherein said cell is an immune cell.

In one embodiment, for use in increasing the secretion of said substance, and the agent is capable of increasing the number and/or function of the intraluminal vesicles in said migrasome.

In one embodiment, the agent is capable of increasing the transportation of the intraluminal vesicles into said migrasome.

In one embodiment, the agent is capable of increasing the amount and/or function of a motor protein in said cell.

In one embodiment, the agent is capable of increasing fusion of the membrane of said migrasome with the intraluminal vesicle therein.

In one embodiment, the agent is capable of increasing the amount and/or function of a SNARE complex in said migrasome.

In one embodiment, the agent is capable of increasing calcium in said migrasome.

In one embodiment, for use in inhibiting the secretion of said substance, and the agent is capable of reducing the number and/or function of the intraluminal vesicles in said migrasome.

In one embodiment, the agent is capable of inhibiting the transportation of the intraluminal vesicles into said migrasome.

In one embodiment, the agent is capable of reducing the amount and/or function of a motor protein in said cell.

In one embodiment, the agent is capable of inhibiting fusion of the membrane of said migrasome with the intraluminal vesicle therein.

In one embodiment, the agent is capable of reducing the amount and/or function of a SNARE complex in said migrasome.

In one embodiment, the agent is capable of inhibiting the expression of one or more components of the SNARE complex.

In one embodiment, the agent is capable of knocking out or knocking down the expression of a gene encoding for one or more components of the SNARE complex.

In one embodiment, the agent is capable of reducing calcium in said migrasome.

In one embodiment, the agent comprises a calcium chelator.

In one embodiment, wherein said calcium chelator comprises BAPTA-AM.

In one embodiment, wherein said motor protein comprises a Myosin.

In one embodiment, wherein said SNARE comprises a v-SNARE and/or a t-SNARE.

In one embodiment, wherein said t-SNARE comprises SNAP23.

In one embodiment, wherein said v-SNARE comprises VAMP2 and/or VAMP7.

In one aspect, the present application provides an engineered cell with altered ability for regulating the secretion of a substance by a migrasome generated by said cell comparing to a corresponding unmodified cell, said engineered cell has been modified to alter the number and/or function of an intraluminal vesicle in said migrasome.

In one embodiment, wherein said cell is a migrating cell.

In one embodiment, wherein said cell is a circulating cell.

In one embodiment, wherein said cell is a cell in or from the blood.

In one embodiment, wherein said cell is an immune cell.

In one embodiment, the engineered cell has increased ability for the secretion of the substance, and said engineered cell has been modified to increase the number and/or function of the intraluminal vesicles in said migrasome.

In one embodiment, the engineered cell has been modified to increase the transportation of the intraluminal vesicles into said migrasome.

In one embodiment, the engineered cell has been modified to increase the amount and/or function of a motor protein in said cell.

In one embodiment, the engineered cell has been modified to increase fusion of the membrane of said migrasome with the intraluminal vesicle therein.

In one embodiment, the engineered cell has been modified to increase the amount and/or function of a SNARE complex in said migrasome.

In one embodiment, the engineered cell has been modified to increase calcium in said migrasome.

In one embodiment, the engineered cell has reduced ability for the secretion of the substance, and said engineered cell has been modified to reduce the number and/or function of the intraluminal vesicles in said migrasome.

In one embodiment, the engineered cell has been modified to inhibit the transportation of the intraluminal vesicles into said migrasome.

In one embodiment, the engineered cell has been modified to reduce the amount and/or function of a motor protein in said cell.

In one embodiment, the engineered cell has been modified to inhibit fusion of the membrane of said migrasome with the intraluminal vesicle therein.

In one embodiment, the engineered cell has been modified to reduce the amount and/or function of a SNARE complex in said migrasome.

In one embodiment, the engineered cell has been modified to inhibit the expression of one or more components of the SNARE complex.

In one embodiment, wherein the expression of a gene encoding for one or more components of the SNARE complex has been knocked down or knocked out.

In one embodiment, the engineered cell has been modified to reduce calcium in said migrasome.

In one embodiment, the engineered cell has been treated with a calcium chelator.

In one embodiment, wherein said calcium chelator comprises BAPTA-AM.

In one embodiment, wherein said motor protein comprises a Myosin.

In one embodiment, wherein said SNARE comprises a v-SNARE and/or a t-SNARE.

In one embodiment, wherein said t-SNARE comprises SNAP23.

In one embodiment, wherein said v-SNARE comprises VAMP2 and/or VAMP7.

In one aspect, the present application provides a use of an agent capable of regulating the number and/or function of an intraluminal vesicle in a migrasome of the present application and/or an engineered cell with altered ability for regulating the secretion of a substance by a migrasome generated by said cell of the present application in the preparation of a regulator for the secretion of a substance by said migrasome.

In one aspect, the present application provides a composition, comprising the agent capable of regulating the number and/or function of an intraluminal vesicle in a migrasome of the present application, and/or the engineered cell with altered ability for regulating the secretion of a substance by a migrasome generated by said cell of the present application.

In one aspect, the present application provides a kit, comprising the agent capable of regulating the number and/or function of an intraluminal vesicle in a migrasome of the present application, the engineered cell with altered ability for regulating the secretion of a substance by a migrasome generated by said cell of the present application, and/or the composition of the present application.

In this application, regulation of migrasome could be used in regulating secretion. Secretory vesicles may be translocated into migrasome by actin-based motor protein, once reach migrasome, secretory vesicles can fuse with migrasome membrane and release its content. Moreover, a package of cytokines may be released from cell as migrasome bound form, thus, migrasome are organelle for localized secretion and packaged releasing of secretory proteins in migrating cells.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWING

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “FIG. ” , “Fig. ” and “FIG. ” herein) , of which:

FIG. 1 illustrates Characterization of intraluminal vesicles of migrasome. (1A) Representative transmission electron microscopy (TEM) images of L929 cells. Scale bar, 10 μm. Middle panel, enlarged region of interesting (ROI) . Scale bar, 500 nm. Right panel, the relationship between the distance from migrasome to cell body with the diameter of migrasome and the number of intraluminal vesicles per migrasome is quantified. n = 30 cells from three independent experiments. (1B) TEM images of high pressure freezing (HPF) samples of L929 cells. Left panel, retraction fiber. Right panel, migrasome. Scale bar, 500 nm. (1C) TEM images of L929 cells. Left panel, the entrance of retraction fibers. Right panel, detached migrasome. Scale bar, 500 nm. (1D) TEM images of L929 cells treated with 10 μM BAPTA-AM. Scale bar, 500 nm. Right panel, statistical analysis of the number of small vesicles per migrasome. Error bars, the mean ± SEM n > 100 migrasomes from three independent experiments. Two-tailed unpaired t tests are used for statistical analyses. (1E) L929-T4-mCherry cells are immunostained with antibody against Rab8a and then visualized. White dashed lines, the outline of the cell body. Scale bar, 20 μm. Right panels, enlarged ROI. Scale bar, 2 μm. (1F) Structured illumination microscopy (SIM) images of L929 cells stably expressing GFP-Rab8a and T4-mCherry. Scale bar, 500 nm. (1G) Representative TEM images of DAB staining pattern in L929 cells stably expressing APEX2-GFP-Rab8 and reacted with diaminobenzidine (DAB) . Scale bar, 100 nm. (1H) Confocal images of GFP-Rab8a-and T4-mCherry-expressing L929 cells treated with 10 μM BAPTA-AM. Scale bar, 20 μm. The right panel shows statistical analysis of the number of GFP-Rab8a-puncta in migrasomes per cell. Error bars, mean ± SEM n > 100 cells from three independent experiments. Two-tailed unpaired t tests are used for statistical analyses.

FIG. 2 illustrates Myosin5a transports Rab8a-labelled intraluminal vesicles into migrasome. (2A) Confocal images of L929 cells stably expressing GFP-Myosin5a (Myo5a) and T4-mCherry. Scale bar, 20 μm. Lower panels, enlarged ROI. Scale bar, 2 μm. (2B) APEX2-based TEM images of L929 cells stably expressing APEX2-GFP-Myo5a. Scale bar, 2 μm. The lower panels show higher magnification images of vesicles from cell body (C) , the base of retraction fiber (T) and migrasome (M) . Scale bar, 200 nm. (2C) Time-lapse images of L929 cells stably expressing GFP-Myo5a and T4-mCherry. Scale bar, 20 μm. Right panels, enlarged ROI. Scale bar, 5 μm. (2D) Grazing Incidence-Structured Illumination Microscopy (GI-SIM) images of L929-GFP-Myo5a cells. Scale bar, 5 μm. Right panels, enlarged ROI. Time-lapse images are acquired at intervals of 30 s. White arrows indicate Myo5a moving into migrasomes. The arrows indicate Myo5a accumulating to the edge of cell and left on retraction fibers. Scale bar, 2 μm. (2E) TEM images of WT, Myosin5a-overexpression (Myo5a OE) and Myosin5a-knockout (Myo5a KO) L929 cells. Scale bar, 500 nm. The right panel shows the statistics of the number of small vesicles per migrasome. The error bars are mean ± SEM of > 100 migrasomes from three independent experiments. Two-tailed unpaired t-tests are used for statistical analyses. (2F) Overexpression of myosin5a increased the number of GFP-Rab8 puncta in migrasomes, indicating that Rab8 positive vesicles were transported into migrasomes by myosin5a.

FIG. 3 illustrates VAMP2, VAMP7 and SNAP23 mediate the fusion of intraluminal vesicles with migrasome membrane. (3A) SIM images of L929 cells stably expressing GFP-VAMP2 and T4-mCherry. Scale bar, 200 nm. (3B) TEM images of L929 cells stably expressing APEX2-GFP-VAMP2 and reacted with DAB. Scale bar, 200 nm. (3C) Immunostaining of endogenous VAMP2 in WT, Myo5a OE and Myo5a KO L929 cells. Scale bar, 20 μm. Right panel, statistical analysis of the number of VAMP2 puncta in migrasomes per cell. Error bars, mean ± SEM n > 100 cells from three independent experiments. Two-tailed unpaired t tests are used for statistical analyses. (3D) L929-T4-mCherry cells are immunostained with SNAP23 antibody and then visualized. Scale bar, 20 μm. (3E) Western blot analysis of total plasma membrane proteins isolated from the cell bodies (C) or migrasomes (M) using the indicated antibodies. (3F) L929-T4-mCherry cells are infected with nonspecific (WT) or SNAP23-shRNA lentiviral constructs. Cells are then immunostained with VAMP2 antibody and subjected to confocal analysis. Scale bar, 20 μm. Right panel, statistical analysis of the number of VAMP2 puncta in migrasomes per cell. Error bars, mean ± SEM n > 100 cells from three independent experiments. Two-tailed unpaired t tests are used for statistical analyses. (3G) L929 cells stably expressing GFP-VAMP2 are subjected to time-lapse imaging. Time-lapse images are acquired at intervals of 5 s. Scale bar, 2 μm. (3H) Confocal images of L929 cells stably expressing VAMP2-PHlourin and T4-mCherry. Scale bar, 20 μm. The right panel shows statistical analysis of the migrasome/cell fluorescence intensity ratio. Data represent the mean ± SEM n > 30 cells from three independent experiments. Two-tailed unpaired t tests are used for statistical analyses.

FIG. 4 illustrates Migrasome mediate localized secretion of cytokines in active monocyte. (4A) Mouse monocytes are cultured in FN-precoated confocal dishes in the presence of 500 ng/mL LPS for 12 hr. Cells are then stained with CCR2 and WGA before visualization. Scale bar, 5 μm. (4B) Representative TEM images of activated monocytes from (A) . Scale bar, 1 μm. Right panels, enlarged migrasomes containing intraluminal vesicles are shown. Scale bar, 200 nm. (4C and 4D) Immunostaining of endogenous TNF-α (4C) and IL-6 (4D) in activated monocytes as shown in (4A) . Scale bar, 5 μm. The lower panels show enlarged migrasomes. Scale bar, 500 nm. (4E) Western blot analysis of migrasomes purified from activated monocytes using the indicated antibodies. CPQ and Itg α5 are used as migrasome markers. Equal amounts of total protein from cell bodies (C) and migrasomes (M) are subjected to western blot analysis. (4F) Confocal images of WT and TSPAN9^-/-(T9 KO) monocytes plated on dishes with or without FN-precoated. Scale bar, 5 μm. Quantification of the number of migrasomes per cell is shown as the mean ± SEM. n> 100 cells from three independent experiments are analysed using two-tailed unpaired t tests (right panel) . (4G) TNF-α and IL-6 secretion in indicated monocytes. Cells are activated, primed with 500 ng/mL LPS, and seeded into control or FN-precoated dishes for 16 hr. Cell lysates and concentrated medium are collected and analyzed by western blot. (4H) L929 cells are cultured in medium containing migrasomes isolated from activated monocytes in the presence of 10 μM zVAD for 18 hr. Cells undergoing necroptosis are detected by propidium iodide (PI) staining coupled with FACS analysis. The right panel shows statistical analysis of cell death. Error bar, mean ± SEM. Experiments were independently repeated three times. Two-tailed unpaired t tests were used for statistical analyses. **p < 0.01, ***p < 0.001.

FIG. 5 illustrates Monocyte derived migrasome is the site for packaged releasing of cytokines in vivo. (5A-5B) Intravital imaging of mouse liver monocyte after LPS stimulation. Labeled with PE anti-mouse CCR2 antibody and APC anti-mouse F4/80 antibody. (5A) Time-lapse images are acquired at intervals of 12 s. Scale bar, 5 μm. (5B) WGA labels blood vessels, and white arrowheads indicate free CCR2 positive migrasomes detached form retraction fibers. Time interval, 45 s. Scale bar, 5 μm. (5C) Schematic illustration of monocyte derived migrasomes purification from mouse blood samples. (5D) Representative scanning electron microscopy (SEM) images of migrasomes isolated from blood monocytes as shown in (5C) . Scale bar, 500 nm. (5E and 5F) Immunofluorescence stained z-stack images of migrasomes purified from blood monocytes. Migrasomes are stained with CCR2, VAMP2, TNF-α (5E) and IL-6 (5F) . Z-stack images are acquired by confocal microscopy. Scale bar, 2 μm. Right panels, 3D reconstruction of enlarged migrasomes. Scale bar, 500 nm. (5G) LPS (12 mg/kg) is injected into mice by intraperitoneal injection (i. p. ) . Cell bodies (C) and monocyte derived migrasomes (M) from mouse with or without LPS treatment are isolated from equal volume of blood, and then analyzed by western blot. (5H) Western blot analysis of migrasomes purified from blood monocytes using the indicated antibodies. The lysates of the cell bodies (C) and migrasomes (M) are normalized to equal total protein loading for western blot analysis. (5I) Equal numbers of WT and T9 KO monocytes labelled with different color conjugated CCR2 antibodies are combined for injection into WT mice. Intravital imaging of mouse liver is performed. WGA labels blood vessels. Scale bar, 10 μm. The right panel shows the statistics of the number of migrasomes per cell. Shown are mean ±SEM of > 100 cells from three independent experiments. Two-tailed unpaired t-tests are used for statistical analyses. (5J) Western blot analysis of monocyte derived migrasomes. Migrasomes are isolated from WT and T9 KO mouse blood in equal volume, and then subjected to western blot analysis. (5K) Inflammatory cytokine protein profiles in WT and T9 KO mouse blood. After i. p. of LPS, inflammatory cytokines (TNF-α, IL-6, IFN-γ, MCP-1, IL-10 and IL-12p70) are measured in blood from WT and T9 KO mouse by cytometric bead array (CBA) . Data are presented as mean ± SEM. Experiments are independently repeated two times (n = 12-18 mice per group) . Two-tailed unpaired t tests are used for statistical analyses. (5L) Model for the role of migrasomes in localized secretion and packaged releasing of signaling ligands.

FIG. 6 illustrates Motor domain may be required for localization of Myosin5a in migrasomes. Confocal images of L929-T4-mCherry cells stably expressing the indicated forms of Myo5a. Full-length (FL) , motor domain (H) , tail domain (T) . Scale bar, 20 μm.

FIG. 7 illustrates SNAP23 and VAMP7 may be enriched in migrasomes. (7A) Confocal images of L929 cells stably expressing GFP-SNAP23 and T4-mCherry. Scale bar, 20 μm. (7B) Immunostaining of endogenous VAMP7 in L929-T4-mCherry and L929-mCherry-Myo5a cells. Scale bar, 20 μm. Right panel, statistical analysis of the number of VAMP7 puncta in migrasomes per cell. Error bars, mean ± SEM n > 100 cells from three independent experiments. Two-tailed unpaired t tests are used for statistical analyses.

FIG. 8 illustrates TACE may be localized on the migrasome membrane. Immunostaining of endogenous TACE in activated monocytes. Scale bar, 5 μm. The lower panels show enlarged migrasomes. Scale bar, 500 nm.

FIG. 9 illustrates mouse neutrophil can generate migrasomes. IL-1b (9A) and MCSF (9B) are enriched in migrasomes.

FIG. 10 illustrates mouse primary natural killer cells can generate migrasomes after IL15 stimulation. GM-CSF and CCL5 are enriched in migrasomes. Scale bars, 10 μm.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

Term

As used herein, the term “antibody” generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen. For example, the antibody can include an immunoglobulin composed of at one or more heavy (H) chains and/or one or more light (L) chains, and include any molecule including its antigen binding portion. The term “antibody" includes monoclonal antibodies, antibodies fragment or antibody derivatives, including but not limited to, human antibodies, humanized antibodies, chimeric antibodies, single-strand antibodies (e.g., scFv) , and antigen-binding fragments of antibodies (e.g., Fab, Fab’, VHH and (Fab) 2 fragments) .

As used herein, the term "functional fragment" generally refers to a fragment having a partial region of a full-length protein or nucleic acid, but retaining or partially retaining the biological activity or function of the full-length protein or nucleic acid.

As used herein, the term "functional variant" generally refers to a nucleic acid molecule, or a polypeptide having similar amino acid or nucleic acid sequences as the parent sequence and retain one or more properties of the parent sequence.

As used herein, the term “intraluminal vesicle” generally refers to a membranous vesicle that is formed or present in a lumen, or inside the space of a luminal or tubular structure, such as an organelle or a larger vesicle. The term lumen is also used herein to describe the inside space of a cellular component or structure, such as the migrasome. For example, intraluminal vesicle may be generated from an organelle. For example, an intraluminal vesicle may refer to an intraluminal vesicle in a migrasome or an intraluminal vesicle that is destinated to be comprised in a migrasome.

As used herein, the term “knock down” generally refers to a measurable reduction in the expression of a target mRNA or the corresponding protein in a genetically modified cell or organism as compared to the expression of the target mRNA or the corresponding protein in a counterpart control cell or organism that does not contain the genetic modification to reduce expression. Those skilled in the art will readily appreciate how to use various genetic approaches, e.g., siRNA, shRNA, microRNA, antisense RNA, or other RNA-mediated inhibition techniques, to knock down a target polynucleotide sequence.

As used herein, the term “knock out” generally includes deleting all or a portion of the target polynucleotide sequence in a way that interferes with the function of the target polynucleotide sequence. For example, a knock-out can be achieved by altering a target polynucleotide sequence by inducing a deletion in the target polynucleotide sequence in a functional domain of the target polynucleotide sequence. Those skilled in the art will readily appreciate how to use various genetic approaches, e.g., CRISPR/Cas systems, ZFN, TALEN, TgAgo, to knock out a target polynucleotide sequence or a portion thereof based upon the details described herein.

As used herein, the term “migrasome” generally refers to a membrane-bound cellular structure derived from or generated by a migrating cell. The term “migrasome” encompasses an organelle (also known as “pomegranate-like structure” or PLS) attached to a retraction fiber generated by a migrating cell. In some cases, the term “migrasome” also refers to a vesicle (e.g., an extracellular vesicle) already detached from the cell generating it. In the present disclosure, the term “migrasome” also refers to a vesicle (e.g., an artificial vesicle) with similar functions and/or compositions as such a vesicle or organelle derived from, and/or generated by migrating cells.

As used herein, the term “migrasome mediated biological process” generally refers to a biological process mediated by the formation, movement, function, degradation, and/or disintegration of a migrasome.

As used herein, the terms “migrating cell” and “circulating cell” are used interchangeably, and generally refer to a cell moving from one location to another location. In some cases, a migrating cell is a cell whose relative position, space, and/or contour has changed or is changing with time. A circulating cell comprises a cell circulating in the body fluid (e.g., blood or lymph) of an organism.

As used herein, the term “motor domain” generally refers to a mechanical element of a motor protein. For example, motor domain may hydrolyze GTP or ATP, and change the conformation of the microtubule-binding domains of the motor protein, which may result in the motion of the motor protein.

As used herein, the term “motor protein” generally refers to a molecular motor that can move along the cytoplasm of cells. For example, motor proteins may play roles in intracellular transportation and/or cell motility. For example, motor proteins may comprise actin motor and microtubule motor. For example, actin motors such as myosin may move along microfilaments through interaction with actin, and microtubule motors such as dynein and kinesin may move along microtubules through interaction with tubulin. For example, the term “motor protein” may encompass various isoforms of the motor protein, as well as naturally-occurring allelic and processed forms thereof.

As used herein, the term “myosin family member” generally refers to a member of the superfamily of Myosin motor protein. Myosins constitute a large superfamily of proteins that share a common domain which has been shown to interact with actin, hydrolyze ATP and produce movement in all cases examined to date. Myosins are typically constructed of three functional subdomains: (1) the motor domain which interacts with actin and binds ATP, (2) the neck domain which binds light chains or calmodulin, and (3) the tail domain which serves to anchor and position the motor domain so that it can interact with actin. The motor domains are relatively conserved. The tail domains are the most diverse domains and vary widely in length and in sequence. Functional motifs, such as SH3 domains, GAP domains, FERM domains, and pleckstrin homology (PH) domains are sometimes found in the tails of myosins. In addition, the tails of many myosins contain coiled-coil forming sequences which allow the molecules to dimerize and produce two-headed molecules. The number of myosin genes present in mammals is conservatively estimated at 25–30 from classes I (such as myosin1c) , II, III, V (such as myosin5a) , VI, VII, IX, X and XV.

As used herein, the term “pharmaceutically acceptable excipient” generally refers to any material, which is inert in the sense that it substantially does not have a therapeutic and/or prophylactic effect per se. Such an excipient is added with the purpose of making it possible to obtain a pharmaceutical composition having acceptable technical properties.

As used herein, the term “signal peptide” generally refers to a peptide linked in frame to a polypeptide (e.g., to the amino terminus of a polypeptide) and directs the encoded polypeptide into a cell's secretory pathway.

As used herein, the term “signaling molecule” generally refers to a molecule that is responsible for transmitting information. For example, signaling molecule may comprise lipids, phospholipids, amino acids, monoamines, proteins, glycoproteins, and/or gases. For example, signaling molecule may interact with a target cell as a ligand to cell surface receptors, and/or by entering the cell through its membrane or endocytosis for intracrine signaling.

As used hererin, the term “SNARE complex” generally refers to a protein complex formed by one or more components (e.g., subunits) that may mediate vesicle fusion. SNARE is also known as soluble N-ethylmaleimide–sensitive factor attachment protein (SNAP) receptor. For example, SNARE may comprise vesicle-SNAREs or v-SNAREs, which are incorporated into the membranes of transport vesicles during budding, and target-SNAREs or t-SNAREs, which are associated with nerve terminal membranes. SNARE complex may be the components of the fusion machinery.

As used hererin, the term “t-SNARE” generally refers to a target-SNARE. For example, t-SNARE may be the part of the SNARE complex that is in the target membrane. For example, t-SNARE may compirse syntaxin, SNAP25, SNAP23, the isoforms thereof, as well as the naturally-occurring allelic and processed forms thereof.

As used herein, the term “v-SNARE” generally refers to a vesicle-SNARE. For example, v-SNARE may be the part of the SNARE complex that is in the vesicle. For example, v-SNARE may comprise VAMP2, VAMP3 and/or VAMP7, the isoforms of the VAMP2, VAMP3 and/or VAMP7, the naturally-occurring allelic and processed forms thereof.

Unless otherwise specified, “a” , “an” , “the” and “at least one” are used interchangeably and refer to one or more than one.

In the present disclosure, the term “comprise” also encompasses “is” , “has” and “consist of” . For example, “a composition comprising X and Y” may be understood to encompass a composition that comprises at least X and Y. It shall also be understood to disclose a composition that only comprises X and Y (i.e., a composition consisting of X and Y) .

Regulating transportation of an intraluminal vesicle into migrasome

For example, the present application provides a method for regulating transportation of an intraluminal vesicle into the migrasome. For example, regulating the transportation of intraluminal vesicle may comprise regulating the amount and/or function of a motor protein in a cell generating the migrasome.

For example, the transportation of an intraluminal vesicle into the migrasome may be promoted. For example, the amount of the intraluminal vesicle transported into the migrasome may be 0.1%higher, 1%higher, 10%higher, 20%higher, 30%higher, 40%higher, 50%higher, 60%higher, 70%higher, 80%higher, 90%higher, 95%higher, 99%higher, 100%higher, 2 times higher, 3 times higher, 4 times higher, 5 times higher, 10 times higher, 50 times higher, 100 times higher, 1000 times higher, or 10000 times higher than the untreated cell or the cell before treating. For example, the amount of the intraluminal vesicle transported into the migrasome may be analyzed by confocal imaging analysis and/or Transmission Electron Microscopy analysis.

For example, the transportation of an intraluminal vesicle into the migrasome may be inhibited. For example, the amount of the intraluminal vesicle transported into the migrasome e may be 0.1%lower, 1%lower, 10%lower, 20%lower, 30%lower, 40%lower, 50%lower, 60%lower, 70%lower, 80%lower, 90%lower, 95%lower, 99%lower, 100%lower, 2 times lower, 3 times lower, 4 times lower, 5 times lower, 10 times lower, 50 times lower, 100 times lower, 1000 times lower, or 10000 times lower than the untreated cell or the cell before treating. For example, the amount of the intraluminal vesicle transported into the migrasome may be analyzed by confocal imaging analysis and/or Transmission Electron Microscopy analysis.

For example, wherein regulating the transportation of said intraluminal vesicle may comprise regulating the amount and/or function of a motor protein in a cell generating said migrasome.

Regulating the amount and/or function of a motor protein

For example, the present application provides a method for regulating the amount and/or function of a motor protein.

For example, increasing the amount and/or function of motor protein may comprise providing motor protein, overexpressing motor protein, and/or activating motor protein. For example, increasing the amount and/or function of motor protein may comprise introducing motor protein and/or a gene encoding for motor protein. For example, increasing the amount and/or function of motor protein may comprise activating the interaction between the motor protein and the intraluminal vesicle.

For example, decreasing the amount and/or function of motor protein may comprise knocking out the expression of a gene encoding for motor protein, knocking down the expression of a gene encoding for motor protein, and/or treating the migrasome with an agent capable of inhibiting the function of motor protein. For example, decreasing the amount and/or function of motor protein may comprise introducing CRISPR Cas9 system and/or miRNA targeting motor protein. For example, decreasing the amount and/or function of motor protein may comprise inhibiting the interaction between the motor protein and the intraluminal vesicle. For example, decreasing the amount and/or function of motor protein may comprise binding to the motor protein.

Knocking out

Knocking out refers to a genetic process in which the target protein encoding gene is made inoperative ( “knocked out” ) . When the encoding gene is knocked out, it may comprise heterozygous knock out or homozygous knock out. In the heterozygous knock out, only one of two gene copies (alleles) is knocked out, in the homozygous knock out, both copies are knocked out.

Knockouts may be accomplished through a variety of techniques. In some cases, the knockouts may be naturally occurring mutations that are screened out or identified (e.g., by DNA sequencing or other methods) .

In some cases, the knockouts are generated by homologous recombination. For example, it may involve creating a nucleic acid (e.g., DNA) construct containing the desired mutation. The construct may also comprise a drug resistance marker in place of the desired knockout gene. The construct may further contain a minimum length (e.g., 2kb or above) of homology to the target sequence. The construct may be delivered to target cells (for example, through microinjection, electroporation, or other methods, such as transfection, using a virus or a non-virus system) . This method then relies on the cell’s own repair mechanisms to recombine the nucleic acid construct into the existing DNA (e.g., the genome of the cell) . This may result in the sequence of the gene being altered, and most cases the gene will be translated into a nonfunctional protein, if it is translated at all. The drug selection marker on the construct may be used to select for cells in which the recombination event has occurred. In diploid organisms, which contain two alleles for most genes, and may as well contain several related genes that collaborate in the same role, additional rounds of transformation and selection may be performed until every targeted gene is knocked out. Selective breeding may be required to produce homozygous knockout animals.

In some cases, the knockouts are generated using site-specific nucleases. Various methods may be used to precisely target a DNA sequence in order to introduce a double-stranded break. Once this occurs, the cell’s repair mechanisms will attempt to repair this double stranded break, often through non-homologous end joining (NHEJ) , which involves directly ligating the two cut ends together. This may be done imperfectly, therefore sometimes causing insertions or deletions of base pairs, which cause frameshift mutations. These mutations can render the gene in which they occur nonfunctional, thus creating a knockout of that gene.

For example, a zinc-finger nuclease may be used to generate such knockouts. Zinc-finger nucleases comprise DNA binding domains that can precisely target a DNA sequence. Each zinc finger can recognize codons of a desired DNA sequence, and therefore can be modularly assembled to bind to a particular sequence. These binding domains are coupled with a restriction endonuclease that can cause a double stranded break (DSB) in the DNA. Repair processes may introduce mutations that destroy functionality of the gene.

As another example, Transcription activator-like effector nucleases (TALENs) may be used to generate such knockouts. TALENs contain a DNA binding domain and a nuclease that can cleave DNA. The DNA binding region may comprise amino acid repeats that each recognize a single base pair of the desired targeted DNA sequence. If this cleavage is targeted to a gene coding region, and NHEJ-mediated repair introduces insertions and deletions, a frameshift mutation often results, thus disrupting function of the gene. As a further example, clustered regularly interspaced short palindromic repeats (CRISPR) system may be used to generate such knockouts. The CRISPR/Cas9 method is a method for genome editing that contains a guide RNA complexed with a Cas9 protein. The guide RNA can be engineered to match a desired DNA sequence through simple complementary base pairing. The coupled Cas9 may cause a double stranded break in the DNA. Following the same principle as zinc-fingers and TALENs, the attempts to repair these double stranded breaks often result in frameshift mutations that result in a nonfunctional gene.

The knockout may also comprise a conditional gene knockout. A conditional gene knockout allows gene deletion in a tissue or cell when certain conditions are fulfilled, for example, in a tissue specific manner. It may be achieved by introducing short sequences called loxP sites around the gene. These sequences will be introduced into the germ-line via the same mechanism as a knock-out. This germ-line can then be crossed to another germline containing Cre-recombinase which is a viral enzyme that can recognize these sequences, recombines them, and deletes the gene flanked by these sites.

Knocking down

Knocking down refers to a process by which the expression of the target protein encoding gene is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.

The knocking down may be through a genetic modification or may be transient. If a DNA of an organism or cell is genetically modified, the resulting organism or cell may be referred to as a “knockdown organism” or a “knockdown cell” . If the change in gene expression is caused by an oligonucleotide binding to an mRNA or temporarily binding to a gene, this leads to a temporary change in gene expression that does not modify the chromosomal DNA, and the result may be referred to as a “transient knockdown” .

In a transient knockdown, the binding of this oligonucleotide to the active gene or its transcripts causes decreased expression through a variety of processes. Binding can occur either through the blocking of transcription (in the case of gene-binding) , the degradation of the mRNA transcript (e.g., by small interfering RNA (siRNA) ) or RNase-H dependent antisense, or through the blocking of either mRNA translation, pre-mRNA splicing sites, or nuclease cleavage sites used for maturation of other functional RNAs, including miRNA (e.g., by morpholino oligos or other RNase-H independent antisense) .

RNA interference (RNAi) is a means of silencing genes by way of mRNA degradation. Gene knockdown by this method is achieved by introducing small double-stranded interfering RNAs (siRNA) into the cytoplasm. Small interfering RNAs can originate from inside the cell or can be exogenously introduced into the cell. Once introduced into the cell, exogenous siRNAs are processed by the RNA-induced silencing complex (RISC) . The siRNA is complementary to the target mRNA to be silenced, and the RISC uses the siRNA as a template for locating the target mRNA. After the RISC localizes to the target mRNA, the RNA is cleaved by a ribonuclease.

For example, decreasing the amount and/or function of an angiogenesis factor in migrasome may comprise decreasing the amount of angiogenesis factor. For example, decreasing the amount and/or function of an angiogenesis factor in migrasome may comprise decreasing the expression of angiogenesis factor. For example, decreasing the amount and/or function of an angiogenesis factor in migrasome may comprise introducing CRISPR Cas9 system and/or miRNA targeting angiogenesis factor. For example, decreasing the amount and/or function of an angiogenesis factor in migrasome may comprise inhibiting the interaction between the angiogenesis factor and the receptor of the angiogenesis factor. For example, decreasing the amount and/or function of an angiogenesis factor in migrasome may comprise binding to the angiogenesis factor.

For example, which increases the transportation of said intraluminal vesicle into said migrasome and may comprise increasing the amount and/or function of said motor protein in said cell.

For example, wherein increasing the amount and/or function of the motor protein may comprise overexpressing said motor protein, a functional fragment thereof, a motor domain thereof, and/or a functional variant thereof in said cell.

For example, which may decrease the transportation of said intraluminal vesicle into said migrasome and may comprise decreasing the amount and/or function of said motor protein in said cell.

For example, wherein decreasing the amount and/or function of said motor protein may comprise knocking out or knocking down the expression of a gene encoding for said motor protein in said cell.

For example, wherein decreasing the amount and/or function of said motor protein may comprise treating said cell with a motor protein inhibitor.

For example, wherein said motor protein may be a Myosin family member.

Regulate myosin family member and motor protein

For example, the motor protein may comprise a myosin family member. For example, the motor protein may comprise Myosin1c, Myosin5a or comprises a motor domain thereof.

For example, increasing the amount and/or function of myosin family member may comprise providing myosin family member, overexpressing myosin family member, and/or activating myosin family member. For example, increasing the amount and/or function of myosin family member may comprise introducing myosin family member and/or a gene encoding for myosin family member. For example, increasing the amount and/or function of myosin family member may comprise activating the interaction between the myosin family member and the intraluminal vesicle.

For example, decreasing the amount and/or function of myosin family member may comprise knocking out the expression of a gene encoding for myosin family member, knocking down the expression of a gene encoding for myosin family member, and/or treating the migrasome with an agent capable of inhibiting the function of myosin family member. For example, decreasing the amount and/or function of myosin family member may comprise introducing CRISPR Cas9 system and/or miRNA targeting myosin family member. For example, decreasing the amount and/or function of myosin family member may comprise inhibiting the interaction between the myosin family member and the intraluminal vesicle. For example, decreasing the amount and/or function of myosin family member may comprise binding to the myosin family member.

For example, wherein said motor protein may be Myosin1c, Myosin5a or may comprise a motor domain thereof.

For example, which may be an in vivo method.

For example, which may be an in vitro method or an ex vivo method.

Agent capable of regulating transportation of an intraluminal vesicle into a migrasome

For example, agent may be capable of regulating the amount and/or function of a motor protein in a cell generating said migrasome.

For example, agent may be capable of increasing the amount and/or function of the motor protein, for use in increasing the transportation of said intraluminal vesicle into said migrasome.

For example, agent may be capable of resulting in overexpression of said motor protein, a motor domain thereof, a functional fragment thereof, and/or a functional variant thereof in said cell.

For example, agent may comprise the motor protein, a motor domain thereof, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.

For example, agent may be capable of decreasing the amount and/or function of the motor protein, for use in decreasing the transportation of said intraluminal vesicle into said migrasome.

For example, agent may be capable of knocking out or knocking down the expression of a gene encoding for said motor protein in said cell.

For example, agent may comprise a motor protein inhibitor.

For example, wherein said motor protein may be a Myosin family member.

Engineered cell with altered ability for forming migrasomes containing intraluminal vesicles

For example, this patent application provides an agent capable of modifying an engineered cell by altering its transportation of the intraluminal vesicles into said migrasome, for use in preparing an engineered cell with altered ability for forming migrasomes containing intraluminal vesicles comparing to a corresponding unmodified cell.

For example, this patent application provides a method for preparing an engineered cell with altered ability for forming migrasomes containing intraluminal vesicles comparing to a corresponding unmodified cell, the method comprises modifying a cell by altering its transportation of the intraluminal vesicles into said migrasome.

For example, wherein the expression and/or function of a motor protein has been modified.

For example, wherein the expression and/or function of the motor protein has been increased.

For example, wherein said motor protein, a motor domain thereof, a functional fragment thereof, and/or a functional variant thereof has been overexpressed.

For example, wherein the expression and/or function of the motor protein has been decreased.

For example, wherein a gene encoding for said motor protein has been knocked out or knocked down.

For example, which has been treated with a motor protein inhibitor.

For example, wherein said motor protein may be a Myosin family member.

Use, composition, and kit

For example, which may be a pharmaceutical composition and optionally may comprise a pharmaceutically acceptable excipient.

For example, the composition may be formulated with a suitable amount of a pharmaceutically acceptable excipient to provide the form for proper administration. For example, the composition may take the form of capsules, tablets, powders, solutions, or any other form suitable for administration. For example, the pharmaceutically acceptable excipient may be the excipient that is approved by a regulatory agency and/or listed in generally recognized pharmacopeia for use in subject, e.g., humans.

For example, dosages and desired concentration of the composition may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well known within the skill of an ordinary artisan. It is within the scope of the present application that different formulations may be effective.

Kit

The kit of the present disclosure may comprise the agent, the engineered cell, and/or the composition according to the present disclosure. The agent, the engineered cell, and/or the composition may be comprised in suitable packaging, and written material that can include instructions for use, discussion of experimental studies (such as clinical studies) , listing of side effects, and the like. Such kits may also include information, such as scientific literature references, package insert materials, experimental results (such as clinical trial results) , and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the agent, the engineered cell and/or the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the users (such as health care provider or consumers) . Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials. The kit may further contain an additional agent. In some embodiments, the agent, engineered cell and/or the composition of the present invention and the additional agent may be provided as separate compositions in separate containers within the kit. In some embodiments, the agent, the engineered cell and/or the composition of the present disclosure and the additional agent are provided as a single composition within a container in the kit. Suitable packaging and additional articles for use (e.g., measuring cup for liquid preparations, foil wrapping to minimize exposure to air, and the like) are known in the art and may be included in the kit. Kits described herein can be provided, marketed and/or promoted to users (such as health providers) , including scientists, physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in some embodiments, be marketed directly to the consumer.

Characterizing a migrasome and/or an intraluminal vesicle in a migrasome

In one aspect, the present application provides a method for characterizing a migrasome and/or an intraluminal vesicle in a migrasome, comprising determining the presence and/or amount of a secretory vesicle marker. For example, said characterizing may comprise monitoring and detecting.

Presence and/or amount of a secretory vesicle marker

For example, the present application provides a method for characterizing a migrasome and/or an intraluminal vesicle in a migrasome, which comprises determining the presence and/or amount of a secretory vesicle marker. For example, the presence and/or amount of a secretory vesicle marker may indicate the presence and/or amount of a migrasome and/or an intraluminal vesicle in a migrasome. For example, the presence and/or amount of a secretory vesicle marker may be used for monitoring the presence and/or amount of a migrasome and/or an intraluminal vesicle in a migrasome.

For example, the secretory vesicle marker may be the marker that is specifically present in the secretory vesicle. For example, the secretory vesicle marker may comprise a Rab, which may be used to regulate Golgi to plasma membrane trafficking. For example, the secretory vesicle marker may comprise a SNAP Receptor (SNARE) , which may be used to mediate vesicle fusion. For example, the Rab protein may comprise Rab6, Rab8, Rab11, Rab17, and/or Rab23. For example, the SNARE may comprise VAMP2, VAMP7, VAMP8, SNAP25 and/or SNAP23. For example, amount of the marker may be analyzed by western blot, Immunofluorescence and/or quantitative mass spectrometry analysis.

For example, comprising determining the presence and/or amount of the secretory vesicle marker in the migrasome.

For example, wherein said determining may comprise using an agent capable of specifically identifying said secretory vesicle marker.

For example, wherein said agent capable of specifically identifying said secretory vesicle marker may comprise an antibody against said marker or an antigen binding fragment thereof.

For example, wherein said antigen binding fragment may comprise Fab, F (ab) 2, F (ab’) , F (ab’) 2, scFv, affibody and/or VHH.

For example, wherein said agent capable of specifically identifying said secretory vesicle marker further may comprise a detectable label.

For example, wherein said determining may comprise detecting the presence and/or amount of a modified secretory vesicle marker containing a detectable label.

For example, wherein said detectable label may comprise a fluorescent label, a luminescent label, and/or a non-optically detectable label.

detectable label

For example, a detectable label may be attached to an analyte to render the reaction of the analyte detectable. For example, the analyte of the present application may be secretory vesicle marker, e.g., Rab protein and/or a SNAP Receptor (SNARE) .

For example, the detectable label may produce a signal that is detectable by visual and/or instrumental means. For example, the detectable label may comprise moieties that produce light, and/or moieties that produce fluorescence. For example, the detectable label may comprise a fluorescent label, a luminescent label, and/or a non-optically detectable label.

For example, the detectable label may be attached to a secretory vesicle marker, and the presence and/or amount of the signal produced by the detectable label may indicate the presence and/or amount of the secretory vesicle marker. For example, one or more analytes may be attached with one or more detectable labels. For example, each of one or more specific analytes may be attached with different detectable label. For example, different detectable label may have different color, or may have different types of labels, e.g., a fluorescent label, a luminescent label, and/or a non-optically detectable label.

For example, a secretory vesicle marker, which is attached with a detectable label, may be expressed in the subject, e.g., a cell. For example, the presence and/or amount of the signal produced by the detectable label may indicate the presence and/or amount of the secretory vesicle marker, and the signal may be used for characterizing a migrasome and/or an intraluminal vesicle in a migrasome.

For example, wherein said secretory vesicle marker may comprise a Rab protein and/or a SNAP Receptor (SNARE) .

For example, wherein said Rab protein may comprise Rab8.

For example, wherein said SNARE may comprise a t-SNARE and/or a v-SNARE.

For example, wherein said t-SNARE may comprise SNAP23.

For example, wherein said v-SNARE may comprise VAMP2 and/or VAMP7.

For example, further comprising determining the presence and/or amount of Tspan4, Integrin, Pleckstrin Homology (PH) domain, NDST1, PIGK, CPQ, and/or EOGT.

For example, further comprising staining with wheatgerm agglutinin (WGA) .

ii) isolating the characterized migrasome and/or the characterized intraluminal vesicle, and/or administering a regulating agent to said characterized migrasome and/or said characterized intraluminal vesicle. For example, said intraluminal vesicle may comprise intraluminal vesicle in a migrasome. For example, said regulating agent may comprise an agent capable of regulating transportation of an intraluminal vesicle into a migrasome.

For example, which may be an in vitro or ex vivo method.

For example, which may be an in vivo method.

Agent capable of determining the presence and/or amount of a secretory vesicle marker

For example, which may be capable of specifically identifying said secretory vesicle marker.

For example, which may comprise an antibody against said marker or an antigen binding fragment thereof.

For example, further comprising a detectable label.

For example, which may comprise a modified secretory vesicle marker containing a detectable label.

For example, wherein said Rab protein may comprise Rab8.

For example, wherein said SNARE may comprise a t-SNARE and/or a v-SNARE.

For example, wherein said t-SNARE may comprise SNAP23.

For example, wherein said v-SNARE may comprise VAMP2 and/or VAMP7.

Use, composition, and kit

For example, the composition further comprises a second agent capable of determining the presence and/or amount of a migrasome.

For example, wherein said second agent may be capable of determining the presence and/or amount of Tspan4, Integrin, Pleckstrin Homology (PH) domain, NDST1, PIGK, CPQ, and/or EOGT.

For example, wherein said second agent may comprise WGA.

For example, wherein said agent of the present application may be not mixed with said second agent.

Regulating the secretion of a substance by a migrasome

For example, the present application provides a method for regulating the secretion of a substance by a migrasome. For example, regulating the secretion of a substance by a migrasome comprises regulating the number and/or function of an intraluminal vesicle in the migrasome.

For example, the secretion of a substance by a migrasome may be promoted. For example, the amount of the substance secreted by a migrasome may be 0.1%higher, 1%higher, 10%higher, 20%higher, 30%higher, 40%higher, 50%higher, 60%higher, 70%higher, 80%higher, 90%higher, 95%higher, 99%higher, 100%higher, 2 times higher, 3 times higher, 4 times higher, 5 times higher, 10 times higher, 50 times higher, 100 times higher, 1000 times higher, or 10000 times higher than the untreated migrasome or the migrasome before treating. For example, the amount of the substance secreted by a migrasome may be analyzed by western blot, Immunofluorescence and/or quantitative mass spectrometry analysis.

For example, the secretion of a substance by a migrasome may be inhibited. For example, the amount of the substance secreted by a migrasome may be 0.1%lower, 1%lower, 10%lower, 20%lower, 30%lower, 40%lower, 50%lower, 60%lower, 70%lower, 80%lower, 90%lower, 95%lower, 99%lower, 100%lower, 2 times lower, 3 times lower, 4 times lower, 5 times lower, 10 times lower, 50 times lower, 100 times lower, 1000 times lower, or 10000 times lower than the untreated migrasome or the migrasome before treating. For example, the amount of the substance secreted by a migrasome may be analyzed by western blot, Immunofluorescence and/or quantitative mass spectrometry analysis.

For example, wherein said migrasome may be generated by a cell.

For example, wherein said cell may be a migrating cell.

For example, wherein said cell may be a circulating cell.

For example, wherein said cell may be a cell in or from the blood.

For example, wherein said cell may be an immune cell.

For example, wherein said immune cell may comprise a monocyte, a macrophage, a neutrophil and/or a T cell.

For example, wherein said substance to be secreted may be comprised in said intraluminal vesicle.

For example, wherein said substance to be secreted may comprise one or more signaling molecules.

Substance to be secreted

For example, the intraluminal vesicle may comprise substance to be secreted. For example, the signaling function of the intraluminal vesicle may be depend on releasing substance to be secreted. For example, substance to be secreted may be expressed and secreted from the cell, the migrasome and/or the intraluminal vesicle. For example, the substance to be secreted may comprise signaling molecule. For example, substance to be secreted may comprise a signal peptide. For example, substance to be secreted may be a cytokine and/or a flavonoid.

Signaling molecules

For example, the intraluminal vesicle may comprise signaling molecule. For example, the signaling function of the intraluminal vesicle may be depend on releasing signaling molecule. For example, signaling molecule may be expressed and secreted from the cell, the migrasome and/or the intraluminal vesicle. For example, signaling molecule may comprise a signal peptide. For example, signaling molecule may be a cytokine and/or a flavonoid.

For example, wherein said substance to be secreted may comprise one or more protein or polypeptide containing a signal peptide.

For example, which increases the secretion of the substance, and may comprise increasing the number and/or function of the intraluminal vesicles in said migrasome.

Increase the number of intraluminal vesicles

For example, the number of intraluminal vesicles that is in the migrasome may be increasing. For example, the number of intraluminal vesicles that has been in the migrasome may be increasing. For example, the number of intraluminal vesicles that would be transported in the migrasome may be increasing.

For example, increasing the number of intraluminal vesicles may comprise increasing the number of intraluminal vesicles in the cell. For example, increasing the number of intraluminal vesicles may comprise increasing the transportation of the intraluminal vesicles into migrasome. For example, increasing the number of intraluminal vesicles may comprise increasing the amount and/or function of a motor protein in the cell.

For example, the number of intraluminal vesicles in the migrasome may be 0.1%higher, 1%higher, 10%higher, 20%higher, 30%higher, 40%higher, 50%higher, 60%higher, 70%higher, 80%higher, 90%higher, 95%higher, 99%higher, 100%higher, 2 times higher, 3 times higher, 4 times higher, 5 times higher, 10 times higher, 50 times higher, 100 times higher, 1000 times higher, or 10000 times higher in migrasome generated by treated cell, than the untreated cell or the cell before treating. For example, the number of intraluminal vesicles in the migrasome may be analyzed by confocal imaging analysis and/or Transmission Electron Microscopy analysis.

For example, wherein said increasing the number and/or function of the intraluminal vesicles in said migrasome may comprise increasing the transportation of the intraluminal vesicles into said migrasome.

For example, wherein said increasing the number and/or function of the intraluminal vesicles in said migrasome may comprise increasing the amount and/or function of a motor protein in said cell.

For example, wherein said increasing the number and/or function of the intraluminal vesicles in said migrasome may comprise increasing fusion of the membrane of said migrasome with the intraluminal vesicle therein.

Increase fusion of the membrane of a migrasome with an intraluminal vesicle therein

For example, the present application provides an engineered cell which has been modified to increase fusion of the membrane of migrasome with the intraluminal vesicle therein. For example, the amount of the intraluminal vesicle, which is fusing or fused with migrasome, may be increasing. For example, the amount of the migrasome, which is fusing or fused with intraluminal vesicle therein, may be increasing. For example, the amount of merged membrane, which is derived from migrasome and the intraluminal vesicle therein, may be increasing. For example, the amount of the marker, which is derived from the intraluminal vesicle, may be increasing on the membrane of migrasome.

For example, the amount of the marker, which is derived from the intraluminal vesicle, may be 0.1%higher, 1%higher, 10%higher, 20%higher, 30%higher, 40%higher, 50%higher, 60%higher, 70%higher, 80%higher, 90%higher, 95%higher, 99%higher, 100%higher, 2 times higher, 3 times higher, 4 times higher, 5 times higher, 10 times higher, 50 times higher, 100 times higher, 1000 times higher, or 10000 times higher on the membrane of migrasome from the engineered cell, than the unmodified cell or the cell before modifying. For example, amount of the marker may be analyzed by western blot, Immunofluorescence and/or quantitative mass spectrometry analysis.

For example, wherein increasing said fusion may comprise increasing the amount and/or function of a SNARE complex in said migrasome.

For example, wherein increasing said fusion may comprise increasing calcium in said migrasome. For example, wherein increasing said fusion may comprise introducing calcium in said migrasome.

For example, which inhibits the secretion of the substance, and may comprise reducing the number and/or function of the intraluminal vesicles in said migrasome.

For example, wherein said reducing the number and/or function of the intraluminal vesicles in said migrasome may comprise inhibiting the transportation of the intraluminal vesicles into said migrasome.

For example, wherein said reducing the number and/or function of the intraluminal vesicles in said migrasome may comprise reducing the amount and/or function of a motor protein in said cell.

For example, wherein said reducing the number and/or function of the intraluminal vesicles in said migrasome may comprise inhibiting fusion of the membrane of said migrasome with the intraluminal vesicle therein.

Inhibit fusion of the membrane of said migrasome with an intraluminal vesicle therein

For example, the present application provides an engineered cell which has been modified to inhibit fusion of the membrane of migrasome with the intraluminal vesicle therein. For example, the amount of the intraluminal vesicle, which is fusing or fused with migrasome, may be decreasing. For example, the amount of the migrasome, which is fusing or fused with intraluminal vesicle therein, may be decreasing. For example, the amount of merged membrane, which is derived from migrasome and the intraluminal vesicle therein, may be decreasing. For example, the amount of the marker, which is derived from the intraluminal vesicle, may be decreasing on the membrane of migrasome.

For example, the amount of the marker, which is derived from the intraluminal vesicle, may be 0.1%lower, 1%lower, 10%lower, 20%lower, 30%lower, 40%lower, 50%lower, 60%lower, 70%lower, 80%lower, 90%lower, 95%lower, 99%lower, 100%lower, 2 times lower, 3 times lower, 4 times lower, 5 times lower, 10 times lower, 50 times lower, 100 times lower, 1000 times lower, or 10000 times lower on the membrane of migrasome from the engineered cell, than the unmodified cell or the cell before modifying. For example, amount of the marker may be analyzed by western blot, Immunofluorescence and/or quantitative mass spectrometry analysis.

For example, wherein inhibiting said fusion may comprise reducing the amount and/or function of a SNARE complex in said migrasome.

For example, wherein reducing the amount and/or function of a SNARE complex may comprise inhibiting the expression of one or more components of the SNARE complex.

For example, wherein reducing the amount and/or function of a SNARE complex may comprise knocking out or knocking down the expression of a gene encoding for one or more components of the SNARE complex.

For example, wherein inhibiting said fusion may comprise reducing calcium in said migrasome.

For example, wherein reducing calcium in said migrasome may comprise administering a calcium chelator.

For example, wherein said calcium chelator may comprise BAPTA-AM.

Reducing calcium in migrasome

For example, reducing calcium in migrasome comprises administering an agent capable of regulating calcium in migrasome. For example, administering a calcium chelator. For example, administering EGTA, EDTA, BAPTA and/or BAPTA-AM or derivative thereof. For example, the amount of calcium in migrasome may be 0.1%lower, 1%lower, 10%lower, 20%lower, 30%lower, 40%lower, 50%lower, 60%lower, 70%lower, 80%lower, 90%lower, 95%lower, 99%lower, 100%lower, 2 times lower, 3 times lower, 4 times lower, 5 times lower, 10 times lower, 50 times lower, 100 times lower, 1000 times lower, or 10000 times lower than the untreated migrasome or the migrasome before treating. For example, the amount of calcium in migrasome may be analyzed by calcium assay kit.

For example, wherein said motor protein may comprise a Myosin.

For example, wherein said SNARE may comprise a v-SNARE and/or a t-SNARE.

For example, wherein said t-SNARE may comprise SNAP23.

For example, wherein said v-SNARE may comprise VAMP2 and/or VAMP7.

SNARE complex and its regulation

For example, intraluminal vesicle may comprise a SNAP Receptor (SNARE) . For example, formation of SNARE complex may mediate the fusion of the membrane of migrasome with the intraluminal vesicle. For example, SNARE may comprise vesicle-SNAREs or v-SNAREs, which are incorporated into the membranes of transport vesicles during budding. For example, the v-SNARE may comprise SNAP23. For example, SNARE may comprise target-SNAREs or t-SNAREs, which are associated with nerve terminal membranes. SNARE complex may be the components of the fusion machinery. For example, the t-SNARE may comprise VAMP2 and/or VAMP7. For example, during membrane fusion, v-SNARE and t-SNARE on separate membranes may combine to form a SNARE complex.

For example, regulating SNARE complex may comprise regulating the amount and/or function of member of SNARE complex family.

For example, increasing the amount and/or function of SNARE complex may comprise providing member of SNARE complex family, overexpressing member of SNARE complex family, and/or activating member of SNARE complex family. For example, increasing the amount and/or function of SNARE complex may comprise introducing member of SNARE complex family and/or a gene encoding for member of SNARE complex family. For example, increasing the amount and/or function of SNARE complex may comprise activating the interaction between the member of SNARE complex family.

For example, decreasing the amount and/or function of SNARE complex may comprise knocking out the expression of a gene encoding for member of SNARE complex family, knocking down the expression of a gene encoding for member of SNARE complex family, and/or treating the migrasome with an agent capable of inhibiting the function of member of SNARE complex family. For example, decreasing the amount and/or function of SNARE complex may comprise introducing CRISPR Cas9 system and/or miRNA targeting member of SNARE complex family. For example, decreasing the amount and/or function of SNARE complex may comprise inhibiting the interaction between the member of SNARE complex family. For example, decreasing the amount and/or function of SNARE complex may comprise binding to the member of SNARE complex family.

Agent capable of regulating the number and/or function of an intraluminal vesicle in a migrasome

For example, wherein said migrasome may be generated by a cell.

For example, wherein said cell may be a migrating cell.

For example, wherein said cell may be a circulating cell.

For example, wherein said cell may be a cell in or from the blood.

For example, wherein said cell may be an immune cell.

For example, wherein said substance to be secreted may comprise a cytokine and/or a flavonoid.

For example, for use in increasing the secretion of said substance, and the agent may be capable of increasing the number and/or function of the intraluminal vesicles in said migrasome.

For example, which may be capable of increasing the transportation of the intraluminal vesicles into said migrasome.

For example, which may be capable of increasing the amount and/or function of a motor protein in said cell.

For example, which may be capable of increasing fusion of the membrane of said migrasome with the intraluminal vesicle therein.

For example, which may be capable of increasing the amount and/or function of a SNARE complex in said migrasome.

For example, which may be capable of increasing calcium in said migrasome.

For example, for use in inhibiting the secretion of said substance, and the agent may be capable of reducing the number and/or function of the intraluminal vesicles in said migrasome.

For example, which may be capable of inhibiting the transportation of the intraluminal vesicles into said migrasome.

For example, which may be capable of reducing the amount and/or function of a motor protein in said cell.

For example, which may be capable of inhibiting fusion of the membrane of said migrasome with the intraluminal vesicle therein.

For example, which may be capable of reducing the amount and/or function of a SNARE complex in said migrasome.

For example, which may be capable of inhibiting the expression of one or more components of the SNARE complex.

For example, which may be capable of knocking out or knocking down the expression of a gene encoding for one or more components of the SNARE complex.

For example, which may be capable of reducing calcium in said migrasome.

For example, which may comprise a calcium chelator.

For example, wherein said calcium chelator may comprise BAPTA-AM.

For example, wherein said motor protein may comprise a Myosin.

For example, wherein said SNARE may comprise a v-SNARE and/or a t-SNARE.

For example, wherein said t-SNARE may comprise SNAP23.

For example, wherein said v-SNARE may comprise VAMP2 and/or VAMP7.

Engineered cell with altered ability for regulating the secretion of a substance by a migrasome generated by said cell

For example, this patent application provides an agent capable of modifying an engineered cell by altering the number and/or function of an intraluminal vesicle in said migrasome, for use in preparing an engineered cell with altered ability for regulating the secretion of a substance by a migrasome generated by said cell comparing to a corresponding unmodified cell.

For example, this patent application provides a method for preparing an engineered cell with altered ability for regulating the secretion of a substance by a migrasome generated by said cell comparing to a corresponding unmodified cell, the method comprises modifying a cell by altering the number and/or function of an intraluminal vesicle in said migrasome.

For example, wherein said cell may be a migrating cell.

For example, wherein said cell may be a circulating cell.

For example, wherein said cell may be a cell in or from the blood.

For example, wherein said cell may be an immune cell.

For example, which has increased ability for the secretion of the substance, and said engineered cell has been modified to increase the number and/or function of the intraluminal vesicles in said migrasome.

For example, which has been modified to increase the transportation of the intraluminal vesicles into said migrasome.

For example, which has been modified to increase the amount and/or function of a motor protein in said cell.

For example, which has been modified to increase fusion of the membrane of said migrasome with the intraluminal vesicle therein.

For example, which has been modified to increase the amount and/or function of a SNARE complex in said migrasome.

For example, which has been modified to increase calcium in said migrasome.

For example, which has reduced ability for the secretion of the substance, and said engineered cell has been modified to reduce the number and/or function of the intraluminal vesicles in said migrasome.

For example, which has been modified to inhibit the transportation of the intraluminal vesicles into said migrasome.

For example, which has been modified to reduce the amount and/or function of a motor protein in said cell.

For example, which has been modified to inhibit fusion of the membrane of said migrasome with the intraluminal vesicle therein.

For example, which has been modified to reduce the amount and/or function of a SNARE complex in said migrasome.

For example, which has been modified to inhibit the expression of one or more components of the SNARE complex.

For example, wherein the expression of a gene encoding for one or more components of the SNARE complex has been knocked down or knocked out.

For example, which has been modified to reduce calcium in said migrasome.

For example, which has been treated with a calcium chelator.

For example, wherein said calcium chelator may comprise BAPTA-AM.

For example, wherein said motor protein may comprise a Myosin.

For example, wherein said SNARE may comprise a v-SNARE and/or a t-SNARE.

For example, wherein said t-SNARE may comprise SNAP23.

For example, wherein said v-SNARE may comprise VAMP2 and/or VAMP7.

Use, composition and kit

Migrasomes are vesicular organelles which grow on retraction fibers of migrating cells and are released when the cell moves away. It shows that secretory vesicles are transported into and released locally from the migrasome via fusion, or released with the migrasome as its intraluminal vesicles. In monocytes, cytokines such as TNF-α and IL-6 are enriched in migrasomes. In vitro and in vivo, cytokine secretion is reduced when migrasome formation is compromised, which suggests that migrasomes are required for efficient cytokine secretion. It shows monocyte-derived, cytokine-enriched migrasomes in blood from LPS-treated mice, which indicates that cytokines can be released en masse in migrasome-bound form. It shows that migrasomes mediate both localized secretion and packaged release of cytokines, and thus are portals for secretion in migrating cells.

Secretion is a fundamental process of cells. Cells communicate with each other by secreting signaling ligands such as cytokines and neurotransmitters. However, highly localized secretion on specialized membrane structures has only been found in neurons, in which secretory vesicles are transported from the cell body to the axon terminal. The basic unit of secretion is the secretory vesicle. Once the secretory vesicle fuses with plasma membrane, the cargos are released from the cell. It is unknown whether there are specialized cellular structures which can package and release secretory cargos en masse.

Migrasomes are newly discovered organelles in migrating cells. When cells migrate, long membrane tethers named retraction fibers are left on the trailing edge of the cell. Large vesicles, named migrasomes, with diameters of around 2 μm grow on the retraction fibers. Inside migrasomes are numerous small intraluminal vesicles of unknown origin. Members of the tetraspanin protein family are key players in migrasome formation. Migrasome formation is enhanced by overexpression of certain tetraspanins such as Tspan4 and Tspan9, and reduced by knockout of migrasome-forming tetraspanins. Migrasomes have been observed in various biological settings and have been shown to play important physiological roles in vivo. How these signaling ligands are transported and released from migrasomes is currently unknown.

In this application, it shows the role of migrasome in secretion. Secretory vesicles may be translocated into migrasome by actin-based motor protein, once reach migrasome, secretory vesicles can fuse with migrasome membrane and release its content. Moreover, it shows a package of cytokines may be released from cell as migrasome bound form, thus, migrasome are organelle for localized secretion and packaged releasing of secretory proteins in migrating cells (Fig. 5L) , in a sense, migrasomes are combination of broadcasting tower which broadcasting signal locally, and satellite which is lunched from ground and broadcasting signal remotely.

It shows migrasome not only mediated localized secretion, but in some case, migrasome may serve as the main site for secretion. It shows both in vivo and in vitro, the secretion of monocyte is coupled with its ability to generate migrasome. One possible explanation is t-SNARE such as SNAP23 are highly enriched on migrasome compare to the cell body, thus, once secretory vesicles are transported to the trail edge of cells, the fusion between these vesicles with plasma membrane is limited due to the insufficient t-SNARE on plasma membrane, instead, it is transported into migrasome and fusion occur there. For example, it shows that 1) t-SNARE are enriched on membrane migrasome than cell body, 2) accumulation cluster of secretory vesicles at plasma membrane to migrasome at the trail edge of cell.

It shows after LPS treatment, monocyte releasing migrasome enriched with cytokines such as TNF-α, IL-6 and IFN-γ □into blood steam. Theoretically, packaged releasing of a set of cytokines by migrasome may have profound difference with releasing cytokines as soluble form. Frist, as μm scaled vesicles, the dynamic of cytokines enriched migrasome may be vastly different from soluble cytokines in blood stream, which will resulting in different spatiotemporal distribution of cytokines in vivo. Secondly, since cytokines are enriched on migrasome, so once reach the site of action, migrasome bound cytokines could reach a much higher local concentration than soluble cytokine in blood stream. Thirdly, since a set of cytokines could pack in a single migrasome and released simultaneously, thus theoretically migrasome may deliver a combination of signals which is synergistic in nature, such as TNF-α and IL-6. Finally, releasing of cytokines from detached migrasome may take time, thus, the migrasome may work as a sustained-release capsule to archive the latency of cytokine releasing. For this reason, migrasome mediated packaged releasing of cytokines may play important roles in immune-response.

Migrasome may be defined as an organelle rather than an extracellular vesicle based on its biogenesis process. It shows before migrasome detached from cell body, migrasome is the site of localized secretion, thus, migrasome carried out important cell autonomous function before it released from cell as extracellular vesicle, it shows that migrasome is an organelle rather than an extracellular vesicle, in this view, detached migrasome may be an extracellular vesicle, migrasome is an organelle for localized secretion, for mitochondria homeostasis and for generating of extracellular vesicles (Fig. 5L) .

Items

Items 1

1.1. A method for regulating the formation and/or function of a migrasome and/or a migrasome mediated biological process, comprising regulating transportation of an intraluminal vesicle into said migrasome.

1.2. The method of item 1.1, wherein regulating the transportation of said intraluminal vesicle comprises regulating the amount and/or function of a motor protein in a cell generating said migrasome.

1.3. The method of item 1.2, which increases the transportation of said intraluminal vesicle into said migrasome and comprises increasing the amount and/or function of said motor protein in said cell.

1.4. The method of item 1.3, wherein increasing the amount and/or function of the motor protein comprises overexpressing said motor protein, a functional fragment thereof, a motor domain thereof, and/or a functional variant thereof in said cell.

1.5. The method of item 1.2, which decreases the transportation of said intraluminal vesicle into said migrasome and comprises decreasing the amount and/or function of said motor protein in said cell.

1.6. The method of item 1.5, wherein decreasing the amount and/or function of said motor protein comprises knocking out or knocking down the expression of a gene encoding for said motor protein in said cell.

1.7. The method of any one of items 1.5-1.6, wherein decreasing the amount and/or function of said motor protein comprises treating said cell with a motor protein inhibitor.

1.8. The method of any one of items 1.2-1.7, wherein said motor protein is a Myosin family member.

1.9. The method of any one of items 1.2-1.8, wherein said motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.

1.10. The method of any one of items 1.1-1.9, which is an in vivo method.

1.11. The method of any one of items 1.1-1.9, which is an in vitro method or an ex vivo method.

1.12. An agent capable of regulating transportation of an intraluminal vesicle into a migrasome, for use in regulating migrasome formation, function, and/or a migrasome-mediated biological process.

1.13. The agent of item 1.12, which is capable of regulating the amount and/or function of a motor protein in a cell generating said migrasome.

1.14. The agent of item 1.13, which is capable of increasing the amount and/or function of the motor protein, for use in increasing the transportation of said intraluminal vesicle into said migrasome.

1.15. The agent of item 1.14, which is capable of resulting in overexpression of said motor protein, a motor domain thereof, a functional fragment thereof, and/or a functional variant thereof in said cell.

1.16. The agent of item 1.15, which comprises the motor protein, a motor domain thereof, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.

1.17. The agent of item 1.13, which is capable of decreasing the amount and/or function of the motor protein, for use in decreasing the transportation of said intraluminal vesicle into said migrasome.

1.18. The agent of item 1.17, which is capable of knocking out or knocking down the expression of a gene encoding for said motor protein in said cell.

1.19. The agent of any one of items 1.13-1.18, which comprises a motor protein inhibitor.

1.20. The agent of any one of items 1.13-1.19, wherein said motor protein is a Myosin family member.

1.21. The agent of any one of items 1.13-1.20, wherein said motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.

1.22. An engineered cell with altered ability for forming migrasomes containing intraluminal vesicles comparing to a corresponding unmodified cell, said engineered cell has been modified to alter transportation of the intraluminal vesicles into said migrasome.

1.23. The engineered cell of item 1.22, wherein the expression and/or function of a motor protein has been modified.

1.24. The engineered cell of any one of items 1.22-1.23, wherein the expression and/or function of the motor protein has been increased.

1.25. The engineered cell of any one of items 1.23-1.24, wherein said motor protein, a motor domain thereof, a functional fragment thereof, and/or a functional variant thereof has been overexpressed.

1.26. The engineered cell of any one of items 1.22-1.23, wherein the expression and/or function of the motor protein has been decreased.

1.27. The engineered cell of any one of items 1.22-1.23 and 1.26, wherein a gene encoding for said motor protein has been knocked out or knocked down.

1.28. The engineered cell of any one of items 1.22-1.23 and 1.26-1.27, which has been treated with a motor protein inhibitor.

1.29. The engineered cell of any one of items 1.23-1.28, wherein said motor protein is a Myosin family member.

1.30. The engineered cell of any one of items 1.23-1.29, wherein said motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.

1.31. Use of the agent according to any one of items 1.12-1.21 and/or the engineered cell according to any one of items 1.22-1.30 in the preparation of a regulator for: i) migrasome formation and/or function; and/or ii) a migrasome-mediated biological process.

1.32. A composition, comprising the agent according to any one of items 1.12-1.21, and/or the engineered cell according to any one of items 1.22-1.30.

1.33. The composition of item 1.32, which is a pharmaceutical composition and optionally comprises a pharmaceutically acceptable excipient.

1.34. A kit, comprising the agent according to any one of items 1.12-1.21, the engineered cell according to any one of items 1.22-1.30, and/or the composition according to any one of items 1.32-1.33.

Items 2

2.1. A method for characterizing a migrasome and/or an intraluminal vesicle in a migrasome, comprising determining the presence and/or amount of a secretory vesicle marker.

2.2. The method of item 2.1, comprising determining the presence and/or amount of the secretory vesicle marker in the migrasome.

2.3. The method of any one of items 2.1-2.2, wherein said determining comprises using an agent capable of specifically identifying said secretory vesicle marker.

2.4. The method of item 2.3, wherein said agent capable of specifically identifying said secretory vesicle marker comprises an antibody against said marker or an antigen binding fragment thereof.

2.5. The method of item 2.4, wherein said antigen binding fragment comprises Fab, F (ab) 2, F (ab’) , F (ab’) 2, scFv, affibody and/or VHH.

2.6. The method of any one of items 2.3-2.5, wherein said agent capable of specifically identifying said secretory vesicle marker further comprises a detectable label.

2.7. The method of any one of items 2.1-2.6, wherein said determining comprises detecting the presence and/or amount of a modified secretory vesicle marker containing a detectable label.

2.8. The method of any one of items 2.6-2.7, wherein said detectable label comprises a fluorescent label, a luminescent label, and/or a non-optically detectable label.

2.9. The method of any one of items 2.1-2.8, wherein said secretory vesicle marker comprises a Rab protein and/or a SNAP Receptor (SNARE) .

2.10. The method of item 2.9, wherein said Rab protein comprises Rab8.

2.11. The method of any one of items 2.9-2.10, wherein said SNARE comprises a t-SNARE and/or a v-SNARE.

2.12. The method of item 2.11, wherein said t-SNARE comprises SNAP23.

2.13. The method of any one of items 2.11-2.12, wherein said v-SNARE comprises VAMP2 and/or VAMP7.

2.14. The method of any one of items 2.1-2.13, further comprising determining the presence and/or amount of Tspan4, Integrin, Pleckstrin Homology (PH) domain, NDST1, PIGK, CPQ, and/or EOGT.

2.15. The method of any one of items 2.1-2.14, further comprising staining with wheatgerm agglutinin (WGA) .

2.16. A method for isolating and/or regulating a migrasome and/or an intraluminal vesicle in a migrasome, comprising:

i) characterizing the migrasome and/or the intraluminal vesicle in a migrasome according to the method of any one of items 2.1-2.15; and

2.17. The method of any one of items 2.1-2.16, which is an in vitro or ex vivo method.

2.18. The method of any one of items 2.1-2.16, which is an in vivo method.

2.19. An agent capable of determining the presence and/or amount of a secretory vesicle marker, for use in characterizing a migrasome and/or an intraluminal vesicle in a migrasome.

2.20. The agent of item 2.19, which is capable of specifically identifying said secretory vesicle marker.

2.21. The agent of any one of items 2.19-2.20, which comprises an antibody against said marker or an antigen binding fragment thereof.

2.22. The agent of item 2.21, wherein said antigen binding fragment comprises Fab, F (ab) 2, F (ab’) , F (ab’) 2, scFv, affibody and/or VHH.

2.23. The agent of any one of items 2.19-2.22, further comprising a detectable label.

2.24. The agent of item 2.23, which comprises a modified secretory vesicle marker containing a detectable label.

2.25. The agent of any one of items 2.23-2.24, wherein said detectable label comprises a fluorescent label, a luminescent label, and/or a non-optically detectable label.

2.26. The agent of any one of items 2.19-2.25, wherein said secretory vesicle marker comprises a Rab protein and/or a SNAP Receptor (SNARE) .

2.27. The agent of item 2.26, wherein said Rab protein comprises Rab8.

2.28. The agent of any one of items 2.26-2.27, wherein said SNARE comprises a t-SNARE and/or a v-SNARE.

2.29. The agent of item 2.28, wherein said t-SNARE comprises SNAP23.

2.30. The agent of any one of items 2.28-2.29, wherein said v-SNARE comprises VAMP2 and/or VAMP7.

2.31. A composition, comprising the agent according to any one of items 2.19-2.30.

2.32. The composition of item 2.31, further comprising a second agent capable of determining the presence and/or amount of a migrasome.

2.33. The composition of item 2.32, wherein said second agent is capable of determining the presence and/or amount of Tspan4, Integrin, Pleckstrin Homology (PH) domain, NDST1, PIGK, CPQ, and/or EOGT.

2.34. The composition of any one of items 2.32-2.33, wherein said second agent comprises WGA.

2.35. The composition of any one of items 2.32-2.34, wherein said agent according to any one of items 2.19-2.30 is not mixed with said second agent.

2.36. A kit, comprising the agent according to any one of items 2.19-2.30, and/or the composition according to any one of items 2.31-2.35.

2.37. Use of the agent according to any one of items 2.19-2.30, the composition according to any one of items 2.31-2.35, and/or the kit according to item 2.36, in the preparation of an indicator for a migrasome and/or an intraluminal vesicle in a migrasome.

Examples

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i.m., intramuscular (ly) ; i.p., intraperitoneal (ly) ; s.c., subcutaneous (ly) ; r.t., room temperature; and the like.

Reagents and antibodies

Fibronectin (PHE0023) , WGA (W7024) , Puromycin (A1113803) , Prolong Live Antifade Reagent (P36975) and Lipofectamine 3000 (L3000001) were purchased from ThermoFisher. BAPTA-AM (A1076) , Diaminobenzidine (D12384) , Propidium Iodide (81845) , LPS (L2630) and Proteinase K (P8811) were purchased from Sigma-Aldrich. Z-VAD-FMK (S7023) was purchased from Selleck. Vigofect (T001) was purchased from Vigorous. G418 (E859) was purchased from Amresco. Hygromycin B (10843555001) was purchased from Roche.

Anti-Rab8a (ab188574) , anti-SNAP23 (ab4114) and anti-TACE (ab2051) antibodies were from Abcam. Anti-Myosin5a (#3402) , anti-Rab8a (#6975) , anti-VAMP2 (#13508) , anti-TNF-α (#11948) , anti-IL-6 (#12912) , anti-Integrin α5 (#4705) , anti-Integrin β1 (#4706) and anti-Atp1α1 (#3010) , anti-Slc1a5 (#8057) antibodies were from Cell Signaling Technology. Anti-GAPDH (60004-1-Ig) and anti-VAMP7 (22268-1-AP) antibodies were from Proteintech Group. Anti-CPQ (HPA023235-100UL) antibody was from Sigma-Aldrich. PE anti-mouse CCR2 (150609) , APC anti-mouse CCR2 (150627) , PE anti-mouse F4/80 (123109) , APC anti-mouse F4/80 (123115) , PE anti-mouse CD115 (135505) , APC anti-mouse CD115 (135509) , APC anti-mouse Ly6C (128015) and Biotin anti-mouse CD115 (135507) antibodies were from BioLegend. Goat anti-Rabbit IgG (111-035-003) and Goat anti-Mouse IgG (115-035-003) were from Jackson.

Cells

L929 cells were grown in DMEM (C11995500BT, Gibco) supplemented with 10%FBS (04-001-1A, Biological Industries) , 2 mM GlutaMAX (35050-061, Gibco) and 100 U/mL penicillin-streptomycin (GNM15140, GENOM) . Cells were cultured at 37 ℃ in an incubator with 5%CO₂.

Mouse bone marrow monocytes (BMMs) were acquired from WT or T9 KO C57BL/6 mice as described in the 10q26 Risk Haplotype of Age-Related using the EasySep mouse monocyte isolation kit (#19861, STEMCELL Technologies) . Briefly, bone marrow cell suspensions (1 × 10⁸ cells/mL) were incubated with isolation cocktail component and dextran rapidspheres sequentially. Unwanted cells (T cells, B cells, NK cells, DCs, etc. ) were labeled with antibodies combined with magnetic particles, and separated from unlabeled cells (monocytes) by a magnet. For purity assessment, cells stained with CD115 and Ly6C antibodies were analyzed by flow cytometry. BMMs were grown in RPMI 1640 (C11875500BT, Gibco) supplemented with 10%FBS, 2 mM GlutaMAX and 100 U/mL penicillin-streptomycin in 5%CO₂ at 37℃. BMMs were activated with 500 ng/mL LPS for 12-24 hr.

Mice

CRISPR/Cas9 was used to generate TSPAN9 KO C57BL/6 mice as described in Mitocytosis, a migrasome-mediated process (Jiao H, Jiang D, Hu X, et al. Cell, 2021, 184 (11) : 2896-2910. e13) . The sgRNA sequence designed to target exon 4 of mouse TSPAN9 was 5’-GAAGGTGGCGAAGTTGCCTT-3’ (SEQ ID NO: 1) . Gender and age matched WT C57BL/6 mice were used as controls for T9 KO animals. WT C57BL/6 mice were from Animal facility at Tsinghua University. Animals were maintained in a light-and temperature-regulated room under specific pathogen-free (SPF) conditions. Animal studies and experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee at Tsinghua University, and were performed following the guidelines of the Laboratory Animal Research Center of Tsinghua University.

Cell transfection and virus infection

Cell transfection was conducted using Vigofect according to the manufacturer’s manual.

The PiggyBac Transposon Vector System had been used to generate stably expressing cell lines as described in Mitocytosis, a migrasome-mediated mitochondrial quality-control process (Jiao H, Jiang D, Hu X, et al. Cell, 2021, 184 (11) : 2896-2910. e13) . Briefly, various proteins were cloned into pB-CAG (transposon vector) as the expressing plasmid backbone. Constructed pB-CAG combined with pBASE (transposase vector) was co-transfected into L929 cells at a ratio of 1: 3 using the above Vigofect transfection protocol. After 24 hr, the cells were treated with 600 μg/mL G418 or 200 μg/mL hygromycin B for selection (3-5 days) . Single cells were sorted in 96-well plates by flow cytometry. These single cell clones were cultured and expanded, followed by confocal analysis.

The lentivirus-based vector pLKO. 1-puro carrying shRNA was used to achieve gene knockdown. Lentiviral transduction and infection were performed as described in Chaperone-like protein p32 regulates. Briefly, for lentiviral production, lentiviral vectors (pLKO. 1, psPAX2 and pMD2. G) were co-transfected into L929 cells at a ratio of 4: 3: 1. After 48 hr, the supernatant was centrifuged at 600 g for 5 min to remove cell debris. Viruses were harvested and used in the following experiments. For virus infection, the indicated cells seeded at 40-60%confluence were co-cultured with virus containing 8 μg/mL polybrene for 24 hr. The cells were replaced with fresh medium containing 5 μg/mL puromycin for selection until drug-resistant colonies became visible. The sequence for constructing siRNAs to mouse SNAP23 in pLKO. 1-puro was 5’-GAACAACTAAATCGCATAGAA-3’ (SEQ ID NO: 2) .

The CRISPR/Cas9-2hitKO system was used to generate gene knockouts in L929 cells. For the generation of Myosin5a KO cell line, two guide RNA (sgRNA) coding sequences were cloned into PX458M. The cells were transfected with PX458M containing Myosin5a targeting sequences (sgRNA sequences: 5’-GTGCCGGTATGCGCCAGGCA-3’ (SEQ ID NO: 3) and 5’-AGTTCGCTTCATCGATTCCA-3’ (SEQ ID NO: 4) ) . After single cell sorting by flow cytometry, these single cell clones were further determined by PCR and Western blot.

Cell imaging and image analysis

10 μg/mL FN was used to pre-coat confocal dishes at 37 ℃ for at least 1 hour. For confocal snapshot images, cells were cultured in FN-precoated confocal dishes for 10-12 hr, and imaged using a NIKON A1RSiHD25 laser scanning confocal microscope at 1024 × 1024 pixels. Z-stack imaging of cells and migrasomes were performed with NIKON A1 microscope. Structured illumination microscopy (SIM) images were acquired using a Nikon N-SIM Super Resolution Microscope.

For long-term time-lapse imaging, cells were grown in FN-precoated confocal dishes for 4-6 hr before imaging. Cells were then maintained in the living cell system (37 ℃, 5%CO₂) , and monitored by NIKON A1 microscope. Ultra-fast super-resolution time-lapse images were collected using a Grazing Incidence-Structured Illumination Microscopy (GI-SIM) . NIS-Elements analysis 5.4 software was used to deconvolute images acquired by NIKON A1 microscope. Z-projection and 3D reconstruction were performed with NIS-Elements 5.4. Images were processed using Image J and Imaris software 8.1.4, and statistical analyses were conducted by Graphpad Prism 8.

Intravital imaging

Spinning disk microscopy (Perkin Elmer) was used to acquire time-lapse multiple-view z-stack intravital imaging as described in Mitocytosis, a migrasome-mediated mitochondrialquality-control process (Jiao H, Jiang D, Hu X, et al. Cell, 2021, 184 (11) : 2896-2910. e13) . Briefly, for circulating monocytes imaging, LPS (12 mg/kg) was injected into mice by intraperitoneal injection (i.p. ) . C57BL6/J mice were injected with 5 mg WGA and 1 mg CCR2 antibody by intravenous injection (i.v. ) at 4-8 hr after LPS stimulation. WGA and CCR2 antibodies were used to label vessels and monocytes, respectively. After 5 min, mice were anaesthetized by i. p injection of avertin (375 mg/kg) . Subsequently, the anesthetized mice were anatomized to expose the liver, and the blood vessels on the surface were monitored by spinning disk microscopy.

For WT combined T9 KO monocytes imaging, monocytes isolated from WT and T9 KO mice were stained with PE anti-mouse CCR2 antibody and APC anti-mouse CCR2 antibody respectively. After washing with PBS, WT and T9 KO monocytes labeled with different color conjugated CCR2 antibodies were combined in equal amounts and injected into the spleen of C57BL6/J mice which had been injected with 5mg WGA by i.v. After anesthetized, the mice were anatomized to expose the liver, and the blood vessels on the surface were monitored by spinning disk microscopy. Images were processed using Image J and Imaris software 8.1.4, and statistical analyses were conducted by Graphpad Prism 8.

Isolation of migrasome from culture cell

Crude migrasomes were collected by differential centrifugation as described in Identification of markers for migrasome detection (Zhao X, Lei Y, Zheng J, et al. Cell Discov, 2019, 5, 27) . Briefly, cells and migrasomes in 15 cm dishes were gently harvested into 50ml tubes after trypsin digestion. All subsequent manipulations were conducted at 4℃. After double centrifugation at 600 g for 10 min at 4 ℃, the supernatant was centrifuged again at 2000 g for 20 min at 4 ℃ to remove the cell body and large debris. Crude migrasomes were then acquired as the pellet by centrifugation at 18,000 g for 30 min at 4 ℃.

High purity migrasome isolation was performed by iodixanol-sucrose density gradient centrifugation following the protocol set up in Lateral transfer of mRNA and protein by migrasomes modifies the recipient cells (Zhu M, Zou Q, Huang R, et al. Cell Res, 2021, 31: 237-240) . Briefly, crude migrasome pellets were resuspended in 800 μl extraction buffer, and then fractionated at 150,000g for 4 h at 4 ℃ in a multistep Optiprep dilution gradient. The step gradient was 50% (500 μl) , 40% (500 μl) , 35% (500 μl) , 30% (500 μl) , 25% (500 μl) , 20% (500 μl) , 15% (500 μl) , 10% (500 μl) , 5% (500 μl) and crude migrasomes (5%, 800 μl) . After centrifugation, samples were collected from top to bottom gently (500 μl per fraction) . Fractions 4, 5 and 6 were mixed with 500 μl PBS, respectively, and then centrifuged at 18,000g for 30 min at 4 ℃. The pellets were washed with PBS and centrifuged again at 18,000g for 30 min to precipitate migrasomes. The samples were immediately available for downstream applications such as Western blot analysis, TEM, and mass spectrometry.

Isolation of migrasome purification from blood

Mice blood was collected by cardiac puncture and mixed with the same volume of blood collection buffer (10 mM EDTA in PBS) on ice. The blood mixture was centrifuged at 600 g for 10 min at 4 ℃, followed by 2000 g, 4 ℃ for 20 min to remove the blood cells. Crude migrasomes were then collected as the pellets by centrifugation at 18,000 g for 30 min at 4 ℃.

To purify monocyte derived migrasomes, crude migrasomes were negatively selected by magnetic sorting using the EasySep mouse monocyte isolation kit. Briefly, the pellets containing the crude migrasome fraction were resuspended and then incubated with isolation cocktail component and dextran rapidspheres sequentially. Unwanted components were labeled with antibodies combined with magnetic particles, and separated from monocyte-derived migrasomes by a magnet.

For confocal imaging analysis, the anti-CCR2 antibody was first coated on cover glass, followed by incubation of the crude migrasome preparation with antibody coated surface, followed by washing and immunostaining.

Transmission electron microscopy

Cells were grown in 35 mm dishes precoated with fibronectin (10 μg/ml) . After 10-12 hr, cells were pre-fixed using a 1: 1 ratio of growth medium to 2.5%glutaraldehyde for 5 min at room temperature. Cells were further fixed with 2.5%glutaraldehyde in PB buffer for 2 hr at room temperature, washed three times with PBS and dehydrated in ascending gradual series of ethanol (50%, 70%, 90%, 95%, and 100%) for 8 min each. Samples were infiltrated with and embedded in SPON12 resin. After polymerization for 48 h at 60 ℃, ultrathin sections of 70 nm thick were cut with a diamond knife, and then collected with Formvar-coated copper grids (100 mesh) . The sections were double stained with uranyl acetate and lead citrate. After air drying, samples were examined with a transmission electron microscope H-7650B at an acceleration voltage of 80 kV.

Western blot

Cells or migrasomes were lysed by 2.5%SDS lysis buffer and boiled for 10-20 min at 95℃. The protein concentration of each sample was determined using the BCA kit. Proteins were separated by appropriate percentage of SDS-PAGE gels according to the MW of the target proteins, followed by electrophoretic transfer onto PVDF membranes. After blocking with 5%non-fat milk in TBST buffer, the membranes were incubated with primary antibody overnight at 4℃. Membranes were then incubated with secondary antibody (HRP) for 1 hr at room temperature, and signals were detected using WESTAR ηC 2.0 kit (CYANAGEN) .

The following primary antibodies were used for Western blot analysis at the indicated dilutions: anti-Myosin5a (1: 1000) , anti-SNAP23 (1: 1000) , anti-Slc1a5 (1: 2000) , anti-Atp1α1 (1: 5000) , anti-TNF α (1: 1000) , anti-IL-6 (1: 1000) , anti-CPQ (1: 1000) , anti-Integrin α5 (1: 1000) , anti-Actin (1: 5000) and anti-GAPDH (1: 5000) .

Statistical analysis

Statistical analyses were conducted using the unpaired two-tailed t test in Graphpad Prism 5 (or 8) software (Graphpad Software) . Error bars represented the mean ± SEM. Significance was indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS, not significant. Statistical parameters and significance were reported in the Figures and the Figure Legends.

Example 1 Characterization of intraluminal vesicles of migrasome

Intraluminal vesicles were present in migrasomes. In order to investigate the origin and identification of these vesicles in detail, transmission electron microscopy study was first carried out on migrasome-forming cells. It showed that the size and the number of intraluminal vesicles were correlated closely with the distance between the migrasome and cell body, the further away from the cells, the larger the migrasomes and the lower the number of intraluminal vesicles (Fig. 1A) . Besides migrasomes, it also showed individual or small clusters of intraluminal vesicles in retraction fiber, in many cases, actin filaments were clearly visible associated with these vesicles (Fig. 1B) . Moreover, it showed large clusters of vesicles at the base of retraction fibers and detached migrasomes containing intraluminal vesicles (Fig. 1C) . The positioning and the distribution of these vesicles suggested that the intraluminal vesicles of the migrasomes might be transported to the base of the retraction fiber and then to migrasomes via the retraction fiber.

The fact that the distal migrasomes had fewer intraluminal vesicles suggested that the intraluminal vesicles might fuse with the migrasome membrane. Calcium was required for SNARE mediated vesicle fusion, it showed that treating cells with BAPTA-AM, a cell-permeant chelator for calcium, significantly increased the number of intraluminal vesicles in migrasomes (Fig. 1D) , supporting our hypothesis that intraluminal vesicles may fuse with migrasome membrane in a calcium-dependent manner.

Example 2 Marker of intraluminal vesicles in migrasome

Mass spectrometry analysis identified that Rab8, which might be used to regulate Golgi to plasma membrane trafficking, was enriched in migrasomes. It showed that endogenous Rab8 was indeed present inside the migrasomes and along the retraction fibers (Fig. 1E) . Structured illumination microscopy (SIM) images showed GFP-Rab8 signal was presented as intraluminal puncta inside the migrasome (Fig. 1F) . To verify these puncta were indeed intraluminal vesicles, APEX2-based intracellular-specific protein imaging was performed by electron microscopy. APEX2 catalyzed the local deposition of DAB, which enhanced the contrast in EM images by binding electron dense osmium. It showed that APEX2-GFP-Rab8 was indeed localized on intraluminal vesicles of the migrasomes (Fig. 1G) , allowing intraluminal vesicles to be labeled by Rab8. Moreover, it showed that BAPTA-AM could significantly increase the number of GFP-Rab8-labeled vesicles in migrasome (Fig. 1H) , which was consistence with TEM analysis.

Example 3 Transporting of migrasome intraluminal vesicles

Next, the motor proteins that could transport intraluminal vesicles to the migrasomes were identified. Since it showed bundled actin inside the retraction fiber, the test was carried out on actin-based motor myosin. Myosin5a, which was known to mediate long distance transportation of vesicles, might be included. To check the localization of Myosin5a, a cell line was generated in which GFP-Myosin5a was stably expressed. It showed that GFP-Myosin5a formed bright puncta along retraction fibers and inside the migrasomes, which was much brighter than GFP-Myosin5a signal inside cells (Fig. 2A) . Moreover, GFP-Myosin5a motor domain was highly enriched in the migrasomes, whereas the GFP-Myosin5a-tail domain was absent in the migrasomes, suggesting that the motor domain was required for localization of Myosin5a in migrasomes (Fig. 6A) . APEX2-based intracellular-specific protein TEM imaging revealed APEX2-GFP-Myosin5a was indeed decorated around intraluminal vesicles and vesicles clustered around the base of retraction fibers, suggesting intraluminal vesicles might be transported to the base of retraction fibers and into migrasomes by Myosin5a (Fig. 2B) . To visualize the movement of intraluminal vesicles, time-lapse imaging is performed. Time lapse imaging showed GFP-Myosin5a signal on retraction fibers became brighter as cell migrated away, eventually became bright puncta, in many cases, migrasome grew around GFP-Myosin5a puncta, eventually enclosing GFP-Myosin5a puncta in migrasome, these data indicated that GFP-Myosin5a was transported to the site of migrasome formation, moreover, the increasing GFP-Myosin5a at the migrasome formation site suggested GFP-Myosin5a could gradually transported to migrasome formation site (Fig. 2C) . To better characterize the movement of Myosin5a, the ultra-fast super-resolution imaging was performed using Grazing Incidence Structured Illumination Microscopy (GI-SIM) , which could achieve100nm resolution with a speed of 200 frames/second. GI-SIM imaging showed that small myosin5a was transported to the edge of cell and accumulated as bright clusters, from these clusters on the base of retraction fibers, a stream of small vesicles rapidly moved into the migrasome, in some cases, the clusters of vesicles remained on the retraction fibers as the cells migrated away (Fig. 2D) . Put together, it showed that intraluminal vesicles could be transported into migrasomes by myosin5a.

Cell lines were established in which myosin5a was stably overexpressed (myosin5a OE) or knocked out (myosin5a KO) , and the number intraluminal vesicles was checked by TEM. It showed that myosin5a overexpression increased, myosin5a knockout decreased the number of intraluminal vesicles, showing that intraluminal vesicles were indeed transported into the migrasomes by myosin5a (Fig. 2E) . Similarly, overexpression of myosin5a increased the number of GFP-Rab8 puncta in migrasomes, indicating that Rab8 positive vesicles were transported into migrasomes by myosin5a (Fig. 2F) .

Example 4 Fusion of intraluminal vesicles with migrasome membrane

Next, the SNAREs required for the fusion of intraluminal vesicles with migrasome membrane were investigated. It showed that VAMP2, a V-SNAREs involved in constitutive exocytosis, was localized inside migrasome as small puncta (Fig. 3A) . APEX2-based TEM revealed VAMP2 was localized on the membrane of intraluminal vesicles (Fig. 3B) . Overexpression of myosin5a significantly increased the number of VAMP2 puncta, while knockout of myosin5a markedly reduced the number of VAMP2 puncta in the migrasomes, indicating that these vesicles were transported by myosin5a (Fig. 3C) . VAMP2 formed SNARE complex with t-SNARE SNAP23, showing that SNAP23 was highly enriched on migrasome (Fig. 3D and Fig. 7A) . The total membrane proteins were isolated from plasma membrane and from migrasome, and it showed that SNAP23 was indeed markedly enriched in migrasome (Fig. 3E) . Finally, SNAP23 knockdown significantly increased the number of VAMP2 vesicles in migrasome, suggesting that VAMP2 vesicles fused with migrasome membrane in a SNAP23-dependent manner (Fig. 3F) . Put together, these data showed that intraluminal vesicles could fuse with migrasome via SNARE.

To directly visualize the fusion of VAMP2 vesicles with the migrasome membrane, the time lapse imaging was performed, it showed that VAMP2 signal started as a cluster of small puncta, as the migrasome grew, VAMP2 signal gradually moved to the migrasome membrane, indicating that the fusion had occurred (Fig. 3G) . To directly detect the fusion between VAMP2 vesicle and the migrasome membrane, a VAMP2-pHlourin expressing cell line was generated. PHlourin was a pH-sensitive green fluorescent protein which had been widely used to visualize vesicle secretion. In contrast to the VAMP2 antibody, the VAMP2-pHlourin signal did not have the vesicular pool inside cell or inside migrasome as these vesicles wereacidic, instead, all the signal were on the plasma membrane or in membrane of migrasome, importantly, the VAMP2-pHlourin signal was more intense on migrasome membrane than on plasma membrane of cell body in an observation plate, indicating that in these cells, the migrasome was preferred secretion site compared to the plasma membrane of cell body (Fig. 3H) . Put together, these data showed that VAMP2 vesicles fuse with migrasome, and in some cells migrasome appeared to be the preferred fusion sites for VAMP2 vesicles. In addition to VAMP2, the signal of VAMP7 was also seen in migrasome, suggesting that migrasome could release secretory vesicles from different origins (Fig. 7B) .

Example 5 Localized secretion of cytokines in active monocyte

Next, a more physiologically relevant model was used to study the physiological role of migrasome-mediated secretion. Monocytes were chosen, as activated monocytes are highly migratory and secretory. It showed that activated monocytes generated large number of migrasomes (Fig. 4A) , TEM analysis showed that there were numerous intraluminal vesicles inside migrasomes (Fig. 4B) .

Activated monocytes secrete TNF-α and IL-6, which played important roles in the innate immune-response. TNF-α is secreted as membrane-bound form, the soluble TNF-α being cleaved from the membrane by a metalloproteinase called TNF-alpha-converting enzyme (TACE) . In contrast, IL-6 is secreted as a soluble factor. Immunostaining of monocytes with antibodies against IL-6 and TNF-α revealed that both TNF-α TACE were localized on the migrasome membrane, whereas IL-6 was localized in intraluminal vesicles inside the migrasome (Figs. 4C and 4D; Fig 8A) . Western blot confirmed TNF-α and IL-6 were not only present, but enriched in the migrasome compare to the cell body (Fig. 4E) .

Next, whether migrasome could contribute to the secretion of TNF-α and IL-6 was tested. Fibronectin can significantly enhance the formation of migrasome on NRK cells. When monocytes were cultured on glass surfaces without fibronectin coating, it showed on this surface, monocytes did not generate migrasomes. Member of tetraspanin family regulated migrasome formation, neutrophils from Tspan9 knockout mouse had impaired migrasome biogenesis. It showed migrasome formation in monocytes from Tspan9-/-mice was also impaired (Fig. 4F) .

To test whether migrasome formation could promote the secretion, cells were cultured on FN coated or control surface for 12 hours, and the secreted TNF-α and IL-6 on the medium were then measured. As expected, it showed that the LPS treatment significantly increased the secretion of TNF-α□and IL-6 (Fig. 4E) , interestingly, compared to the FN coated surface, secreted soluble TNF-α□and IL-6 were significantly reduced in monocytes cultured on the controlled surface, which might not support migrasome formation (Fig. 4G) . Similarly, the level of secreted TNF-α, IL-6 was also measured in the medium of wild type and Tspan9-/-monocytes cultured on FN-coated surface. It showed that soluble TNF-α□and IL-6 were significantly reduced in Tspan9-/-monocytes (Fig. 4G) . Put together, these data showed that migrasomes not only mediated localized secretion, but also contributed to the overall secretion of monocytes.

Example 6 Function of migrasome bound TNF-α

It showed that TNF-α and IL-6 could be localized in detached migrasomes. After detachment, the migrasome might be the vesicular carrier for signaling ligands. Since TNF-α was present on the surface of migrasomes, to test whether migrasome bound cytokines were functional, it was tested whether migrasomes could transmit TNF-α□signaling. As the fact that the combination of TNF-α□and the caspase 8 inhibitor zVAD could induce necroptosis in L929 cells, it showed that adding the isolated monocyte-derived migrasomes and ZVAD toL929 cells could indeed effectively kill L929 cells (Fig. 4H) , indicationg that the migrasome-bound TNF-α was functional.

Example 7 Cytokines containing migrasome released by monocyte in vivo

Using the in vivo migrasome imaging protocol, whether monocytes could generate migrasome in vivo was investigated. Using fluorophores conjugated anti-CCR2 antibody, monocytes were successfully labeled. It showed that monocytes could be difficult to find in the control mouse. After LPS stimulation, monocytes could be easily observed in blood vessels and it showed robust migrasome formation in circulating monocytes (Fig. 5A) . Free CCR2 positive migrasomes were observed in blood vessels (Fig. 5B) . Next, a crude migrasome preparation was isolated from blood. For confocal imaging analysis, the anti-CCR2 was first coated on cover glass, then the crude migrasome preparation was incubated with the antibody-coated surface, followed by washing and immunostaining (Fig. 5C) . Our isolation protocol yielded CCR2 positive vesicles with morphological hallmarks of migrasome (Fig. 5D) , moreover, these vesicles contained VAMP2 positive intraluminal vesicles, indicating that these vesicles were indeed monocyte-derived migrasomes. Importantly, both TNF-α and IL-6 could be found in migrasomes (Figs. 5E and 5F) . Next, the monocyte derived migrasome was analyzed by Western blot. The blood was first centrifuged at low speed to remove the cell, then monocyte-derived migrasome was isolated from an equal volume of blood using a protocol based on negative selection. Next, the sample was analyzed using antibodies against CPQ, a protein presented on migrasomes but not in exosomes, and antibodies against TNF-α and IL-6. It showed that LPS treatment increased the amount of CPQ, TNF-α and IL-6 in the cell fraction (Fig. 5G) , which was likely due to the increased recruitment of immune cells into the circulation, resulting in more immune cells in the same volume of blood. This observation was consistent with in vivo imaging data. Similarly, LPS treatment increased the amount of CPQ in the migrasome fraction, suggesting that the monocyte-derived migrasome formation was enhanced, which could result from more migrasome-generating monocytes being recruited into the circulation, as a consequence, LPS treatment also significantly enhanced the amount of TNF-α and IL-6 in the migrasome fraction. (Fig. 5G)

To compare the relative abundance of CPQ, TNF-α and IL-6 in cell and in migrasome, equal amounts of total protein from cell and from migrasome were loaded and the amount of CPQ, TNF-αand IL-6 were analyzed. It showed that CPQ, TNF-α and IL-6 were highly enriched in monocyte-derived migrasome compared to cell body (Fig. 5H) , moreover, it is worth noting that although LPS treatment increased the TNF-α and IL-6 expression level in monocytes, the fold of increasing was much more dramatic in migrasome (Fig. 5H) . Put together, these data suggested that circulating monocytes released migrasome-bound cytokines, and LPS treatment dramatically enhanced the release of cytokines-enriched migrasomes into blood steam.

Example 8 Monocyte derived migrasome reduced in Tspan9-/-mouse

To test the role of Tspan9 in monocyte migrasome formation in vivo, monocytes were isolated from both wild type and Tspan9-/-mice, labeled with CCR2 antibodies with different color, and then injected into wild type mice, it showed, similar to what it had been showed in vitro, monocytes from Tspan9-/-mice produced less migrasomes in vivo (Fig. 5I) . Next, equal volume of blood was taken from wild type and Tspan9-/-mice, and isolated monocyte-derived migrasome as described above, it showed that the amount of CPQ was significantly reduced in the sample from Tspan9-/-mice, suggesting that monocyte-derived migrasomes were indeed reduced in Tspan9-/-mice. Consistently, it showed that the amount of migrasomes bound TNF-α and IL-6 was reduced in the same volume of blood from Tspan9-/-mice (Fig. 5J) .

Example 9 Reduced soluble cytokine level in blood by knocking out Tspan9

As shown above, VAMP2 positive secretory vesicles could fused with migrasome membrane, and secrete the secretory proteins into medium, moreover, it showed in in vitro cultured active monocytes, the secretion of TNF-α and IL-6 were reduced, suggesting that blocking migrasome formation might cause reduced secretion. Next, the levels of a panel of soluble cytokines were analyzed in the blood of wild type and Tspan9-/-mice using a cytokine array, consistent with what it showed in in vitro cultured monocytes, it showed TNF-α, IL-6, IL-10, IL-12 and IFN-γ levels were also significantly reduced in Tspan9-/-mice (Fig. 5K) . Thus, both soluble and migrasome-bound cytokines were reduced in Tspan9-/-mice.

Example 10 Characterization of intraluminal vesicles of migrasome in a neutrophil, a T cell and/or a NK cell

Similar test was perfpormerd on neutrophils, T cells and/or NK cells. It showed that intraluminal vesicles could be transported into the migrasome of neutrophil, T cell and/or NK cell. And the intraluminal vesicles in the migrasome of neutrophil, T cell and/or NK cell could be fused to the migrasome.

While exemplary embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

A method for regulating the secretion of a substance by a migrasome, comprising regulating the number and/or function of an intraluminal vesicle in said migrasome.
The method of claim 1, wherein said migrasome is generated by a cell.
The method of claim 2, wherein said cell is a migrating cell.
The method of any one of claims 2-3, wherein said cell is a circulating cell.
The method of any one of claims 2-4, wherein said cell is a cell in or from the blood.
The method of any one of claims 2-5, wherein said cell is an immune cell.
The method of claim 6, wherein said immune cell comprises a monocyte, a macrophage, a neutrophil and/or a T cell.
The method of any one of claims 1-7, wherein said substance to be secreted is comprised in said intraluminal vesicle.
The method of any one of claims 1-8, wherein said substance to be secreted comprises one or more signaling molecules.
The method of any one of claims 1-9, wherein said substance to be secreted comprises one or more protein or polypeptide containing a signal peptide.
The method of any one of claims 1-10, wherein said substance to be secreted comprises a cytokine and/or a flavonoid.
The method of any one of claims 1-11, which increases the secretion of the substance, and comprises increasing the number and/or function of the intraluminal vesicles in said migrasome.
The method of claim 12, wherein said increasing the number and/or function of the intraluminal vesicles in said migrasome comprises increasing the transportation of the intraluminal vesicles into said migrasome.
The method of any one of claims 12-13, wherein said increasing the number and/or function of the intraluminal vesicles in said migrasome comprises increasing the amount and/or function of a motor protein in said cell.
The method of any one of claims 12-14, wherein said increasing the number and/or function of the intraluminal vesicles in said migrasome comprises increasing fusion of the membrane of said migrasome with the intraluminal vesicle therein.
The method of claim 15, wherein increasing said fusion comprises increasing the amount and/or function of a SNARE complex in said migrasome.
The method of any one of claims 15-16, wherein increasing said fusion comprises increasing calcium in said migrasome.
The method of any one of claims 1-11, which inhibits the secretion of the substance, and comprises reducing the number and/or function of the intraluminal vesicles in said migrasome.
The method of claim 18, wherein said reducing the number and/or function of the intraluminal vesicles in said migrasome comprises inhibiting the transportation of the intraluminal vesicles into said migrasome.
The method of any one of claims 18-19, wherein said reducing the number and/or function of the intraluminal vesicles in said migrasome comprises reducing the amount and/or function of a motor protein in said cell.
The method of any one of claims 18-20, wherein said reducing the number and/or function of the intraluminal vesicles in said migrasome comprises inhibiting fusion of the membrane of said migrasome with the intraluminal vesicle therein.
The method of claim 21, wherein inhibiting said fusion comprises reducing the amount and/or function of a SNARE complex in said migrasome.
The method of claim 22, wherein reducing the amount and/or function of a SNARE complex comprises inhibiting the expression of one or more components of the SNARE complex.
The method of any one of claims 22-23, wherein reducing the amount and/or function of a SNARE complex comprises knocking out or knocking down the expression of a gene encoding for one or more components of the SNARE complex.
The method of any one of claims 21-24, wherein inhibiting said fusion comprises reducing calcium in said migrasome.
The method of claim 25, wherein reducing calcium in said migrasome comprises administering a calcium chelator.
The method of claim 26, wherein said calcium chelator comprises BAPTA-AM.
The method of any one of claims 14 and 20, wherein said motor protein comprises a Myosin.
The method of any one of claims 14, 20 and 28, wherein said motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.
The method of any one of claims 12 and 22-24, wherein said SNARE comprises a v-SNARE and/or a t-SNARE.
The method of claim 30, wherein said t-SNARE comprises SNAP23.
The method of any one of claims 30-31, wherein said v-SNARE comprises VAMP2 and/or VAMP7.
An agent capable of regulating the number and/or function of an intraluminal vesicle in a migrasome, for use in regulating the secretion of a substance by said migrasome.
The agent of claims 33, wherein said migrasome is generated by a cell.
The agent of claim 34, wherein said cell is a migrating cell.
The agent of any one of claims 34-35, wherein said cell is a circulating cell.
The agent of any one of claims 34-36, wherein said cell is a cell in or from the blood.
The agent of any one of claims 34-37, wherein said cell is an immune cell.
The agent of claim 38, wherein said immune cell comprises a monocyte, a macrophage, a neutrophil and/or a T cell.
The agent of any one of claims 33-39, wherein said substance to be secreted is comprised in said intraluminal vesicle.
The agent of any one of claims 33-40, wherein said substance to be secreted comprises one or more signaling molecules.
The agent of any one of claims 33-41, wherein said substance to be secreted comprises one or more protein or polypeptide containing a signal peptide.
The agent of any one of claims 33-42, wherein said substance to be secreted comprises a cytokine and/or a flavonoid.
The agent of any one of claims 33-43, for use in increasing the secretion of said substance, and the agent is capable of increasing the number and/or function of the intraluminal vesicles in said migrasome.
The agent of claim 44, which is capable of increasing the transportation of the intraluminal vesicles into said migrasome.
The agent of any one of claims 44-45, which is capable of increasing the amount and/or function of a motor protein in said cell.
The agent of any one of claims 44-46, which is capable of increasing fusion of the membrane of said migrasome with the intraluminal vesicle therein.
The agent of claim 47, which is capable of increasing the amount and/or function of a SNARE complex in said migrasome.
The agent of any one of claims 47-48, which is capable of increasing calcium in said migrasome.
The agent of any one of claims 33-43, for use in inhibiting the secretion of said substance, and the agent is capable of reducing the number and/or function of the intraluminal vesicles in said migrasome.
The agent of claim 50, which is capable of inhibiting the transportation of the intraluminal vesicles into said migrasome.
The agent of any one of claims 50-51, which is capable of reducing the amount and/or function of a motor protein in said cell.
The agent of any one of claims 50-52, which is capable of inhibiting fusion of the membrane of said migrasome with the intraluminal vesicle therein.
The agent of claim 53, which is capable of reducing the amount and/or function of a SNARE complex in said migrasome.
The agent of claim 54, which is capable of inhibiting the expression of one or more components of the SNARE complex.
The agent of any one of claims 54-55, which is capable of knocking out or knocking down the expression of a gene encoding for one or more components of the SNARE complex.
The agent of any one of claims 53-56, which is capable of reducing calcium in said migrasome.
The agent of claim 57, which comprises a calcium chelator.
The agent of claim 58, wherein said calcium chelator comprises BAPTA-AM.
The agent of any one of claims 46 and 52, wherein said motor protein comprises a Myosin.
The agent of any one of claims 46, 52 and 60, wherein said motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.
The agent of any one of claims 48 and 54-56, wherein said SNARE comprises a v-SNARE and/or a t-SNARE.
The agent of claim 62, wherein said t-SNARE comprises SNAP23.
The agent of any one of claims 62-63, wherein said v-SNARE comprises VAMP2 and/or VAMP7.
An engineered cell with altered ability for regulating the secretion of a substance by a migrasome generated by said cell comparing to a corresponding unmodified cell, said engineered cell has been modified to alter the number and/or function of an intraluminal vesicle in said migrasome.
The engineered cell of claim 65, wherein said cell is a migrating cell.
The engineered cell of any one of claims 65-66, wherein said cell is a circulating cell.
The engineered cell of any one of claims 65-67, wherein said cell is a cell in or from the blood.
The engineered cell of any one of claims 65-68, wherein said cell is an immune cell.
The engineered cell of claim 69, wherein said immune cell comprises a monocyte, a macrophage, a neutrophil and/or a T cell.
The engineered cell of any one of claims 65-70, wherein said substance to be secreted is comprised in said intraluminal vesicle.
The engineered cell of any one of claims 65-71, wherein said substance to be secreted comprises one or more signaling molecules.
The engineered cell of any one of claims 65-72, wherein said substance to be secreted comprises one or more protein or polypeptide containing a signal peptide.
The engineered cell of any one of claims 65-73, wherein said substance to be secreted comprises a cytokine and/or a flavonoid.
The engineered cell of any one of claims 65-74, which has increased ability for the secretion of the substance, and said engineered cell has been modified to increase the number and/or function of the intraluminal vesicles in said migrasome.
The engineered cell of claim 75, which has been modified to increase the transportation of the intraluminal vesicles into said migrasome.
The engineered cell of any one of claims 75-76, which has been modified to increase the amount and/or function of a motor protein in said cell.
The engineered cell of any one of claims 75-77, which has been modified to increase fusion of the membrane of said migrasome with the intraluminal vesicle therein.
The engineered cell of claim 78, which has been modified to increase the amount and/or function of a SNARE complex in said migrasome.
The engineered cell of any one of claims 78-79, which has been modified to increase calcium in said migrasome.
The engineered cell of any one of claims 65-74, which has reduced ability for the secretion of the substance, and said engineered cell has been modified to reduce the number and/or function of the intraluminal vesicles in said migrasome.
The engineered cell of claim 81, which has been modified to inhibit the transportation of the intraluminal vesicles into said migrasome.
The engineered cell of any one of claims 81-82, which has been modified to reduce the amount and/or function of a motor protein in said cell.
The engineered cell of any one of claims 81-83, which has been modified to inhibit fusion of the membrane of said migrasome with the intraluminal vesicle therein.
The engineered cell of claim 84, which has been modified to reduce the amount and/or function of a SNARE complex in said migrasome.
The engineered cell of claim 85, which has been modified to inhibit the expression of one or more components of the SNARE complex.
The engineered cell of any one of claims 85-86, wherein the expression of a gene encoding for one or more components of the SNARE complex has been knocked down or knocked out.
The engineered cell of any one of claims 84-87, which has been modified to reduce calcium in said migrasome.
The engineered cell of claim 88, which has been treated with a calcium chelator.
The engineered cell of claim 89, wherein said calcium chelator comprises BAPTA-AM.
The engineered cell of any one of claims 77 and 83, wherein said motor protein comprises a Myosin.
The engineered cell of any one of claims 77, 83 and 91, wherein said motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.
The engineered cell of any one of claims 79 and 85-87, wherein said SNARE comprises a v-SNARE and/or a t-SNARE.
The engineered cell of claim 93, wherein said t-SNARE comprises SNAP23.
The engineered cell of any one of claims 93-94, wherein said v-SNARE comprises VAMP2 and/or VAMP7.
Use of an agent according to any one of claims 33-64 and/or an engineered cell according to any one of claims 65-95 in the preparation of a regulator for the secretion of a substance by said migrasome.
A composition, comprising the agent according to any one of claims 33-64, and/or the engineered cell according to any one of claims 65-95.
The composition of claim 97, which is a pharmaceutical composition and optionally comprises a pharmaceutically acceptable excipient.
A kit, comprising the agent according to any one of claims 33-64, the engineered cell according to any one of claims 65-95, and/or the composition according to any one of claims 97-98.