WO2018226776A1

WO2018226776A1 - Assay for monitoring autophagosome completion

Info

Publication number: WO2018226776A1
Application number: PCT/US2018/036184
Authority: WO
Inventors: Yoshinori Takahashi; Hong-gang WANG
Original assignee: The Penn State Research Foundation
Priority date: 2017-06-08
Filing date: 2018-06-06
Publication date: 2018-12-13

Abstract

Provided are compositions and methods for autophagosome completion assays useful for detecting and monitoring phagophore closure and autophagosome maturation, and for screening for compounds effective for the treatment of diseases or disorders associated with defects in phagophore closure and autophagosome maturation.

Description

TITLE OF THE INVENTION

Assay for Monitoring Autophagosome Completion

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No.

62/516,756 filed June 08, 2017, the contents of which are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

Autophagy is an intracellular catabolic process where cytoplasmic material is sequestered into double-membrane autophagosomes for lysosomal degradation

(Eskelinen, 2008, Int Rev Cell Mol Biol, 266:207-247). A series of AuTophaGy-related (ATG) proteins coordinate the initiation, nucleation, and elongation of crescent-shaped isolation membranes or phagophores during autophagosome biogenesis (Mizushima et al., 2011, Annu Rev Cell Dev Biol, 27: 107-132; Choi et al., 2013, N Engl J Med, 368:651-662). However, how the phagophore undergoes membrane remodeling to generate the inner and outer membranes of the completed autophagosome remains far from clear (Lamb et al., 2013, NatRevMol Cell Biol, 14:759-774) and has been hindered by technical challenges associated with distinguishing unclosed and closed

autophagosomal membranes (Klionsky et al., 2016, Autophagy 12: 1-222).

The endosomal sorting complex required for transport (ESCRT) proteins were originally identified as regulators of ubiquitinated cargo sorting into multivesicular bodies (MVBs) but have since extended to mediate reverse-topology membrane scission in a variety of cellular processes (Christ et al., 2017, Trends Biochem Sci, 42:42-56; Hurley, 2015, EMBO J, 34:2398-2407). While ESCRTs failed to be identified in yeast screens for essential ATG genes, ESCRT defects in C elegans, Drosophila and mammals accumulate autophagosome-like structures (Lee et al., 2007, Curr Biol, 17: 1561-1567; Rusten et al., 2007, Curr Biol, 17: 1817-1825; Filimonenko et al., 2007, J Cell Biol, 179:485-500; Roudier et al., 2005, Traffic, 6:695-705). Interestingly, the topological membrane transformation that occurs during phagophore closure resembles that of

ESCRT-mediated intraluminal vesicle formation (Hurley, 2015, EMBO J, 34:2398-2407; Knorr et al., 2015, Autophagy, 11 :2134-2137; Rusten and Stenmark, 2009, J Cell Sci, 122:2179-2183).

Microtubule-associated protein 1 light chain 3 (LC3) is a mammalian ortholog of yeast Atg8 that is conjugated with phosphatidylethanolamine to form LC3-II at the phagophore during autophagosome biogenesis (Kabeya et al., 2000, EMBO J, 19:5720-5728; Mizushima et al., 2001, J Cell Biol, 152:657-668). Upon closure, LC3-II on the outer autophagosomal membrane (OAM) is delipidated and released to the cytosol, while LC3-II associated with the inner autophagosomal membrane (IAM) is degraded upon autophagosome-lysosome fusion (Tanida et al., 2005, Autophagy, 1 :84- 91).

There is a need in the art for compositions and methods to assay phagophore closure and autophagosome completion. The present invention satisfies this unmet need.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to an autophagosome maturation assay vector comprising a nucleotide sequence encoding an autophagy-related 8 (Atg8) family protein operably linked to a nucleotide sequence encoding a modified haloalkane dehalogenase (HaloTag).

In one embodiment, the Atg8 family protein is γ-aminobutyric acid receptor-associated protein (GABARAP), γ-aminobutyric acid receptor-associated like protein (GABARAPLl), Golgi-associated ATPase enhancer of 16 kDa (GATE- 16), microtubule-associated protein light chain 3 A (LC3 A), microtubule-associated protein light chain 3B (LC3B), microtubule-associated protein light chain 3B2 (LC3B2) or microtubule-associated protein light chain 3C (LC3C). In one embodiment, the vector comprises a nucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO:2. In one embodiment, the vector comprises a nucleotide sequence as set forth in SEQ ID NO: 1.

In one embodiment, the invention relates to a cell comprising an autophagosome maturation assay vector comprising a nucleotide sequence encoding an Atg8 family protein operably linked to a nucleotide sequence encoding a HaloTag. In one embodiment, the invention relates to an autophagosome maturation assay that provides superior signal-to-noise ratio and high reproducibility to distinguish phagophores, nascent autophagosomes, and mature autophagosomal structures. In one embodiment, the method comprises a) contacting a cell comprising an autophagosome maturation assay vector comprising a nucleotide sequence encoding an Atg8 family protein operably linked to a nucleotide sequence encoding a HaloTag with at least one detectable membrane impermeable haloalkane dehalogenase ligand (MIL), b) contacting the cell of a) with at least one detectable membrane permeable haloalkane dehalogenase ligand (MPL), and c) determining the level of at least one of a MIL+ MPL+

autophagosome, a MIL+ MPL- phagophore and a MIL- MPL+ organelle in the cell.

In one embodiment, the MIL comprises a membrane-impermeable AlexaFluor488-HaloTag ligand. In one embodiment, the MPL comprises a membrane- permeable tetramethylrhodamine-HaloTag ligand.

In one embodiment, the method further comprises comprising contacting the cell with a compound or treatment prior to step a).

In one embodiment, the method further comprises contacting the cell with a plasma membrane permeabilization agent prior to step a).

In one embodiment, the plasma membrane permeabilization agent is a cholesterol-complexing agent, recombinant perfringolysin (rPFO/XF-MPM), or digitonin.

In one embodiment, the method further comprises inducing autophagy in the cell prior to step a). In one embodiment, the method comprises culturing the cell in starvation medium.

In one embodiment, step c) is performed using a method selected from the group consisting of immunoelectron microscopy, wide-field fluorescence microscopy, flow cytometry, confocal microscopy, fluorimetry, microplate-based cytometry, high- content cell analysis, cell microarray analysis, high-content cell screening, and laser- scanning cytometry.

In one embodiment, the invention relates to a method of screening for the effect of a compound on the closure of autophagosomes or the progression of the lysosomal-dependent catabolic pathway comprising: a) contacting a cell comprising an autophagosome maturation assay vector comprising a nucleotide sequence encoding an Atg8 family protein operably linked to a nucleotide sequence encoding a HaloTag with at least one compound or treatment; b) inducing autophagy in the cell; c) contacting the cell with a plasma membrane permeabilization agent; d) contacting the cell with at least one detectable MIL, e) contacting the cell of a) with at least one detectable MPL, and f) determining the level of at least one of a MIL+ MPL+ autophagosome, a MIL+ MPL- phagophore and a MIL- MPL+ organelle in the cell.

In one embodiment, the invention relates to a method of treating a disease or disorder associated with a deficiency in the closure of autophagosomes, or the progression of the lysosomal-dependent catabolic pathway, comprising administering to a subject in need thereof an activator of at least one ESCRT component selected from the group consi sting of CHMP2 A and VP S4.

In one embodiment, the disease is Crohn's disease, Vici syndrome, cancers such as breast, ovarian, prostate, liver, colorectal and hematologic cancers, systemic lupus erythematosus, neurodegenerative diseases such as static encephalopathy of childhood with neurodegeneration in adulthood (SEND A) or Parkinson's disease, and phospholipidosis.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1, comprising Figure 1 A through Figure ID, depicts the results of experiments demonstrating that the HaloTag-LC3 autophagosome completion assay distinguishes unclosed and closed autophagosomal membranes. Figure 1 A depicts a schematic strategy of the HT-LC3 autophagosome completion assay. The assay is performed by the following procedures: step 1, after the induction of autophagy, HT- LC3 -expressing cells are treated with cholesterol-complexing agents including recombinant perfringolysin (rPFO/XF-MPM) or digitonin to permeabilize the plasma membrane (PM) and release HT-LC3-I from the cytosol; step 2, cells are incubated with a saturating dose of membrane-impermeable HT ligand (MIL) to stain membrane-bound HT-LC3-II that is accessible to the cytoplasmic region (MIL also diffuses into nucleus and stains nuclear LC3); step 3, cells are incubated with membrane-permeable HT ligand (MPL) to stain LC3-II that is sequestered within membranes. Figure IB depicts exemplary experimental results in which HT-LC3 U-2 OS cells were incubated in starvation medium (SM) or control complete medium (CM) in the presence or absence of 100 nM BafAl for 4 hours and subjected to the HT-LC3 autophagosome completion assay followed by confocal microscopy. Magnified images of the boxed and arrow- indicated areas are shown in the middle and right panels, respectively. The scale bars represent 10 μπι and 1 μπι in the magnified images. Figure 1C depicts exemplary experimental results demonstrating that HT-LC3 U-2 OS cells were starved for 3 hours and subjected to 3D-deconvolution fluorescence microscopy. LC3 signals on the phagophore or the outer autophagosomal membrane, and in the autophagosome lumen were stained using Alexa Fluor 488 (AF488)-conjugated MIL and tetramethylrhodamine (TMR)-conjugated MPL, respectively, xz and yz images at the dash-lined area in Figure 2B are shown to the right and bottom, respectively; arrows, indicate MIL⁺MPL^", MIL⁺MPL⁺, and MIL^"MPL⁺ structures The scale bars represent 1 μπι in the magnified images. Figure ID depicts the results of exemplary experiments in which the cytoplasmic fluorescence intensities of MIL and MPL in each cell in Figure IB were quantified and normalized to the respective mean fluorescence intensities of the cells starved in the presence of BafAl (n > 100). Data shown are representative of three independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. All values are mean ± SD. ns, not significant; ****, p<0.0001. Figure 2, comprising Figure 2A through Figure 2E, depicts the results of experiments demonstrating establishment of the HT-LC3 autophagosome completion assay. Figure 2A depicts the results of exemplary experiments in which U-2 OS cells were transduced with lentiviruses encoding HT-LC3 and selected with puromycin for 5 days. The resultant stable transfectants and the parental wild-type cells were incubated in starvation medium (SM) or control complete medium (CM) in the presence or absence of 100 nM BafAl for 3 hours and subjected to immunoblotting using the indicated antibodies. Figure 2B depicts the results of exemplary experiments in which HT-LC3 U-2 OS cells were starved in the presence of 100 nM BafAl for 1.5 hours, stained with TMR- MPL, and treated in lxMAS containing 3 nM XF-PMP at 37°C for the indicated periods of time (minutes). Fluorescence images were acquired every 1 minute for 30 minutes using a Leica AOBS SP8 laser-scanning confocal microscope. Magnified images of the boxed areas are shown in the middle panels. Figure 2C depicts the results of exemplary experiments in which HT-LC3 U-2 OS and wild-type U-2 OS cells were starved for 2 hours, permeabilized with digitonin (PM Perm) and subjected to the HT-LC3

autophagosome completion assay. The fluorescence images were obtained by 3D deconvolution microscopy. Magnified images of the boxed areas are shown in Figure 1C. Figure 2D depicts the results of exemplary experiments in which HT-LC3 U-2 OS cells were starved for 3 hours, incubated with 3 nM XF-PMP in the presence or absence of AF488-MIL at 37°C for 15 minutes, fixed, then incubated with AF660-MIL at RT for 30 minutes followed by TMR-MPL at RT for 30 minutes, and subjected to confocal microscopy. Figure 2E depicts the results of exemplary experiments in which HeLa cells stably expressing HT-LC3 were incubated in CM or SM in the presence or absence of lysosomal inhibitors (100 nM Baf Al or protease inhibitors (Pis; 10 μg/ml pepstatin A, 1 μΜ leupeptin, 10 μΜ E64d) for 2 hours and subjected to the HT-LC3 autophagosome completion assay followed by confocal microscopy. The scale bars represent 10 μπι and 1 μπι in the magnified images in Figure 2B and Figure 2E.

Figure 3, comprising Figure 3 A through Figure 3G, depicts the results of experiments demonstrating that the cytoplasmic HT-LC3 positive foci specifically represent autophagic structures. Figure 3A depicts the results of exemplary experiments in which ATG7-deficient U-2 OS cells were generated using the Crispr-Cas9 gene editing system and subjected to starvation for 3 hours followed by immunoblotting using the indicated antibodies. Figure 3B depicts the results of exemplary experiments in which ATG7-deficient HT-LC3 U-2 OS cells were starved in the presence of lysosomal protease inhibitors (Pis) and subjected to the HT-LC3 autophagosome completion assay followed by deconvolution fluorescence microscopy. Figure 3C and Figure 3D depict the results of exemplary experiments in which ATGl 3 -deficient U-2 OS cells were generated using the Crispr-Cas9 gene editing system. Figure 3C depicts the results of exemplary experiments in which the resultant cells were subjected to immunoblotting using the indicated antibodies. Figure 3D depicts the results of exemplary experiments in which the resultant cells were starved in the presence or absence of 100 nM BafAl followed by immunoblot analysis. Figure 3E depicts the results of exemplary experiments in which ATGl 3 -deficient HT-LC3 U-2 OS cells were starved in the presence or absence of Pis and subjected to the HT-LC3 autophagosome completion assay followed by confocal microscopy. Figure 3F depicts the results of exemplary experiments in which HT-LC3 U- 2 OS cells were starved for 2 hours, stained with MIL and MPL, and subjected to immunofluorescence microcopy using anti-Atgl6L antibody. Figure 3G depicts the results of exemplary experiments in which HT-LC3 U-2 OS cells were starved for 2 hours, stained with MIL and MPL, and subjected to correlative electron microscopy using anti-Atgl6L antibody. Arrows in Figure 3F and arrowheads in Figure 3G indicate ATG16L-positive MIL⁺MPL^" foci and phagophore-associated endoplasmic reticulum (ER), respectively. In Figure 3F, the number of ATG16L-positive MIL⁺MPL^",

MIL⁺MPL⁺ and MIL^"MPL⁺ foci per cell was quantified and shown (n = 45 from two independent experiments). Statistical significance was determined b one-way ANOVA followed by Tukey's multiple comparison test. All values are mean ± SD. ns, not significant; ****, p<0.0001. The scale bars represent 10 μπι, and 1 μπι in the magnified images in Figure 3F.

Figure 4, comprising Figure 4A through Figure 4B, depicts the results of experiments demonstrating screening of ESCRT components and their regulatory proteins using the HT-LC3 autophagosome completion assay. Figure 4A depicts the results of exemplary experiments in which HT-LC3 U-2 OS cells were cultured in serum- free Accell siRNA delivery medium (DM) for 48 hours or CM for 45 hours followed by SM or CM for 3 hours, subjected to the HT-LC3 autophagosome completion assay, and analyzed by confocal microscopy. Figure 4B depicts the results of exemplary

experiments in which HT-LC3 U-2 OS cells were incubated with the indicated Accell SMRT Pool siRNAs for 72 hours, subjected to the HT-LC3 autophagosome completion assay, and analyzed by confocal microscopy. The scale bars represent 10 μιη. Data shown are representative of two independent experiments (at least 5 images were taken at 63x magnification at each experiment).

Figure 5, comprising Figure 5A through Figure 5B, depicts the results of experiments demonstrating that depletion of ESCRT components leads to the

accumulation MIL⁺. Figure 5 A depicts the results of exemplary experiments in which HT-LC3 U2-OS cells were transfected with indicated ON-TARGETplus SMART Pool siRNAs for 48 hours, and subjected to immunoblotting using the indicated antibodies. Figure 5B depicts the results of exemplary experiments in which HT-LC3 U2-OS cells were starved for 3 hours in the presence or absence of 100 nM BafAl and subjected to the HT-LC3 autophagosome completion assay followed by confocal microscopy. In Figure 5A, the asterisks indicate non-specific bands. In Figure 5B, the cytoplasmic fluorescence intensities of MIL and MPL in each cell were quantified and normalized to the respective mean fluorescence intensities of control siNT transfected cells starved in the presence of BafAl (n > 100). Statistical significance was determined by Kruskal- Wallis one-way ANOVA on ranks followed by Dunn's multiple comparison test. All values are mean ± SD. ****, p<0.0001.

Figure 6, comprising Figure 6A through Figure 6E, depicts the results of experiments demonstrating that CHMP2A depletion results in the accumulation of unclosed autophagosomal membranes. Figure 6A depicts the results of exemplary experiments in which HT-LC3 -expressing U-2 OS cells were transfected with the indicated ON-TARGETplus SMART Pool siRNAs for 48 hours and subjected to the HT- LC3 autophagosome completion assay followed by confocal microscopy. Figure 6B depicts the results of exemplary experiments in which cells were incubated in CM or SM in the presence or absence of 100 nM BafAl for 3 hours and subjected to the HT-LC3 autophagosome completion assay. Representative images are shown in Figure 7B. The cytoplasmic fluorescence intensities of MIL and MPL in each cell were quantified and normalized to the respective mean fluorescence intensities of control siNT transfected cells starved in the presence of BafAl (n > 100). Data shown are representative of three independent experiments. Statistical significance was determined by Kruskal-Wallis oneway ANOVA on ranks followed by Dunn' s multiple comparison test. All values are mean ± SD. **, p<0.01 ; ****, p<0.0001. Figure 6C depicts the results of exemplary experiments in which GFP-Atg5-expressing U-2 OS cells were transfected with the indicated ON-TARGETplus SMART Pool siRNAs for 48 hours. Cells were stained with TMR-conjugated MPL, starved for 2 hours and subjected to confocal microscopy. Figure 6D depicts the results of exemplary experiments in which parental wild-type U-2 OS cells were transfected with the indicated ON-TARGETplus SMART Pool siRNAs for 48 hours. Cells were starved for 3 hours and subjected to electron microscopy. The samples were processed in the absence of potassium ferrocyanide. Arrows, two-headed arrow, and arrowhead indicate phagophore-like (clearly opened in 2D micrographs),

autophagosome-like, and autolysosome-like structures, respectively. Figure 6E depicts the results of exemplary experiments in which the number of total and unclosed autophagic structures per cytoplasmic area was quantified and shown (n = 26). Statistical significance was determined by two-way ANOVA followed by Sidak' s multiple comparison test. All values are mean ± SD. ****, p<0.0001. The scale bars represent 10 μπι and 1 μπι in the magnified images.

Figure 7, comprising Figure 7A through Figure 7E, depicts the results of experiments demonstrating that depletion of CFDVIP2A results in the accumulation of LC3 -positive immature autophagosomal membranes. Figure 7 A depicts the results of exemplary experiments in which U-2 OS and HeLa cells were transfected with the indicated ON-TARGETplus SMART Pool siRNAs for the indicated periods of time and subjected to immunoblotting using the indicated antibodies. Figure 7B depicts the results of exemplary experiments in which HT-LC3 U-2 OS cells were transfected with the indicated siRNAs for 48 hours, incubated in CM or SM in the presence or absence of 100 nM BafAl for 2 hours and subjected to the HT-LC3 autophagosome completion assay. Figure 7C depicts the results of exemplary experiments in which HT-LC3 HeLa cells were transfected with the indicated siRNAs for 48 hours, incubated in CM or SM in the presence or absence of 100 nM BafAl for 2 hours and subjected to the HT-LC3 autophagosome completion assay. Figure 7D depicts the results of exemplary

experiments in which U-2 OS cells expressing GFP-LC3 were transfected with the indicated siRNAs for 48 hours and incubated in CM or SM in the presence or absence of 100 nM Baf Al for 2 hours. Figure 7E depicts the results of exemplary experiments in which wild-type U-2 OS cells were transfected with the indicated siRNAs for 48 hours, starved for 2 hours and subjected to immunofluorescence microscopy using anti-LC3B antibodies. All fluorescence images were obtained by confocal microscopy. The scale bars represent 10 μιη.

Figure 8, comprising Figure 8A through Figure 8C, depicts the results of experiments demonstrating that CFIMP2B is dispensable for autophagosome formation. Figure 8A depicts the results of exemplary experiments in which wild-type U-2 OS cells were transfected with the indicated ON-TARGETplus SMART Pool siRNAs for 48 hours and subjected to immunoblotting using the indicated antibodies. Figure 8B depicts the results of exemplary experiments in which HT-LC3 U-2 OS cells were transfected with the indicated ON-TARGETplus SMART Pool siRNAs for 48 hours and incubated in CM or SM in the presence or absence of 100 nM Baf Al for 2 hours, and subjected to the autophagosome completion assay followed by confocal microscopy. The cytoplasmic fluorescence intensities of MIL and MPL in each cell were quantified and normalized to the respective mean fluorescence intensities in the control siNT transfected cells starved in the presence of Baf Al (n > 100). The MIL/MPL ratio in each cell was calculated and shown at the right. Statistical significance was determined using two-way ANOVA with Sidak' s multiple comparison test. All values are mean ± SD. ns, not significant. Figure 8C depicts the results of exemplary experiments in which wild-type U-2 OS cells were transfected with the indicated ON-TARGETplus SMART Pool siRNAs for 48 hours and incubated in CM or SM in the presence or absence of 100 nM Baf Al for 3 hours and subjected to immunoblotting using the indicated antibodies.

Figure 9, comprising Figure 9A through Figure 9E, depicts the results of experiments demonstrating that CFDVIP2A depletion accumulates ATG2, LC3, ATG9A, and p62-positive immature autophagosomal membranes and impairs autophagic flux. Figure 9A depicts the results of exemplary experiments in which U-2 OS cells stably expressing GFP-LC3 were transfected with the indicated ON-TARGETplus SMART Pool siRNAs for 48 hours, starved for 2 hours, stained with the indicated antibodies and subjected to confocal microscopy. Nuclei were stained with DAPI. Figure 9B depicts the results of exemplary experiments in which U-2 OS cells stably expressing ATG2A/B- deficient U-2 OS cells stably expressing GFP-ATG2A were transfected with the indicated ON-TARGETplus SMART Pool siRNAs for 48 hours, starved for 2 hours, stained with the indicated antibodies and subjected to confocal microscopy. Nuclei were stained with DAPI. Figure 9C depicts the results of exemplary experiments in which wild-type U-2 OS cells were transfected with the indicated siRNAs for 48 hours, starved for 2 hours, stained with the indicated antibodies and subjected to electron microscopy. Asterisks and arrowheads indicate oval-shaped phagophore-like structures,

autolysosomes, atypical autolysosomes, and phagophore-associated ER (arrowheads). Magnified images in the indicated areas are shown in Figure 14 A. Figure 9D depicts the results of exemplary experiments in which CFDVIP2A knockdown or control siNT transfected U-2 OS cells expressing mRFP-GFP-LC3 were starved for 3 hours and subjected to confocal microscopy. Magnified images in the indicated areas are shown in Figure 10A. The scale bars represent 10 μπι, and 1 μπι in the magnified images.

Figure 10, comprising Figure 10A through Figure 10D, depicts the results of experiments demonstrating that CFDVIP2A depletion impairs autophagic flux. Figure 10A depicts the results of exemplary experiments in which U-2 OS cells were transfected with the indicated ON-TARGETplus SMART Pool siRNAs. Eight hours after transfection, cells were transduced with lentiviruses encoding mRFP-GFP-LC3, cultured for 40 hours, incubated in CM or SM for 3 hours, and subjected to confocal microscopy. The images shown are representative from the starved groups shown in Figure 9D. The scale bars represent 10 μπι. Figure 10B depicts the results of exemplary experiments in which the acidification of autophagic structures in Figure 10A was assessed by Pearson's correlation coefficient. Fifty cells from each group were analyzed. Statistical significance was determined by Mann-Whitney nonparametric t-test. All values are mean ± SD. ****, p<0.0001. Figure IOC depicts the results of exemplary experiments in which U-2 OS cells were transfected with the indicated ON-TARGETplus SMART Pool siRNAs.

Forty-eight hours after transfection, cells were incubated in CM or SM in the presence or absence of 100 nM BafAl for 3 hours and subjected to immunoblotting using the indicated antibodies. Representative blots from 4 independent experiments are shown. Figure 10D depicts the results of exemplary experiments in which the LC3-II levels relative to respective β-actin in Figure IOC were quantified and normalized to the value of starved, siNT-transfected cells. Statistical significance was determined by two-way ANOVA followed by Tukey's multiple comparison test. All values are mean ± SD. ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001.

Figure 11, comprising Figure 11 A through Figure 1 IB, depicts the results of experiments demonstrating that overexpression of CFDVIP2A-GFP results in the accumulation of immature autophagosomal structures. Figure 11A depicts the results of exemplary experiments in which HT-LC3 U-2 OS cells were transiently transfected with CFDVIP2A-GFP for 24 hours and subjected to the HT-LC3 autophagosome completion assay. Figure 1 IB depicts the results of exemplary experiments in which HT-LC3 U-2 OS cells were transiently transfected with CHMP2A-GFP for 24 hours and subjected to immunoelectron microcopy using anti-GFP antibody. The scale bars represent 10 μπι in Figure 11 A, and 1 μπι in Figure 1 IB.

Figure 12, comprising Figure 12A through Figure 12C, depicts the results of experiments demonstrating that CFDVIP2A translocates to the phagophore in response to nutrient starvation. Figure 12A depicts the results of exemplary experiments in which HT-LC3 U-2 OS cells were transduced with lentiviruses encoding CFDVIP2A-GFP and cultured for 6 days. The resultant cells stably expressing CFDVIP2A-GFP were incubated in CM or SM for 2 hours and subjected to the HT-LC3 autophagosome completion assay using AF660-conjugated MIL and TMR-conjugated MPL followed by confocal microscopy. Magnified images in the boxed areas are shown in the right panels.

Arrowheads and arrows indicate colocalization of CFDVIP2A with a MIL⁺MPL^" phagophore-like and a MIL⁺MPL⁺ immature autophagosome-like structures. Figure 12B depicts the results of exemplary experiments in which the number of total and LC3- associated CFDVIP2A-GFP-positive foci per cell in Figure 12A was quantified and shown as dot plots {n > 80). Statistical significance was determined using two-way ANOVA with Sidak's multiple comparison test. All values are mean ± SD. ****, p<0.0001. Data shown are representative of two independent experiments. Figure 12C depicts the results of exemplary experiments in which CFDVIP2A-GFP-expressing U-2 OS cells were starved for 2 hours and subjected to immunoelectron microscopy using anti-GFP antibody. The scale bars represent 10 μιη and 1 μιη in the magnified images in Figure 12A, and 500 nm in Figure 12C.

Figure 13, comprising Figure 13 A through Figure 13D, depicts the results of experiments demonstrating that inhibition of VPS4 accumulates MTL⁺MPL^" phagophores and impairs autophagic flux. Figure 13 A depicts the results of exemplary experiments in which HT-LC3 U-2 OS were transfected with GFP-VPS4A^E228Q or control GFP. Twelve hours after the transfection, cells were incubated in CM or SM for 3 hours and subjected to the HT-LC3 autophagosome completion assay followed by confocal microscopy. The scale bars represent 10 μπι and 1 μπι. Figure 13B depicts the results of exemplary experiments in the cytoplasmic fluorescence intensities of MIL and MPL per cell in Figure 13 A were quantified and normalized to the respective mean fluorescence intensities of control GFP transfected cells starved in the presence of BafAl {n > 81). Data shown are representative of two independent experiments. Figure 13C depicts the results of exemplary experiments in which the colocalization coefficient of GFP-VPS4A^E228Q with MIL or MPL-labeled HT-LC3 per cell in Figure 13 A were quantified and shown (n > 72). In Figure 13B and Figure 13C, statistical significance was determined by Kruskal-Wallis one-way ANOVA on ranks followed by Dunn's multiple comparison test. All values are mean ± SD. ns, not significant; **, p<0.01; ****, p<0.0001. Figure 13D depicts the results of exemplary experiments in which wild-type U2-OS cells were transfected with GFP-VPS4A^E228Q or control GFP. Six hours after transfection (0 hour time point), cells were incubated in the presence or absence of 100 nM BafA for 6 hours and subjected to immunoblotting using the indicated antibodies. The LC3-II levels relative to respective β-actin were quantified and normalized to the value of control GFP cells at time 0.

Figure 14, comprising Figure 14A through Figure 14E, depicts the results of experiments demonstrating that phagophore closure is a critical step in functional autolysosome formation. Figure 14A depicts exemplary images of atypical autolysosome- like structures in CHMP2A-depleted cells and normal autophagosome (AP)- and autolysosome (AL)-like structures in starved, control siNT transfected cells. Asterisks and double-asterisks indicate lysosomal (and endolysosomal) contents and IAM-intact autophagosome-like structures, respectively. Figure 14B depicts the results of exemplary experiments in which the number of atypical autolysosomes per cytoplasmic area in Figure 14A was quantified and shown (n = 35). Statistical significance was determined by Mann-Whitney nonparametric t-test. All values are mean ± SD. ****, p<0.0001. Figure 13C depicts the results of exemplary experiments in which U-2 OS cells stably expressing GFP-STX17TM were transfected with the indicated ON-TARGETplus SMART Pool siRNAs for 48 hours. Cells were starved for 2 hours, stained for endogenous LC3B, and subjected to confocal microscopy. Figure 13D depicts the results of exemplary experiments in which U-2 OS cells stably expressing GFP-STX17TM were transfected with the indicated ON-TARGETplus SMART Pool siRNAs for 48 hours. Cells were starved for 1.5 hours, incubated with 100 nM LysoTracker Deep Red, and subjected to confocal microscopy. Figure 13E depicts the results of exemplary experiments in which wild-type U-2 OS cells were starved for 3 hours and subjected to immunoelectron microscopy using anti-LAMPl antibody. Asterisks and arrowheads indicate lysosomal/endosomal LAMP 1 -positive structures and LAMPl signals on the IAM, respectively. The scale bars represent 1 μπι in Figure 14A, 10 μπι in Figure 14C, 1 μπι in the magnified images in Figure 14C, and 0.5 μπι in Figure 14D.

Figure 15, depicts the results of exemplary experiments demonstrating immunoelectron micrographs of CFDVIP2A knockdown and control siNT -transfected, starved U-2 OS cells labelled with anti-LAMPl antibody. Wild-type U-2 OS cells were the indicated ON-TARGETplus SMART Pool siRNAs for 48 hours, starved for 3 hours, and subjected to immunoelectron microscopy using anti-LAMPl antibody. Arrowheads indicate LAMPl signals on the IAM. The scale bars represent 1 μπι.

Figure 16, comprising Figure 16A through Figure 16D, depicts the results of experiments demonstrating, that the SERCA inhibitor thapsigargin further accumulates unclosed autophagosomal membranes in CFDVIP2A-depleted cells. Figure 16A depicts the results of exemplary experiments in which HT-LC3 -expressing U-2 OS cells were transfected with the indicated ON-TARGETplus SMART Pool siRNAs for 48 hours. The indicated inhibitors (100 nM thapsigargin (TG); 100 nM BafAl) were added in the last 18 hours of culture. Cells were subjected to the HT-LC3 autophagosome completion assay followed by confocal microscopy. The scale bars represent 10 μπι, and 1 μπι in the magnified images. Figure 16B depicts the results of exemplary experiments in which the cytoplasmic fluorescence intensities of MIL and MPL per cell in Figure 16A were quantified and normalized to the respective mean fluorescence intensities of control siNT transfected cells (n > 130). Data shown are representative of two independent experiments. Statistical significance was determined by Kruskal-Wallis one-way

ANOVA on ranks followed by Dunn' s multiple comparison test. All values are mean ± SD. ns, not significant; ****, p<0.0001. GFP-LC3 -expressing U-2 OS cells were transfected with the indicated ON-TARGETplus SMART Pool siRNAs for 48 hours. Figure 16C and Figure 16D depict the results of exemplary experiments in which the low-speed pellet (LSP) and high-speed pellet (HSP) prepared from the postnuclear supernatant were resuspended in homogenate buffer in the presence or absence of proteinase K (ProK) and Triton X-100 (TX-100), and subjected to the proteinase K protection assay followed by immunoblotting using an anti-LC3 antibody. The LC3-II levels were quantified and normalized to the respective non-treatment control.

DETAILED DESCRIPTION

During autophagy, the cytosolic form of microtubule-associated protein light chain 3 (LC3), LC3-I, is conjugated with phosphatidylethanolamine to from the membrane bound form of LC3 (LC3-II) which translocates to the membrane of phagophores and autophagosomes. The LC3-II is one of the mammalian orthologues of autophagy-related 8 (Atg8), a family of proteins which includes, but is not limited to, γ- aminobutyric acid receptor-associated protein (GABARAP), γ-aminobutyric acid receptor-associated like protein (GABARAPLl), Golgi-associated ATPase enhancer of 16 kDa (GATE-16) and four LC3 proteins (LC3A, LC3B, LC3B2 and LC3C). All of the Atg8 family members are known to be lipidated and translocate to autophagosomal membrane during autophagy. The invention is based, in part, on a modification of an Atg8 family protein with a tag, which then is specifically and sequentially targeted by ligands having a differential ability to cross cellular membranes. The assay of the invention provides a method of differentially labeling Atg8 family protein generated following closure of autophagosomes and Atg8 family protein that has been generated prior to complete closure of autophagosomes. In one embodiment, the Atg8 family protein is modified with a halo tag and sequentially targeted with membrane permeable and membrane impermeable halo tag ligands (MPL and MIL respectively). In such an embodiment, can be used to detect at least one of a MIL+ MPL- structure, a MIL+ MPL+ structure or a MIL- MPL+ structure in the sample, representing phagophores, nascent autophagosomes, and mature autophagosomes/autolysosomes respectively. In one embodiment, the assay of the invention provides superior signal-to-noise ratio and high reproducibility to distinguish phagophores, nascent autophagosomes, and mature autophagosomal structures.

The invention generally relates to an assay suitable for applications involving immunoelectron microscopy, wide-field fluorescence microscopy, flow cytometry, confocal microscopy, fluorimetry, microplate-based cytometry, high-content cell analysis, cell microarray analysis, high-content cell screening, laser-scanning cytometry and other imaging and detection modalities.

In one embodiment, the invention relates to a system comprising a modified Atg8 family protein and detectable ligands for detection of the modified Atg8 family protein. In one embodiment, the modified Atg8 family protein comprises a fusion of Atg8 family protein to a modified haloalkane dehalogenase (HaloTag) to generate a HaloTag-Atg8 family fusion protein that can covalently bind to a synthetic ligand comprising a reactive chloroalkane linker bound to a functional group. In various embodiments, the functional group is Coumarin, Oregon Green, Alexa Fluor 488, Alexa Fluor 660, diAcFAM, or TMR. In one embodiment, the synthetic ligand is a membrane impermeable ligand (MIL). The MIL of the invention can be used to label membrane bound Atg8 family protein on the surface of phagophores and on the outer membrane of closed autophagosome vacuoles, but does not label membrane bound Atg8 family protein on the inner surface of closed autophagosome vacuoles. In one embodiment, the synthetic ligand is a membrane permeable ligand (MPL). The MPL of the invention can be used to label membrane bound Atg8 family protein on the inner surface of closed autophagosome vacuoles. The MPL and MIL can be employed for distinguishing organelles of the lysosome catabolic pathway, including phagophores, nascent autophagosomes, and mature autophagosomes/autolysosomes. Moreover, the assay can be used to monitor the process of Atg8 family protein delipidation on the outer autophagosome membrane (OAM) which occurs around the autophagosome completion step for autophagosome maturation. The assay can also be used to measure dysfunction of the lysosomal- dependent catabolic pathway, including dysfunction that leads to the accumulation of phagophores or incomplete closure of autophagosomes, and thus is relevant to

macroautophagy in general (herein referred to as autophagy), as well as to pexophagy, xenophagy and other related autophagic pathways or any combination thereof and to diseases and disorders associated with the lysosome catabolic pathway including, but not limited to, Crohn's disease, Vici syndrome, cancers such as breast, ovarian, prostate, liver, colorectal and hematologic cancers, systemic lupus erythematosus,

neurodegenerative diseases such as static encephalopathy of childhood with

neurodegeneration in adulthood (SEND A) and Parkinson's disease, and phospholipidosis. One potential application of the new assay is in preclinical drug safety assessment using in vitro cell culture models to aid in the drug development process.

The HaloTag autophagosome detection assays disclosed can be used to assess the impact of compounds or therapeutics on the lysosomal-dependent catabolic pathway and related compartments. In one embodiment, the assay of the invention can be performed as a semi-automated or automated multi-well cell-based assay to provide rapid and quantitative high-throughput data for determining drug- or toxic agent-induced response. Secondary screening of candidate drugs for potentially adverse cell activity in the drug discovery phase could predict later risks in drug development arising from drug safety issues. Such a screening approach could aid in selecting the best candidate compounds for further drug development efforts, as well as provide preliminary benchmarking of dosing limits in preclinical toxicity studies. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%), ±5%), ±1%), or ±0.1%) from the specified value, as such variations are appropriate to perform the disclosed methods.

The term "abnormal" when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal" (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.

A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.

An "effective amount" or "therapeutically effective amount" of a compound is that amount of a compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. An "effective amount" of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.

As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of a compound, composition, vector, or delivery system of the invention in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material can describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit of the invention can, for example, be affixed to a container which contains the identified compound, composition, vector, or delivery system of the invention or be shipped together with a container which contains the identified compound, composition, vector, or delivery system. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

The terms "patient," "subject," "individual," and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in vivo, amenable to the methods described herein.

A "therapeutic" treatment is a treatment administered to a subject who exhibits signs or symptoms of pathology, for the purpose of diminishing or eliminating those signs or symptoms.

As used herein, "treating a disease or disorder" means reducing the severity and/or frequency with which a sign or symptom of the disease or disorder is experienced by a patient. Disease and disorder are used interchangeably herein.

As used herein, an "immunoassay" refers to any binding assay that uses an antibody capable of binding specifically to a target molecule to detect and quantify the target molecule.

A "coding region" of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.

A "coding region" of a mRNA molecule also consists of the nucleotide residues of the mRNA molecule which are matched with an anti-codon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon. The coding region may thus include nucleotide residues comprising codons for amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

"Complementary" as used herein to refer to a nucleic acid, refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%), or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

The term "DNA" as used herein is defined as deoxyribonucleic acid. "Isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in its normal context in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural context is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

An "isolated nucleic acid" refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified

polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

The term "RNA" as used herein is defined as ribonucleic acid.

The term "recombinant DNA" as used herein is defined as DNA produced by joining pieces of DNA from different sources.

The term "recombinant polypeptide" as used herein is defined as a polypeptide produced by using recombinant DNA methods.

As used herein, "conjugated" refers to covalent attachment of one molecule to a second molecule.

"Variant" as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.

By fluorescence is meant the emission of light as a result of absorption of light-emission, occurring at a longer wavelength than the incident light.

By fluorophore is meant a component of a molecule which causes a molecule to be fluorescent.

By autophagy is meant an evolutionarily conserved subcellular degradation process decomposing folded proteins, protein complexes and entire organelles, such as aggregates of misfolded proteins or damaged mitochondria, involving the import of cytoplasmic components into the lysosome.

By membrane-impermeable ligand (MIL) is meant a member of a class of compounds which do not have the ability to cross an intact autophagosome membrane either through a passive or active transport mechanism.

By membrane-permeable ligand (MPL) is meant a member of a class of compounds which have the ability to cross an intact autophagosome membrane either through a passive or active transport mechanism. The MPLs are sequestered into cells by a variety of metabolically-driven, but receptor-independent means including

mitochondrial membrane potential-driven concentration, nuclear concentration via DNA affinity and vacuolar- ATPase-driven trapping. MPL concentration within cells may arise from the innate membrane permeability of the MPL, as well as by means of specific transporters (organic cation transporters, choline transporters, etcetera).

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Description

The invention is based, in part on the development of an assay for Atg8 family protein localization and turnover that can be used to distinguish between an Atg8 family protein unsequestered in the autophagosome which is accessible to a membrane- impermeable detectable ligand and Atg8 family protein sequestered in the

autophagosome which is accessible to a membrane-permeable detectable ligand. In one embodiment, the assay of the invention provides superior signal-to-noise ratio and high reproducibility to distinguish phagophores, nascent autophagosomes, and mature autophagosomal structures.

Compositions

In one embodiment, the invention relates to vectors for use in the method of the invention. In another embodiment, the invention relates to cells expressing a tagged LC3-II protein generated using the method of the invention.

HaloTag Autophagosome Detection Assay Vector

A HaloTag autophagosome detection assay vector of the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an Atg8 family protein or a variant thereof operably linked to a nucleotide sequence encoding a HaloTag. In one embodiment, the Atg8 family protein is a human ortholog of Atg8. In one embodiment, the Atg8 family protein is GABARAP, GABARAPLl, GATE- 16, LC3 A, LC3B, LC3B2 or LC3C. In one embodiment, the vector encodes a HaloTag operably linked to LC3B, having an amino acid sequence as set forth in SEQ ID NO:2, or a variant thereof. In one embodiment, the vector comprises a nucleotide sequence as set forth in SEQ ID NO: l, or a variant thereof, encoding a HaloTag operably linked to LC3B.

In one embodiment, the vector further comprises one or more additional functional element such as a reporter marker, a linker, a protease cleavage site, an antibiotic resistance gene, origin of replication, cell selection marker sequences, mRNA stabilization sequence, exogenous gene sequence, termination sequence, internal ribosomal entry sequence (IRES), promoter sequences, translation initiation sequences, recombinase recognition sites, and other functional elements.

A reporter marker is a molecule, including polypeptide as well as polynucleotide, expression of which in a cell confers a detectable trait to the cell. In various embodiments, reporter markers include, but are not limited to, chloramphenicol- acetyl transferase(CAT), β-galactosyltransferase, horseradish peroxidase, luciferase, NanoLuc®, alkaline phosphatase, and fluorescent proteins including, but not limited to, green fluorescent proteins (e.g. GFP, TagGFP, T-Sapphire, Azami Green, Emerald, mWasabi, mClover3), red fluorescent proteins (e.g. mRFPl, JRed, HcRedl, AsRed2, AQ143, mCherry, mRuby3, mPlum), yellow fluorescent proteins (e.g. EYFP, mBanana, mCitrine, PhiYFP, TagYFP, Topaz, Venus), orange fluorescent proteins (e.g. DsRed, Tomato, Kusabria Orange, mOrange, mTangerine, TagRFP), cyan fluorescent proteins (e.g. CFP, mTFPl, Cerulean, CyPet, AmCyanl), blue fluorescent proteins (e.g. Azurite, mtagBFP2, EBFP, EBFP2, Y66H), near-infrared fluorescent proteins (e.g. iRFP670, iRFP682, iRFP702, iRFP713 and iRFP720), infrared fluorescent proteins (e.g. IFP1.4) and photoactivatable fluorescent proteins (e.g. Kaede, Eos, IrisFP, PS-CFP).

Tags for use in the methods of the invention include, but are not limited to, chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His), biotin/streptavidin, V5-tag, Myc-tag, HA-tag, E-tag, His-tag, Flag tag, Halo-tag, Snap-tag, Fc-tag, Nus-tag, BCCP, Thioredoxin, SnooprTag, SpyTag, Isopeptag, SBP-tag, S-tag, AviTag, Calmodulin, or any combination of sequences appropriate for use in a method of tagging a protein. The target protein and associated tag can be purified from target cells or target cell culture medium by any method known in the art for purifying polypeptides. Examples of such methods include salt fractionation, high pressure liquid chromatography, antibody column chromatography, affinity tag column chromatography, and acrylamide gel electrophoresis. Such methods are well known to those skilled in the art.

A selection marker sequence can be used to eliminate target cells in which an insertion cassette has not been properly inserted or to eliminate host cells in which the FIDR vector has not been properly transfected. A selection marker sequence can be a positive selection marker reporter marker or negative selection marker. Positive selection markers permit the selection for cells in which the gene product of the marker is expressed. This generally comprises contacting cells with an appropriate agent that, but for the expression of the positive selection marker, kills or otherwise selects against the cells. For suitable positive and negative selection markers, see Table I in U.S. Pat. No. 5,464,764.

Examples of selection markers also include, but are not limited to, proteins conferring resistance to compounds such as antibiotics, proteins conferring the ability to grow on selected substrates, proteins that produce detectable signals such as

luminescence, catalytic RNAs and antisense RNAs. A wide variety of such markers are known and available, including, for example, a Zeocin™ resistance marker, a blasticidin resistance marker, a neomycin resistance (neo) marker (Southern & Berg, J. Mol. Appl. Genet. 1 : 327-41 (1982)), a puromycin (puro) resistance marker; a hygromycin resistance (hyg) marker (Te Riele et al., Nature 348:649-651 (1990)), thymidine kinase (tk), hypoxanthine phosphoribosyltransferase (hprt), and the bacterial guanine/xanthine phosphoribosyltransferase (gpt), which permits growth on MAX (mycophenolic acid, adenine, and xanthine) medium. See Song et al., Proc. Nat'l Acad. Sci. U.S.A. 84:6820- 6824 (1987). Other selection markers include histidinol-dehydrogenase,

chloramphenicol-acetyl transferase (CAT), dihydrofolate reductase (DHFR), β- galactosyltransferase and fluorescent proteins such as GFP.

Expression of a fluorescent protein can be detected using a fluorescent activated cell sorter (FACS). Expression of β-galactosyltransferase also can be sorted by FACS, coupled with staining of living cells with a suitable substrate for β-galactosidase. A selection marker also may be a cell-substrate adhesion molecule, such as integrins which normally are not expressed by the mouse embryonic stem cells, miniature swine embryonic stem cells, and mouse, porcine and human hematopoietic stem cells. Target cell selection marker can be of mammalian origin and can be thymidine kinase, aminoglycoside phosphotransferase, asparagine synthetase, adenosine deaminase or metallothionien. The cell selection marker can also be neomycin phosphotransferase, hygromycin phosphotransferase or puromycin phosphotransferase, which confer resistance to G418, hygromycin and puromycin, respectively.

Suitable prokaryotic and/or bacterial selection markers include proteins providing resistance to antibiotics, such as kanamycin, tetracycline, and ampicillin. In one embodiment, a bacterial selection marker includes a protein capable of conferring selectable traits to both a prokaryotic host cell and a mammalian target cell.

In accordance with the present invention, the selection marker usually is selected based on the type of the cell undergoing selection. For instance, it can be eukaryotic (e.g., yeast), prokaryotic (e.g., bacterial) or viral. In such an embodiment, the selection marker sequence is operably linked to a promoter that is suited for that type of cell. A promoter can be selected based on the type of host or target cell or the desired level of expression of an exogenous gene. Suitable promoters include but are not limited to the ubiquitin promoters, the herpes simplex thymidine kinase promoters, human cytomegalovirus (CMV) promoters/enhancers, EF-1 alpha promoters, SV40 promoters, β-actin promoters, immunoglobulin promoters, regulatable promoters such as metallothionein promoters, adenovirus late promoters, and vaccinia virus 7.5K

promoters. The promoter sequence also can be selected to provide tissue-specific transcription.

In certain embodiments, an IRES sequence may be included in the insertion cassette to improve the translation of a downstream gene. In one embodiment, the IRES may improve the translation of an exogenous gene sequences, a target cell selection marker sequence or a reporter marker sequence. The IRES site can be located within the insertion cassette and may be a mammalian internal ribosome entry site, such as an immunoglobulin heavy chain binding protein internal ribosome binding site. In one embodiment, the IRES sequence is selected from encephalomyocarditis virus, poliovirus, piconaviruses, picorna-related viruses, and hepatitis A and C. Examples of suitable IRES sequences can be found in U.S. Pat. No. 4,937, 190, in European patent application 585983, and in PCT applications W09611211, WO09601324, and WO09424301, respectively.

In one embodiment, an autophagosome maturation assay vector comprises a translational initiation sequence or enhancer, such as the so-called "Kozak sequence" (Kozak, J. Cell Biol. 108: 229-41 (1989)) or "Shine-Delgarno" sequence. These sequences may be located in the insertion cassette, 3' to an IRES site but 5' to an endogenous gene sequence, reporter marker sequence or selection marker sequence.

In one embodiment, an insertion cassette of the invention comprises one or more mRNA stabilization sequence. An mRNA stabilization sequence may alter the half- life of an mRNA molecule encoding a target gene and fused to the sequence such that the reading frame is maintained. In one embodiment the mRNA stabilization sequence is a polynucleotide sequence that increases the half-life of a linked mRNA. In one

embodiment the mRNA stabilization sequence is a polynucleotide sequence that decreases the half-life of a linked mRNA. In one embodiment, a mRNA stabilization sequence is a poly(A) tail which protects the mRNA molecule from enzymatic degradation in the cytoplasm. In one embodiment, a mRNA stabilization sequence is a MALAT1 3' stabilization sequence.

In one embodiment, an autophagosome maturation assay vector comprises a transcription termination sequence. A typical transcriptional termination sequence includes a polyadenylation site (poly A site). In one embodiment, a poly A site is the SV40 poly A site. These sequences may be located in the insertion cassette, 3' to an endogenous gene sequence, reporter marker sequence or selection marker sequence.

In one embodiment, an autophagosome maturation assay vector comprises one or more termination/stop codon(s) in one or more reading frames at the 3' end of an endogenous gene sequence, reporter marker sequence or selection marker sequence, such that translations of these sequences, if they encode polypeptides, are terminated at the stop codon(s).

In one embodiment, an autophagosome maturation assay vector comprises an origin of replication capable of initiating DNA synthesis in a suitable host cell.

Preferably, the origin of replication is selected based on the type of host cell. For instance, it can be eukaryotic (e.g., yeast) or prokaryotic (e.g., bacterial) or a suitable viral origin of replication may be used.

Cells

An autophagosome maturation assay vector of the present invention can be used to express tagged LC3-II in a target cell. An autophagosome maturation assay vector can be introduced into a target cell by any methods as appreciated in the art, including but not limited to, electroporation, viral infection, retrotransposition, microinjection, lipofection, liposome-mediated transfection, calcium phosphate precipitation, DEAE-dextran, and ballistic or "gene gun" penetration.

In one embodiment, target cells are prokaryotic cells. In one embodiment, target cells are eukaryotic cells. In one embodiment, a target cell is a mammalian cell, such as a murine or human cell. The target cell may be a somatic cell or a germ cell. The germ cell may be a stem cell, such as embryonic stem cells (ES cells), including murine embryonic stem cells. The target cell may be a non-dividing cell, such as a neuron, or alternatively, the target cell can proliferate in vitro under certain culturing conditions.

The target cell may be chosen from commercially available mammalian cell lines. The target cell may be a primary cell isolated from a subject. A target cell may be any type of diseased cell, including cells with abnormal phenotypes that can be identified using biological or biochemical assays. For instance, the diseased cell may be a cell from a subject with a disease or disorder associated with autophagosomal closure.

Halo ligands

The LC3-II targeting MPL and MIL molecules for use in the invention may be generated by attaching a detectable molecule to a membrane permeable or membrane impermeable target-specific moiety. Thus, binding between the target specific moiety and LC3-II, or modified LC3-II, may be monitored by essentially determining the presence or amount of detectable molecule that is bound to LC3-II. In one embodiment, the detectable molecules of the MPL and MIL are different dyes, allowing for differential labeling of LC3-II. In one embodiment, the detectable label of the MIL is detectable at a different wavelength than the detectable label of the MPL, allowing for detection of both labels in the same sample.

In one embodiment, the MIL comprises a Halo ligand which does not have the ability to cross an intact autophagosome membrane either through a passive or active transport mechanism. In one embodiment, the detectable label of the MIL comprises Coumarin, Oregon Green, Alexa Fluor 488, Alexa Fluor 660, diAcFAM, or

tetramethylrhodamine (TMR). In one embodiment, the MIL comprises G1001 or G8471.

In one embodiment, the MIL comprises a Halo ligand which has the ability to cross an intact autophagosome membrane either through a passive or active transport mechanism. The MPLs can be sequestered into cells by a variety of

metabolically-driven, but receptor-independent means including mitochondrial membrane potential-driven concentration, nuclear concentration via DNA affinity and vacuolar-ATPase-driven trapping. MPL concentration within cells may arise from the innate membrane permeability of the MPL, as well as by means of specific transporters (organic cation transporters, choline transporters, etcetera). In one embodiment, the detectable label of the MPL comprises Coumarin, Oregon Green, Alexa Fluor 488, Alexa Fluor 660, diAcFAM, or TMR. In one embodiment, the MIL comprises G8251.

Autophagosome Maturation Assay Method

The autophagosomal maturation assay is particularly well suited for detecting or identifying a gene, protein or pathway associated with the presence of a disease or disorder associated with a defect in autophagosome closure and for evaluating the effects of compounds or treatment on autophagosome closure. In various

embodiments, a disease or disorder associated with a defect in autophagosome closure is Crohn's disease, Vici syndrome, cancers such as breast, ovarian, prostate, liver, colorectal and hematologic cancers, systemic lupus erythematosus, neurodegenerative diseases such as static encephalopathy of childhood with neurodegeneration in adulthood (SEND A) and Parkinson's disease, or phospholipidosis

As discussed above, the invention provides a method of distinguishing between specific vacuoles of cells. Such vacuoles include, for example, lysosomes, phagophores, autophagosomes or autophagolysosomes, and combinations of the foregoing. The assay of the invention comprises the use of a first membrane permeable detectable ligand and a second membrane impermeable detectable ligand which are specific for LC3-II.

In one embodiment, the present disclosure relates to an assay to monitor autophagosome completion within a cell. In one embodiment, the assay comprises contacting a cell expressing an autophagosome maturation assay vector with a detectable MIL and a detectable MPL and determining the level of at least one of a MIL+ MPL+ autophagosome, a MIL+ MPL- phagophore and a MIL- MPL+ organelle in the cell.

In one embodiment, the assay further comprises inducing autophagy in the cell prior to contacting the cell with at least one of a detectable MIL and a detectable MPL. Autophagy can be induced chemically or through culturing cells in starvation medium or under low oxygen (hypoxic) conditions. In one embodiment, a starvation medium comprises 140 mM NaCl, 1 mM CaCh, 1 mM MgCh, 5 mM glucose, 20 mM Hepes, pH 7.4, and 1% BSA. Alternatively a starvation medium may comprise Dubelcco modified eagle medium (DMEM) without amino acids, and optionally without glucose, Earle's Balance Salt Solution (EBSS) or other starvation media known and used in the art for induction of autophagy. Chemical means for inducing autophagy include, but are not limited to, STF-62247, Akebia saponin, Amiodarone hydrochloride, Cobalt Chloride, Deferoxamine mesylate, SRP5341, G2911, GF 109203X hydrochloride, N-Hexanoyl-D- sphingosine, MRT68921 dihydrochloride, Niclosamide, Qcl (a reversible inhibitor of threonine dehydroxygenase), Rapamycin, Rottlerin, Tamoxifen, Temsirolimus, unc-51 like autophagy activating kinase 1 and Z36.

In one embodiment, the assay further comprises contacting the cell with a plasma membrane permeabilization agent prior to contacting the cell with at least one of a detectable MIL and a detectable MPL, for example, to remove cytosolic LC3-I and allow access of the MIL to membrane bound LC3-II. Permeabilization agents that can be utilized according to the method of the invention, include, but are not limited to, cholesterol-complexing agents including recombinant perfringolysin (rPFO/XF-MPM) or digitonin.

The present disclosure also relates to a method of identifying compound having or suspected of having an effect on autophagy. A drug or compound library may be applied to a cell of the invention to screen for candidates that may regulate the autophagocytosis pathway. In an embodiment of the present disclosure, the method of identifying a compound having or suspected of having an effect on autophagy comprises performing the steps:

a) inserting a vector of the present invention into a cell for expression of a HaloTag-Atg8 family fusion protein;

b) contacting the host cell with a compound having or suspected of having an effect on autophagy;

c) culturing the transformed host cell in medium comprising MIL;

d) culturing the transformed host cell in medium comprising MPL; and e) detecting at least one of a MIL+ MPL- vacuole, a MIL+ MPL+ vacuole and a MIL- MPL+ vacuole in the host cell.

In one embodiment, the methods further comprise determining the relative level of closure of autophagosomes or the progression of the lysosomal-dependent catabolic pathway. In one embodiment, the method comprises detecting a decreased level of mature autophagosomes in a sample as compared to a comparator control. In one embodiment, the method comprises detecting an increased level of mature

autophagosomes in a sample as compared to a comparator control. For example, in one embodiment, a deficiency in the lysosomal-dependent catabolic pathway can be is characterized by the failure of phagophores to close, resulting in a decreased level of mature autophagosomes in a sample as compared to a comparator control. In one embodiment, a deficiency in the lysosomal-dependent catabolic pathway can be characterized by the failure of autophagosomes to mature into autolysomes, resulting in an increased level of mature autophagosomes in a sample as compared to a comparator control. In one embodiment, the method comprises evaluating the amount of MIL+ MPL+ autophagosomes in a sample. In one embodiment, the method comprises evaluating the amount of MIL+ MPL- phagophores in a sample. In one embodiment, the method comprises evaluating the amount of MIL- MPL+ organelles in a sample. Thus, in one embodiment, the level or amount of at least one of MIL+ MPL+ autophagosomes, MIL+ MPL- phagophores and MIL- MPL+ organelles is used to determine the effect of a treatment or compound on the closure of autophagosomes or the progression of the lysosomal-dependent catabolic pathway. In one embodiment, the method can be used to evaluate the potential effectiveness or toxicity of a treatment for a disease or disorder associated the closure of autophagosomes or the progression of the lysosomal-dependent catabolic pathway.

Methods of Identifying a Modulator of Autophagosome Closure

In one embodiment, the current invention relates to methods of identifying a compound that modulates the level of autophagosome closure or the progression of the lysosomal-dependent catabolic pathway. In some embodiments, the method of identifying of the invention identifies an inhibitor compound that decreases the level of autophagosome closure or the progression of the lysosomal-dependent catabolic pathway. In other embodiments, the method of identifying of the invention identifies an activator compound that increases autophagosome closure or the progression of the lysosomal- dependent catabolic pathway. In one embodiment, the method comprises contacting a cell comprising an autophagosome maturation assay vector of the invention with a test compound, at least one MIL and at least one MPL and evaluating the effect of the test compound on the level of at least one of a MIL+ MPL+ autophagosome, a MIL+ MPL- phagophore and a MIL- MPL+ organelle in the cell. Methods of evaluating the effect of the test compound on the level of at least one of a MIL+ MPL+ autophagosome, a MIL+ MPL- phagophore and a MIL- MPL+ organelle in the cell are well known in the art and include, but are not limited to, immunoelectron microscopy, wide-field fluorescence microscopy, flow cytometry, confocal microscopy, fluorimetry, microplate-based cytometry, high-content cell analysis, cell microarray analysis, high-content cell screening, laser-scanning cytometry and other imaging and detection modalities.

Other methods, as well as variation of the methods disclosed herein will be apparent from the description of this invention. In various embodiments, the test compound concentration in the screening assay can be fixed or varied. A single test compound, or a plurality of test compounds, can be tested at one time. In some embodiments, the method of identifying is a high-throughput screen. Suitable test compounds that may be used include, but are not limited to, proteins, nucleic acids, antisense nucleic acids, shRNA, small molecules, antibodies and peptides.

The invention relates to a method for screening test compounds to identify a modulator compound by its ability to modulate (i.e., increase or decrease) the level of at least one of a MIL+ MPL+ autophagosome, a MIL+ MPL- phagophore and a MIL- MPL+ organelle, in the presence and absence of the test compound. Test compounds that can be assessed in the methods of the invention include a chemical compound, a protein, a peptide, a peptidomemetic, an antibody, a nucleic acid, an antisense nucleic acid, an siRNA, a miRNA, a shRNA, a ribozyme, an allosteric modulator, and a small molecule chemical compound.

The test compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam et al., 1997, Anticancer Drug Des. 12:45).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example, in: DeWitt et al., 1993, Proc. Natl. Acad. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91 : 11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al., 1993, Science 261 : 1303; Carrell et al., 1994, Angew. Chem. Int. Ed. Engl. 33 :2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33 :2061; and Gallop et al., 1994, J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (e.g., Houghten,

1992, Biotechniques 13 :412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; and Ladner supra).

In situations where "high-throughput" modalities are preferred, it is typical to that new chemical entities with useful properties are generated by identifying a chemical compound (called a "lead compound") with some desirable property or activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds.

In one embodiment, high throughput screening methods involve providing a library containing a large number of test compounds potentially having the desired activity. Such "combinatorial chemical libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.

In one embodiment, a compound identified as a potential therapeutic using a screen of the invention is a compound that increases the level of MIL- MPL+ organelles. In one embodiment, the compound that increases the level of MIL- MPL+ organelles is one of a chemical compound, a protein, a peptide, a peptidomemetic, an antibody, a nucleic acid, an antisense nucleic acid, an siRNA, a miRNA, a shRNA, a ribozyme, an allosteric modulator, and a small molecule chemical compound. Use of Compounds

In one embodiment, compounds or molecules identified in a screen using the autophagosome maturation assay of the invention are useful for treatment of a disease associated with a disease or disorder associated the closure of autophagosomes or the progression of the lysosomal-dependent catabolic pathway. Therefore, the invention relates to a method of treating a disease comprising administering to a subject in need thereof a compound identified by the method of screening for the effect of a compound of the invention. In one embodiment, the compound was identified in the screen as having an effect on the level of at least one of MIL+ MPL+ autophagosomes, MIL+ MPL- phagophores and MIL- MPL+ organelles. In one embodiment, the effect in an increase in the level of at least one of MIL+ MPL+ autophagosomes, MIL+ MPL- phagophores and MIL- MPL+ organelles. In one embodiment, the effect is a decrease in the level of at least one of MIL+ MPL+ autophagosomes, MIL+ MPL- phagophores and MIL- MPL+ organelles. Modulation of Genes Identified in an Autophagosome Maturaton Assay

In one embodiment, the invention relates to modulation of one or more gene identified in a screen using the autophagosome maturation assay of the invention are useful for treatment of a disease or disorder associated with the closure of

autophagosomes or the progression of the lysosomal-dependent catabolic pathway.

Therefore, the invention relates to a method of treating a disease comprising

administering to a subject in need thereof a modulator of one or more gene identified by the method of invention as having an effect on the level of at least one of MIL+ MPL+ autophagosomes, MIL+ MPL- phagophores and MIL- MPL+ organelles. In one embodiment, the effect in an increase in the level of at least one of MIL+ MPL+ autophagosomes, MIL+ MPL- phagophores and MIL- MPL+ organelles. In one embodiment, the effect is a decrease in the level of at least one of MIL+ MPL+ autophagosomes, MIL+ MPL- phagophores and MIL- MPL+ organelles.

Exemplary genes identified by the methods of the invention as having an effect on the level of at least one of MIL+ MPL+ autophagosomes, MIL+ MPL- phagophores and MIL- MPL+ organelles include, but are not limited to, CHMP2A, VPS4, and ESCRT subunits.

Therefore, in various embodiments, the invention relates to the use of an activator of at least one ESCRT component for the treatment of a disease or disorder associated with reduced or deficient levels of autophagosome completion. In one embodiment, at least one ESCRT component is CHMP2A or VPS4.

An ESCRT activator can include, but should not be construed as being limited to, a chemical compound, a protein, a peptidomemetic, an antibody, a nucleic acid molecule. One of skill in the art would readily appreciate, based on the disclosure provided herein, that a ESCRT activator encompasses a chemical compound that increases the level, enzymatic activity, or the like of at least one ESCRT component. In some embodiments, the enzymatic activity is regulation of autophagosome closure.

Additionally, an ESCRT activator encompasses a chemically modified compound, and derivatives, as is well known to one of skill in the chemical arts.

It will be understood by one skilled in the art, based upon the disclosure provided herein, that an increase in the level of at least one ESCRT component encompasses the increase in expression, including transcription, translation, or both of at least one ESCRT component. The skilled artisan will also appreciate, once armed with the teachings of the present invention, that an increase in the level of at least one ESCRT component includes an increase in at least one ESCRT component activity (e.g., enzymatic activity, etc.). Thus, increasing the level or activity of at least one ESCRT component includes, but is not limited to, increasing the amount of polypeptide of at least one ESCRT component, increasing transcription, translation, or both, of a nucleic acid encoding at least one ESCRT component; and it also includes increasing any activity of at least one ESCRT component as well. In one embodiment, the ESCRT activator compositions and methods of the invention can selectively activate CHMP2A or VPS4. Further, one of skill in the art would, when equipped with this disclosure and the methods exemplified herein, appreciate that a ESCRT activator includes such activators as discovered in the future, as can be identified by well-known criteria in the art of pharmacology, such as the physiological results of activation of at least one ESCRT component as described in detail herein and/or as known in the art. Therefore, the present invention is not limited in any way to any particular ESCRT activator as exemplified or disclosed herein; rather, the invention encompasses those activators that would be understood by the person of skill in the art to be useful as are known in the art and as are discovered in the future.

Pharmaceutical Compositions

The present invention includes pharmaceutical compositions comprising one or more modulators of the closure of autophagosomes or the progression of the lysosomal-dependent catabolic pathway of the invention. The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts.

Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs. Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intratumoral, epidural, intracerebral, intracerebroventricular, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.

Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a

preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems.

Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Kits

The present invention also pertains to kits useful in the methods of the invention. Such kits comprise various combinations of components useful in any of the methods described elsewhere herein, including for example an autophagosome maturation assay vector or expression construct or a cell comprising an autophagosome maturation assay vector as above, optionally along with components selected from a group comprising MIL, MPL and instruction manual or any combination thereof.

In various embodiments, to determine whether the level of at least one of a

MIL+ MPL+ autophagosome, a MIL+ MPL- phagophore and a MIL- MPL+ organelle is modulated in a sample, the level of at least one of a MTL+ MPL+ autophagosome, a MIL+ MPL- phagophore and a MIL- MPL+ organelle is compared with the level of at least one comparator control contained in the kit, such as a positive control, a negative control, a historical control, a historical norm, or a detectable molecule specific for a reference molecule in a biological sample.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1 : A novel autophagosome completion assay reveals the ESCRT-III component CHMP2A as a key regulator of phagophore closure

The ESCRT components have been suggested as potential regulators of autophagosomal membrane closure (Hurley, 2015, EMBO J, 34:2398-2407; Knorr et al., 2015, Autophagy, 11 :2134-2137; Rusten and Stenmark, 2009, J Cell Sci, 122:2179- 2183). However, characterization of the roles of each ESCRT protein in phagophore closure is technically challenging due to the lack of a robust assay that can distinguish phagophores and nascent autophagosomes. In this study, a novel HT-LC3

autophagosome completion assay is developed and used to demonstrate a role for ESCRT proteins in phagophore closure. By sequentially labelling membrane-unenclosed and - enclosed HT-LC3-II with saturated doses of MIL and MPL, respectively, MIL+MPL- phagophores, MIL+MPL+ nascent autophagosomes, and MIL-MPL+ mature autophagic vacuoles are distinguished. This assay provides a superior signal-to-noise ratio and high reproducibility with a semi-quantitative output and high-throughput adaptability by performing a siRNA screen of the ESCRT components and identifying several subunits, including the ESCRT-III CHMP2A, as critical regulators of phagophore closure. Further, this study demonstrates that phagophore closure is regulated by the ESCRT machinery.

The data presented herein shows that CHMP2A translocates to the phagophore during autophagy. The observation that CHMP2A depletion dramatically increases MIL+MPL- signals in the HT-LC3 autophagosome completion assay is consistent with the electron microscopy and proteinase K protection assay results that demonstrate the accumulation of unclosed phagophore-like structures and protease- unprotected GFP-LC3-II, respectively. The accumulation of MIL+MPL- signals also occurs upon the inhibition of VPS4 by expression of the ATPase-deficient mutant VPS4AE228Q. Moreover, VPS4AE228Q accumulates on MIL+MPL- structures, suggesting that VPS4 functions together with CHMP2A at the phagophore closure site to drive the membrane fission and generate the OAM and IAM. As the accumulation of phagophore/immature autophagosome-like structures is observed under non-starved condition and nutrient starvation only marginally increases MIL+ signals in CHMP2A- depleted and VPS4AE228Q-expressing cells, one can speculate that inhibition of the ESCRT subunits may also promote the induction of autophagy. Indeed, it has been reported that ESCRT defects activate JNK (a potent autophagy inducer; Zhou et al., 2015, Biosci Rep, 35) in Drosophila (Herz et al., 2006, Development, 133 : 1871-1880; Rodahl et al., 2009, PLoS One, 4:e4354) and increase autophagic flux in C. elegans (Djeddi et al., 2012, J Cell Sci, 125:685-694). However, the autophagic flux is inhibited by

CHMP2A depletion and that the rate of LC3-I to LC3-II conversion is not enhanced by VPS4 inhibition. The observations that TG treatment significantly reduces the MPL signals and enhances the sensitivity of GFP-LC3-II to proteinase K upon CHMP2A depletion further indicate the role of ESCRT in phagophore closure. Therefore, without being bound by theory, although upregulation of autophagy upon ESCRT inhibition may contribute to the increase in phagophores, it is hypothesized that the accumulation of immature autophagosomal structures by ESCRT inhibition is mainly due to the impairment of membrane abscission required for the OAM and IAM separation.

While the current study focuses on the function of CHMP2A in autophagy, other ESCRT -III proteins including CHMP3 and CHMP7 may also function as potential regulators of phagophore closure. However, unlike CHMP2A, knockdown of these proteins only moderately accumulates MIL+ immature autophagosome-like structures and many of the other components of the ESCRT machinery appear to be dispensable for phagophore closure. This observation is not surprising, since it has previously been reported that, unlike the canonical ESCRT-mediated membrane fission requiring ESCRT-0, -I, -II, and -III subunits, only CHMP2 and CHMP4 are found to be critical ESCRT-III components during HIV budding (Morita et al., 2011, Cell Host Microbe, 9:235-242). Moreover, it has recently been shown that nuclear envelope reformation is regulated by the ESCRT-III subunits CHMP2A and CHMP7 in a manner that is independent of canonical upstream targeting and bridging molecules (Gu et al., 2017, Proc Natl Acad Sci U S A, 114:E2166-E2175; Olmos et al., 2015, Nature, 522:236- 239; Vietri et al., 2015, Nature, 522:231-235). Thus, the phagophore closure may also be regulated by a noncanonical ESCRT pathway. Moreover, as the present siRNA screening only targets single ESCRT genes, it is important in the future to examine the effects of combinational targeting of functionally redundant ESCRT subunits (e.g. VPS4A/B, CHMP4A-C, and ALIX/ESCRT-II) on phagophore closure to fully characterize the ESCRT machinery for autophagosome completion.

The data also show that, while phagophore closure is important for lysosome recruitment and fusion, a small fraction of unclosed autophagosomal membranes still can undergo lysosomal fusion. However, this event appears to be nonproductive, as LAMPl is distributed on both the OAM and IAM and the lysosomal contents are accumulated in the intermembrane space rather than the lumen of the autophagosome-like structure. Moreover, the observation that nearly all of

phagophore/immature autophagosome-like structures in CHMP2A-depeleted cells are positive for STX17 is consistent with a recent report showing that STX17 dissociation is triggered by IAM degradation (Tsuboyama et al, 2016, Science, 354: 1036-1041). Without being bound by theory, it was hypothesize that the generation of the OAM and IAM by ESCRT-mediated membrane fission prior to lysosomal fusion is critical to prevent the misdistribution of glycosylated lysosomal membrane proteins on the IAM and the failure of which limits access to lysosomal proteases and lipases to impair degradation. The materials and methods employed in the experiments are now described.

Reagents

The following antibodies were used for immunoblotting (IB), immunofluorescence (IF) and immunoelectron microscopy (IEM): ATG7 (IB; Santa

Cruz, SC-8668, 1 :300); ATG9A (IF; Cell Signaling, 13509, 1 :300); ATG16L (IF, MBL, PM040, 1 :400); β-ACTIN (IB, Sigma-Aldrich, A5441, 1 : 10,000); CD107a/LAMPl (IEM, BD Biosciences, 555798, 1 : 100); CEP55 (IB; Santa Cruz, SC- 377018, 1 :200); CHMP2A (IB, Proteintech, 104771-AP, 1 : 1,000); CHMP2B (IB, Cell Signaling, 76173, 1 : 1,000); CHMP3 (IB; Santa Cruz, SC-166361, 1 :200); CHMP7 (IB; Santa Cruz, SC- 271805, 1 :200); GFP (IEM, Abeam, ab6556, 1 :500); MAP1LC3B (IB, Novus, NBIOO- 2220, 1 :3,000; IF, Cell Signaling, 3868, 1 :200); p62 (IB, IF; American Research

Products, 03-GP62-C, 1 :4,000 (IB), 1 :400 (IF)). Accell SMART Pool siRNAs and ON- TARGETplus SMART Pool siRNAs listed in Figure 17 were purchased from GE

Healthcare Dharmacon. All other reagents were obtained from the following sources: Bafilomycin Al (LC Laboratories, B-1080); bovine serum albumin (BSA) (EMD

Millipore, 126575); Membrane-impermeable HaloTag Ligand (MIL) (Promega, Alexa Fluor 488-conjugated, G1001; Alexa Fluor 660-conjugated, G8471); Membrane- permeable HaloTag Ligand (MPL) (Promega, tetramethylrhodamine-conjugated, G8251); digitonin (SIGMA, D141); Nucleofector Kit V (Lonza, VCA-1003); Nucleofector Kit R (Lonza, VCA-1001); recombinant human epidermal growth factor (EGF) (Thermo-Fisher Scientific, #PHG0311); XF Plasma Membrane Permeabilizer (XF-PMP) (Seahorse Bioscience, 102504-100); thapsigargin (Sigma-Aldrich, T9033); proteinase K

(Invitrogen, 25530-049). pHaloTag-human MAP1LC3-Lvl 10 (HT-LC3) was custom- made by GeneCopoeia. The human CHMP2A cDNA (Addgene#31805) was amplified by PCR using a primer set (5'-TTTGCTAGCGCCACCATGGACCTATTGTTCGGGC- 3' (SEQ ID NO:3); 5 ' -TTGAATTCGGTCCCTCCGC AGGTTCTTAA-3 ' (SEQ ID NO:4)) and subcloned into the Nhe I-EcoRI site of pCDHl-EGFP(Nl)-EFl-puro. The linker sequence between CHMP2A and GFP is N'-(CHMP2A)-

RILQSTVPRARDPPVAT-(GFP)-C (SEQ ID NO:8). The pCDHl-EGFP(Nl)-EFl-puro vector was generated by subcloning the MCS-EGFP sequence of pEGFP-Nl (Clontech, #6085-1) into the Nhe I-Notl site of pCDHl-MCSl-EFl-Puro (System Biosciences, #CD510A-1). The following plasmids were used: pMXs-IP-EGFP-LC3

(Addgene#38195) (Hara et al., 2008, J Cell Biol, 181 :497-510); pMXs-IP-EGFP-mAtg5 (Addgene#38196) (Hara et al., 2008, J Cell Biol, 181 :497-510); pMRXIP GFP- STX17TM (Addgene#45910) (Itakura et al., 2012, Cell, 151 : 1256-1269). pEGFP-Cl- human ATG2A (Addgene#36456) 29. pCDHl -EGFP-human ATG2A, lentiCRISPR v2- human ATG2A sgRNA, pLX-human ATG2B sgRNA, lentiCRISPR v2-human ATG7 sgRNA were generated as previously described (Tang et al., 2017, Cell Death Differ, 24:2127-2138). Human ATG13 sgRNAs (5'-TCTTTTCACCAAGCCGAGCC-3' (SEQ ID NO: 5), 5'-CACATGGACCTCCCGACTGC-3' (SEQ ID NO: 6), and 5'-

CAGTCTGTTGTACACCGTGT-3 ' (SEQ ID NO:7)) were sub-cloned into LRG

(Lenti sgRN A EF S GFP) (Addgene 65656).

Cell culture, transfection and viral transduction

HeLa cells and U-2 OS cells were obtained from American Type Culture

Collection and maintained in Dulbecco's Modification of Eagle's Medium (DMEM) and McCoy's 5 A Medium, respectively, supplemented with 10% fetal bovine serum and lx Antibiotic Antimycotic Solution (Corning, 30-004-CI). Retrovirus- and lentivirus- mediated gene transduction were performed as described previously (Young et al., 2012, J Biol Chem, 287: 12455-12468). To generate HT-LC3 U-2 OS and HeLa cells, cells were transduced with lentiviruses encoding HT-LC3 and selected with 1 μg/ml puromycin for 5 days. ATG2A/B double-knockout and ATG7 knockout U-2 OS cells were generated as previously described (Tang et al., 2017, Cell Death Differ, 24:2127-2138). To generate ATG13 knockout U-2 OS cells, cells were transfected with an equal amount (6 μg each per a 10-cm dish) of three human ATG13 gRNA for 48 hours and sorted for GFP- positive transfected cells. Fourteen days after transfection, the cells were re-sorted for GFP-negative population to eliminate Cas9 stable transfectants and used for experiments. For siRNA screening, cells were grown overnight on the Lab-Tekll 8-well Chambered Coverglass (Nunc, 155409) and incubated in Accell siRNA Delivery Medium

(Dharmacon, B-005000-100) containing Ι μΜ Accell siRNA for 72 hours. siRNA- mediated gene silencing was performed by nucleofection as described previously

(Takahashi et al., 2007, Nat Cell Biol, 9: 1142-1151).

Fluorescence microscopy and electron microscopy

Cells were grown on Lab-Tekll Chambered Coverglass, Chamber Slide (Nunc, 154941) or Glass Bottom Dish (MatTek, P35GCOL-0-14-C).

Immunofluorescence was performed as follows: for the detection of p62 and ATG9A, cells were fixed in 4% paraformaldehyde (PFA)-phosphate-buffered saline (PBS) for 10 minutes and permeabilized with 0.1% Triton X-100 for 3 minutes. For LC3, cells were permeabilized and fixed in methanol at -20°C for 10 minutes. Cells were then incubated in 10% normal goat serum for 1 hour followed by the primary and the secondary antibodies and mounted with ProLong Gold Antifade Mountant (Thermo Scientific, P10144 or P36941 (with DAPI)). Fluorescent images were obtained using a Leica AOBS SP8 laser-scanning confocal microscope (63x water or oil-immersion lens), or an

OLYMPUS 1X81 deconvolution microscope (63x oil-immersion lens), deconvolved using Huygens deconvolution software (Scientific Volume Imaging) or SlideBook software (Intelligent Imaging Innovations), and analyzed using Imaris software

(Bitplane), Volocity software (PerkinElmer) or SlideBook software. Electron microscopy was performed as previously described (Takahashi et al., 2011, Autophagy, 7:61-73; Takahashi et al., 2013, Blood 121, 1622-1632). Briefly, cells were grown on thermanox plastic coverslips (Thermo Scientific, 174950) overnight, incubated in SM for 2-3 hours, fixed in 2% paraformaldehyde-2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3, for 1.5 hours at room temperature followed by post-fixation buffer (1% osmium tetr oxide/ 1.5% potassium ferrocyanide-0.1 M sodium cacodylate, pH 7.3) overnight, dehydrated in a graded series of ethanol, embedded in EMbed 812 resin (Electron Microscopy Sciences, 14120), sectioned at a thickness of 70 nm, mounted on mesh copper grids, stained with aqueous uranyl acetate and lead citrate and analyzed using a JEOL JEM 1400 transmission electron microscope. For the quantification of autophagic structures, samples were post-fixed in the absence of potassium ferrocyanide.

Immunoelectron microscopy was performed as described previously (Takahashi et al., 2007, Nat Cell Biol, 9: 1142-1151; Takahashi et al., 2016, Oncotarget 7, 20855-20868 ).

HaloTag-LC3 autophagosome completion assay and correlative light electron microscopy (CLEM)

HaloTag-LC3 expressing cells were incubated in lx MAS buffer (220 mM mannitol, 70 mM sucrose, 10 mM KH2PO4, 5 mM MgCh, 2 mM HEPES, 1 mM EGTA) containing XF-PMP (2-3 nM for U-2 OS and 3 nM for HeLa cells) and MIL at 37°C for 15 minutes. Alternatively, cells were permeabilized with 20 μΜ digitonin at 37°C for 2 minutes and incubated with MIL at 37°C for 15 minutes (Figure 1C; Figure 2C). Cells were then fixed in 4% PFA for 5 minutes, washed three times in PBS, and incubated with MPL for 30 minutes. After washing three times in PBS, cells were analyzed by fluorescence deconvolution or confocal microscopy. For CLEM, cells were grown overnight on Gridded Glass Bottom Dish (MatTek, P35G-1.5-14-C-GRID), starved for 2 hours and fixed in 4% PFA-PBS for 5 minutes, and incubated with lx MAS containing XF-PMP and MIL for 30 minutes followed by MPL for 30 minutes. Cells of interest were identified by correlating the grid and three-dimensional images were obtained by confocal microscopy before processing for electron microscopy.

Immunoblotting

Total cell lysates were prepared in radio-immunoprecipitation assay buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% SDS, 1% Triton X-100, 1%

Deoxycholate, 5 mM EDTA, pH 8.0) containing protease and phosphatase inhibitors and subjected to immunoblotting as described previously 45. The signal intensities were quantified using the Image Studio version 5 software (LI-COR Biotechnology).

Protease protection assay

Cells were resuspended in ice-cold homogenization buffer (HB: 0.25 M sucrose, 140 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH8.0), passed 10 times through a 27-guage syringe needle and then centrifuged at 300 x g at 4°C for 5 minutes to obtain post-nuclear supernatant (PNS). The PNS was centrifuged at 7,600 x g at 4°C for 5 minutes to obtain low-speed pellet (LSP) and supernatant (S76). The S76 was then centrifuged at 100,000 x g at 4°C for 30 minutes to obtain high-speed pellet (HSP) and supernatant. Each pellet fraction was resuspended in ice-cold HB, equally divided into three tubes and incubated with or without 100 μg/ml proteinase K and 0.5% Triton X-100 on ice for 30 minutes. After the addition of 1 mM phenylmethylsulfonyl fluoride to stop the reaction, the reaction mixture was subjected to immunoblotting.

Statistical analyses

Statistical significance was determined using Graph Pad Prism 7.0.

Threshold for statistical significance for each test was set at 95% confidence (p<0.05).

The results of the experiments are now described.

The HaloTag-LC3 assay distinguishes phagophores, nascent autophagosomes, and mature autophagosomal structures

To distinguish unclosed and closed autophagosomal membranes, a novel reporter HaloTag-LC3 (HT-LC3) was used in combination with membrane-impermeable Alexa Fluor (AF) 488 or AF660 HaloTag ligand (MIL) and membrane-permeable tetramethylrhodamine HaloTag ligand (MPL) (Figure 1 A). U-2 OS cells stably expressing HT-LC3 were generated and it was confirmed that starvation-induced HT- LC3-I lipidation and HT-LC3-II turnover were comparable to endogenous LC3 (Figure 2A). To perform the autophagosome completion assay, cells were starved to induce autophagy followed by permeabilization of the plasma membrane to release cytosolic HT-LC3-I and sequential labeling of HT-LC3-II with MIL and MPL (Figure 1 A). While cytosolic HT signals were lost during permeabilization, HT-labeled autophagic structures were retained in the cytoplasmic region throughout the assay process (Figure 2B). The specificity of each HaloTag ligand and the saturation of phagophore- and OAM- associated HT-LC3-II by the initial MIL labeling were verified during assay optimization (Figure 2C and Figure 2D). As expected, three populations of HT-LC3-II structures were detected in the cytoplasmic region of starved cells: 1) MIL+MPL-, representing phagophores, 2) MIL+MPL+, representing nascent autophagosomes, and 3) MIL-MPL+, representing mature autophagosomes, amphisomes, and autolysosomes (Figure IB). Moreover, the MIL+MPL- signals displayed cup- or oval-shaped structures (Figure IB, i- iii; arrows in Figure 1C) in agreement with phagophore morphology 17, while

MIL+MPL+ signals formed nascent autophagosome-like structures in which MIL signals (OAM-associated HT-LC3-II) surrounded MPL signals (IAM-associated HT-LC3-II) (Figure IB, i and iv; Figure 1C) and MIL-MPL+ puncta were consistent with mature autophagosomal structures in which OAM-associated LC3-II has been delipidated (Figure IB, i and v; Figure 1C). While nutrient starvation significantly increased cytoplasmic MIL and MPL HT-LC3 signals, only MPL signals were strongly

accumulated upon lysosomal inhibition (Figure ID) to indicate lysosomal degradation of IAM- but not OAM-associated HT-LC3-II. Similar results were obtained in HeLa cells stably expressing HT-LC3 in the presence or absence of BafAl or lysosomal protease inhibitors (Pis) (Figure 2E).

LC3 is an aggregation-prone protein that can be incorporated into protein aggregates independent of autophagy (Kuma et al., 2007, Autophagy, 3 :323-328). To confirm that the HT-LC3 -positive foci represent autophagic structures, LC3-I lipidati on- defective U-2 OS cells were generated by disrupting the ATG7 gene (Figure 3 A) and performed the HT-LC3 autophagosome completion assay. The cytoplasmic region of Atg7-depleted cells was not labeled by MIL or MPL, thus demonstrating the successful removal of soluble HT-LC3-I and the specificity of HaloTag ligands for membrane- bound LC3-II rather than LC3 aggregates (Figure 3B). To exclude the possibility that HL-LC3-II localizes on non-autophagic membranes, similar experiments were performed in the absence of ATG13, which is dispensable for LC3-II conversion but required for autophagosome formation (Kaizuka and Mizushima, 2015, Mol Cell Biol, 36:585-595). Indeed, knockout of ATG13 did not affect LC3 lipidati on but suppressed starvation- induced LC3-II turnover (Figure 3C and Figure 3D). Importantly, the cytoplasmic HT- LC3 foci formation was severely impaired in ATG13 -deficient cells (Figure 3E), indicating that the cytoplasmic HL-LC3 puncta are autophagosomal structures.

Immunofluorescence staining for the phagophore marker ATG16L further validated the assay specificity, as the majority of ATG16L-positive HT-LC3 puncta were MIL+MPL- rather than MIL+MPL+ or MIL-MPL+ (Figure 3F). Moreover, correlative electron microscopy confirmed that MIL+MPL- and MIL+MPL+ signals represent ER-associated phagophores (Figure 3G, i) and autophagosome-like structures (Figure 3G, ii), respectively. Collectively, these data demonstrate the successful development of a novel autophagosome completion assay that distinguishes phagophores, nascent

autophagosomes, and mature autophagic vacuoles.

Screening of ESCRT components using the HT-LC3 autophagosome completion assay for potential regulators of autophagosome closure

The topological membrane transformation that occurs during phagophore closure resembles that of ESCRT-mediated intraluminal vesicle formation (Hurley, 2015, EMBO J, 34:2398-2407; Knorr et al., 2015, Autophagy, 11 :2134-2137; Rusten and Stenmark, 2009, J Cell Sci, 122:2179-2183). To determine if the ESCRT machinery regulates phagophore closure, a siRNA library targeting 40 different ESCRT -related genes (Figure 17) was screened using the HT-LC3 autophagosome completion assay. The screening was performed using serum-free Accell siRNA delivery medium in which autophagy was induced comparable to SM (Figure 4A). Among the strongest hits for the accumulation of MIL+MPL- structures compared to siNT control were several ESCRT- III siRNAs including siCHMP2A, siCHMP3 and siCHMP7 (Figure 4B). Increased

MIL+MPL- signals were also detected in cells treated with targeting (CEP55, PDCD6), bridging (VP S37 A), and remodeling (AURKB) ESCRT siRNAs. The screening results were validated using independent siRNA pools targeting CHMP2A, CHMP3, CHMP7 and CEP55. Knockdown of the ESCRT components was confirmed by immunoblotting (Figure 5A). Consistent with the screening results, depletion of each ESCRT, but not lysosomal inhibition, resulted in a significant increase in MIL+ immature

autophagosomal membranes under starved conditions (Figure 5B) to suggest a role of the ESCRT machinery in phagophore closure. Among the hits, siCHMP2A displayed the strongest accumulation of MIL signals.

CHMP2A deficiency accumulates phagophores CHMP2A and 2B are the components of ESCRT-III that form capping assemblies with CHMP3 to drive membrane scission (Christ et al., 2017, Trends

Biochem Sci, 42:42-56; Alonso et al., 2016, FEBS J 283 :3288-3302). The data showed that depletion of CFDVIP2A (Figure 6A, Figure 6B, and Figure 7A through Figure 7C) but not CFDVIP2B (Figure 8A and Figure 8B) resulted in a dramatic increase in MIL+ immature autophagosomal membranes in both U-2 OS and HeLa cells under basal and starved conditions. Many of the MIL+ structures in CFDVIP2A-depleted cells were negative for MPL (magnified images in Figure 6A and Figure 7C), indicating the accumulation of phagophores. Accumulation of LC3 -positive structures by CFDVIP2A depletion was also observed using GFP-LC3 (Figure 7D) or by staining for endogenous LC3 (Figure 7E) to demonstrate that the phenotype is not due to ectopic expression of HT-LC3. Moreover, the accumulated LC3 structures in siCFDVIP2A cells contained not only an autophagic substrate p62 but also the phagophore markers ATG5, ATG2A, and ATG9A (Figure 6C; Figure 9A and Figure 9B); a result consistent with the failed dissociation of ATG machinery in the absence of closure (Alonso et al., 2016, FEBS J 283 :3288-3302). Next, electron microscopy was performed to determine the

ultrastructure of LC3 -positive foci accumulated in CFDVIP2A-depleted cells. Consistently, depletion of CFDVIP2A accumulated immature autophagosomal structures, some of which appeared to be unclosed (asterisks in Figure 9C). To quantify this observation, a similar experiment was performed in the absence of potassium ferrocyanide, which artificially enlarges the intermembrane space of phagophores and autophagosomes (Eskelinen, 2008, Methods Mol Biol, 445: 11-28; Kishi-Itakura et al., 2014, J Cell Sci, 127:4089-4102) to allow us to easily detect immature autophagic structures. As expected, a significantly increased number of unclosed autophagosomal membranes was observed by CFDVIP2A depletion in 2D electron micrographs (Figure 6D and Figure 6E). However, as 2D electron microscopy is limited in the ability to distinguish closed and unclosed autophagosomal membranes, the actual number of 'unclosed' membranes is likely more than shown. Collectively, these results demonstrate that CFDVIP2A depletion results in the accumulation of phagophores.

CFiMP2A is required for basal and starvation-induced autophagy The increase in LC3-II-positive foci is attributed either to the inhibition of autophagic flux or to the promotion of autophagosome biogenesis (Klionsky et al., 2016, Autophagy 12: 1-222). To determine the effect of CHMP2A depletion on autophagic flux, a tandem fluorescent-tagged LC3 assay (Kimura et al., 2007, Autophagy, 3 :452-460) was performed. This assay is based on the difference in pKa values between RFP and GFP to distinguish non-degradative (GFP+RFP+) and degradative (GFP-RFP+) autophagic structures. While starvation induced both GFP+RFP+ and GFP-RFP+ structures in control cells, CFDVIP2A depletion resulted in the accumulation of GFP+RFP+, but not GFP-RFP+, structures under both starved and non-starved conditions, indicating the impairment of autophagic flux (Figure 10A, Figure 10B, and Figure 9D). Consistently, both basal and starvation-induced lysosomal turnover of LC3-II was found to be impaired by the depletion of CHMP2A (Figure IOC, Figure 10D), but not CHMP2B (Figure 8C). These results indicate an indispensable role of CFDVIP2A in basal and induced autophagy. CHMP2A translocates to the phagophore during autophagy

To determine if CFDVIP2A localizes on the phagophore during autophagy, as the commercially available CFDVIP2A antibodies failed to show the specificity, a GFP- tagged CFDVIP2A was generated. It was found that overexpression of CFDVIP2A-GFP accumulated MIL+MPL- structures in a similar manner to CFDVIP2A depletion (Figure 11), which is in agreement with the previous observation that overexpression of several ESCRT-III components acts as a dominant-negative to inhibit MVB sorting (Teis et al., 2008, Dev Cell, 15:578-589). HT-LC3 U-2 OS cells were generated that stably express CFDVIP2A-GFP but do not accumulate autophagosomal membranes under basal conditions (Figure 12 A). Strikingly, nutrient starvation induced CFDVIP2A-GFP foci formation in the cytoplasm with about half of the signals positive for LC3 (Figure 12B). Importantly, CHMP2A signals were detected adjacent to LC3 -positive puncta and not in the MPL+ autophagosomal lumen (Figure 12 A, i-iv) with a subset of CHMP2A signals located at the edge of phagophore-like structures (arrowheads in Figure 12A).

Consistently, CHMP2A signals were detected on phagophore/immature autophagosome- like structures by immunoelectron microscopy (arrows in Figure 12C, i-iii). Collectively, the data demonstrate that CHMP2A translocates to the phagophore during autophagy. Inhibition of the AAA-ATPase VPS4 activity impairs autophagosome completion

The AAA-ATPase VPS4 hydrolyzes ATP to depolymerize ESCRT-III assemblies from membranes (Christ et al., 2017, Trends Biochem Sci, 42:42-56; Peel et al., 2011, Trends Biochem Sci, 36: 199-210). As the function of VPS4 is indispensable for ESCRT-mediated membrane fission (Adell et al., 2014, J Cell Biol, 205:33-49), the ability of inhibition of the VPS4 activity to impair phagophore closure was examined using the HT-LC3 autophagosome completion assay. Similar to CHMP2A depletion, the expression of an ATPase-deficient dominant-negative mutant of VPS4A (VPS4AE228Q) resulted in the accumulation of MIL+ structures under both starved and non-starved conditions (Figure 13 A and Figure 13B). Moreover, VPS4AE228Q signals were frequently detected on MIL+MPL- phagophore-like structures (Figure 13 A, i-iv) and the OAM of MIL+MPL+ nascent autophagosome-like structures (Figure 13 A, v).

Quantification analysis revealed that nearly 80% of VPS4AE228Q signals were associated with MIL+ immature autophagosomal membranes (Figure 13C). Since CHMP2A depletion or VPS4 inhibition strongly accumulated phagophores even under non-starved condition, impairment of the ESCRT machinery might not only inhibit phagophore closure but also enhance autophagosome biogenesis. To explore this possibility, autophagic flux was measured upon the expression of VPS4AE228Q. In comparison to control GFP transfected cells, the protein levels of LC3-II and p62 were increased in VPS4AE228Q-transfected cells but did not further increase upon lysosomal inhibition with BafAl (Figure 13D, lanes 2, 5, 6), suggesting that disruption of the ESCRT machinery impacts autophagy flux rather than induction. Indeed, VPS4 inhibition had minimal effect on the induction of basal autophagy as the rates of LC3-II increase during the 6 hour period of BafAl treatment were comparable between GFP (2.22 = 3.22 - 1.00) and VPS4AE228Q (2.41 = 4.82 - 2.41) transfectants to indicate that VPS4 inhibition does not affect LC3-I to LC3-II conversion. Collectively, these results demonstrate that VPS4 activity is required for phagophore closure. CHMP2A-mediated phagophore closure is a critical step in functional autolysosome formation

Despite impaired autophagosome completion, the number of MPL+ structures were slightly but significantly increased by CHMP2A depletion or VPS4 inhibition (Figure 6B, Figure 13B). Interestingly, electron dense lysosomal structures were occasionally observed between the outer and inner membrane space of

autophagosome-like structures in CHMP2A-depleted cells (Figure 14A, Figure 14B, Figure 9C). These structures are quite different from a typical autolysosome, in which the IAM is digested to distribute electron dense lysosomal material throughout the vacuole (left panel in Figure 14A). Notably, it was found that CHMP2A depletion did not block the recruitment of the autophagosome-lysosome fusion mediator, syntaxin 17 (STX17) (Itakura et al., 2012, Cell, 151 : 1256-1269), to LC3-positive membranes (Figure 14C). Moreover, while the majority of the STX17-positive structures in CFIMP2A-depleted cells were negative for LysoTracker, a small portion of the autophagic structures were labelled by LysoTracker (Figure 14D). These results suggest that lysosomal fusion can still proceed in the absence of autophagosome completion. However, in stark contrast to control cells where LysoTracker was primarily diffuse in the vacuole lumen, the

LysoTracker signals in CHMP2A-depleted cells formed ring-shaped structures (arrows in Figure 14D). This is similar to that reported in ATG3 -deficient cells (Tsuboyama et al., 2016, Science, 354: 1036-1041) and suggests the impairment of IAM degradation to form a functional autolysosome.

As the inhibition of phagophore closure prevents the separation of the OAM from the IAM, aberrant fusion of an unclosed autophagosomal membrane with lysosomes/late endosomes would result in the distribution of glycosylated lysosomal membrane proteins throughout the autophagosomal membrane. To determine if this occurs in CHMP2A-depleted cells, immunoelectron microscopy was performed using an anti-lysosome-associated membrane glycoprotein 1 (LAMPl) antibody. LAMPl signals were detected on the OAM, but not the IAM, in control siNT transfected cells (Figure 14E; Figure 15), indicating that lysosomal fusion occurs after the completion of autophagosome formation. In contrast, while the majority of autophagic structures in CHMP2A-depleted cells were negative for LAMPl, some of the phagophore/immature autophagosome-like structures were found to be LAMPl positive and contained LAMPl signals in both OAM and IAM (Figure 14E; Figure 15), indicating the importance of phagophore closure for the proper autolysosome formation.

Finally, it was determined whether the atypical phagophore-lysosome fusion contributes to the increase in MPL+ structures upon CFDVIP2A depletion. To this end, cells were depleted of CFDVIP2A in the presence or absence of the sarco/ER Ca2+ ATPase (SERCA) inhibitor thapsigargin (TG), which has been shown to arrest autophagy by blocking autophagosome fusion with endosomes and lysosomes (Ganley et al., 2011, Mol Cell, 42:731-743). As expected, MPL but not MIL signals were significantly decreased in siCFDVIP2A-transfected cells while TG increased both MIL and MPL in control cells (Figure 16A and Figure 16B). To verify if the TG-induced MPL signal reduction is due to the inhibition of atypical phagophore closure in CHMP2A-depleted cells, the GFP-LC3 protease protection assay was performed (Velikkakath et al., 2012, Mol Biol Cell, 23 :896-909), which is based on the accessibility of protease to

autophagosome-sequestered (inaccessible) and -unsequestered (accessible) LC3.

Consistent with the HT-LC3 assay results, the sensitivity of GFP-LC3-II to proteinase K (ProK) was enhanced by CHMP2A depletion in both low-speed and high-speed pellet fractions (Figure 16C) and that TG treatment further increased the proportion of protease- accessible GFP-LC3-II in CHMP2A-depleted cells (Figure 16D). Collectively, these results further demonstrate the role of CHMP2A in proper phagophore closure during autophagosome biogenesis.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:

1. A autophagosome maturation assay vector comprising a nucleotide sequence encoding an autophagy-related 8 (Atg8) family protein operably linked to a nucleotide sequence encoding haloalkane dehalogenase (HaloTag).

2. The vector of claim 1, wherein the Atg8 family protein is selected from the group consisting of γ-aminobutyric acid receptor-associated protein

(GABARAP), γ-aminobutyric acid receptor-associated like protein (GABARAPLl), Golgi-associated ATPase enhancer of 16 kDa (GATE- 16), microtubule-associated protein light chain 3 A (LC3 A), microtubule-associated protein light chain 3B (LC3B), microtubule-associated protein light chain 3B2 (LC3B2) and microtubule-associated protein light chain 3C (LC3C).

3. The vector of claim 1, encoding an amino acid sequence as set forth in SEQ ID NO:2.

4. The vector of claim 1, comprising a nucleotide sequence as set forth in SEQ ID NCv l .

5. A cell comprising an autophagosome maturation assay vector of any of claims 1-4.

6. An autophagosome maturation assay that provides superior signal- to-noise ratio and high reproducibility to distinguish phagophores, nascent

autophagosomes, and mature autophagosomal structures, the method comprising:

a) contacting a cell of claim 5 with at least one detectable membrane impermeable haloalkane dehalogenase ligand (MIL),

b) contacting the cell of a) with at least one detectable membrane permeable haloalkane dehalogenase ligand (MPL), and c) determining the level of at least one of a MIL+ MPL+ autophagosome, a MIL+ MPL- phagophore and a MIL- MPL+ organelle in the cell.

7. The method of claim 6, wherein the MIL comprises a membrane- impermeable AlexaFluor488-HaloTag ligand.

8. The method of claim 6, wherein the MPL comprises a membrane- permeable tetramethylrhodamine-HaloTag ligand.

9. The method of claim 6, further comprising contacting the cell with a compound or treatment prior to step a).

10. The method of claim 6, further comprising contacting the cell with a plasma membrane permeabilization agent prior to step a).

11. The method of claim 10, wherein the plasma membrane permeabilization agent is selected from the group consisting of a cholesterol-complexing agent, recombinant perfringolysin (rPFO/XF-MPM), and digitonin.

12. The method of claim 6, further comprising inducing autophagy in the cell prior to step a).

13. The method of claim 12 comprising culturing the cell in starvation medium.

14. The method of claim 6, wherein step c) is performed using a method selected from the group consisting of immunoelectron microscopy, wide-field fluorescence microscopy, flow cytometry, confocal microscopy, fluorimetry, microplate- based cytometry, high-content cell analysis, cell microarray analysis, high-content cell screening, and laser-scanning cytometry.

15. A method of screening for the effect of a compound on the closure of autophagosomes or the progression of the lysosomal-dependent catabolic pathway comprising: a) contacting a cell of claim 5 with at least one compound or treatment;

b) inducing autophagy in the cell;

c) contacting the cell with a plasma membrane permeabilization agent;

d) contacting the cell with at least one detectable membrane impermeable haloalkane dehalogenase ligand (MIL),

e) contacting the cell of a) with at least one detectable membrane permeable haloalkane dehalogenase ligand (MPL), and

f) determining the level of at least one of a MIL+ MPL+

autophagosome, a MIL+ MPL- phagophore and a MIL- MPL+ organelle in the cell.

16. The method of claim 15, wherein the MIL comprises a membrane- impermeable AlexaFluor488-HaloTag ligand.

17. The method of claim 15, wherein the MPL comprises a membrane- permeable tetramethylrhodamine-HaloTag ligand.

18. A method of treating a disease or disorder associated with a deficiency in the closure of autophagosomes, or the progression of the lysosomal- dependent catabolic pathway, comprising administering to a subject in need thereof an activator of at least one ESCRT component selected from the group consisting of

CHMP2A and VPS4.

19. The method of claim 18, wherein the disease is selected from the group consisting of Crohn's disease, Vici syndrome, cancers such as breast, ovarian, prostate, liver, colorectal and hematologic cancers, systemic lupus erythematosus, neurodegenerative diseases such as static encephalopathy of childhood with

neurodegeneration in adulthood (SEND A) and Parkinson's disease, and phospholipidosis.