CN103160569A - Discovery for new mechanism for regulating dynamic state of cell microfilaments - Google Patents

Abstract

The invention discloses discovery for a new mechanism for regulating the dynamic state of cell microfilaments, wherein nerve cell is an important polar cell composed of axons, dendrons and cytons; the formation of axons is the morphological index of the differentiation and formation of nerve cells; the dynamic change of cytoskeletons are necessary to the formation of axons; and microfilament formed by polymerizing actins is one of the important cytoskeletons, however, the molecular mechanism of the dynamic change of the microfilament is not clear yet. The research discovers for the first time that UNC-51 protein kinase with conservation property from caenorhabditis elegans to mammals is capable of both binding and phosphorylating actins, enabling microfilaments to form granular distribution, and conveying the granular microfilaments into a cytoplasmic matrix; UNC-51 protein kinase is also capable of inducing N1E115 nerve-tumour cells into the cells which are in the shape of furcal nerve; and UNC-51 mutant (K39M) proteins without kinase activity form accumulated body structures around cell nucleuses together with microfilaments, and are incapable of inducing N1E115 cells into the cells which are in the shape of furcal nerve. According to the discovery result aforementioned, such a model is put forward: UNC-51 protein kinase is used for regulating the dynamic change of cytoskeletons, and then further regulating the formation and function of nerve cell axons via the molecular mechanism of directly phosphorylating actins and conveying the granular microtubules into the cytoplasmic matrix.

Description

Regulate the discovery of the dynamic new mechanism of Microfilaments In Cells

Technical field:

The invention belongs to the Basic Life science, biological chemistry, cytobiology, molecular biology, brain science, the muscle science, the new mechanism of Microfilaments In Cells dynamic change has been disclosed in a plurality of fields such as neurocyte skeleton and neurocyte signal transduction innovatively and neurocyte forms and the up-to-date principle of work.

Background technology:

Formation and the function of dynamic and neurocyte aixs cylinder and the muscle cell of Microfilaments In Cells, cell fission and cell movement etc. has close relationship.It has been generally acknowledged that, the formation of the interior microfilament of cell and function are indirectly by the phosphorylation of microfilament in conjunction with albumen (nucleation albumin A rp2/3 mixture, Cofilin cofilin etc.), but the dynamic Basic Mechanism of microfilament is still unclear.Also do not affect the dynamic kinase whose report of microfilament with its phosphorylation mechanism about both Direct Phosphorylation Actin muscles yet.Model animals C. Elegans Automatic Screening and mammiferous neural system are similar on morphology and biological chemistry; Keeping quality gene can be expressed identical effect in C. Elegans Automatic Screening and mammiferous different experiments system.Having conservatory protein serine/threonine in evolution from the nematode to the mankind of the unc-51 genes encoding of C. Elegans Automatic Screening, is that neural formation is necessary; The sudden change of this gene causes that the nerve of nematode forms and shortcoming and the short and thick bodily form of motion.The substrate of the protein kinase of announcement unc-51 coded by said gene and the meaning of its biochemical function are important.

Summary of the invention:

The object of the invention is to disclose important substrate and its biochemical function of the protein kinase of unc-51 coded by said gene, understand the molecule mechanism that neurocyte forms, for the treatment of N﹠M disease provides brand-new principle and method.Content of the present invention is:

(1) disclose first Actin muscle can with the combination of UNC-51 kinases, more can be phosphorylated as this kinase whose substrate

The kinase whose combination of Actin muscle and UNC-51 such as Fig. 1, Fig. 2 and shown in Figure 5.Fig. 1 is the result after a lysate of the Human Embryonic Kidney HEK s293 cell of expressing the UNC-51 kinase protein is used for immunoblotting, and this figure is presented in transfected HEKs293 cell, and the UNC-51 kinase protein is in conjunction with Actin muscle.The vitro enzyme linked immunosorbent assay result demonstration of Fig. 2 can both be in conjunction with the UNC-51 kinase protein from the Actin muscle of C. Elegans Automatic Screening and rabbit flesh.Fig. 5 is the image of laser scanning fluorescence confocal microscope, this image is presented in non-state green monkey kidney cell (COS-7) matter matrix, redly the particulate state UNC-51 protein kinase of fluorescence and with the particulate state Actin muscle of green fluorescent in conjunction with having produced granular yellow fluorescence.

Fig. 3 show the UNC-51 kinases external can phosphorylation from Actin muscle and the self-phosphorylation of rabbit flesh, ATP and GTP all can be used as phosphodonor.Fig. 4 shows that the Serine of Actin muscle of C. Elegans Automatic Screening and rabbit flesh is by the UNC-51 tyrosine phosphorylation.

(2) disclose first UNC-51 protein kinase activity energy conveying granular microfilament in cytoplsma matrix

Compare at the aggregation that nucleus forms on every side with the UNC-51 mutain (K39M) without kinase activity in Fig. 6, Fig. 5 shows, in the COS-7 cell of overexpression UNC-51 kinase protein (redness), the UNC-51 kinase activity can carry granular Actin muscle in cytoplsma matrix.

(3) disclosing first the UNC-51 protein kinase activity can induce neuroblastoma N1E115 cell to become the nervosa cell of multi-branched

Fig. 7 shows, the UNC-51 kinase activity can induce neuroblastoma N1E115 cell to become cell like furcal nerve.

Fig. 8 represents that the UNC-51 kinase activity is by to the phosphorylation of Actin muscle in cell with transport the particulate state Actin muscle in cytoplsma matrix, the model of inducing neural axon to form.

Description of drawings:

The kinase whose Binding in vivo of Fig. 1 Actin muscle and UNC-51

The kinase whose external combination of Fig. 2 Actin muscle and UNC-51

Fig. 3 UNC-51 kinases is at phosphorylation in vitro Actin muscle and self-phosphorylation

Fig. 4 UNC-51 kinases is at the Serine of the Actin muscle of phosphorylation in vitro C. Elegans Automatic Screening and rabbit flesh

Fig. 5 UNC-51 kinase activity can carry granular Actin muscle in cytoplsma matrix

Fig. 6 forms aggregation without UNC-51 mutain (K39M) and the Actin muscle of kinase activity around nucleus

Fig. 7 UNC-51 kinase activity can induce neuroblastoma N1E115 cell to become cell like furcal nerve

The model of Fig. 8 UNC-51 kinase activity by inducing neural axon to form to phosphorylation and the granular Actin muscle of conveying of Actin muscle in cell

Embodiment:

[1] mensuration of UNC-51 protein kinase and Actin muscle combination

Express the HEK293 cell of UNC-51 protein kinase with the dissolving of cytolysis damping fluid, the supernatant of the lysate that obtains is used for immunoblotting.Utilize affinity chromatography to obtain the UNC-51 protein kinase from the supernatant purifying of above lysate.Use solid-phase enzyme-linked immune assay method (ELISA) to analyze the external combination of Actin muscle and UNC-51 protein kinase.

[2] mensuration of kinase activity

The adenosine triphosphate that contains 50 μ M end radioactivity souvenirs [γ- ³²P]-ATP or guanosine triphosphate [γ- ³²P]-the Hepes kinase buffer reaction solution (pH7.4) of GTP in, carried out the reaction of UNC-51 protein kinase phosphorylation Actin muscle.Kinase activity has been measured in SDS-PAGE with 6.5% and the analysis of X-line film radioautography.Two-way separation electrophoresis method on thin layer cellulose plate (TLC) is used for the same fixed of phosphorylation Serine.

The observation of [3] laser scanning fluorescence confocal microscope

DAPI is used for nucleus dyeing, and TexasRed and FITC are respectively used to the dyeing of UNC-51 and Actin muscle.The ZeissLSM510 confocal microscope is used to cell observation and record.

Claims (3)

1. The kinase activity that the present invention has found UNC-51 first directly the phosphorylation Actin muscle and and Microfilaments In Cells be distributed to cytoplsma matrix with grain structure together.UNC-51 is first both phosphorylation Actin muscle kinases of also directly affecting microfilament structure and distributing.Its key is found to be:

   1. in vitro biochemical test, the kinase activity of UNC-51 is phosphorylation nonmuscle cells Actin muscle directly, muscle cell Actin muscle and C. Elegans Automatic Screening Actin muscle, in vitro and in biochemical test in vivo, UNC-51 is in conjunction with the nonmuscle cells Actin muscle, muscle cell Actin muscle or C. Elegans Automatic Screening Actin muscle.
2. 2. in vivo biochemical test, UNC-51 and Microfilaments In Cells become grain structure and transportation grain structure microfilament together to cytoplsma matrix.

   Fibrous microfilament+UNC-51+ATP (GTP) → granular microfilament+ADP of phosphorylation (GDP)
3. 3. remember the know-why of binomial requirement in life science, brain science, tumour science, computer science, space science, traffic science, medical science, pharmacy and pharmacopedics, the Chinese medicine science, the therapeutics of various diseases, the application of medical skill and every other aspect and popularization.

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