Common Regulatory Pathways for the Genesis of Lysosome-Related Organelles and Dynamics of Microtubules during Development

溶酶体相关细胞器的起源和发育过程中微管动力学的常见调控途径

基本信息

批准号：
10684931
负责人：
David M Glover
金额：
$ 43.79万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-30 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10684931
关键词：
Affect Antigen-Presenting Cells Behavior Biogenesis Biological Blastoderm Blood Platelets CHS1 gene Cell Nucleus Cells Centrosome Co-Immunoprecipitations Coma Complex Cytoplasm Cytoplasmic Granules Cytotoxic T-Lymphocytes Defect Development Dissociation Dominant-Negative Mutation Drosophila genus Dynein ATPase Embryo Enabling Factors Endosomes Failure Female Film Genes Genetic Human Immunologics Immunoprecipitation In Vitro Knowledge Lysosomes Lytic Mass Spectrum Analysis Mediating Melanosomes Microtubule-Associated Proteins Microtubules Mitosis Modeling Molecular Mothers Mutate Mutation Oocytes Organelles Pathway interactions Patients Pharmaceutical Preparations Phenotype Pilot Projects Polyploidy Positioning Attribute Process Proteins Regulatory Pathway Reporting Research Resolution Role SNAP receptor System Testing Translating Vesicle cell type chediak-higashi syndrome dynactin gamma Tubulin gene product immunological synapse lysosomal proteins mutant protein transport recruit therapeutic development trafficking

项目摘要

PROJECT SUMMARY Lysosome-Related Organelles (LROs) contain both lysosomal proteins and cell-type specific proteins in an acidic lumen. They are enlarged in Chediak-Higashi Syndrome (CHS) patients resulting from either excessive fusion or inhibition of their fission. The mutated gene in CHS encodes the lysosomal trafficking regulator (LYST) protein, whose function is poorly understood. Defects in microtubule behavior and centrosome behavior are seen at the immunological synapse of CHS patients but whether microtubule nucleation is affected directly in CHS cells is controversial. To determine LYST's function in LROs and clarify its requirements at microtubules, we will use a Drosophila model in which mutants of the LYST counterpart, encoded by the mauve (mv) gene, show enlarged LROs (yolk granules) and microtubule defects in mitosis and in maintaining nuclei at the correct position in the embryo. Mauve co-immunoprecipitates from Drosophila embryos with factors involved in maturation of endosomes; a factor enabling dissociation of the SNARE complex from mature vesicles; Dynein/Dynactin, which have roles in vesicle trafficking and at microtubules; and several centrosome-associated molecules. Thus, this stage of Drosophila development is highly amenable to study the role of LYST/Mauve in the biogenesis of LROs and at microtubules and centrosomes. To establish the role of the Mauve/LYST complex in regulating LRO size and trafficking, we will follow yolk granule biogenesis in wild-type and mv-mutant females; determine the effects of constitutively active and dominant-negative forms of the enodcytotic regulators Rab5, Rab7 and NSF1. To discover the role of Mauve/LYST complex in regulating microtubule dynamics, we will determine microtubule defects in mv- derived embryos and establish the genetic interactions between mv and genes for microtubule associated proteins with which it associates and physical interactions between these gene products. By determining how Mauve directs the centrosomal association of Minispindles protein; how together with Rab5 and Dynein, it promotes accumulation of microtubule associated proteins at the centrosome; and how Mauve's partner proteins participate in recruitment of microtubule organizing molecules at centrosomes we will uncover how vesicle trafficking associated proteins can participate in promoting centrosomal maturation. We anticipate that this will define the dual role of Mauve/LYST in regulating vesicle fission/fusion and in the trafficking of proteins important for microtubule nucleation and centrosome maturation. We anticipate our findings will translate to human cells where they will have potential to unlock doors for the development of therapeutic agents to treat the immunological defects of CHS patients.

项目摘要溶酶体相关细胞器（LROS）含有溶酶体蛋白和细胞型特异性蛋白在酸性管腔中。它们在Chediak-Higashi综合征（CHS）患者中被扩大过度融合或抑制其裂变。 CHS中的突变基因编码溶酶体贩运调节剂（LYST）蛋白，其功能知之甚少。微管中的缺陷在CHS患者的免疫突触中可以看到行为和中心体行为微管成核直接在CHS细胞中受到影响是有争议的。要确定LYST在LROS中的功能并阐明其在微管上的要求，我们将使用一个果蝇模型，其中lyst的突变体由淡紫色（MV）基因编码 LROS（蛋黄颗粒）和微管缺陷的有丝分裂和维持核的微管缺陷在胚胎中正确位置。淡紫色共免疫沉淀从果蝇胚胎与因子参与内体的成熟；使军鼓复合物从成熟的囊泡； Dynein/dynactin，在囊泡运输和微管中具有作用；和几个与中心体相关的分子。因此，果蝇发展的这一阶段很高可以研究lyst/提的作用在LROS和微管的生物发生中中心体。为了确定淡紫色/莱斯特综合体在调节LRO规模和贩运方面的作用，我们将跟随蛋黄野生型和MV突变女性的颗粒生物发生；确定组成性活动的影响，并 Enodcytotic调节剂RAB5，RAB7和NSF1的主要阴性形式。发现的作用在调节微管动力学时，我们将确定MV-中的微管缺陷衍生的胚胎并建立了与微管相关的MV与基因之间的遗传相互作用它与这些基因产物之间的蛋白质和物理相互作用。通过确定 Mauve如何指导Minispindles蛋白的中心体关联；如何与rab5和dynein在一起，它促进了微管相关蛋白在中心体上的积累。以及梅夫的伴侣蛋白质参与募集微管组织分子在中心体的分子我们将发现如何揭示囊泡运输相关蛋白可以参与促进中心体成熟。我们预计这将定义紫菜/lyst在调节囊泡裂变/融合和的双重作用在运输蛋白质中，对微管成核和中心体成熟很重要。我们预见我们的发现将转化为人类细胞，在那里它们有可能解锁门开发治疗剂以治疗CHS患者的免疫缺陷。