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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.

项目总结溶酶体相关细胞器(LRO)既含有溶酶体蛋白，又含有细胞类型特异性蛋白在酸性的管腔中。Chediak-Higashi综合征(CHS)患者因以下原因而增大要么过度融合，要么抑制它们的裂变。CHS中突变的基因编码溶酶体运输调节蛋白(Lyst)，其功能尚不清楚。微管缺陷在CHS患者的免疫突触中可以看到行为和中心体行为，但是否微管成核在CHS细胞中的直接作用是有争议的。为了确定Lyst在LROS中的功能并阐明其在微管中的要求，我们将使用果蝇模型，在该模型中，由紫色(MV)基因编码的Lyst对应的突变体显示卵黄颗粒增大，有丝分裂和维持细胞核的微管缺陷。在胚胎中的正确位置。果蝇胚胎与因子的紫红色免疫共沉淀物参与内小体的成熟；一种使SNARE复合体从成熟囊泡；Dynein/dynactin，在囊泡运输和微管中发挥作用；以及几个中心体相关的分子。因此，果蝇发育的这一阶段是高度能够研究Lyst/Mauve在LROS的生物发生中的作用以及在微管和中心体。为了确定Mauve/Lyst复合体在调节LRO大小和贩运方面的作用，我们将跟踪蛋黄野生型和MV突变型雌性的颗粒生物发生；确定构成活性和显性-阴性形式的内皮细胞调节因子Rab5、Rab7和NSF1。发现……的作用 MUVE/LYST复合体在微管动力学调节中的作用，我们将确定MV- 获得胚胎并建立MV与微管相关基因的遗传互作与之相关的蛋白质以及这些基因产物之间的物理相互作用。通过确定 Mauve如何指导Minispindles蛋白的中心体关联；如何与Rab5和Dynein一起，它促进中心体微管相关蛋白的积累；以及Mauve的合作伙伴如何蛋白质参与中心体上微管组织分子的募集，我们将揭示囊泡运输相关蛋白可参与促进中心体成熟。我们预计这将定义Mauve/Lyst在调节囊泡分裂/融合和在蛋白质的运输中，对微管成核和中心体成熟很重要。我们预计我们的发现将转化为人类细胞，在那里它们将有可能打开治疗慢性阻塞性肺病患者免疫缺陷的治疗药物的开发。