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

• 批准号: 10099427
• 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
• 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/10099427
• 关键词: Affect; Antigen-Presenting Cells; Behavior; Biogenesis; Biological; Blastoderm; Blood Platelets; CHS1 gene; Cell Nucleus; Cells; Centrosome; 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; 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; delta protein; dynactin; gamma Tubulin; gene product; immunological synapse; lysosomal proteins; mutant; protein transport; recruit; therapeutic development; trafficking

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（LRO）包含溶酶体蛋白和细胞类型特异性蛋白 在一个酸性的管腔里。在Chediak-Higashi综合征（CHS）患者中， 要么过度融合要么抑制它们的裂变。CHS中的突变基因编码溶酶体 运输调节蛋白（LYST），其功能知之甚少。微管缺陷 行为和中心体行为在CHS患者的免疫突触中被观察到，但是， 微管成核直接影响CHS细胞是有争议的。 为了确定LYST在LRO中的功能并阐明其在微管中的要求，我们将使用 果蝇模型中，由紫红色（mv）基因编码的LYST对应物的突变体显示， 扩大的LRO（卵黄颗粒）和微管缺陷，在有丝分裂和维持核在 在胚胎中的正确位置来自果蝇胚胎的紫红色共免疫沉淀物与因子 参与核内体的成熟;一种使SNARE复合物从核内体中解离的因子 动力蛋白/动力肌动蛋白，其在囊泡运输和微管中起作用;以及 几个中心体相关分子因此，果蝇发育的这一阶段是非常重要的。 适合研究LYST/Mauve在LRO生物发生和微管中的作用， 中心体 为了确定Mauve/LYST复合物在调节LRO大小和运输中的作用，我们将跟踪卵黄 野生型和mv突变型雌性中的颗粒生物发生;确定组成性活性和 内吞调节因子Rab 5、Rab 7和NSF 1的显性负性形式。去发现 Mauve/LYST复合物在调控微管动力学中的作用，我们将确定mv中的微管缺陷。 并建立了mv和微管相关基因之间的遗传相互作用 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蛋白的中心体结合;如何与Rab 5和动力蛋白一起， 它促进了微管相关蛋白在中心体的积累， 蛋白质参与微管组织分子在中心体的募集，我们将揭示如何 囊泡运输相关蛋白可参与促进中心体成熟。 我们预计这将确定Mauve/LYST在调节囊泡分裂/融合中的双重作用， 在微管成核和中心体成熟的重要蛋白质的运输中。我们 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为人类细胞打开大门。 开发治疗CHS患者免疫缺陷的治疗剂。