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: 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
• 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

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.

项目概要 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 在酸性管腔中。 Chediak-Higashi 综合征 (CHS) 患者的肿块增大，原因是 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），其功能尚不清楚。微管缺陷 在 CHS 患者的免疫突触中观察到行为和中心体行为，但是否 CHS 细胞中微管成核是否受到直接影响尚存在争议。 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 增大的 LRO（卵黄颗粒）以及有丝分裂和维持细胞核的微管缺陷 在胚胎中的正确位置。果蝇胚胎与因子的淡紫色共免疫沉淀 参与内体的成熟；使 SNARE 复合体从 成熟的囊泡；动力蛋白/动力蛋白，在囊泡运输和微管中发挥作用；和 一些中心体相关分子。因此，果蝇发育的这个阶段是高度 适合研究 LYST/Mauve 在 LRO 和微管生物发生中的作用 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，我们将跟踪蛋黄 野生型和 mv 突变雌性中的颗粒生物发生；确定本构活性和 内吞调节因子 Rab5、Rab7 和 NSF1 的显性失活形式。去发现角色 Mauve/LYST 复合物在调节微管动力学中的作用，我们将确定 mv- 中的微管缺陷 衍生胚胎并建立 mv 和微管相关基因之间的遗传相互作用 与其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 如何指导小纺锤体蛋白的中心体关联；如何与 Rab5 和 Dynein 一起使用， 它促进微管相关蛋白在中心体的积累；以及 Mauve 的搭档如何 蛋白质参与中心体微管组织分子的招募，我们将揭示如何 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 Mauve/LYST 在调节囊泡裂变/融合和 对微管成核和中心体成熟很重要的蛋白质运输。我们 预计我们的发现将转化为人类细胞，它们将有可能为人类打开大门 开发治疗中枢性低通气综合症患者免疫缺陷的治疗药物。

项目成果

Mauve/LYST limits fusion of lysosome-related organelles and promotes centrosomal recruitment of microtubule nucleating proteins.

Mauve/LYST 限制溶酶体相关细胞器的融合并促进微管成核蛋白的中心体募集。

• 发表时间: 2021-04-05
• 影响因子: 11.8
• 作者: Lattao, Ramona;Rangone, Hélène;Llamazares, Salud;Glover, David M
• 通讯作者: Glover, David M