Molecular and cellular mechanisms regulating actin dynamics
调节肌动蛋白动力学的分子和细胞机制
基本信息
- 批准号:10549331
- 负责人:
- 金额:$ 106.73万
- 依托单位:
- 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:2020-02-01 至 2025-01-31
- 项目状态:未结题
- 来源:
- 关键词:AccelerationActinsAffectAlzheimer&aposs DiseaseAmyotrophic Lateral SclerosisAnimalsBindingBiochemicalBiologicalBiological ProcessCardiovascular DiseasesCell divisionCell physiologyCellsComplexCryoelectron MicroscopyCytoskeletonCytosolDefectDeformityDiseaseDissociationEndocytosisF-ActinFilamentFundingGeneticGlia Maturation FactorGoalsGrowthHuntington DiseaseIn VitroIndividualInfertilityIntracellular TransportKnowledgeLeadLimb structureMalignant NeoplasmsMammalian CellMediatingMicrofilamentsMolecularMorphogenesisNational Institute of General Medical SciencesNucleotidesOrganismParkinson DiseaseProcessProteinsRegulationResearchRoleStructureSystemTestingTimeTissuesTotal Internal Reflection FluorescentWorkYeastscell motilitycellular imagingcofilincoronin proteindepolymerizationdevelopmental diseasegenetic regulatory proteinhearing impairmenthuman diseasein vivoinhibitorinsightmutantnew technologynovel strategiesprotein structurereconstitutionsingle molecule
项目摘要
Project Summary
The overall goal of the NIGMS-funded research in my lab is to define the molecular and cellular mechanisms
underlying dynamic rearrangements of the actin cytoskeleton, and to explore how these mechanisms are
harnessed in vivo (in yeast and animal cells) to control diverse actin-based processes such as cell motility,
endocytosis, intracellular transport, and cell morphogenesis. Genetic and biochemical research has been
rapidly producing a ‘molecular parts list’ for the actin cytoskeleton, and many of the components have been
characterized individually for their biochemical effects on actin filaments and their genetic effects on cellular
actin organization and function. However, it is becoming clear that most of these proteins do not function alone,
but rather in groups to perform their biological roles, and thus, new approaches are needed to define how they
work in concert to perform their cellular functions. Our lab is tackling this problem using advanced single
molecule TIRF microscopy to directly observe multi-component actin regulatory mechanisms in real time, and
testing these mechanisms using genetic, cell biological, biochemical, and structural approaches. Through this
approach, we have made fundamental new insights into actin regulation. For instance, we defined the first
collaborative actin nucleation mechanisms of formins (with Bud6 & APC). We discovered that formins and
Capping Protein can bind simultaneously at filament ends to accelerate each other’s dissociation. We showed
that Cofilin, AIP1, and Coronin work together via an ordered mechanism to sever and disassemble F-actin. We
discovered that Srv2/CAP works in conjunction with Cofilin and Twinfilin to depolymerize filament ends. In
parallel, we have combined genetics, cellular imaging, and separation-of-function mutants to dissect the
contributions of these mechanisms to actin-based processes in yeast and mammalian cells.
Moving forward, we will ask the following questions: what are the complete regulatory cycles of the two yeast
formins (Bni1 and Bnr1)? How is Arp2/3 complex-mediated actin nucleation balanced by its inhibitors (Coronin
and GMF) and activators (Las17/WASP and Abp1)? How is actin nucleation at the leading edge of motile cells
controlled by interactions among IQGAP1, APC and formins? How do interactions at filament ends between
Capping Protein and formins (and their in vivo binding partners) control actin network growth? How do the
filament severing and depolymerization mechanisms (Cofilin, AIP1, Coronin, Twinfilin, and Srv2/CAP) drive net
disassembly of actin under the assembly-promoting conditions of the cytosol? Are there actin-associated
proteins that accelerate the nucleotide state transition on F-actin to promote disassembly? In addition, we will
introduce new technologies and directions to our research, including in vitro reconstitution of cellular actin
structures, cryo-EM to study protein structure, cell-free extracts to genetically-biochemically dissect actin
mechanisms, and a systems-level approach to determine how genetic disruptions in individual actin regulators
affect the cellular levels, localization, and functions of the remaining actin-associated proteins.
项目摘要
我实验室的NIGMS资助研究的总体目标是确定分子和细胞机制
潜在的动态重排的肌动蛋白细胞骨架,并探讨这些机制是如何
在体内(在酵母和动物细胞中)控制多种基于肌动蛋白的过程,如细胞运动,
内吞作用、细胞内转运和细胞形态发生。基因和生物化学研究已经
迅速产生肌动蛋白细胞骨架的“分子部件清单”,许多成分已经被
其特征分别为它们对肌动蛋白丝的生化作用和它们对细胞增殖的遗传作用。
actin组织和功能。然而,越来越清楚的是,这些蛋白质中的大多数并不单独起作用,
而是以群体的形式发挥它们的生物学作用,因此,需要新的方法来定义它们是如何
协同工作来执行它们的细胞功能。我们的实验室正在使用先进的单
分子TIRF显微镜直接观察真实的时间多组分肌动蛋白调节机制,
使用遗传学、细胞生物学、生物化学和结构方法测试这些机制。通过这个
通过这种方法,我们对肌动蛋白的调控有了新的认识。例如,我们定义了第一个
协作肌动蛋白成核机制的formins(与Bud 6和APC)。我们发现,
加帽蛋白可以同时结合在细丝末端以加速彼此的解离。我们展示
Cofilin、AIP 1和Coronin通过有序的机制共同作用,切断和分解F-肌动蛋白。我们
发现Srv 2/CAP与Cofilin和Twinfilin一起工作以去除细丝末端。在
与此同时,我们结合遗传学、细胞成像和功能分离突变体来剖析
这些机制对酵母和哺乳动物细胞中基于肌动蛋白的过程的贡献。
展望未来,我们将提出以下问题:什么是完整的监管周期的两种酵母
formins(Bni 1和Bnr 1)?Arp 2/3复合物介导的肌动蛋白成核如何被其抑制剂平衡(Coronin
和GMF)和激活剂(Las 17/WASP和Abp 1)?肌动蛋白如何在运动细胞的前沿成核
控制IQGAP 1,APC和formins之间的相互作用?细丝末端的相互作用
加帽蛋白和formins(以及它们在体内的结合伙伴)控制肌动蛋白网络的生长?是如何
丝切断和解聚机制(Cofilin、AIP 1、Coronin、Twinfilin和Srv 2/CAP)驱动网络
肌动蛋白的拆卸下组装促进条件下的胞质溶胶?是否存在肌动蛋白相关的
加速F-肌动蛋白上核苷酸状态转换以促进分解的蛋白质?此外,我们将
介绍新的技术和方向,我们的研究,包括在体外重建细胞肌动蛋白
结构,cryo-EM研究蛋白质结构,无细胞提取物对肌动蛋白进行遗传生物化学解剖
机制和系统水平的方法来确定如何在个别肌动蛋白调节基因中断
影响剩余肌动蛋白相关蛋白的细胞水平、定位和功能。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Bruce L Goode其他文献
Bruce L Goode的其他文献
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{{ truncateString('Bruce L Goode', 18)}}的其他基金
Molecular and cellular mechanisms regulating actin dynamics
调节肌动蛋白动力学的分子和细胞机制
- 批准号:
10091492 - 财政年份:2020
- 资助金额:
$ 106.73万 - 项目类别:
Molecular and cellular mechanisms regulating actin dynamics
调节肌动蛋白动力学的分子和细胞机制
- 批准号:
10343858 - 财政年份:2020
- 资助金额:
$ 106.73万 - 项目类别:
FORMINS AND NATIVE COMPLEXES: REGULATION AND FUNCTION
福尔明和天然复合物:调节和功能
- 批准号:
8171242 - 财政年份:2010
- 资助金额:
$ 106.73万 - 项目类别:
Regulation of formins and cell polarity in yeast
酵母中福尔明和细胞极性的调节
- 批准号:
8126615 - 财政年份:2010
- 资助金额:
$ 106.73万 - 项目类别:
Novel mechanisms regulating formins and cell polarity
调节福尔明和细胞极性的新机制
- 批准号:
8610321 - 财政年份:2008
- 资助金额:
$ 106.73万 - 项目类别:
FORMINS AND NATIVE COMPLEXES: REGULATION AND FUNCTION
福尔明和天然复合物:调节和功能
- 批准号:
7723632 - 财政年份:2008
- 资助金额:
$ 106.73万 - 项目类别:
Novel mechanisms regulating formins and cell polarity
调节福尔明和细胞极性的新机制
- 批准号:
8292733 - 财政年份:2008
- 资助金额:
$ 106.73万 - 项目类别:
Regulation of formins and cell polarity in yeast
酵母中福尔明和细胞极性的调节
- 批准号:
7354201 - 财政年份:2008
- 资助金额:
$ 106.73万 - 项目类别:
Novel mechanisms regulating formins and cell polarity
调节福尔明和细胞极性的新机制
- 批准号:
8449132 - 财政年份:2008
- 资助金额:
$ 106.73万 - 项目类别:
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