Arg and cortactin regulation of the Arp2/3 complex and its role in dendritic spine stability
Arp2/3 复合物的 Arg 和 cortactin 调节及其在树突棘稳定性中的作用
基本信息
- 批准号:9794652
- 负责人:
- 金额:$ 2.94万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2018
- 资助国家:美国
- 起止时间:2018-09-14 至 2020-04-30
- 项目状态:已结题
- 来源:
- 关键词:ActinsAddressAdolescenceAffectAffinityBehaviorBindingBiochemicalBiological AssayChemicalsColorComplementComplexConfocal MicroscopyCytoskeletonDataDendritic SpinesDiseaseEMS1 geneEconomic BurdenF-ActinFilamentFluorescence Recovery After PhotobleachingFoundationsHippocampus (Brain)LearningLeftMaintenanceMeasuresMediatingMental disordersMethodsMicroscopyModelingNeuronsPathologyPhotobleachingProtein Tyrosine KinaseProteinsRecoveryRegulationResearchRoleStructureSynapsesTestingTherapeutic InterventionTimeTotal Internal Reflection FluorescentVertebral columnWorkbasecohortcrosslinkexperimental studyinterdisciplinary approachknock-downmutantnervous system disorderneuron lossprematurerecruitsingle moleculesocial
项目摘要
Project Summary
Dendritic spine stability is disrupted in psychiatric and neurological disorders. Dendritic spines are enriched in a
highly branched cytoskeleton, containing stable and dynamic filamentous- (F-) actin pools and monomeric actin.
Loss of the Abl2/Arg non-receptor tyrosine kinase (Arg) and its interaction partner cortactin from dendritic spines
causes widespread spine loss in late adolescence, but the mechanisms of how these two proteins support
dendritic spine stability remain elusive. Our lab recently showed, using total internal reflection microscopy
(TIRFM) single-filament based assays, that Arg and cortactin synergize to regulate Arp2/3-mediated actin
branching. Coordinated regulation of the Arp2/3 complex via Arg and cortactin is a plausible mechanism by
which these proteins support spine stability. In my research plan, I will test the hypothesis that Arg and cortactin
interact to confer dendritic spine stability via regulation of the Arp2/3 complex and maintenance of the spine
stable actin pool.
Aim 1. To determine how Arg interacts with cortactin and the Arp2/3 complex to promote actin branch
nucleation. It remains unresolved how Arg regulates the Arp2/3 complex and how cortactin coordinates with
Arg to enhance this effect. To address how Arg regulates the complex, I will learn how to conduct functional
TIRFM single- filament based assays to define the minimal domain of Arg that is sufficient to activate the Arp2/3
complex. My preliminary data indicate that Arg can directly bind the Arp2/3 complex. I will expand these binding
assays and learn how to conduct chemical crosslinking experiments to determine (1) the minimal Arg fragment
that can make a high affinity interaction with the Arp2/3 complex and (2) the subdomain contacts through which
Arg interacts with the Arp2/3 complex, respectively. Finally, I will develop two methods to determine how cortactin
can coordinate with Arg to stimulate Arp2/3 activation. Two-color single molecule TIRF assays will address if
cortactin recruits Arg to branch points and competition binding assays will determine if cortactin functions
(mechanistically) to release Arg from nascent branch points, allowing filament elongation.
Aim 2. To determine the mechanisms through which Arg and cortactin regulate spine stability. Arg,
cortactin and the Arp2/3 complex are concentrated in dendritic spines and each is required for spine stability,
but how they interact to confer this stability is not understood. Preliminary data collected in the lab suggests that
loss of cortactin initially reduces stable F-actin prior to dendritic spine destabilization. Using well-defined Arg or
cortactin mutants, I will perform knockdown/complementation and confocal microscopy in cultured hippocampal
neurons to determine how disruptions of Arp2/3 complex regulation affect spine dynamic behavior and stability.
I will use GFP-actin fluorescent recovery after photobleaching (FRAP), employing the approaches used by my
collaborator, with the same mutant cohort to determine how these proteins impact spine stability via control of
the stable and dynamic actin pools.
项目摘要
树突棘的稳定性在精神和神经系统疾病中被破坏。
高度分支的细胞骨架,包含稳定和动态的丝状肌动蛋白(F-actin)池和单体肌动蛋白。
树突棘中的β 2/Arg非β受体酪氨酸激酶(Arg)及其相互作用伴侣cornein的缺失
导致青春期后期广泛的脊柱缺失,但这两种蛋白质如何支持的机制
树突棘的稳定性仍然难以捉摸。我们的实验室最近发现,使用全内反射显微镜,
(TIRFM)基于单丝的测定,Arg和cornein协同调节Arp 2/3-actin介导的肌动蛋白
Arp 2/3复合物通过Arg和cornein的协调调节是一种合理的机制,
在我的研究计划中,我将测试精氨酸和胶原蛋白
相互作用,通过调节Arp 2/3复合物和维持树突棘的稳定性,
稳定肌动蛋白池。
目的:1.确定精氨酸与coronin和Arp 2/3复合物相互作用促进肌动蛋白分支的机制
Arg如何调节Arp 2/3复合物以及corneumen如何与Arp 2/3复合物协调仍然没有解决。
为了解决Arg如何调节复合物,我将学习如何进行功能性
基于TIRFM单丝的测定,以确定足以激活Arp 2/3的Arg的最小结构域
我的初步数据表明,Arg可以直接结合Arp 2/3复合物。
分析和学习如何进行化学交联实验,以确定(1)最小的精氨酸片段
可以与Arp 2/3复合物进行高亲和力相互作用的亚结构域,以及(2)通过其
最后,我将开发两种方法来确定Corp 2/3复合物是如何与Arg相互作用的。
可与Arg协同刺激Arp 2/3的活化,双色单分子TIRF检测可检测
corneumn将Arg募集到分支点,竞争结合试验将确定corneumn是否起作用
(机械地)从新生分支点释放Arg,允许细丝伸长。
目的2.确定精氨酸和皮质醇调节脊柱稳定性的机制。
coronin和Arp 2/3复合物集中在树突棘中并且每一个都是棘稳定性所需的,
但尚不清楚它们如何相互作用以赋予这种稳定性。实验室收集的初步数据表明,
在树突棘不稳定之前,coronin的缺失首先降低了稳定的F-肌动蛋白。
corptin突变体,我将进行敲除/互补和共聚焦显微镜培养海马
神经元,以确定Arp 2/3复合物调节的中断如何影响脊柱动态行为和稳定性。
我将使用绿色荧光蛋白-肌动蛋白荧光恢复后光漂白(FRAP),采用我的方法,
合作者,与相同的突变队列,以确定这些蛋白质如何影响脊柱稳定性,通过控制
稳定和动态肌动蛋白池。
项目成果
期刊论文数量(0)
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