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.
项目总结:
树突状脊椎的稳定性在精神疾病和神经功能障碍中被破坏。树突状脊椎在一年内也被丰富。
高度分枝的肌动蛋白细胞骨架,包含稳定的肌动蛋白和动态的丝状(F-)肌动蛋白池,以及单体肌动蛋白。
失去了Abl2/Arg的非受体酪氨酸氨基转移酶(Arg),并失去了其与树突状突起的合作伙伴Cortactin的相互作用。
导致青春期后期广泛的脊椎缺失,但这两种主要蛋白质如何支持的主要机制。
树突和脊柱的稳定性仍然难以捉摸。正如我们的实验室最近所显示的那样,使用的是全内反射显微镜。
(TIRFM)是一种基于单丝的分析方法,表明精氨酸和皮质肌动蛋白可以协同作用,进一步调节Arp2/3介导的肌动蛋白。
分支。通过精氨酸(Arg)和抗坏血酸(Cortactin)对Arp2/3复合体进行协调监管,是一种可行的监管机制。
这些蛋白质将支持脊柱的稳定性。在我的研究计划中,我将进一步测试精氨酸和皮质肌动蛋白的假设。
通过对Arp2/3复合体的监管和脊椎的维护,互动以增强树突和脊椎的稳定性。
稳定的肌动蛋白池。
目的:1.进一步确定精氨酸如何与皮质肌动蛋白相互作用,以及如何通过Arp2/3复合体来促进肌动蛋白在分支中的作用。
对于Arg如何调节Arp2/3复合体,以及Cortactin如何与Arp2/3协调,目前仍未解决。
Arg希望进一步增强这一效果。为了解决Arg如何监管金融复合体,我将进一步学习如何进行有效的监管。
TIRFM是一种基于单丝的分析方法,用于定义Arg的最小域值,即它没有足够的空间来激活Arp2/3。
复杂性。我的初步数据表明,Arg可以直接或绑定到Arp2/3的建筑群。我不会扩大这些具有约束力的项目。
化验人员和他们学习如何进行化学和交联实验,以确定最小的Arg片段。
这也可以使它成为与ARP2/3的最高亲和力和互动的基础上,并(2)通过它的主要子域进行联系。
Arg将分别与Arp2/3复合体相互作用。最后,我将进一步开发两种新的方法,以进一步确定Cortactin是如何产生的。
我们可以与精氨酸进行协调,以进一步刺激Arp2/3的激活。TIRF表示,双色和单分子TIRF检测将无法解决这一问题。
Cortactin将从Arg招聘到其分支机构,而具有约束力的检测结果的竞争将不会决定Cortactin是否发挥作用。
(机械上)允许将Arg从新生的分支上释放到点上,并允许细丝伸长。
目的:2.进一步确定Arg和Cortactin调节脊柱稳定性的机制。
Cortactin和Arp2/3复合体通常集中在树突和脊椎中,每一个都是脊椎稳定所必需的。
但是,他们将如何互动,以确保这一稳定机制,目前尚不清楚。从实验室收集的初步数据来看,这一点似乎是错误的。
皮质肌动蛋白的丢失最初会减少稳定的F-肌动蛋白,而不是在树突状突起和脊柱不稳定之前。现在使用的是定义明确的F-肌动蛋白或。
Cortactin是一种突变体,我想他们将在培养的新海马区进行基因敲除/互补和共聚焦显微镜观察。
神经元需要确定Arp2/3基因复杂调控的干扰将如何影响脊柱的动态行为和神经稳定性。
我将不会使用GFP-肌动蛋白来进行光漂白治疗(FRAP)后的荧光治疗,并使用我的治疗方法中最常用的方法。
合作者,与另一个相同的基因突变体研究队列,以确定这些蛋白质如何通过对基因的控制影响脊柱的稳定性。
稳定的肌动蛋白和动态的肌动蛋白池。
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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