Defining the Role of Dynamin in Endocytosis

描述(申请人提供)：Dynamin是一个约100kD的多结构域GTP酶，需要网状蛋白介导的内吞作用，但其确切作用尚不清楚。它自组装成环和螺旋堆叠的连接，自组装刺激动力蛋白的GTP酶活性-100倍。在GTPGammaS存在的情况下，动力蛋白螺旋(称为‘环’)聚集在深度凹陷的涂层坑的颈部。GTP结合和/或水解缺陷的动力蛋白突变体的过表达可抑制细胞内吞作用。在这些观察的基础上，我们首次提出了Dynamin作为机械力化学‘夹击’的作用模型，在该模型中，GTP水解驱动协同构象变化，以收缩组装的Dynamin环，并驱动膜分裂。在探索协调自组装和刺激GTP水解的机制时，我们发现Dynamin中的GTP酶效应域或GED是自组装所必需的，并作为组装依赖的间隙发挥作用。令人惊讶的是，在GED突变的情况下，内吞作用在过度表达Dynamin的细胞中被刺激，该突变损害了自组装，从而削弱了组装依赖的GAP活性。这一意想不到的结果导致我们提出了另一个模型，认为Dynamin(像所有其他GTPase超家族成员一样)作为一种调节酶发挥作用，控制囊泡形成所需的下游效应器。最近的证据表明，Dynamin的表达可以改变肌动蛋白的动力学，影响细胞形状的变化，并触发导致P53激活和凋亡的信号级联反应，这与Dynamin在细胞信号传递中的更广泛作用是一致的。然而，关于Dynamin在网状蛋白介导的内吞作用中的确切作用仍有相当大的争议。从根本上说，关键问题变成了：动力是CCV形成背后的“大脑”还是“斗殴”？我们建议通过追求以下特定目标来回答这个问题：1)分析shibire-TS2等位基因和shibire(寿司)的3个等位基因内抑制基因的人类同源物的生化特性；2)产生和表型表征Dynamin-2基因敲除细胞和可能的小鼠；3)利用基于荧光的显微分析来监测Dynamin在导致包被坑成熟和包被囊泡形成的一系列事件中的作用。4)利用一种新开发的从分离的质膜中形成CCV的方法来体外剖析动力蛋白的功能5)确定细胞中动力蛋白功能所需的下游效应分子。

DESCRIPTION (provided by applicant): Dynamin is an approximately 100 kD multidomain GTPase required for clathrin mediated endocytosis, but its exact role remains unknown. It self-assembles into rings and helical stacks of tings and self-assembly stimulates dynamin's GTPase activity -100-fold. In the presence of GTPgammaS, dynamin spirals (called 'collars') accumulate around the necks of deeply invaginated coated pits. Endocytosis is inhibited by overexpression of dynamin mutants defective in GTP binding and/or hydrolysis. Based on these observations, we first proposed a model for dynamin function as a mechanochemical 'pinchase', in which GTP hydrolysis drives a concerted conformational change to constrict the assembled dynamin collar and drive membrane fission. In probing the mechanisms that coordinate self-assembly with stimulated GTP hydrolysis we discovered that the GTPase effector domain, or GED, in dynamin is required for self-assembly and functions as an assembly-dependent GAP. Surprisingly, endocytosis was stimulated in cells overexpressing dynamin with a mutation in GED that impairs self-assembly and consequently assembly-dependent GAP activity. This unexpected result led us to propose an alternate model suggesting that dynamin functions (like all other GTPase superfamily members) as a regulatory enzyme controlling downstream effectors required for vesicle formation. Recent evidence that dynamin expression can alter actin dynamics, affect cell shape changes and trigger a signaling cascade leading the p53 activation and apoptosis is consistent with a broader role for dynamin in cellular signaling. However, considerable controversy exists as to the exact function of dynamin in clathrin-mediated endocytosis. Fundamentally, the critical question becomes: Is dynamin the 'brains' or the 'brawns' behind CCV formation? We propose to answer this question by pursuing the following Specific Aims: 1) Analyze the biochemical properties of the human homologues of the shibire-ts2 allele and 3 intra-allelic suppressors of shibire (sushi) 2) Generate and phenotypically characterize dynamin-2 knock-out cells and possibly mice 3) Utilize fluorescence-based microscopic assays to monitor dynamin's effects on the hierarchy of events leading to coated pit maturation and coated vesicle formation. 4) Utilize a newly developed assay for CCV formation from isolated plasma membranes to dissect dynamin function in vitro 5) Identify downstream effector molecules required for dynamin function in the cell.