Defining the Role of Dynamin in Endocytosis

描述（由申请人提供）：Dynamin是一种约100 kD的多结构域GTPase，用于网格蛋白介导的内吞作用，但其确切作用尚不清楚。它自我组装成环状和螺旋状的东西，自我组装刺激动力蛋白的GTPase活性-100倍。在gtpgamma存在的情况下，动力蛋白螺旋（称为“项圈”）在深内陷的涂膜凹陷周围积聚。胞吞作用被GTP结合和/或水解缺陷的动力蛋白突变体过度表达所抑制。基于这些观察，我们首先提出了动力蛋白作为机械化学“挤压”功能的模型，其中GTP水解驱动一致的构象变化，以收缩组装的动力蛋白环并驱动膜裂变。在探索协调自组装与受刺激GTP水解的机制时，我们发现动力蛋白中的GTP酶效应域（GED）是自组装所必需的，并作为组装依赖的GAP发挥作用。令人惊讶的是，在过度表达动力蛋白的细胞中，内吞作用受到刺激，而GED突变会损害自组装，从而损害组装依赖的GAP活性。这一意想不到的结果使我们提出了另一种模型，表明动力蛋白的功能（像所有其他GTPase超家族成员一样）是一种调节酶，控制着囊泡形成所需的下游效应物。最近的证据表明，动态蛋白表达可以改变肌动蛋白动力学，影响细胞形状变化并触发信号级联导致p53激活和凋亡，这与动态蛋白在细胞信号传导中的广泛作用是一致的。然而，对于动力蛋白在网格蛋白介导的胞吞作用中的确切作用，存在相当大的争议。从根本上说，关键问题变成了：在CCV形成背后，动力是“大脑”还是“肌肉”？我们建议通过以下具体目标来回答这个问题：1)分析shibiire -ts2等位基因的人类同源物和shibiire （sushi）的3个等位基因内抑制子的生化特性；2)生成dynamin-2敲除细胞和可能的小鼠的表型特征；3)利用基于荧光的显微分析来监测dynamin对导致包被坑成熟和包被泡形成的事件层次的影响。4)利用新开发的分离质膜形成CCV的实验来解剖体外动力蛋白功能5)鉴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.