Defining the Role of Dynamin in Endocytosis

描述（由申请方提供）：发动蛋白是网格蛋白介导的内吞作用所需的约100 kD多结构域GT酶，但其确切作用仍未知。它可以自我组装成环状和螺旋状的环，自我组装可以刺激发动蛋白的GT3活性100倍。在GTP γ S的存在下，发动蛋白螺旋（称为“项圈”）聚集在深深内陷的被膜小凹的颈部周围。内吞作用被GTP结合和/或水解缺陷的发动蛋白突变体的过表达抑制。基于这些观察，我们首先提出了一个模型的动力功能作为一个机械化学的“pinchase”，其中GTP水解驱动一个协调的构象变化，以压缩组装的动力衣领和驱动膜裂变。在探索协调自组装与刺激GTP水解的机制时，我们发现发动蛋白中的GTP酶效应结构域或GED是自组装所需的，并作为组装依赖性GAP发挥作用。令人惊讶的是，内吞刺激过表达发动蛋白的细胞与GED的突变，损害自组装，因此组装依赖性GAP活性。这个意想不到的结果使我们提出了一种替代模型，表明发动蛋白功能（像所有其他GTdR超家族成员）作为一种调节酶控制囊泡形成所需的下游效应子。最近的证据表明，发动蛋白的表达可以改变肌动蛋白动力学，影响细胞形状的变化，并触发信号级联导致p53激活和凋亡是一致的发动蛋白在细胞信号转导中的作用更广泛。然而，关于发动蛋白在网格蛋白介导的内吞作用中的确切功能存在相当大的争议。从根本上说，关键的问题变成了：动力蛋白是CCV形成背后的“大脑”还是“肌肉”？我们建议通过追求以下具体目标来回答这个问题：1）分析shibire-ts 2等位基因和shibire（sushi）的3个等位基因内抑制子的人类同源物的生物化学性质2）产生并表型表征动力蛋白-2敲除细胞和可能的小鼠3）利用荧光-基于显微镜分析，以监测发动蛋白的层次结构的事件，导致包被坑成熟和包被囊泡形成的影响。4)利用新开发的用于从分离的质膜形成CCV的测定来剖析发动蛋白的体外功能5）鉴定细胞中发动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.