Mechanisms and regulation of macropinosome closure

描述（由申请人提供）：本项目将定义大肌肽体闭合的基本机制。巨噬细胞作用和膜和肌动蛋白骨架的形态类似运动在上皮生理和宿主防御多种感染中起重要作用。巨饮作用的成功完成总是需要闭合：细胞表面的一个区域收缩，将一个杯状的质膜内陷转化为细胞内离散的膜结合细胞器。尽管它在健康和疾病中无处不在和重要，但关闭的机制和规则在很大程度上仍然无法解释。形态学预测了大肌醇小体闭合的两个不同阶段：在杯状细胞表面褶皱的远端边缘收缩，这需要磷酸肌醇3'-激酶（PI3K），随后是将大肌醇小体从质膜分离的断裂。巨饮杯可以被认为是细胞表面的一种短暂的、自组织的收缩结构。我们假设PI3K组织的基于肌动蛋白-肌球蛋白的收缩活动局限于巨红细胞杯的内膜，这是由远端边缘的屏障维持的，该屏障限制了3'磷酸肌苷向杯外的横向扩散。这一假设将通过在巨噬细胞中精确地确定巨量酶体的关闭点，以及在巨量酶体形成的各个阶段定位扩散障碍来验证。特异性目标1将确定闭合的时间和形态。将通过测量外部添加的膜外荧光探针对大酶体的访问来确定断裂点。本实验室开发的仪器和方法将用于获得表达荧光探针的细胞收缩和断裂的时间分辨、三维（4D）重建。特异性目标2将确定巨红细胞杯中横向扩散的障碍。形态学研究表明，在闭合期间，杯的边缘阻止了内小叶磷脂的扩散出杯。杯缘的扩散屏障可以促进3'磷酸肌苷的局部积累，并限制杯内膜的收缩活动。为了识别这些屏障，光激活GFP （paGFP）与膜锚定蛋白结构域（paGFP- mem）的荧光嵌合体将在巨小体或杯内或附近被局部光激活。如果paGFP-MEM在双分子层平面上的扩散受到杯缘的限制，则在杯内产生的荧光paGFP-MEM应被限制在质膜的杯域内。通过表达荧光磷脂结合结构域的细胞的定量显微镜，可以测量杯内和杯缘的磷酸肌苷浓度梯度。因此，通过定义关闭过程中细胞质的时空组织，这些研究将为分析大体积小体和吞噬体关闭的分子机制创建一个框架。

DESCRIPTION (provided by applicant): This project will define the essential mechanics of macropinosome closure. Macropinocytosis and morphologically analogous movements of membrane and the actin cytoskeleton are important in epithelial physiology and host defense against many infections. The successful completion of macropinocytosis always requires closure: the constriction of a region of the cell surface that transforms a cup-shaped invagination of plasma membrane into a discrete membrane-bounded organelle inside the cell. Despite its ubiquity and importance in health and disease, the mechanics and regulation of closure remain largely unexplained. Morphology predicts two distinct stages of macropinosome closure: constriction at the distal margin of cup-shaped, cell surface ruffles, which requires phosphoinositide 3'- kinase (PI3K), followed by a scission that separates the macropinosome from the plasma membrane. The macropinocytic cup can be considered as a transient, self-organized contractile structure at the cell surface. We hypothesize that PI3K organizes actin-myosin-based contractile activities localized to the inner membrane of the macropinocytic cup, which are maintained by a barrier at the distal rim that restricts lateral diffusion of 3' phosphoinositides out of the cup. This hypothesis will be tested by identifying precisely the point of macropinosome closure in macrophages and by localizing barriers to diffusion at various stages of macropinosome formation. Specific Aim 1 will identify the timing and morphology of closure. The point of scission will be identified by measuring access to macropinosomes of externally added, membrane-impermeant fluorescent probes. Instruments and methods developed in this lab will be used to acquire time-resolved, 3-dimensional (4D) reconstructions of constriction and scission in cells expressing fluorescent probes. Specific Aim 2 will identify barriers to lateral diffusion in macropinocytic cups. Morphological studies suggest that during closure the rim of the cup prevents diffusion of inner-leaflet phospholipids out of the cup. A barrier to diffusion at the cup rim could facilitate the local accumulation of 3' phosphoinositides and constrain contractile activities to the inner membrane of the cup. To identify such barriers, fluorescent chimeras of photoactivatable GFP (paGFP) with membrane-anchoring protein domains (paGFP-MEM) will be locally photoactivated in or near macropinosomes or cups. If diffusion of paGFP-MEM in the plane of the bilayer is restricted by cup rims, then fluorescent paGFP-MEM generated inside cups should be constrained to the cup domain of the plasma membrane. Concentration gradients of phosphoinositides within cups and across the cup rim will be measured by quantitative microscopy of cells expressing fluorescent, phospholipid-binding domains. Thus, by defining the spatiotemporal organization of cytoplasm during closure, these studies will create a framework for analyzing molecular mechanisms of closure in macropinosomes and phagosomes. PUBLIC HEALTH RELEVANCE: Pinocytosis is a microscopic drinking activity, present in nearly all living cells, which is especially important in immunity and in kidney function. This work will analyze an unexplained but essential last step in the process, in which an invagination of the cell's plasma membrane closes to form a fluid-filled vesicle inside the cell. The results should have implications for how cells coordinate large-scale activities in their cytoplasm.