Mechanisms and regulation of macropinosome closure

描述(由申请人提供)：这个项目将定义大针体关闭的基本机制。巨噬细胞吞噬作用以及膜和肌动蛋白细胞骨架的形态相似运动在上皮生理学和宿主对多种感染的防御中具有重要作用。成功地完成巨噬细胞吞噬总是需要闭合：细胞表面的一个区域的收缩，将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，也很重要，但关闭的机制和规则在很大程度上仍然不清楚。形态分析表明，大管胞小体的闭合有两个不同的阶段：杯状细胞表面皱纹的远端边缘的收缩，这需要磷脂酰肌醇3‘-激酶(PI3K)，然后是将大管胞小体从质膜上分离出来的断裂。巨饮细胞杯可以被认为是细胞表面的一种瞬时的、自组织的收缩结构。我们假设PI3K组织定位于巨饮细胞杯内膜的肌动蛋白-肌球蛋白收缩活动，这种收缩活动由远端边缘的屏障维持，该屏障限制3‘-磷酸肌醇向杯外的横向扩散。这一假说将通过准确地确定巨噬细胞中巨噬细胞巨噬体闭合点和定位巨噬细胞形成不同阶段的扩散障碍来检验。具体目标1将确定关闭的时间和形态。断裂点将通过测量外部添加的、膜无关的荧光探针的大针体的通达性来确定。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内收缩和断裂的时间分辨、三维(4D)重建。具体目标2将确定巨饮细胞杯的侧向扩散障碍。形态研究表明，在闭合过程中，杯子的边缘阻止了小叶内的磷脂扩散出杯子。杯缘的扩散屏障可促进3‘-磷酸肌醇类物质的局部蓄积，并将收缩活动限制在杯内膜上。为了识别这种屏障，带有膜锚定蛋白结构域的可光激活绿色荧光蛋白(PaGFP)嵌合体(paGFP-MEM)将在大胞体或杯状体内或其附近被局部光激活。如果paGFP-MEM在双层平面上的扩散受到杯缘的限制，那么在杯内产生的荧光paGFP-MEM应该被限制在质膜的杯区。通过对表达荧光磷脂结合区的细胞的定量显微镜观察，将测量杯体内和杯缘上的磷脂酰肌醇的浓度梯度。因此，通过定义闭合过程中细胞质的时空组织，这些研究将创建一个框架，用于分析大针体和吞噬体闭合的分子机制。 与公共卫生相关：胞饮是一种微小的饮用活动，几乎存在于所有活细胞中，这对免疫和肾脏功能特别重要。这项工作将分析这一过程中一个无法解释但必不可少的最后步骤，在这个过程中，细胞质膜的内陷关闭，在细胞内形成充满液体的小泡。这一结果应该对细胞如何协调其细胞质中的大规模活动具有一定的意义。

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.