Microfilament Function in Cell Polarity

项目摘要/摘要 细胞通过极化其内部产生生化和形态上不同的质膜域 结构和生物合成途径。如果没有这种极性，细胞就不能执行运输等功能 跨上皮细胞和神经元中的信号传递。此外，对细胞极性的不正确调节可能会引发 癌症转移，如在不受调控的上皮细胞间质转化(EMT)。多个进程 细胞极化的交织，包括极化细胞骨架的组装，脂类和蛋白质的合成 随着它们的运输到适当的表面，以及胞吐和内吞。虽然远距离运输经常 涉及微管、局部运输和质膜的形态特征一般都涉及 信号通路、微丝和膜交通之间的相互作用。虽然人们对每一种情况都很了解 在各个领域，我们的两个项目解决了在理解如何协调它们方面的严重差距。首先，我们 目的了解结构元素和信号通路如何汇聚以定义细胞的形态。 以上皮细胞顶端的微绒毛作为我们的模型。我们有 定义了主要的结构成分，并提供了对关键微丝的调节的洞察- 膜连接蛋白Ezrin。我们将阐明影响Ezrin和其他因素的信号通路 将微绒毛限制在顶端表面，确定微绒毛如何影响膜蛋白质组，并 确定Ezrin的其他功能和调节器。其次，我们研究了以汽车为基础的运输如何 微丝与膜的运输是协调的。我们使用酵母菌，其中微丝作为轨道 基于肌球蛋白V的分泌囊泡运输对芽生长和母体细胞器分离的影响 和女儿在细胞分裂期间。我们将通过研究细胞器运输的基本方面来定义 肌球蛋白-V与囊泡的生物发生相协调地拾取、运输和输送分泌囊泡 和胞吐。我们已经建立了一个高时间和空间精度的投递成像系统 分子马达的循环，以及胞吐作用的步骤，并表明马达的释放与， 并依赖于胞吐作用。我们将对时间、依赖关系、 以及利用现有的和新产生的突变体，协调胞吐和运动释放的步骤。AS 所涉及的分子在酵母和脊椎动物之间是保守的，最明显的是从广泛的 研究神经递质在突触的释放，这一发现将具有普遍意义。重要的是要 请注意，酵母中的胞吐作用比神经肌肉接头处慢几个数量级，允许远 更高的时间分辨率，以及更易于实验访问的系统。此外，许多疾病 与分子马达缺陷有关，包括肌球蛋白-V和参与胞吐的成分。 这项拟议的研究将为细胞的两个关键方面提供基本的和医学上相关的见解 极性，细胞形态的局部调节和细胞器的传递。

Project Summary/Abstract Cells generate biochemically and morphologically distinct plasma membrane domains by polarizing their internal structure and biosynthetic pathways. Without this polarity, cells could not perform functions such as transport across epithelia and signal transmission in neurons. Further, improper regulation of cell polarity can initiate cancer metastasis, as in unregulated epithelial the mesenchymal transition (EMT). Multiple processes are interwoven for cell polarization, including assembly of a polarized cytoskeleton, synthesis of lipids and proteins with their transport to the appropriate surface, and exocytosis and endocytosis. While long-range transport often involves microtubules, local transport and morphological features of the plasma membrane generally involves an interplay between signaling pathways, microfilaments and membrane traffic. While much is known about each individual area, our two projects address the critical gap in understanding of how they are coordinated. First, we aim to understand how structural elements and signaling pathways converge to define the morphology of a specific membrane domain, using the microvilli on the apical aspect of epithelial cells as our model. We have defined the major structural components and provided insight into regulation of the critical microfilament- membrane linking protein ezrin. We will elucidate the signaling pathways that impinge on ezrin and other factors to restrict microvilli to the apical surface, to determine how microvilli impact the membrane proteome, and to identify the additional functions and regulators of ezrin. Second, we study how motor-based transport along microfilaments is coordinated with membrane traffic. We utilize yeast where microfilaments serve as tracks for the myosin-V based transport of secretory vesicles for bud growth and in organelle segregation between mother and daughter during cell division. We will define fundamental aspects of organelle transport by investigating how the myosin-V picks up secretory vesicles, transports and delivers them in coordination with vesicle biogenesis and exocytosis. We have established a system with high temporal and spatial precision for imaging the delivery cycle of a molecular motor, as well as steps in exocytosis, and shown that motor release is coordinated with, and dependent on, exocytosis. We will undertake an extensive mechanistic analysis of the timing, dependencies, and coordination of steps in exocytosis and motor release exploiting available and newly generated mutants. As the molecules involved are conserved between yeast and vertebrates, most notably identified from the extensive studies of neurotransmitter release at the synapse, the findings will be of general significance. It is important to note that exocytosis in yeast is orders of magnitude slower that at the neuromuscular junction, permitting far greater temporal resolution, and in a much more experimentally accessible system. Moreover, many diseases are associated with defects in molecular motors, including myosin-Vs, and components involved in exocytosis. The proposed research will provide fundamental and medically relevant insights into two critical aspects of cell polarity, local regulation of cell morphology and organelle delivery.