Pathways and mechanisms of clathrin-independent endocytosis

网格蛋白依赖性内吞作用的途径和机制

• 批准号: 8149571
• 负责人: Julie G Donaldson
• 金额: $ 91.99万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149571
• 关键词: Clathrin; Endocytosis; Pathway interactions

The laboratory is interested in understanding the clathrin-independent endocytosis pathway that is associated with Arf6. We have shown that this pathway is responsible for internalizing plasma membrane (PM) proteins that lack sequences that allow recognition by the clathrin and adaptor protein machinery. Among the proteins that enter cells via this mode of endocytosis are the major histocompatibility complex Class I protein (MHCI) and the lipid anchored protein CD59. After endocytosis, MHCI and CD59 in vesicles are delivered to and fuse with endosomes containing cargo proteins from the clathrin-dependent endocytosis pathway such as transferrin receptor. From there, MHCI and CD59 can proceed on to late endosomal compartments where they are degraded or they can be recycled back out to the PM via unique tubular recycling endosomes. In HeLa cells these recycling endosomes contain only cargo that had entered via clathrin-independent endocytosis and their return to the PM is dependent upon the activity of Arf6 and several other regulators including Rab11 and 22. We have identified 7 new cargo proteins that enter cells and traffic along this clathrin-independent pathway: CD44, CD55, CD98, CD147, Lat1, ICAM1 and the non-insulin stimulated glucose transporter Glut1. We have confirmed that these proteins enter cells by clathrin-independent endocytosis in many human cell lines and in addition, in HeLa cells, we see a difference in intracellular itinerary followed by these proteins. CD55 and Glut1 follow a similar itinerary as MHCI, merging with endosomes containing the transferrin receptor before routing to the tubular recycling endosomes. By contrast, CD44, 98 and 147 after endocytosis do not enter transferrin-containing endosomes but are routed directly to the recycling endosome. This divergence in itinerary suggests that clathrin-independent cargo proteins are sorted within the cell and we are studying whether signals contained on these cargo proteins (extracellular, transmembrane or cytoplasmic) are directing their trafficking. We have found that sequences in the cytoplasmic tail of CD147 are responsible for this altered itinerary and through a yeast two-hybrid screen are looking for proteins that recognize these cytoplasmic sorting sequences. We are also examining the action of the MARCH E-3 ubiquitin ligases, which when overexpressed in cells leads to the downregulation of a number of clathrin-independent cargo proteins including MHCI. The laboratory has also been developing new probes for following endocytosis using SNAP-tag technology and has filed a provisional patent application for making releasable SNAP-tag ligands. This technique could be useful for quantitative evaluation of endocytosis and recycling of specifically labeled cell surface proteins. In addition to regulating recycling of endosomal membrane back to the cell surface, Arf6 has also been implicated in some forms of regulated secretion. Both of these processes generally require the actin cytoskeleton for exocytosis of the membrane back to the plasma membrane. In a collaborative study, it was found that myosin Vc is associated with tubular endosomes in breast cancer cell line MCF7 and also with secretory granules (Jacobs et al 2009). Although it is not clear whether myosin Vc is associated with the recycling endosomes in HeLa cells, we are investigating roles for other motor proteins, including microtubular-based motors, in this process. Finally, we have found that clathrin-independent endocytosis is observed in a variety of cell types and found evidence that this form of endocytosis participates in lumen formation observed in human vascular endothelial cells cultured in collagen gels, a model for vasculogenesis.

该实验室有兴趣了解与Arf 6相关的网格蛋白独立内吞途径。 我们已经表明，这一途径是负责内化质膜（PM）蛋白，缺乏允许识别的网格蛋白和衔接蛋白机器的序列。 通过这种内吞作用进入细胞的蛋白质包括主要组织相容性复合物I类蛋白（MHCI）和脂质锚定蛋白CD 59。 在胞吞作用之后，囊泡中的MHCI和⑶ 59被递送至含有来自网格蛋白依赖性胞吞作用途径的货物蛋白（如转铁蛋白受体）的内体并与之融合。 从那里，MHCI和CD 59可以继续进入晚期内体区室，在那里它们被降解，或者它们可以通过独特的管状再循环内体再循环回到PM。 在HeLa细胞中，这些再循环内体仅包含通过网格蛋白非依赖性内吞作用进入的货物，并且它们返回PM取决于Arf 6和包括Rab 11和22在内的几种其他调节剂的活性。 我们已经鉴定了7种新的货物蛋白，它们进入细胞并沿着沿着这种网格蛋白非依赖性途径运输：CD 44、CD 55、CD 98、CD 147、Lat 1、ICAM 1和非胰岛素刺激的葡萄糖转运蛋白Glut 1。 我们已经证实，这些蛋白质进入细胞的网格蛋白独立的内吞作用在许多人类细胞系，此外，在HeLa细胞中，我们看到细胞内行程的差异，其次是这些蛋白质。 CD 55和Glut 1遵循与MHCI类似的路线，在路由到管状再循环内体之前与含有转铁蛋白受体的内体合并。 相比之下，内吞作用后的CD 44、98和147不进入含有转铁蛋白的内体，而是直接路由到再循环内体。 行程的这种分歧表明，网格蛋白独立货物蛋白在细胞内进行分类，我们正在研究这些货物蛋白（细胞外，跨膜或细胞质）上包含的信号是否指导其运输。 我们已经发现，在CD 147的胞质尾序列是负责这个改变行程，并通过酵母双杂交筛选正在寻找蛋白质，识别这些细胞质排序序列。 我们还研究了MARCH E-3泛素连接酶的作用，当其在细胞中过表达时，会导致包括MHCI在内的许多网格蛋白非依赖性货物蛋白的下调。 该实验室还一直在开发新的探针，用于使用SNAP标签技术跟踪内吞作用，并已提交了一项临时专利申请，用于制造可释放的SNAP标签配体。 该技术可用于定量评价特异性标记的细胞表面蛋白的内吞和再循环。 除了调节内体膜再循环回到细胞表面之外，Arf 6还涉及某些形式的调节分泌。这两个过程通常需要肌动蛋白细胞骨架的胞吐作用的膜回到质膜。 在一项合作研究中，发现肌球蛋白Vc与乳腺癌细胞系MCF 7中的管状内体以及分泌颗粒相关（Jacobs et al 2009）。 虽然目前还不清楚肌球蛋白Vc是否与HeLa细胞中的内体回收有关，但我们正在研究其他马达蛋白（包括微管马达）在此过程中的作用。 最后，我们发现，网格蛋白独立的内吞作用是在各种类型的细胞中观察到的，并发现证据表明，这种形式的内吞作用参与管腔形成中观察到的人血管内皮细胞培养在胶原凝胶，模型血管生成。