Wang Yingchun, Klemke Richard L
Department of Pathology and MooresCancer Center, University of California, San Diego, La Jolla, CA, USA.
Methods Mol Biol. 2007;370:55-66. doi: 10.1007/978-1-59745-353-0_5.
Cell migration requires the formation of a leading pseudopodium (lamellipodium) in the direction of movement. This process requires signal amplification to facilitate directional sensing mechanisms that lead to actin-mediated membrane extension. However, it has been difficult to study pseudopodia formation because it has not been possible to purify this structure for biochemical analysis. Here we describe a method to biochemically purify the protruding pseudopodium from the cell body compartment using polycarbonate microporous filters. Cells are cultured on top of 3.0-microm porous filters and allowed to extend pseudopodia through the small pores to the undersurface in response to a gradient of either chemokine or extracellular matrix (ECM) protein. Pseudopodia and cell bodies are then differentially scraped from the filter surface into lysis buffer for biochemical analysis. Using this method, it is possible to identify novel pseudopodium and cell body proteins as well as study the spatiotemporal organization of signaling processes that regulate pseudopodium formation and cell polarity. This method will help facilitate our understanding of how cells protrude pseudopodia through small openings in the ECM and vasculature during cancer cell invasion, immune cell surveillance, and embryonic development.
细胞迁移需要在运动方向上形成一个领先的伪足（片状伪足）。这个过程需要信号放大，以促进导致肌动蛋白介导的膜延伸的方向感测机制。然而，研究伪足形成一直很困难，因为无法纯化这种结构进行生化分析。在这里，我们描述了一种使用聚碳酸酯微孔滤膜从细胞体区室中生化纯化突出伪足的方法。将细胞培养在3.0微米的微孔滤膜顶部，并使其响应趋化因子或细胞外基质（ECM）蛋白梯度，通过小孔向底面延伸伪足。然后将伪足和细胞体从滤膜表面分别刮入裂解缓冲液中进行生化分析。使用这种方法，可以鉴定新的伪足和细胞体蛋白，以及研究调节伪足形成和细胞极性的信号传导过程的时空组织。这种方法将有助于促进我们对癌细胞侵袭、免疫细胞监测和胚胎发育过程中细胞如何通过ECM和脉管系统中的小开口伸出伪足的理解。
