The function of Arp2/3 in motility and actin branch stability

描述（由申请人提供）：细胞迁移对于从胚胎形态发生到成人伤口愈合的许多不同生理过程是必不可少的，并且是包括肿瘤转移在内的病理过程中的主要异常行为。在过去的四十年里，在理解支持正常发育和肿瘤发生的基本遗传和信号线索方面取得了巨大进步。然而，相比之下，在这些背景下对细胞迁移的理解仍然是基本的。基于肌动蛋白的突起长期以来被认为是细胞在2D基底上迁移的关键物理要求。Arp 2/3复合物是一种特征良好的大分子机器，其在这些突起内组装分支肌动蛋白，并且是细胞迁移所需的。该提案旨在通过确定负责招募和稳定Arp 2/3复合物的因素，以及复合物中允许其结合肌动蛋白的特定氨基酸，更好地了解正常和异常细胞迁移。利用新产生的Arp 2/3缺陷细胞的另一个目的是了解不同肌动蛋白组织范式之间的相互作用及其对高阶细胞过程（包括体内细胞运动）的影响。的 将使用多种经典和新颖的细胞生物学方法来评估上述目的。Arp 2/3缺陷或敲除细胞已经通过标记到荧光蛋白GFP的野生型或突变亚基的再表达而被拯救，并且将用于通过FRAP测定Arp 2/3动力学，通过记录图测定活细胞中的前沿突出率，以随时间追踪随机细胞运动性，并测试响应于施加的梯度而诱导的定向迁移 生长因子或粘附的基质线索。进一步的分析将涉及通过WT与Arp 2/3缺陷细胞中的电子显微镜直接评估肌动蛋白细胞骨架，或通过活细胞成像和FRAP直接评估GFP-肌动蛋白动力学。最后，稳定的shRNA或瞬时siRNA介导的其他肌动蛋白相关蛋白的耗竭将使我们深入了解Arp 2/3缺失时肌动蛋白的聚合。这项工作将确定影响肌动蛋白分支稳定性的因素，并提高Arp 2/3功能的理解和阿普和非阿普肌动蛋白聚合蛋白在细胞运动过程中的相互作用。此外，先进的成像方法和这些研究期间建立的遗传p34敲除小鼠将使我们更好地了解Arp 2/3和非Arp肌动蛋白成核在其他情况下的重要性，例如肿瘤细胞侵袭和转移。

DESCRIPTION (provided by applicant): Cell migration is essential for many diverse physiological processes from embryonic morphogenesis to adult wound healing, as well as being a primary aberrant behavior in pathological processes including tumor metastasis. Great strides have been made over the past four decades in understanding basic genetic and signaling cues underpinning both normal development and tumorigenesis. Yet the understanding of cell migration in these contexts remains rudimentary in comparison. Actin-based protrusion has long been identified as a critical physical requirement for cell migration over a 2D substrate. The Arp2/3 complex is a well-characterized macromolecular machine that assembles branched actin within these protrusions and is required for cell migration. This proposal aims to better understand normal and aberrant cell migration by identifying the factors responsible for recruiting and stabilizing the Arp2/3 complex, as well as the specific amino acids in the complex that allow it to bind actin. An additional aim, making use of newly generated Arp2/3-deficient cells, is to understand the interplay between distinct actin organization paradigms and their impact on higher order cellular processes, including cell motility in vivo. The objectives outlined above will be assessed using a variety of classic and novel cell biological approaches. Arp2/3 deficient or knockout cells have been rescued via re-expression of wildtype or mutant subunits tagged to the fluorescent protein GFP, and will be used to assay Arp2/3 dynamics via FRAP, leading edge protrusion rates in live cells via kymography, to track random cell motility over time, and to test directional migration induced in response to applied gradients of growth factors or adhered matrix cues. Further analyses will involve direct assessment of the actin cytoskeleton via electron microscopy in WT vs. Arp2/3-deficient cells, or GFP-actin dynamics via live cell imaging and FRAP. Finally, stable shRNA or transient siRNA-mediated depletion of other actin-associated proteins will give insight into actin polymerization in the absence of Arp2/3. This work will identify factors that impact actin branch stability and enhance the understanding of Arp2/3 function and the interplay between Arp and non-Arp actin polymerizing proteins during cell motility. Furthermore, advanced imaging approaches and genetic p34 knockout mice established during these studies will lead to a better understanding of the importance of Arp2/3 and non-Arp actin nucleation in additional contexts, such as tumor cell invasion and metastasis.