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

描述(申请人提供)：细胞迁移在许多不同的生理过程中是必不可少的，从胚胎形态形成到成人伤口愈合，也是包括肿瘤转移在内的病理过程中的一种主要异常行为。在过去的40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长足的进步。然而，与之相比，对这些背景下的细胞迁移的理解仍然是初级的。长期以来，基于肌动蛋白的突起一直被认为是细胞在2D基质上迁移的关键物理要求。Arp2/3复合体是一种具有良好特性的大分子机器，它在这些突起中组装分支肌动蛋白，是细胞迁移所必需的。这一建议旨在通过确定负责招募和稳定Arp2/3复合体的因素，以及复合体中使其与肌动蛋白结合的特定氨基酸来更好地了解正常和异常的细胞迁移。另一个目的是利用新产生的Arp2/3缺陷细胞，了解不同的肌动蛋白组织范例之间的相互作用，以及它们对包括体内细胞运动在内的高级细胞过程的影响。这个 上面概述的目标将使用各种经典和新颖的细胞生物学方法进行评估。Arp2/3缺陷或敲除细胞已通过标记到荧光蛋白GFP的野生型或突变亚基的重新表达而被拯救，并将被用于通过FRAP分析Arp2/3动力学，通过细胞分裂图分析活细胞的前沿突出率，跟踪随时间的随机细胞运动，并测试对施加梯度的反应所诱导的定向迁移 生长因子或附着的基质线索。进一步的分析将包括通过电子显微镜直接评估WT和Arp2/3缺陷细胞中的肌动蛋白细胞骨架，或者通过活细胞成像和FRAP来评估GFP-肌动蛋白的动力学。最后，稳定的shRNA或瞬时siRNA介导的其他肌动蛋白相关蛋白的缺失将有助于深入了解在没有Arp2/3的情况下肌动蛋白的聚合。这项工作将识别影响肌动蛋白分支稳定性的因素，并加深对Arp2/3功能以及Arp和非Arp肌动蛋白聚合蛋白在细胞运动过程中的相互作用的理解。此外，在这些研究中建立的先进的成像方法和遗传的p34基因敲除小鼠将有助于更好地理解Arp2/3和非Arp肌动蛋白在其他环境中的重要性，例如肿瘤细胞的侵袭和转移。

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.