Spatial Segregation of Cell Functioning during Motility

描述（由申请人提供）：我们的长期目标是确定细胞如何建立和维持前向运动，以响应外部信号重新填充组织。在伤口修复中，可溶性生长因子指导成纤维细胞和内皮细胞重新填充未成熟基质以形成再生组织结构所需的支持基质和脉管系统。最初的迁移是由伤口床内产生的刺激线索驱动的。然而，一旦在伤口床内，细胞必须经常在没有刺激梯度的情况下分布。因此，一个核心问题是细胞如何建立渐进迁移所需的持续不对称性。细胞迁移需要生物物理力相关过程的不对称性;在真核细胞中，这可能是由细胞内信号控制的。在前部，细胞必须延伸板状伪足并形成新的粘附以稳定主导突起，而后部的去粘附和回缩则需要使其能够进行性运动。在这两个细胞区域之间，发生收缩以使细胞体向前移动。为了有效地编排这些过程，细胞必须建立持久的方向性。在最初的授权期间，我们已经发现，在由几乎无处不在的趋化动力学EGFR配体诱导的运动期间，PLC γ（磷脂酶-Cy）的初始活性建立了重要的膜部分PIP 2（磷酸肌醇二磷酸）的不对称性。我们的初步数据表明，导致生物物理过程的生化级联至少部分地由该膜部分和对接位点调节。因此，我们假设，生产细胞运动所需的关键生化信号级联的局部激活的结果从整合的磷酸肌醇不对称性的质膜。我们建议测试以下假设：/。运动期间后部释放所需的m-钙蛋白酶（CAPN 2）通过与PIP 2结合而定位于可激活的质膜周围区域。二. PKCS介导的收缩性是将细胞体和尾部向前拉动所需的，其定位于与磷酸肌醇周转相关的PLC γ活性区域。三. PI 3激酶指定并稳定主导的，占主导地位的片足类动物，以提供方向性。我们专注于人成纤维细胞和内皮细胞，并扩展到间充质干细胞。这些研究将定义受体信号传导的空间限制和由此产生的生物物理反应的分子基础，为组织工程的“智能”支架设计提供承诺，以支持组织功能。

DESCRIPTION (provided by applicant): Our long-term goal is to determine how cells establish and maintain progressive motility to repopulate tissues in response to external signals. In wound repair, soluble growth factors direct fibroblasts and endothelial cells repopulate the immature matrix to form both the supporting matrix and vasculature required to regenerate the tissue structures. The initial migration is driven by stimulatory cues arising from within the wound bed. However, once within the wound bed, the cells must distribute often in the absence of stimuli gradients. A central question is therefore how cells establish the sustained asymmetry required for progressive migration. Cell migration requires asymmetry of biophysical force-related processes; in eukaryotic cells this is likely governed by intracellular signals. At the front, the cells must extend lamellipodia and form new adhesions to stabilize the dominant protrusion, while rear de-adhesion and retraction is required to enable progressive movement. Between these two cell regions, contractility occurs to bring the cell body forward. To productively choreograph these processes, a cell must establish persistent directionality. During the initial grant period we have found that during motility induced by near ubiquitous chemokinetic EGFR ligands, that the initial actviation of PLCy (phospholipase-Cy) establishes an asymmetry of an important membrane moiety, PIP2 (phosphoinositide bisphosphate). Our preliminary data suggest that the biochemical cascades leading to the biophysical processes are regulated at least in part by this membrane moiety and docking site. Thus, we hypothesize that the localized activation of key biochemical signaling cascades required for productive cell motility results from the integration of phospho-inositide asymmetry in the plasma membrane. We propose to test the following postulates: /. That m-calpain (CAPN2), required for rear release during motility, is localized to an activatable peri-plasma membrane locale by binding to PIP2. II. That PKCS-mediated contractility, required to pull the cell body and tail forward, is localized to regions of PLCy activity linked by phospho-inositide turnover. III. That PI3 kinase designates and stabilizes the leading, dominant lamellipod to provide directionality. We focus on human fibroblasts and endothelial cells, with extension to mesenchymal stem cells. These studies will define molecular bases for spatial restriction of receptor signaling and resultant biophysical responses, offering promise for design of 'smart' scaffolds for tissue engineering to support tissue function.