Spatial Segregation of Cell Functioning during Motility

描述(申请人提供)：我们的长期目标是确定细胞如何建立和保持渐进的运动，以重新填充组织对外部信号的反应。在伤口修复中，可溶性生长因子引导成纤维细胞和内皮细胞重新填充未成熟的基质，形成支持基质和再生组织结构所需的血管系统。最初的迁移是由伤口床内产生的刺激性线索驱动的。然而，一旦进入伤口床，细胞必须经常在没有刺激梯度的情况下分布。因此，一个中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，细胞必须延伸板脂并形成新的粘连，以稳定主要的突起，而后部则需要去粘连和回缩以实现渐进运动。在这两个细胞区域之间，发生收缩以使细胞体向前移动。为了高效地编排这些流程，单元必须建立持久的方向性。在最初的授权期，我们发现在几乎无处不在的化学动力学的EGFR配体诱导的运动过程中，PLCy(磷脂酶-Cy)的初始作用建立了重要的膜部分PIP2(磷脂酰二磷酸)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受到这个膜部分和对接位置的调节。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是质膜中磷酸肌醇不对称整合的结果。我们建议测试以下假设：/.运动过程中后向释放所需的m-calain(CAPN2)通过与PIP2结合定位于可激活的质膜周围区域。第二，PKCS介导的伸缩性，需要将细胞体和尾巴向前拉，定位于通过磷酸肌醇转换连接的PLCy活性区域。PI3激酶指定并稳定了主导的、占主导地位的蛋白，以提供方向性。我们专注于人类成纤维细胞和内皮细胞，并延伸到间充质干细胞。这些研究将确定受体信号和由此产生的生物物理反应的空间限制的分子基础，为组织工程支持组织功能的“智能”支架的设计提供希望。

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.