Spatial Segregation of Cell Functioning during Cell Motility

描述（由申请人提供）：在器官发生和伤口修复过程中诱导细胞运动功能（重新）填充靶空间，这种情况在许多临床条件和衰老个体中都是缺乏的。在组织再生过程中，间质成纤维细胞、内皮细胞和可能的循环干细胞从周围组织迁移到临时基质中，形成支撑基质和脉管系统。最初的细胞迁移是有方向性的，由信号驱动，即来自伤口床内部的“线索”。然而，一旦进入伤口床，细胞必须在没有明显刺激梯度的情况下分布。因此，我们的长期目标是确定细胞如何在外部刺激下建立并保持进行性运动以重新填充组织。细胞运动需要生物物理细胞过程的不对称性，这在真核细胞中是由细胞内信号建立的，以响应环境中的细胞因子“线索”，主要通过基质和干细胞的EGF受体和内皮细胞的VEGF，尽管通过其他受体的信号也遵循相同的细胞内级联。细胞必须扩展板足并稳定主要的突出，而后方的去粘连和回缩则需要使其能够渐进运动。在这两个细胞区域之间，发生收缩使细胞体向前移动。在这些过程之上，细胞必须建立其方向性，本质上没有明显的外部梯度。在最初的两个拨款期间，发现一个关键是PI(4,5)P2（磷酸肌肽4,5二磷酸；为了便于阅读，我们将不使用下标形式）的不对称分布及其处理。有趣的是，越来越明显的是，PIP2不仅在其已知的对接位点和活性代谢物的前体中起作用，而且在其本身中起主动信号换能器和辅助因子的新作用。最近的研究表明，PIP2直接激活calpain 2来驱动后释放，可能有助于指导细胞体向前移动所需的跨细胞收缩性，并可能调节肌动蛋白细胞骨架捆绑。因此，我们的发现超越了我们最初的假设，即细胞内信号级联是亚细胞定位的，假设了生长因子诱导运动过程中不对称的统一机制。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由磷酸肌苷的不对称作用引起的，通过对接/局部化和直接调节效应蛋白。我们的Specific Aims拟验证以下假设：1 . m-calpain （calpain-2）直接被PIP2激活。2。磷酸肌苷对1-肌动蛋白-4 （ACTN4）的调节控制了细胞骨架和膜之间的联系。3。磷酸肌苷的分布和磷酸肌苷的周转直接影响收缩性。这些研究的完成将定义受体信号通路空间限制的分子基础，以及由此产生的对人体组织细胞迁移至关重要的生物物理反应，为细胞功能的基本生物学提供缺失的信息。这些知识将使亚细胞尺度的“智能”支架设计成为可能，用于细胞和组织工程，指导基质和血管床的合成，以支持组织功能。

DESCRIPTION (provided by applicant): Induced cell motility functions during organogenesis and wound repair to (re)populate the target space, a situation that is deficient in a number of clinical conditions and in aging individuals. During tissue (re)generation stromal fibroblasts, endothelial cells, and possibly circulating stem cells migrate into the provisional matrix from surrounding tissues to form both the supporting matrix and vasculature. The initial cell immigration is directional, being driven by signals, 'cues' that arise from within the wound bed. However, once within the wound bed, the cells must distribute without evident stimuli gradients. Thus, our long-term goal is to determine how cells establish and then maintain progressive motility to repopulate tissues in response to external stimuli. Cell motility requires asymmetry of biophysical cell processes, which in eukaryotic cells is established by intracellular signals, in response to cytokine 'cues' in the environment, mainly via the EGF receptor for stromal and stems cells and VEGF for endothelial cells, though signaling via other receptors also follows the same intracellular cascades. Cells must extend lamellipodia and 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. Overriding these processes, a cell must establish its directionality, intrinsically in absence of significant external gradients. During the initial two grant periods it was found that a key is the asymmetric distribution of PI(4,5)P2 (phosphoinositide 4,5 bisphosphate; for ease of reading we will not be using the subscript form) and its processing. Interestingly, it is becoming increasingly evident that PIP2 functions not only in its known role as a docking site and precursor to active metabolites but in the novel role of active signal transducer and cofactor in and of itself. Recent work suggests that PIP2 directly activates calpain 2 to actuate rear release, may serve to direct the transcellular contractility needed to move the cell body forward, and may regulate actin cytoskeleton bundling. Thus, our findings allow advance beyond our original hypothesis that intracellular signaling cascades were subcellularly localized to positing a unifying mechanism for asymmetry during growth factor-induced motility. We hypothesize that the localized activation of key biochemical signaling cascades required for productive cell motility results from the asymmetric actions of phospho-inositide, by both docking/localizing and directly modulating effector proteins. Our Specific Aims propose to test the following postulates: I. That m-calpain (calpain-2) is directly activated by PIP2. II. That 1-actinin-4 (ACTN4) regulation by phospho-inositides controls the linkage between the cytoskeleton and the membrane. III. That distribution of phospho-inositides and phospho-inositide turnover direct contractility. The completion of these investigations will define molecular bases of the spatial restriction of receptor signaling pathways and resultant biophysical responses critical to human tissue cell migration, providing missing information for basic biology of cell functioning. This knowledge will enable the design on a subcellular scale of 'smart' scaffolds for cell and tissue engineering directing the synthesis of both the matrix and the vascular bed that is required to support tissue function.