Polarization and directed cell movements in engineered cellular environments

描述：该研究项目解决了对人类健康至关重要的问题——生物设计规则，决定细胞是否通过破坏细胞间接触来响应趋化信号，从而从根本上影响组织的组织和完整性。本研究利用软光刻和活细胞成像的工具来定义控制细胞决策的定量生物设计规则。具体而言，我们将(i)定量定义固定的细胞间黏附线索钙粘蛋白的浓度分布如何控制正常和恶性乳腺上皮细胞的差异迁移反应（趋向性），（ii）确定趋化梯度对上皮细胞极性和迁移的影响，以及（iii）确定钙粘蛋白连接和趋化剂如何协调调节引导细胞迁移的GTPase活性的时空分布。软光刻技术能够以在体内或标准组织培养格式中无法获得的方式建立粘附和趋化线索的定义领域。为此，我们将独特地量化细胞对粘附和可溶性线索模式的迁移反应-单独或组合-并定义调节细胞结果的协同或拮抗相互作用。然后，活细胞成像将直接确定在空间和时间上分布的细胞外信号如何改变单个细胞内的信号活动区域，从而弥合外部刺激、整体细胞反应和基本细胞内生化变化之间的差距。如果成功，R21提案中提出的策略将独特地确定癌症和发展过程中控制细胞粘附和趋化性的机制。这些方法的验证将为未来研究其他因素（如整合素、生长因子、基质刚度或与人类发育和疾病相关的任何数量的参数）奠定基础。这个多学科团队拥有微加工（Nuzzo）、表面修饰和蛋白质固定化（Leckband）、生化/细胞生物学技术（Wang）的核心能力，以及钙粘蛋白生物学（Leckband）和趋化性（Wang）方面的专业知识，这对这个项目的成功至关重要。Nuzzo和Leckband多年来一直在多个项目上合作。莱克班德和王正在合作开展一个涉及钙粘蛋白和干细胞分化的独立项目。Wang经常就细胞工作的实验方案向Leckband小组成员提出建议，Wang和Leckband定期举行联合小组会议。这三家pi的实验室和办公室都在相邻的大楼里。该提案是pi之间多次对话的结果。

DESCRIPTION: The research program addresses questions of fundamental importance to human health-biological design rules that determine whether cells respond to chemotactic signals by disrupting intercellular contacts in ways that fundamentally impact the organization and integrity of tissues. This research exploits the enabling tools of soft lithography and live cell imaging to define quantitative biological design rules that control cellular decisions. Specifically, we will (i) define quantitatively how concentration profiles of an immobilized, intercellular adhesive cue, cadherin govern the differential migratory response (haptotaxis) of normal and malignant mammary epithelial cells, (ii) identify the impact of chemotactic gradients on epithelial cell polarity and migration, and (iii) determine how cadherin ligation and chemoattractants coordinately regulate the spatiotemporal distributions of GTPase activities that direct cell migration. Soft lithography enables the establishment of defined fields of adhesive and chemotactic cues in ways that are not accessible in vivo or in standard tissue culture format. To this end, we will uniquely quantify cell migratory responses to patterns of adhesive and soluble cues-individually or in combination-and define synergistic or antagonistic interactions regulating cell outcomes. Live cell imaging will then directly determine how spatially and temporally distributed extracellular cues alter zones of signaling activities within single cells and thus bridge the gap between external stimuli, global cell response, and fundamental intracellular biochemical changes. If successful, the proposed strategies in this R21 proposal will uniquely identify mechanisms governing cell adhesion and chemotaxis in cancer and in development. The validation of these approaches will lay the foundation for future investigations of additional factors such as integrins, growth factors, substrate stiffness, or any number of parameters relevant to human development and disease. This multidisciplinary team possesses core competencies in microfabrication (Nuzzo), surface modification and protein immobilization (Leckband), biochemical/cell biological techniques (Wang), as well as expertise in cadherin biology (Leckband) and chemotaxis (Wang) essential for the success of this program. Nuzzo and Leckband have worked together on multiple projects over several years. Leckband and Wang are collaborating on an independent project involving cadherins and stem cell differentiation. Wang frequently advises Leckband group members on experimental protocols for cell work, and Wang and Leckband periodically hold joint group meetings. The labs and offices of all three PIs are in adjacent buildings. This proposal results from several conversations between the PIs. Public Health Relevance: Cell migration is an essential morphogenetic process in tissue formation, repair, and regeneration. It also drives disease progression in cancer, mental retardation, atherosclerosis, and arthritis. In tissues, cell movements in both normal and diseased tissues involve the coordinated regulation of cell motility and cell-cell adhesion. Cell motility machinery is often triggered by soluble growth factors and chemoattractants, with a concomitant destabilization of intercellular adhesion. Furthermore, gradients of both chemoattractants and adhesion are thought to guide cell movements in tissues. It is the interplay between signals governing chemotaxis or cell migration versus cell-cell adhesion that ultimately governs the formation and structural integrity of tissues. Understanding these processes in both normal development and disease progression is predicated on elucidating the biological design rules that regulate the balance between firm intercellular adhesion and migration. Despite the importance of this issue to human health, these questions have received very little attention. We postulate that the current knowledge gap is due to the limitations inherent in current approaches used to study cell adhesion, migration, and chemotaxis as well as the lack of approaches capable of quantitatively evaluating the impact of adhesive and diffusive signals on cell behavior. This multidisciplinary team embodies the core capabilities needed to address this complex problem. Specifically, current competencies in soft lithography, cell adhesion, and cell migration enable the generation of quantitatively defined biochemical cues-both in immobilized and diffusive form-in order to quantitatively define the balance of different signals that mutually regulate cell behavior. Importantly, these devices enable the quantitative definition of biochemical signals in ways that are not accessible in vivo or in standard tissue culture format. This level of control is essential for defining, for example, chemoattractant in uniform or gradient forms that destabilize cell-cell junctions to promote migration. To this end, this proposed research will use these devices to uniquely quantify cell migratory responses to patterns of adhesive and soluble cues-individually or in combination-in order to define synergistic or antagonistic interactions between chemotactic and adhesive biochemical cues that regulate cell outcomes. We will combine these patterning tools with live cell imaging to directly bridge the gap between external stimuli and the fundamental intracellular biochemical processes that underlie global cell response. This unique complementation of engineered cellular environments and real-time spatiotemporal imaging of biochemical processes should establish causal relationships between spatially and temporally distributed extracellular cues, the intracellular coordination of zones of signaling activities and protein distributions within single cells, and global cell response. Importantly, such real-time spatiotemporal information is also not accessible with standard biochemical assays. The knowledge generated by these studies will be directly relevant to human health and may identify effective therapies for the treatment and prevention of cancer, for example. Furthermore, if successful, the general validation of these approaches will lay the foundation for further studies of additional biochemical factors or environmental parameters-as single components or in various combinations-including integrins, growth factors, substrate stiffness, or any number of parameters relevant to human development and disease.