Decoding dynamic interplay between signaling and membranes in chemotaxis by molecular actuators

趋化作用发生在许多关键的生理过程中，包括血管生成、胚胎 发展和伤口愈合。在病理情况下，它也会导致疾病的进展，例如 癌症转移和关节炎。目前这项提议的目标是揭示生化反应和 物理特性，如膜曲率、变形和组装阶段，相互作用 在实现动态、准确而高效的细胞迁移方面的另一项成就。趋化性已经被理解为 主要是从信号转导的角度，而膜的物理性质是否以及如何发挥作用， 它们如何与信号转导相互作用在很大程度上仍不清楚。通过新开发和实施 一系列可高时空精度直接探测膜性质的分子致动器 在活跃的迁移细胞中，我们将揭示信号转导和膜力学之间的相互作用。 是什么分子机制导致局部的膜曲率发育成丝状足孔和 片状脂溢症？在感应化学诱导剂时，细胞通过经历不对称的膜变形而极化 由前部的丝状伪足和片状伪足组成，后部由膜回缩组成。我们最近发现， 曲率敏感蛋白是肌动蛋白细胞骨架和膜之间缺失的一环。结果让我们 假设肌动蛋白机制以及曲率感知和重塑蛋白，当在一个适当的 反馈环足以产生所需类型的膜变形，例如片状脂膜和 丝状伪足。因此，我们将确定Rho GTP酶、肌动蛋白调节蛋白和bar蛋白的特定组合，以及 其分子逻辑，负责丝足和片状脂胞的形成。 迁移细胞中的信号成分如何对膜变形做出反应？迁移细胞 表现出质膜和核膜的动态形态变化 细胞骨架重排受信号成分调控。探索一种膜变形的可能性 谈到细胞骨架和信号组件，我们将部署可以直接变形的分子致动器 膜。然后我们将量化随后出现的信号组件的活动，如受体 酪氨酸激酶、PI3K和小GTP酶，以及转录因子，如YAP和ELK。 相分离的细胞骨架生物分子缩合物是如何在膜中发挥作用的 变形？肌动蛋白网络可以在质膜上形成生物分子凝聚体 与肌动蛋白调节器之间的弱多价相互作用有关。检查这一阶段的生理重要性 在分离事件中，我们将采用分子技术来组装或拆解凝析油。这些 运算将唯一地实现函数操作的得失，而不会改变 分子组成；改变的是它们的物理组装状态。我们将描述细胞迁移的特征 部署阶段操作前后的表型。

Chemotaxis occurs during a number of key physiological events including angiogenesis, embryonic development and wound healing. It also contributes to disease progression in pathological conditions such as cancer metastasis and arthritis. The goal of the current proposal is to reveal how biochemical reactions and physical characteristics, such as membrane curvature, deformation, and assembly phase, interact with one another in achieving dynamic, accurate yet highly efficient cell migration. Chemotaxis has been understood mainly in the perspective of signal transduction, while if and how physical properties of membranes play a role, and how they interact with signal transduction remain largely unknown. By newly developing and implementing a series of molecular actuators that can directly probe membrane properties with high spatio-temporal precision inside lively migrating cells, we will reveal an interplay between signal transduction and membrane mechanics. What molecular mechanisms generate local membrane curvatures developing into filopodia and lamellipodia? In sensing chemoattractants, cells polarize by undergoing asymmetric membrane deformation consisting of filopodia and lamellipodia at the front, and membrane retraction at the rear. We recently found that curvature-sensitive proteins are a missing link between actin cytoskeleton and membranes. The result made us hypothesize that actin machinery and curvature sensing and remodeling proteins, when properly modulated in a feedback loop, are sufficient to produce desired types of membrane deformations such as lamellipodia and filopodia. We will thus identify a particular combination of Rho GTPases, actin regulators, and BAR proteins, and the molecular logic thereof, that are responsible for formation of filopodia and lamellipodia. How do signaling components in migrating cells respond to membrane deformation? Migrating cells exhibit dynamic morphological changes at plasma membranes and nuclear envelopes “as a consequence” of cytoskeletal rearrangement regulated by signal components. To explore a possibility that membrane deformation talks back to cytoskeletal and signal components, we will deploy molecular actuators that can directly deform membranes. We will then quantify subsequently emerging activity of signaling components such as receptor tyrosine kinases, PI3K, and small GTPases, as well as transcription factors such as YAP and Elk. How does the phase-separated cytoskeletal biomolecular condensate play a role in membrane deformation? Actin networks can undergo formation of biomolecular condensates at the plasma membrane due to weak multivalent interactions among actin regulators. To examine the physiological importance of such phase separation events, we will adapt molecular techniques to assemble or disassemble the condensates. These operations will uniquely achieve gain- or loss-of function manipulations without altering an amount of the molecular constituents; what is altered is their physical assembly status. We will characterize cell migration phenotypes before and after deploying phase manipulations.