Decoding dynamic interplay between signaling and membranes in chemotaxis bymolecular actuators

父级R35拨款 从分子水平解读趋化作用中信号与膜的动态相互作用 致动器“ 建议书摘要 趋化作用发生在许多关键的生理事件期间，包括血管生成， 胚胎发育和伤口愈合。它也促进了疾病的进展 病理情况，如癌症转移和关节炎。当前提案的目标是 是为了揭示生化反应和物理特性，如膜曲率， 变形和装配阶段相互作用，以实现动态、准确 高效的细胞迁移。趋化性主要是从信号的角度来理解的 转导，膜的物理性质是否和如何发挥作用，以及它们是如何相互作用的 信号转导在很大程度上仍不清楚。通过新开发和实施一系列 可直接探测高时空性质的分子致动器 精确度在活跃的迁移细胞中，我们将揭示信号转导和 膜力学。 产生局部膜曲率的分子机制是什么？ 丝状伪足和片状伪足？在感知化学诱导剂时，细胞通过经历 不对称的膜变形，由丝状伪足和片状伪足组成； 膜在后方回缩。我们最近发现对曲率敏感的蛋白质缺失 肌动蛋白细胞骨架和膜之间的联系。这一结果让我们假设肌动蛋白 机械、曲率感知和重塑蛋白质，当在一个适当的 反馈环足以产生所需类型的膜变形，例如 片状和丝状伪足。因此，我们将确定Rho GTP酶的特定组合，肌动蛋白 和bar蛋白，以及它们的分子逻辑，它们负责形成 丝状伪足和片状伪足。 迁移细胞中的信号成分如何对膜变形做出反应？ 迁移细胞在质膜和核中表现出动态的形态变化 受信号成分调控的细胞骨架重排的“结果”包膜。 探索一种膜变形与细胞骨架和信号回传的可能性 对于组件，我们将部署可以直接使膜变形的分子致动器。我们会 然后量化随后出现的信号组件的活性，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凝聚物是如何发挥作用的 膜变形？肌动蛋白网络可以在 由于肌动蛋白调节剂之间的弱多价相互作用而导致的质膜。检视 对于这种相分离事件的生理重要性，我们将采用分子技术 将冷凝物组装或拆卸。这些操作将独一无二地实现收益-或 在不改变分子组成数量的情况下失去功能的操纵；什么是 更改的是它们的物理装配状态。我们将在之前描述细胞迁移表型 并在部署了阶段操作之后。

Parent R35 grant “Decoding dynamic interplay between signaling and membranes in chemotaxis by molecular actuators” Proposal Summary 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.