The coordination of lipid messenger signaling with cytoskeletal regulation is central to many organelle-specific regulatory processes. This coupling often depends on the function of multidomain scaffolds that orchestrate transient interactions among multiple signaling intermediates and regulatory proteins on organelles. The number of possible scaffold interaction partners and the ability for these interactions to occur at different timescales makes investigations of scaffold functions challenging. This work employs live cell imaging to probe how the multidomain scaffold IQ motif containing GTPase activating protein 1 (IQGAP1) coordinates the activities of proteins affecting local actin polymerization, membrane processing, and phosphoinositide signaling. Using endosomes that are confined by a local actin network as a model system, we demonstrate that IQGAP1 can transition between different actin and endosomal membrane tethered states. Fast scaffold binding/disassociation transitions are shown to be driven by interactions between C-terminal scaffold domains and Rho GTPases at the membrane. Fluctuations in these binding modes are linked to negative regulation of actin polymerization. Although this control governs core elements of IQGAP1 dynamics, actin binding by the N-terminal calponin homology domain of the scaffold is shown to help the scaffold track the temporal development of endosome membrane markers, implying actin associations bolster membrane and actin coordination. Importantly, these effects are not easily distilled purely through standard (static) co-localization analyses or traditional pathway perturbations methods and were resolved by performing dynamic correlation and multiple regression analyses of IQGAP1 scaffold mutants. Using these capabilities with pharmacological inhibition, we provide evidence that membrane tethering is dependent on the activities of the lipid kinase phosphoinositide 3-kinase in addition to the Rho GTPases Rac1 and Cdc42. Overall, these methods and results point to a scaffold tethering mechanism that allows IQGAP1 to help control the amplitude of phosphoinositide lipid messenger signaling by coordinating signaling intermediate activities with the development and disassembly of local actin cytoskeletal networks.

中文翻译：

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膜和肌动蛋白束缚转变有助于 IQGAP1 协调 GTP 酶和脂质信使信号传导


脂质信使信号传导与细胞骨架调节的协调是许多细胞器特异性调节过程的核心。这种耦合通常取决于多域支架的功能，该支架协调细胞器上多个信号中间体和调节蛋白之间的瞬时相互作用。可能的支架相互作用伙伴的数量以及这些相互作用在不同时间尺度发生的能力使得支架功能的研究具有挑战性。这项工作采用活细胞成像来探讨含有 GTP 酶激活蛋白 1 (IQGAP1) 的多域支架 IQ 基序如何协调影响局部肌动蛋白聚合、膜加工和磷酸肌醇信号传导的蛋白质活性。使用受局部肌动蛋白网络限制的内体作为模型系统，我们证明 IQGAP1 可以在不同的肌动蛋白和内体膜束缚状态之间转换。快速支架结合/解离转变被证明是由 C 端支架结构域和膜上的 Rho GTPases 之间的相互作用驱动的。这些结合模式的波动与肌动蛋白聚合的负调控有关。尽管这种控制控制着 IQGAP1 动力学的核心元件，但支架的 N 端钙调蛋白同源结构域与肌动蛋白的结合有助于支架跟踪内体膜标记物的时间发育，这意味着肌动蛋白关联增强了膜和肌动蛋白的协调。重要的是，这些效应不容易纯粹通过标准（静态）共定位分析或传统通路扰动方法来提取，而是通过对 IQGAP1 支架突变体进行动态相关和多元回归分析来解决。 利用这些功能和药理抑制作用，我们提供了证据，证明除了 Rho GTPases Rac1 和 Cdc42 之外，膜束缚还依赖于脂质激酶磷酸肌醇 3-激酶的活性。总的来说，这些方法和结果指出了一种支架束缚机制，该机制允许 IQGAP1 通过协调信号中间活动与局部肌动蛋白细胞骨架网络的发育和分解来帮助控制磷酸肌醇脂质信使信号传导的幅度。