Cell and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Chemotaxis Signal Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rockville, MD 20852, USA.
J Cell Sci. 2022 Jun 15;135(12). doi: 10.1242/jcs.258704. Epub 2022 Jun 20.
Capping protein Arp2/3 myosin I linker (CARMIL) proteins are multi-domain scaffold proteins that regulate actin dynamics by regulating the activity of capping protein (CP). Here, we characterize CARMIL-GAP (GAP for GTPase-activating protein), a Dictyostelium CARMIL isoform that contains a ∼130 residue insert that, by homology, confers GTPase-activating properties for Rho-related GTPases. Consistent with this idea, this GAP domain binds Dictyostelium Rac1a and accelerates its rate of GTP hydrolysis. CARMIL-GAP concentrates with F-actin in phagocytic cups and at the leading edge of chemotaxing cells, and CARMIL-GAP-null cells exhibit pronounced defects in phagocytosis and chemotactic streaming. Importantly, these defects are fully rescued by expressing GFP-tagged CARMIL-GAP in CARMIL-GAP-null cells. Finally, rescue with versions of CARMIL-GAP that lack either GAP activity or the ability to regulate CP show that, although both activities contribute significantly to CARMIL-GAP function, the GAP activity plays the bigger role. Together, our results add to the growing evidence that CARMIL proteins influence actin dynamics by regulating signaling molecules as well as CP, and that the continuous cycling of the nucleotide state of Rho GTPases is often required to drive Rho-dependent biological processes.
衔接蛋白 Arp2/3 肌球蛋白 I 连接蛋白（CARMIL）是多结构域支架蛋白，通过调节衔接蛋白（CP）的活性来调节肌动蛋白动力学。在这里，我们描述了 Dictyostelium CARMIL 同种型的 CARMIL-GAP（GAP 代表 GTPase 激活蛋白），它包含一个约 130 个残基的插入片段，通过同源性赋予 Rho 相关 GTP 酶的 GTPase 激活特性。与这一观点一致，这个 GAP 结构域结合了 Dictyostelium Rac1a 并加速了其 GTP 水解的速度。CARMIL-GAP 在吞噬杯中以及趋化细胞的前缘与 F-肌动蛋白集中，并且 CARMIL-GAP 缺失细胞在吞噬作用和趋化性流中表现出明显的缺陷。重要的是，在 CARMIL-GAP 缺失细胞中表达 GFP 标记的 CARMIL-GAP 可以完全挽救这些缺陷。最后，对缺乏 GAP 活性或调节 CP 能力的 CARMIL-GAP 版本的挽救表明，尽管这两种活性对 CARMIL-GAP 功能都有重要贡献，但 GAP 活性起着更大的作用。总之，我们的研究结果增加了越来越多的证据，表明 CARMIL 蛋白通过调节信号分子和 CP 来影响肌动蛋白动力学，并且 Rho GTP 酶的核苷酸状态的连续循环通常是驱动 Rho 依赖性生物过程所必需的。
