Department of Pathology and Laboratory Medicine, Rutgers-Robert Wood Johnson Medical School, Piscataway, New Jersey, United States of America.
PLoS Genet. 2024 Jul 31;20(7):e1011330. doi: 10.1371/journal.pgen.1011330. eCollection 2024 Jul.
Coordinated activation and inhibition of F-actin supports the movements of morphogenesis. Understanding the proteins that regulate F-actin is important, since these proteins are mis-regulated in diseases like cancer. Our studies of C. elegans embryonic epidermal morphogenesis identified the GTPase CED-10/Rac1 as an essential activator of F-actin. However, we need to identify the GEF, or Guanine-nucleotide Exchange Factor, that activates CED-10/Rac1 during embryonic cell migrations. The two-component GEF, CED-5/CED-12, is known to activate CED-10/Rac1 to promote cell movements that result in the engulfment of dying cells during embryogenesis, and a later cell migration of the larval Distal Tip Cell. It is believed that CED-5/CED-12 powers cellular movements of corpse engulfment and DTC migration by promoting F-actin formation. Therefore, we tested if CED-5/CED-12 was involved in embryonic migrations, and got a contradictory result. CED-5/CED-12 definitely support embryonic migrations, since their loss led to embryos that died due to failed epidermal cell migrations. However, CED-5/CED-12 inhibited F-actin in the migrating epidermis, the opposite of what was expected for a CED-10 GEF. To address how CED-12/CED-5 could have two opposing effects on F-actin, during corpse engulfment and cell migration, we investigated if CED-12 harbors GAP (GTPase Activating Protein) functions. A candidate GAP region in CED-12 faces away from the CED-5 GEF catalytic region. Mutating a candidate catalytic Arginine in the CED-12 GAP region (R537A) altered the epidermal cell migration function, and not the corpse engulfment function. We interfered with GEF function by interfering with CED-5's ability to bind Rac1/CED-10. Mutating Serine-Arginine in CED-5/DOCK predicted to bind and stabilize Rac1 for catalysis, resulted in loss of both ventral enclosure and corpse engulfment. Genetic and expression studies strongly support that the GAP function likely acts on different GTPases. Thus, we propose CED-5/CED-12 support the cycling of multiple GTPases, by using distinct domains, to both promote and inhibit F-actin nucleation.
协调的 F-肌动蛋白的激活和抑制支持形态发生的运动。了解调节 F-肌动蛋白的蛋白质非常重要，因为这些蛋白质在癌症等疾病中失调。我们对秀丽隐杆线虫胚胎表皮形态发生的研究确定 GTPase CED-10/Rac1 是 F-肌动蛋白的必需激活剂。然而，我们需要确定在胚胎细胞迁移过程中激活 CED-10/Rac1 的鸟嘌呤核苷酸交换因子（GEF）。双组分 GEF，CED-5/CED-12，已知可激活 CED-10/Rac1，以促进细胞运动，导致胚胎发生期间死亡细胞的吞噬，以及幼虫远端尖端细胞的后期细胞迁移。据信，CED-5/CED-12 通过促进 F-肌动蛋白的形成来为尸体吞噬和 DTC 迁移提供细胞运动的动力。因此，我们测试了 CED-5/CED-12 是否参与胚胎迁移，得到了一个矛盾的结果。CED-5/CED-12 确实支持胚胎迁移，因为它们的缺失导致表皮细胞迁移失败而导致胚胎死亡。然而，CED-5/CED-12 在迁移的表皮中抑制 F-肌动蛋白，这与预期的 CED-10 GEF 相反。为了解释 CED-12/CED-5 如何在尸体吞噬和细胞迁移过程中对 F-肌动蛋白产生两种相反的影响，我们研究了 CED-12 是否具有 GAP（GTPase 激活蛋白）功能。CED-12 中的候选 GAP 区域背离 CED-5 GEF 催化区域。突变 CED-12 GAP 区域中的候选催化精氨酸（R537A）改变了表皮细胞迁移功能，而不改变尸体吞噬功能。我们通过干扰 CED-5 结合 Rac1/CED-10 的能力来干扰 GEF 功能。突变 CED-5/DOCK 中预测与 Rac1 结合并稳定其用于催化的丝氨酸-精氨酸，导致腹侧包封和尸体吞噬均丧失。遗传和表达研究强烈支持 GAP 功能可能作用于不同的 GTPases。因此，我们提出 CED-5/CED-12 通过使用不同的结构域来支持多种 GTPases 的循环，以促进和抑制 F-肌动蛋白的成核。
