Šoštar Marko, Marinović Maja, Filić Vedrana, Pavin Nenad, Weber Igor
Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia.
Department of Physics, Faculty of Science, University of Zagreb, Zagreb, Croatia.
PLoS Comput Biol. 2024 Dec 9;20(12):e1012025. doi: 10.1371/journal.pcbi.1012025. eCollection 2024 Dec.
Small GTPases of the Rho family play a central role in the regulation of cell motility by controlling the remodeling of the actin cytoskeleton. In the amoeboid cells of Dictyostelium discoideum, the active form of the Rho GTPase Rac1 regulates actin polymerases at the leading edge and actin filament bundling proteins at the posterior cortex of polarized cells. We monitored the spatiotemporal dynamics of Rac1 and its effector DGAP1 in vegetative amoebae using specific fluorescent probes. We observed that plasma membrane domains enriched in active Rac1 not only exhibited stable polarization, but also showed rotations and oscillations, whereas DGAP1 was depleted from these regions. To simulate the observed dynamics of the two proteins, we developed a mass-conserving reaction-diffusion model based on the circulation of Rac1 between the membrane and the cytoplasm coupled with its activation by GEFs, deactivation by GAPs and interaction with DGAP1. Our theoretical model accurately reproduced the experimentally observed dynamic patterns, including the predominant anti-correlation between active Rac1 and DGAP1. Significantly, the model predicted a new colocalization regime of these two proteins in polarized cells, which we confirmed experimentally. In summary, our results improve the understanding of Rac1 dynamics and reveal how the occurrence and transitions between different regimes depend on biochemical reaction rates, protein levels and cell size. This study not only expands our knowledge of the behavior of Rac1 GTPases in D. discoideum amoebae but also demonstrates how specific modes of interaction between Rac1 and its effector DGAP1 lead to their counterintuitively anti-correlated dynamics.
Rho家族的小GTP酶通过控制肌动蛋白细胞骨架的重塑，在细胞运动的调节中发挥核心作用。在盘基网柄菌的变形虫细胞中，Rho GTP酶Rac1的活性形式调节极化细胞前缘的肌动蛋白聚合酶和后皮质的肌动蛋白丝束蛋白。我们使用特异性荧光探针监测了营养型变形虫中Rac1及其效应器DGAP1的时空动态。我们观察到，富含活性Rac1的质膜结构域不仅表现出稳定的极化，还表现出旋转和振荡，而DGAP1则从这些区域耗尽。为了模拟这两种蛋白质观察到的动态，我们基于Rac1在膜和细胞质之间的循环，以及其被鸟嘌呤核苷酸交换因子（GEFs）激活、被GTP酶激活蛋白（GAPs）失活和与DGAP1相互作用，开发了一个质量守恒反应扩散模型。我们的理论模型准确地再现了实验观察到的动态模式，包括活性Rac1和DGAP1之间主要的反相关关系。值得注意的是，该模型预测了这两种蛋白质在极化细胞中的一种新的共定位模式，我们通过实验证实了这一点。总之，我们的结果增进了对Rac1动态的理解，并揭示了不同模式的出现和转变如何依赖于生化反应速率、蛋白质水平和细胞大小。这项研究不仅扩展了我们对盘基网柄菌变形虫中Rac1 GTP酶行为的认识，还展示了Rac1与其效应器DGAP1之间特定的相互作用模式如何导致它们反直觉的反相关动态。
