Dissecting the role of formins in large-scale endocytosis

吞噬作用和巨噬细胞吞噬作用是密切相关且进化保守的不依赖于胞质蛋白的内吞过程。两者都是RAS和RAC信号下游肌动蛋白驱动的过程，需要质膜重排来吞噬细胞外物质，然后将摄入的物质输送到溶酶体中提取营养物质。然而，大规模内吞作用中肌动蛋白组装的调控机制仍然不清楚。本项目旨在阐明Forin家族成员在巨噬细胞吞噬和吞噬作用中的具体作用，并阐明它们在RAS和RAC信号转导下游大规模内吞过程中相互之间以及与肌动蛋白组装中Arp2/3复合体的潜在协同作用。在以前发表的工作中，我们证实了透明相关的福明福林受到RASB和RasG的调节，并且它表现出相当差的成核但有效的肌动蛋白-细丝伸长活性。Forg有助于产生定位于杯基的肌动蛋白细丝，它的活性是产生该结构中约50%的肌动蛋白细丝所必需的。由于Arp2/3复合体显然只在杯状扩张的边缘被激活，目前尚不清楚哪些因素在起作用，这些因素负责碱基中另一半肌动蛋白细丝的合成以及更接近边缘的细丝的合成。以前对荧光标记生物传感器的研究实际上显示，整个外周都装饰着活性的RAC，这表明除了RAC结合的SCAR/WAVE复合体之外，还有其他因素的存在，这似乎需要激活尖端中的Arp2/3复合体。值得注意的是，对于Forb，我们最近发现了第二种透明相关的Forin，它以其活跃的形式显著积累在大管胞的整个外围。由于这种类型的Forins在大多数情况下是由Rho家族的GTP酶成员激活的，并且因为在生长阶段表达的所有其他Dictyostelialformin可以在功能上被排除，我们假设forb可能是内吞胞杯中大多数肌动蛋白细丝组装所必需的缺失因子。分析大规模内吞作用中的Forin功能无疑是一项具有挑战性的任务，但我们预计，通过使用各种补充方法，包括对Forb进行彻底的生化分析，鉴定其调控Rac蛋白，以及工作计划中指定的单基因敲除和双基因敲除细胞系的全面细胞生物学分析，最终将揭示这些大型内吞结构中肌动蛋白组装调控的分子活动和机制。

Phagocytosis and macropinocytosis are closely related and evolutionary conserved clathrin-independent endocytic processes. Both are actin-driven processes downstream of Ras and Rac signaling that entail rearrangements of the plasma membrane to engulf extracellular material followed by delivery of the ingested material into lysosomes for extraction of nutrients. However, the regulatory mechanisms underlying actin assembly in large-scale endocytosis still remain elusive. This project aims to clarify the specific contribution of formin family members to both macropinocytosis and phagocytosis, and elucidate their prospective synergy to each other and with the Arp2/3 complex in actin assembly during large-scale endocytosis downstream of Ras and Rac signalling. In previously published work, we established that the Diaphanous-related formin ForG is regulated by RasB and RasG and that it exhibits rather poor nucleation but effective actin-filament elongation activities. ForG contributes to the generation of actin filaments localized at the cup base and its activity is required for the generation of about 50% of the actin filaments in this structure. Since the Arp2/3 complex is apparently activated only at the expanding rims of the cups, it remains currently unclear which factors are at play that are responsible for the synthesis of the other half of the actin filaments in the base as well as for the filaments closer to the rim. Previous studies with fluorescently tagged biosensors in fact revealed that the entire periphery is decorated with active Rac, suggesting the presence of other factors than the Rac-binding Scar/WAVE complex, which appears to be required activating Arp2/3 complex in the tips. Of note, with ForB we have recently identified a second Diaphanous-related formin that prominently accumulates in its active form at the entire periphery of macropinosomes. Since this type of formins is in most cases activated by Rho-family GTPase members and because all other Dictyostelium formins that are expressed during the growth phase can be functionally excluded, we hypothesize that ForB could be the missing factor which is required together with ForG for the assembly of the majority of actin filaments in endocytic cups. The dissection of formin function in large-scale endocytosis is undoubtedly a challenging task, but we anticipate that by using various complementary approaches including thorough biochemical analysis of ForB, the identification of its regulatory Rac protein as well as the comprehensive cell biological analyses of single- and double-knockout cell lines as specified in the work programme will finally reveal the molecular activities and mechanisms underlying regulation of actin assembly in these large endocytic structures.