Sequential recruitment of Rab GTPases during early stages of phagocytosis

Abstract

The phagocytosis and destruction of pathogens and dead cells by macrophages is important for innate immunity and tissue maintenance. Multiple Rab family GTPases engage effector molecules to coordinate the early stages of phagocytosis, which include rapid changes in actin polymerization, membrane phospholipids, trafficking and the activation of receptors. Defining the spatiotemporal, sequential recruitment of these Rabs is critical for insights into how phagocytosis is initiated and coordinated. Here, we screened GFP-tagged Rabs expressed in fixed and live cells to identify and stratify those recruited to early phagocytic membranes at stages defined by phospholipid transitions. We propose a sequence of Rabs 35, 13, 8a, 8b, 27a, 10, and 31 that precedes and accompanies phagocytic cup closure, followed after closure by recruitment of endosomal Rabs 5a, 5b, 5c, 14, and 11. Reducing the expression of individual Rabs by siRNA knockdown, notably Rabs 35 and 13, disrupts phagocytosis prior to phagocytic cup closure, confirming a known role for Rab35 and revealing anew the involvement of Rab13. The results enhance our understanding of innate immune responses in macrophages by revealing the sequence of Rabs that initiates phagocytosis.