Phagocytosis of invading pathogens or cellular debris requires a dramatic change in cell shape driven by actin polymerization. For antibody-covered targets, phagocytosis is thought to proceed through the sequential engagement of Fc-receptors on the phagocyte with antibodies on the target surface, leading to the extension and closure of the phagocytic cup around the target. We find that two actin-dependent molecular motors, class 1 myosins myosin 1e and myosin 1f, are specifically localized to Fc-receptor adhesions and required for efficient phagocytosis of antibody-opsonized targets. Using primary macrophages lacking both myosin 1e and myosin 1f, we find that without the actin-membrane linkage mediated by these myosins, the organization of individual adhesions is compromised, leading to excessive actin polymerization, slower adhesion turnover, and deficient phagocytic internalization. This work identifies a role for class 1 myosins in coordinated adhesion turnover during phagocytosis and supports a mechanism involving membrane-cytoskeletal crosstalk for phagocytic cup closure.

How proteins in the bacterial cell division complex (the divisome) coordinate to divide bacteria remains unknown. To explore how these proteins collectively function, we conducted a complete dynamic characterization of the proteins involved, and then examined the function of FtsZ binding proteins (ZBPs) and their role in cytokinesis. We find that the divisome consists of two dynamically distinct subcomplexes: stationary ZBPs that transiently bind to treadmilling FtsZ filaments, and a directionally-moving complex that includes cell wall synthases. FtsZ filaments treadmill at steady state and the ZBPs have no effect on filament dynamics. Rather, ZBPs bundle FtsZ filaments, condensing them into Z rings. Z ring condensation increases the recruitment of cell wall synthesis enzymes to the division site, and this condensation is necessary for cytokinesis. Main Text:The mechanism by which bacteria divide remains poorly understood. In most bacteria, division begins when filaments of FtsZ, a tubulin homolog, form a "Z ring" at midcell (1). The Z ring then recruits other cell division proteins, collectively called the divisome (Fig 1A). The first group of these proteins (early proteins) arrives concurrently with FtsZ and includes the actin homolog FtsA and several other FtsZ binding proteins (ZBPs): EzrA, SepF, and ZapA. The second group of integral membrane proteins (late proteins) is then recruited, including DivIB, DivIC, and FtsL, and the cell wall synthesis enzymes Pbp2B and FtsW (2, 3). During cytokinesis, the Z ring constricts while the associated cell wall synthesis enzymes build a septum that divides the cell in half (4). Recent work has shown that FtsZ filaments treadmill around the division plane, moving at the same rate as the transpeptidase Pbp2B. (5, 6) (Movie S1). FtsZ treadmilling dynamics are critical for cell division: In Bacillus subtilis, the rate of treadmilling limits Pbp2B motion, the rate of septal cell wall synthesis, and the overall rate of septation (5).To understand how these proteins work to divide cells, we sought to build a dynamic characterization of how this multi-component machine functions in B. subtilis. We first worked to identify groups of divisome proteins that move together, then investigated how the FtsZ-.

Phagocytosis requires rapid actin reorganization and spatially controlled force generation to ingest targets ranging from pathogens to apoptotic cells. How actomyosin activity directs membrane extensions to engulf such diverse targets remains unclear. Here, we combine lattice light-sheet microscopy (LLSM) with microparticle traction force microscopy (MP-TFM) to quantify actin dynamics and subcellular forces during macrophage phagocytosis. We show that spatially localized forces leading to target constriction are prominent during phagocytosis of antibody-opsonized targets. This constriction is largely driven by Arp2/3-mediated assembly of discrete actin protrusions containing myosin 1e and 1f (‘teeth’) that appear to be interconnected in a ring-like organization. Contractile myosin-II activity contributes to late-stage phagocytic force generation and progression, supporting a specific role in phagocytic cup closure. Observations of partial target eating attempts and sudden target release via a popping mechanism suggest that constriction may be critical for resolving complex in vivo target encounters. Overall, our findings present a phagocytic cup shaping mechanism that is distinct from cytoskeletal remodeling in 2D cell motility and may contribute to mechanosensing and phagocytic plasticity.

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Phagocytosis is a receptor-mediated, actin-dependent process of internalization of large extracellular particles, such as pathogens or apoptotic cells. Engulfment of phagocytic targets requires activity of myosins, actin-dependent molecular motors, which perform a variety of functions at distinct steps during phagocytosis. By applying force to actin filaments, plasma membrane, and intracellular proteins and organelles, myosins can generate contractility, directly regulate actin assembly to ensure proper phagocytic internalization, and translocate phagosomes or other cargo to appropriate cellular locations. Recent studies using engineered microenvironments and phagocytic targets have demonstrated how altering the actomyosin cytoskeleton affects phagocytic behavior. Here, we discuss how studies using genetic and biochemical manipulation of myosins, force measurement techniques, and live cell imaging have advanced our understanding of how specific myosins function at individual steps of phagocytosis. Keywords phagocytosis; myosins; actin; macrophagePhagocytosis is an ancient biological process, utilized initially for nutrient uptake and now a critical activity for immune defense [1]. Professional phagocytes, including neutrophils, dendritic cells, and macrophages, regularly rid the body of pathogens, apoptotic cells and cellular debris. These phagocytic targets are recognized by specific cell surface receptors, which relay distinct downstream signals through protein and lipid kinases and phosphatases, small GTPases, and other signaling proteins [2]. Targets coated by plasma-or cell-derived components (opsonins) are recognized by opsonic receptors, including Fc receptors (FcR) that bind the conserved domain of immunoglobulins and complement receptors (CR), which respond to targets coated in the complement derivative iC3b [1]. Non-opsonic receptors (receptors binding to specific motifs on phagocytic targets) include Dectin-1 receptors that recognize fungal β-glucan and scavenger receptors that interact with both apoptotic and microbial ligands [1,3]. For internalization, phagocytic receptors can work in concert, with

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AbstractHow proteins in the bacterial cell division complex (the divisome) coordinate to divide bacteria remains unknown. To explore how these proteins collectively function, we conducted a complete dynamic characterization of the proteins involved, and then examined the function of FtsZ binding proteins (ZBPs) and their role in cytokinesis. We find that the divisome consists of two dynamically distinct subcomplexes: stationary ZBPs that transiently bind to treadmilling FtsZ filaments, and a directionally-moving complex that includes cell wall synthases. FtsZ filaments treadmill at steady state and the ZBPs have no effect on filament dynamics. Rather, ZBPs bundle FtsZ filaments, condensing them into Z rings. Z ring condensation increases the recruitment of cell wall synthesis enzymes to the division site, and this condensation is necessary for cytokinesis.

Phagocytosis is the process of engulfment and internalization of comparatively large particles by the cell, that plays a central role in the functioning of our immune system. We study the...