Replication Protein A (RPA) is a critical complex that acts in replication and promotes homologous recombination by allowing recombinase recruitment to processed DSB ends. Most organisms possess three RPA subunits (RPA1, RPA2, RPA3) that form a trimeric complex critical for viability. The Caenorhabditis elegans genome encodes RPA-1, RPA-2 and an RPA-2 paralog RPA-4. In our analysis, we determined that RPA-2 is critical for germline replication and normal repair of meiotic DSBs. Interestingly, RPA-1 but not RPA-2 is essential for somatic replication, in contrast to other organisms that require both subunits. Six different hetero- and homodimeric complexes containing permutations of RPA-1, RPA-2 and RPA-4 can be detected in whole animal extracts. Our in vivo studies indicate that RPA-1/4 dimer is less abundant in the nucleus and its formation is inhibited by RPA-2. While RPA-4 does not participate in replication or recombination, we find that RPA-4 inhibits RAD-51 filament formation and promotes apoptosis of a subset of damaged nuclei. Altogether these findings point to sub-functionalization and antagonistic roles of RPA complexes in C. elegans.
Vinculin is an actin-binding protein present at cell-matrix and cell-cell adhesions, which plays a critical role in bearing force experienced by cells and dissipating it onto the cytoskeleton. Recently, we identified a key tyrosine residue, Y822, whose phosphorylation plays a critical role in force transmission at cell-cell adhesions. The role of Y822 in human cancer remains unknown, even though Y822 is mutated to Y822C in uterine cancers. Here, we investigated the effect of this amino acid substitution and that of a phosphodeficient Y822F vinculin in cancer cells. We observed that the presence of the Y822C mutation led to cells that proliferate and migrate more rapidly and contained smaller focal adhesions when compared to cells with wild-type vinculin. In contrast, the presence of the Y822F mutation led to highly spread cells with larger focal adhesions and increased contractility. Furthermore, we provide evidence that Y822C vinculin forms a disulfide bond with paxillin, accounting for some of the elevated phosphorylated paxillin recruitment. Taken together, these data suggest that vinculin Y822 modulates the recruitment of ligands.
Abstract By visualizing the movements of Rituximab during Antibody dependent cellular phagocytosis (ADCP) of B lymphoma cells by macrophages, we found that Fcγ receptors (FcγR) on the macrophage surface microcluster, recruit Syk and undergro large-scale reorganization at the phagocytic synapse prior to and during engulfment of the target cell. Given these dramatic rearrangements, we analyzed how the surface mobility of Rituximab contributes to FcγR signal amplification and ADCP efficiency. Depolymerization of the target cell actin cytoskeleton resulted in free diffusion of Rituximab docked to CD20, enhanced microcluster reorganization, Syk recruitment and ADCP. Conversely, immobilization of Rituximab by chemical fixation impaired microcluster formation and diminished Syk recruitment and ADCP. In macrophages lacking Syk, Rituximab accumulated at the base of the phagosome and were trogocytosed, consistent with Syk kinase activity being necessary to trigger redistribution of Rituximab-FcγR during engulfment and to prevent antigenic modulation of the target. Total internal reflection fluorescence analysis of FcγR-IgG on fluid supported lipid bilayers revealed a membrane topography displaying inward reaching leading edges and protruding contact sites reminiscent of podosomes. This topography was distinct from the closely apposed macrophage/target membranes observed during engagement of IgG displayed on immobile supported lipid bilayers. The organization of this contact, pseudopod extension and the rearrangement of microclusters depended critically on Arp 2/3. Thus, Syk and Arp2/3 coordinate actin rearrangements and FcγR-IgG complexes that were of previously unrecognized complexity for the clearance of cells displaying surface-mobile antigens. Significance Statement ADCP is an important effector mechanism for the removal of malignant, immunologically aberrant, and infected cells during treatment with therapeutic antibodies or adaptive immune responses. Most transmembrane protein antigens are mobile with transient confinement from the actin of the target cell. This work demonstrates that macrophage forces overcome these confinements to rearrange FcγR-IgG-antigen complexes before and during ADCP. Thus, new paradigms are needed as ADCP has largely been studied using model target particles that display immobile antigens. Moreover, we found that the mobility of the therapeutic antibody, Rituximab, on the surface of B lymphoma cells foretells ADCP efficacy, with lower densities of IgG mediating ADCP on increasingly mobile antigens.
Endothelial cells respond to mechanical force by stimulating cellular signaling, but how these pathways are linked to elevations in cell metabolism and whether metabolism supports the mechanical response remains poorly understood. Here, we show that the application of force to endothelial cells stimulates VE-cadherin to activate liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis. VE-cadherin stimulated AMPK increases eNOS activity and localization to the plasma membrane, reinforcement of the actin cytoskeleton and cadherin adhesion complex, and glucose uptake. We present evidence for the increase in metabolism being necessary to fortify the adhesion complex, actin cytoskeleton, and cellular alignment. Together these data extend the paradigm for how mechanotransduction and metabolism are linked to include a connection to vasodilation, thereby providing new insight into how diseases involving contractile, metabolic, and vasodilatory disturbances arise.
Antibody dependent cellular phagocytosis (ADCP) removes malignant, immunologically aberrant, and infected cells in response to therapeutic antibodies and vaccines by recognition of cell surface antigens. Using lattice light sheet and TIRF microscopies we show that the phagocyte overcomes target cell restrictions on antigen mobility and drives microclustering of FcγR-IgG-antigen complexes and then reorganizes them into large patches that recruit Syk kinase, promote phosphorylation and trigger engulfment. Increased mobility of CD20-Rituximab in actin-disrupted B lymphoma cells enhanced microcluster reorganization, Syk recruitment and ADCP. Conversely, immobilization of CD20-Rituximab by chemical fixation had the opposite effects. Polarized-TIRF analysis revealed dynamic membrane topography between leading edge and podosome-like structures when IgG was presented fluid bilayers versus flat surfaces on immobile bilayers. The microcluster organization and engulfment depended on Syk and Arp2/3, indicating previously unrecognized complexity for the clearance of cells displaying surface-mobile antigens.
The shape of cells is altered to allow cells to adapt to their changing environments, including responding to internally generated and externally applied force. Force is sensed by cell surface adhesion proteins that are enriched in sites where cells bind to the extracellular matrix (focal adhesions) and neighboring cells (cell–cell or adherens junctions). Receptors at these adhesion sites stimulate intracellular signal transduction cascades that culminate in dramatic changes in the actin cytoskeleton. New actin filaments form, and/or new and existing filaments can be cleaved, branched, or bundled. Here, we discuss the actin cytoskeleton and its functions. We will examine the current understanding for how the actin cytoskeleton is tethered to adhesion sites. Finally, we will highlight recent studies describing how the actin cytoskeleton at these adhesion sites is remodeled in response to force.
Endothelia cells respond to mechanical force by stimulating cellular signaling, but how these pathways are linked to elevations in cell metabolism and whether metabolism supports the mechanical response remains poorly understood. Here, we show that application of force to VE-cadherin stimulates liver kinase B1 (LKB1) to activate AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis. VE-cadherin stimulated AMPK increases eNOS activity and localization to the plasma membrane as well as reinforcement of the actin cytoskeleton and cadherin adhesion complex, and glucose uptake. We present evidence for the increase in metabolism being necessary to fortify the adhesion complex, actin cytoskeleton, and cellular alignment. Together these data extend the paradigm for how mechanotransduction and metabolism are linked to include a connection to vasodilation, thereby providing new insight into how diseases involving contractile, metabolic, and vasodilatory disturbances arise.
Abstract Replication Protein A (RPA) is critical complex that acts in replication and promotes homologous recombination by allowing recombinase recruitment to processed DSB ends. Most organisms possess three RPA subunits (RPA1, RPA2, RPA3) that form a trimeric complex critical for viability. The Caenorhabditis elegans genome encodes for RPA-1, RPA-2 and an RPA-2 paralog RPA-4. In our analysis, we determine that RPA-2 is critical for germline replication, and normal repair of meiotic DSBs. Interestingly, RPA-1 but not RPA-2 is essential for replication, contradictory to what is seen in other organisms, that require both subunits. In the germline, both RPA-1/2 and RPA-1/4 complexes form, but RPA-1/4 is less abundant and its formation is repressed by RPA-2. While RPA-4 does not participate in replication or recombination, we find that RPA-4 inhibit RAD-51 filament formation and promotes apoptosis on a subset of damaged nuclei. Altogether these findings point to sub-functionalization and antagonistic roles of RPA complexes in C. elegans .
