Search for: All records

Different actin nucleation-promoting factors (NPFs) orchestrate different patterns of cell protrusions, likely reflecting their distinct patterns of self-organization. Here, we leveraged in vivo biochemical approaches to investigate how the WAVE complex instructs the formation of sheet-like lamellipodia. We show that the WAVE complex is a core constituent of a linear multilayered protein array at the plasma membrane, expected for an NPF that builds sheet-like actin-based protrusions. Negative membrane curvature is both necessary and sufficient for WAVE complex linear membrane association in the presence of upstream activators (Rac, Arf1/6, and PIP3) and the PRDs of both WAVE2 and Abi2, providing a potential mechanistic basis for templating of lamellipodia and their emergent behaviors, including barrier avoidance. Through computational modeling, we demonstrate that WAVE complex’s linear organization and preference for negative curvature both play important roles in robust lamellipodia formation. Our data reveal key features of mesoscale WAVE complex patterning and highlight an integral relation between NPF self-organization and cell morphogenesis. 

While the involvement of actin polymerization in cell migration is well-established, much less is known about the role of transmembrane water flow in cell motility. Here, we investigate the role of water influx in a prototypical migrating cell, the neutrophil, which undergoes rapid, directed movement to sites of injury, and infection. Chemoattractant exposure both increases cell volume and potentiates migration, but the causal link between these processes are not known. We combine single-cell volume measurements and a genome-wide CRISPR screen to identify the regulators of chemoattractant-induced neutrophil swelling, including NHE1, AE2, PI3K-gamma, and CA2. Through NHE1 inhibition in primary human neutrophils, we show that cell swelling is both necessary and sufficient for the potentiation of migration following chemoattractant stimulation. Our data demonstrate that chemoattractant-driven cell swelling complements cytoskeletal rearrangements to enhance migration speed. 

In migrating cells, the GTPase Rac organizes a protrusive front, whereas Rho organizes a contractile back. How these GTPases are appropriately positioned at the opposite poles of migrating cells is unknown. Here we leverage optogenetics, manipulation of cell mechanics, and mathematical modeling to reveal a surprising mechanochemical long-range mutual activation of the front and back polarity programs that complements their well-known local mutual inhibition. Rac-based protrusion stimulates Rho activation at the opposite side of the cell via membrane tension-based activation of mTORC2. Conversely, Rho-based contraction induces cortical-flow-based regulation of phosphoinositide signaling to trigger Rac activation at the opposite side of the cell. We develop a minimal unifying mechanochemical model of the cell to explain how this long-range facilitation complements local inhibition to enable robust Rho and Rac partitioning. We show that this long-range mutual activation of Rac and Rho is conserved in epithelial cells and is also essential for efficient polarity and migration of primary human T cells, indicating the generality of this circuit. Our findings demonstrate that the actin cortex and plasma membrane function as an integrated mechanochemical system for long-range partitioning of Rac and Rho during cell migration and likely other cellular contexts. 

Abstract Computational protein design is advancing rapidly. Here we describe efficient routes starting from validated parallel and antiparallel peptide assemblies to design two families of α-helical barrel proteins with central channels that bind small molecules. Computational designs are seeded by the sequences and structures of defined de novo oligomeric barrel-forming peptides, and adjacent helices are connected by loop building. For targets with antiparallel helices, short loops are sufficient. However, targets with parallel helices require longer connectors; namely, an outer layer of helix–turn–helix–turn–helix motifs that are packed onto the barrels. Throughout these computational pipelines, residues that define open states of the barrels are maintained. This minimizes sequence sampling, accelerating the design process. For each of six targets, just two to six synthetic genes are made for expression inEscherichia coli. On average, 70% of these genes express to give soluble monomeric proteins that are fully characterized, including high-resolution structures for most targets that match the design models with high accuracy. 

To migrate efficiently, neutrophils must polarize their cytoskeletal regulators along a single axis of motion. This polarization process is thought to be mediated through local positive feedback that amplifies leading edge signals and global negative feedback that enables sites of positive feedback to compete for dominance. Though this two-component model efficiently establishes cell polarity, it has potential limitations, including a tendency to “lock” onto a particular direction, limiting the ability of cells to reorient. We use spatially defined optogenetic control of a leading edge organizer (PI3K) to probe how neutrophil-like HL-60 cells balance “decisiveness” needed to polarize in a single direction with the flexibility needed to respond to new cues. Underlying this balancing act is a local Rac inhibition process that destabilizes the leading edge to promote exploration. We show that this local inhibition enables cells to process input signal dynamics, linking front stability and orientation to local temporal increases in input signals. 

Abstract Oxidative protein folding in the endoplasmic reticulum (ER) is essential for all eukaryotic cells yet generates hydrogen peroxide (H₂O₂), a reactive oxygen species (ROS). The ER-transmembrane protein that provides reducing equivalents to ER and guards the cytosol for antioxidant defense remains unidentified. Here we combine AlphaFold2-based and functional reporter screens inC. elegansto discover a previously uncharacterized and evolutionarily conserved protein ERGU-1 that fulfills these roles. DeletingC. elegansERGU-1 causes excessive H₂O₂and transcriptional gene up-regulation through SKN-1, homolog of mammalian antioxidant master regulator NRF2. ERGU-1 deficiency also impairs organismal reproduction and behavioral responses to H₂O₂. BothC. elegansand human ERGU-1 proteins localize to ER membranes and form network reticulum structures. Human andDrosophilahomologs of ERGU-1 can rescueC. elegansmutant phenotypes, demonstrating evolutionarily ancient and conserved functions. In addition, purified ERGU-1 and human homolog TMEM161B exhibit redox-modulated oligomeric states. Together, our results reveal an ER-membrane-specific protein machinery for peroxide detoxification and suggest a previously unknown and conserved mechanisms for antioxidant defense in animal cells. 

By acting both upstream and downstream of biochemical organizers of the cytoskeleton, physical forces function as central integrators of cell shape and movement. Here we use a combination of genetic, pharmacological, and optogenetic perturbations to probe the role of the conserved mechanosensitive mTORC2 programs in neutrophil polarity and motility. We find that the tension-based inhibition of leading edge signals (Rac, F-actin) that underlies protrusion competition is gated by the kinase-independent role of the complex, whereas the regulation of RhoA and Myosin II-based contractility at the trailing edge depend on mTORC2 kinase activity. mTORC2 is essential for spatial and temporal coordination of the front and back polarity programs for persistent migration under confinement. This mechanosensory pathway integrates multiple upstream signals, and we find that membrane stretch synergizes with biochemical co-input PIP3 to robustly amplify mTORC2 activation. Our results suggest that different signalling arms of mTORC2 regulate spatially and molecularly divergent cytoskeletal programs for efficient coordination of neutrophil shape and movement. [Media: see text] [Media: see text] 

Professional phagocytes like neutrophils and macrophages tightly control what they consume, how much they consume, and when they move after cargo uptake. We show that plasma membrane abundance is a key arbiter of these cellular behaviors. Neutrophils and macrophages lacking the G protein subunit Gβ₄exhibited profound plasma membrane expansion, accompanied by marked reduction in plasma membrane tension. These biophysical changes promoted the phagocytosis of bacteria, fungus, apoptotic corpses, and cancer cells. We also found that Gβ₄-deficient neutrophils are defective in the normal inhibition of migration following cargo uptake. Sphingolipid synthesis played a central role in these phenotypes by driving plasma membrane accumulation in cells lacking Gβ₄. In Gβ₄knockout mice, neutrophils not only exhibited enhanced phagocytosis of inhaled fungal conidia in the lung but also increased trafficking of engulfed pathogens to other organs. Together, these results reveal an unexpected, biophysical control mechanism central to myeloid functional decision-making.