YAP charge patterning mediates signal integration through transcriptional co-condensates.

Transcription factor dynamics are used to selectively engage gene regulatory programs. Biomolecular condensates have emerged as an attractive signaling substrate in this process, but the underlying mechanisms are not well-understood. Here, we probed the molecular basis of YAP signal integration through transcriptional condensates.

The WAVE complex forms linear arrays at negative membrane curvature to instruct lamellipodia formation.

Cells generate a wide range of actin-based membrane protrusions for various cell behaviors. These protrusions are organized by different actin nucleation promoting factors. For example, N-WASP controls finger-like filopodia, whereas the WAVE complex controls sheet-like lamellipodia.

Follow the flow: Actin and membrane act as an integrated system to globally coordinate cell shape and movement.

Migratory cells are polarized with protrusive fronts and contractile rears. This spatial organization necessitates long-range coordination of the signals that organize protrusions and contractions. Cells leverage reciprocal interactions of short-range biochemical signals and long-range mechanical forces for this integration.

Self-extinguishing relay waves enable homeostatic control of human neutrophil swarming.

Neutrophils collectively migrate to sites of injury and infection. How these swarms are coordinated to ensure the proper level of recruitment is unknown. Using an ex vivo model of infection, we show that human neutrophil swarming is organized by multiple pulsatile chemoattractant waves.

Neutrophils actively swell to potentiate rapid migration.

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.

ER-GUARD: an evolutionarily conserved antioxidant defense system at ER membranes.

Oxidative protein folding in the endoplasmic reticulum (ER) is essential for all eukaryotic cells yet generates hydrogen peroxide (HO), 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 in to identify a previously uncharacterized and evolutionarily conserved protein ERGU-1 that fulfills these roles.

Rationally seeded computational protein design of ɑ-helical barrels.

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.

Plasma membrane abundance dictates phagocytic capacity and functional cross-talk in myeloid cells.

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 Gβ exhibited profound plasma membrane expansion, accompanied by marked reduction in plasma membrane tension.

Early-life stress triggers long-lasting organismal resilience and longevity via tetraspanin.

Early-life stress experiences can produce lasting impacts on organismal adaptation and fitness. How transient stress elicits memory-like physiological effects is largely unknown. Here, we show that early-life thermal stress strongly up-regulates , a gene encoding the conserved transmembrane tetraspanin in .

Optogenetic control of YAP reveals a dynamic communication code for stem cell fate and proliferation.

YAP is a transcriptional regulator that controls pluripotency, cell fate, and proliferation. How cells ensure the selective activation of YAP effector genes is unknown. This knowledge is essential to rationally control cellular decision-making.

Local negative feedback of Rac activity at the leading edge underlies a pilot pseudopod-like program for amoeboid cell guidance.

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.

Plasma membrane abundance dictates phagocytic capacity and functional crosstalk in myeloid cells.

Professional phagocytes like neutrophils and macrophages tightly control what they eat, how much they eat, and when they move after eating. We show that plasma membrane abundance is a key arbiter of these cellular behaviors. Neutrophils and macrophages lacking the G-protein subunit Gb4 exhibit profound plasma membrane expansion due to enhanced production of sphingolipids.

Early-life stress triggers long-lasting organismal resilience and longevity via tetraspanin.

Early-life stress experiences can produce lasting impacts on organismal adaptation and fitness. How transient stress elicits memory-like physiological effects is largely unknown. Here we show that early-life thermal stress strongly up-regulates , a gene encoding the conserved transmembrane tetraspanin in .

Molecular mechanism of GPCR spatial organization at the plasma membrane.

G-protein-coupled receptors (GPCRs) mediate many critical physiological processes. Their spatial organization in plasma membrane (PM) domains is believed to encode signaling specificity and efficiency. However, the existence of domains and, crucially, the mechanism of formation of such putative domains remain elusive.

Self-extinguishing relay waves enable homeostatic control of human neutrophil swarming.

Neutrophils exhibit self-amplified swarming to sites of injury and infection. How swarming is controlled to ensure the proper level of neutrophil recruitment is unknown. Using an model of infection, we find that human neutrophils use active relay to generate multiple pulsatile waves of swarming signals.

Cell protrusions and contractions generate long-range membrane tension propagation.

Membrane tension is thought to be a long-range integrator of cell physiology. Membrane tension has been proposed to enable cell polarity during migration through front-back coordination and long-range protrusion competition. These roles necessitate effective tension transmission across the cell.

Neutrophils actively swell to potentiate rapid migration.

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 is not known.

Mechanosensitive mTORC2 independently coordinates leading and trailing edge polarity programs during neutrophil migration.

By acting both upstream of and downstream from 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 mTOR complex 2 (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.

From peptides to proteins: coiled-coil tetramers to single-chain 4-helix bundles.

The design of completely synthetic proteins from first principles- protein design-is challenging. This is because, despite recent advances in computational protein-structure prediction and design, we do not understand fully the sequence-to-structure relationships for protein folding, assembly, and stabilization. Antiparallel 4-helix bundles are amongst the most studied scaffolds for protein design.

Progressive enhancement of kinetic proofreading in T cell antigen discrimination from receptor activation to DAG generation.

T cells use kinetic proofreading to discriminate antigens by converting small changes in antigen-binding lifetime into large differences in cell activation, but where in the signaling cascade this computation is performed is unknown. Previously, we developed a light-gated immune receptor to probe the role of ligand kinetics in T cell antigen signaling. We found significant kinetic proofreading at the level of the signaling lipid diacylglycerol (DAG) but lacked the ability to determine where the multiple signaling steps required for kinetic discrimination originate in the upstream signaling cascade (Tiseher and Weiner, 2019).

WASP integrates substrate topology and cell polarity to guide neutrophil migration.

To control their movement, cells need to coordinate actin assembly with the geometric features of their substrate. Here, we uncover a role for the actin regulator WASP in the 3D migration of neutrophils. We show that WASP responds to substrate topology by enriching to sites of inward, substrate-induced membrane deformation.

The WAVE complex associates with sites of saddle membrane curvature.

How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. Here we investigate how the WAVE complex organizes sheet-like lamellipodia. Using super-resolution microscopy, we find that the WAVE complex forms actin-independent 230-nm-wide rings that localize to regions of saddle membrane curvature.

Optogenetic Tuning of Protein-protein Binding in Bilayers Using LOVTRAP.

Modern microscopy methods are powerful tools for studying live cell signaling and biochemical reactions, enabling us to observe when and where these reactions take place from the level of a cell down to single molecules. With microscopy, each cell or molecule can be observed both before and after a given perturbation, facilitating better inference of cause and effect than is possible with destructive modes of signaling quantitation. As many inputs to cell signaling and biochemical systems originate as protein-protein interactions near the cell membrane, an outstanding challenge lies in controlling the timing, location and the magnitude of protein-protein interactions in these unique environments.

Cell confinement reveals a branched-actin independent circuit for neutrophil polarity.

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic.

Live-cell imaging reveals enhancer-dependent transcription in the absence of enhancer proximity.

Enhancers are important regulatory elements that can control gene activity across vast genetic distances. However, the underlying nature of this regulation remains obscured because it has been difficult to observe in living cells. Here, we visualize the spatial organization and transcriptional output of the key pluripotency regulator and its essential enhancer Control Region (SCR) in living embryonic stem cells (ESCs).