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. However, conflicting observations have left the field divided as to whether cell membranes support or resist tension propagation. This discrepancy likely originates from the use of exogenous forces that may not accurately mimic endogenous forces. We overcome this complication by leveraging optogenetics to directly control localized actin-based protrusions or actomyosin contractions while simultaneously monitoring the propagation of membrane tension using dual-trap optical tweezers. Surprisingly, actin-driven protrusions and actomyosin contractions both elicit rapid global membrane tension propagation, whereas forces applied to cell membranes alone do not. We present a simple unifying mechanical model in which mechanical forces that engage the actin cortex drive rapid, robust membrane tension propagation through long-range membrane flows.

Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module.

The complex, interconnected architecture of cell-signaling networks makes it challenging to disentangle how cells process extracellular information to make decisions. We have developed an optogenetic approach to selectively activate isolated intracellular signaling nodes with light and use this method to follow the flow of information from the signaling protein Ras. By measuring dose and frequency responses in single cells, we characterize the precision, timing, and efficiency with which signals are transmitted from Ras to Erk. Moreover, we elucidate how a single pathway can specify distinct physiological outcomes: by combining distinct temporal patterns of stimulation with proteomic profiling, we identify signaling programs that differentially respond to Ras dynamics, including a paracrine circuit that activates STAT3 only after persistent (>1 hr) Ras activation. Optogenetic stimulation provides a powerful tool for analyzing the intrinsic transmission properties of pathway modules and identifying how they dynamically encode distinct outcomes.

Network crosstalk dynamically changes during neutrophil polarization.

How complex signaling networks shape highly coordinated, multistep cellular responses is poorly understood. Here, we made use of a network-perturbation approach to investigate causal influences, or "crosstalk," among signaling modules involved in the cytoskeletal response of neutrophils to chemoattractant. We quantified the intensity and polarity of cytoskeletal marker proteins over time to characterize stereotyped cellular responses. Analyzing the effects of network disruptions revealed that, not only does crosstalk evolve rapidly during polarization, but also that intensity and polarity responses are influenced by different patterns of crosstalk. Interestingly, persistent crosstalk is arranged in a surprisingly simple circuit: a linear cascade from front to back to microtubules influences intensities, and a feed-forward network in the reverse direction influences polarity. Our approach provided a rational strategy for decomposing a complex, dynamically evolving signaling system and revealed evolving paths of causal influence that shape the neutrophil polarization response.

Membrane tension maintains cell polarity by confining signals to the leading edge during neutrophil migration.

Little is known about how neutrophils and other cells establish a single zone of actin assembly during migration. A widespread assumption is that the leading edge prevents formation of additional fronts by generating long-range diffusible inhibitors or by sequestering essential polarity components. We use morphological perturbations, cell-severing experiments, and computational simulations to show that diffusion-based mechanisms are not sufficient for long-range inhibition by the pseudopod. Instead, plasma membrane tension could serve as a long-range inhibitor in neutrophils. We find that membrane tension doubles during leading-edge protrusion, and increasing tension is sufficient for long-range inhibition of actin assembly and Rac activation. Furthermore, reducing membrane tension causes uniform actin assembly. We suggest that tension, rather than diffusible molecules generated or sequestered at the leading edge, is the dominant source of long-range inhibition that constrains the spread of the existing front and prevents the formation of secondary fronts.

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. Here, live-cell imaging (corrected for topography-induced imaging artifacts) conclusively established the existence of PM domains for GPCRs. Paradoxically, energetic coupling to extremely shallow PM curvature (<1 µm-1) emerged as the dominant, necessary and sufficient molecular mechanism of GPCR spatiotemporal organization. Experiments with different GPCRs, H-Ras, Piezo1 and epidermal growth factor receptor, suggest that the mechanism is general, yet protein specific, and can be regulated by ligands. These findings delineate a new spatiomechanical molecular mechanism that can transduce to domain-based signaling any mechanical or chemical stimulus that affects the morphology of the PM and suggest innovative therapeutic strategies targeting cellular shape.

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. 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 in Escherichia 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.

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. 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.

Illuminating cell signalling with optogenetic tools.

The light-based control of ion channels has been transformative for the neurosciences, but the optogenetic toolkit does not stop there. An expanding number of proteins and cellular functions have been shown to be controlled by light, and the practical considerations in deciding between reversible optogenetic systems (such as systems that use light-oxygen-voltage domains, phytochrome proteins, cryptochrome proteins and the fluorescent protein Dronpa) are well defined. The field is moving beyond proof of concept to answering real biological questions, such as how cell signalling is regulated in space and time, that were difficult or impossible to address with previous tools.

Sequence-dependent sorting of recycling proteins by actin-stabilized endosomal microdomains.

The functional consequences of signaling receptor endocytosis are determined by the endosomal sorting of receptors between degradation and recycling pathways. How receptors recycle efficiently, in a sequence-dependent manner that is distinct from bulk membrane recycling, is not known. Here, in live cells, we visualize the sorting of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, from bulk recycling proteins and the degrading delta-opioid receptor. Our results reveal a remarkable diversity in recycling routes at the level of individual endosomes, and indicate that sequence-dependent recycling is an active process mediated by distinct endosomal subdomains distinct from those mediating bulk recycling. We identify a specialized subset of tubular microdomains on endosomes, stabilized by a highly localized but dynamic actin machinery, that mediate this sorting, and provide evidence that these actin-stabilized domains provide the physical basis for a two-step kinetic and affinity-based model for protein sorting into the sequence-dependent recycling pathway.

Compete globally, bud locally.

How cells generate a single axis of polarity for mating, division, and movement is unknown. In this issue, Howell et al. (2009) use a synthetic biology approach to demonstrate that rapid competition for a soluble signaling component (Bem1) is essential to ensure a unique axis of polarity in budding yeast.

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. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments.

Nodal signaling has dual roles in fate specification and directed migration during germ layer segregation in zebrafish.

During gastrulation, endodermal cells actively migrate to the interior of the embryo, but the signals that initiate and coordinate this migration are poorly understood. By transplanting ectopically induced endodermal cells far from the normal location of endoderm specification, we identified the inputs that drive internalization without the confounding influences of fate specification and global morphogenic movements. We find that Nodal signaling triggers an autocrine circuit for initiating endodermal internalization. Activation of the Nodal receptor directs endodermal specification through sox32 and also induces expression of more Nodal ligands. These ligands act in an autocrine fashion to initiate endodermal cell sorting. Our work defines an 'AND' gate consisting of sox32-dependent endodermal specification and Nodal ligand reception controlling endodermal cell sorting to the inner layer of the embryo at the onset of gastrulation.

TAEL: a zebrafish-optimized optogenetic gene expression system with fine spatial and temporal control.

Here, we describe an optogenetic gene expression system optimized for use in zebrafish. This system overcomes the limitations of current inducible expression systems by enabling robust spatial and temporal regulation of gene expression in living organisms. Because existing optogenetic systems show toxicity in zebrafish, we re-engineered the blue-light-activated EL222 system for minimal toxicity while exhibiting a large range of induction, fine spatial precision and rapid kinetics. We validate several strategies to spatially restrict illumination and thus gene induction with our new TAEL (TA4-EL222) system. As a functional example, we show that TAEL is able to induce ectopic endodermal cells in the presumptive ectoderm via targeted sox32 induction. We also demonstrate that TAEL can be used to resolve multiple roles of Nodal signaling at different stages of embryonic development. Finally, we show how inducible gene editing can be achieved by combining the TAEL and CRISPR/Cas9 systems. This toolkit should be a broadly useful resource for the fish community.

Chick cranial neural crest cells use progressive polarity refinement, not contact inhibition of locomotion, to guide their migration.

To move directionally, cells can bias the generation of protrusions or select among randomly generated protrusions. Here we use 3D two-photon imaging of chick branchial arch 2 directed neural crest cells to probe how these mechanisms contribute to directed movement, whether a subset or the majority of cells polarize during movement, and how the different classes of protrusions relate to one another. We find that, in contrast to Xenopus, cells throughout the stream are morphologically polarized along the direction of overall stream movement and do not exhibit contact inhibition of locomotion. Instead chick neural crest cells display a progressive sharpening of the morphological polarity program. Neural crest cells have weak spatial biases in filopodia generation and lifetime. Local bursts of filopodial generation precede the generation of larger protrusions. These larger protrusions are more spatially biased than the filopodia, and the subset of protrusions that are productive for motility are the most polarized of all. Orientation rather than position is the best correlate of the protrusions that are selected for cell guidance. This progressive polarity refinement strategy may enable neural crest cells to efficiently explore their environment and migrate accurately in the face of noisy guidance cues.

Membrane Tension Acts Through PLD2 and mTORC2 to Limit Actin Network Assembly During Neutrophil Migration.

For efficient polarity and migration, cells need to regulate the magnitude and spatial distribution of actin assembly. This process is coordinated by reciprocal interactions between the actin cytoskeleton and mechanical forces. Actin polymerization-based protrusion increases tension in the plasma membrane, which in turn acts as a long-range inhibitor of actin assembly. These interactions form a negative feedback circuit that limits the magnitude of membrane tension in neutrophils and prevents expansion of the existing front and the formation of secondary fronts. It has been suggested that the plasma membrane directly inhibits actin assembly by serving as a physical barrier that opposes protrusion. Here we show that efficient control of actin polymerization-based protrusion requires an additional mechanosensory feedback cascade that indirectly links membrane tension with actin assembly. Specifically, elevated membrane tension acts through phospholipase D2 (PLD2) and the mammalian target of rapamycin complex 2 (mTORC2) to limit actin nucleation. In the absence of this pathway, neutrophils exhibit larger leading edges, higher membrane tension, and profoundly defective chemotaxis. Mathematical modeling suggests roles for both the direct (mechanical) and indirect (biochemical via PLD2 and mTORC2) feedback loops in organizing cell polarity and motility-the indirect loop is better suited to enable competition between fronts, whereas the direct loop helps spatially organize actin nucleation for efficient leading edge formation and cell movement. This circuit is essential for polarity, motility, and the control of membrane tension.

Gß Regulates Coupling between Actin Oscillators for Cell Polarity and Directional Migration.

For directional movement, eukaryotic cells depend on the proper organization of their actin cytoskeleton. This engine of motility is made up of highly dynamic nonequilibrium actin structures such as flashes, oscillations, and traveling waves. In Dictyostelium, oscillatory actin foci interact with signals such as Ras and phosphatidylinositol 3,4,5-trisphosphate (PIP3) to form protrusions. However, how signaling cues tame actin dynamics to produce a pseudopod and guide cellular motility is a critical open question in eukaryotic chemotaxis. Here, we demonstrate that the strength of coupling between individual actin oscillators controls cell polarization and directional movement. We implement an inducible sequestration system to inactivate the heterotrimeric G protein subunit Gß and find that this acute perturbation triggers persistent, high-amplitude cortical oscillations of F-actin. Actin oscillators that are normally weakly coupled to one another in wild-type cells become strongly synchronized following acute inactivation of Gß. This global coupling impairs sensing of internal cues during spontaneous polarization and sensing of external cues during directional motility. A simple mathematical model of coupled actin oscillators reveals the importance of appropriate coupling strength for chemotaxis: moderate coupling can increase sensitivity to noisy inputs. Taken together, our data suggest that Gß regulates the strength of coupling between actin oscillators for efficient polarity and directional migration. As these observations are only possible following acute inhibition of Gß and are masked by slow compensation in genetic knockouts, our work also shows that acute loss-of-function approaches can complement and extend the reach of classical genetics in Dictyostelium and likely other systems as well.

A naturally monomeric infrared fluorescent protein for protein labeling in vivo.

Infrared fluorescent proteins (IFPs) provide an additional color to GFP and its homologs in protein labeling. Drawing on structural analysis of the dimer interface, we identified a bacteriophytochrome in the sequence database that is monomeric in truncated form and engineered it into a naturally monomeric IFP (mIFP). We demonstrate that mIFP correctly labels proteins in live cells, Drosophila and zebrafish. It should be useful in molecular, cell and developmental biology.

Synthetic control of mammalian-cell motility by engineering chemotaxis to an orthogonal bioinert chemical signal.

Directed migration of diverse cell types plays a critical role in biological processes ranging from development and morphogenesis to immune response, wound healing, and regeneration. However, techniques to direct, manipulate, and study cell migration in vitro and in vivo in a specific and facile manner are currently limited. We conceived of a strategy to achieve direct control over cell migration to arbitrary user-defined locations, independent of native chemotaxis receptors. Here, we show that genetic modification of cells with an engineered G protein-coupled receptor allows us to redirect their migration to a bioinert drug-like small molecule, clozapine-N-oxide (CNO). The engineered receptor and small-molecule ligand form an orthogonal pair: The receptor does not respond to native ligands, and the inert drug does not bind to native cells. CNO-responsive migration can be engineered into a variety of cell types, including neutrophils, T lymphocytes, keratinocytes, and endothelial cells. The engineered cells migrate up a gradient of the drug CNO and transmigrate through endothelial monolayers. Finally, we demonstrate that T lymphocytes modified with the engineered receptor can specifically migrate in vivo to CNO-releasing beads implanted in a live mouse. This technology provides a generalizable genetic tool to systematically perturb and control cell migration both in vitro and in vivo. In the future, this type of migration control could be a valuable module for engineering therapeutic cellular devices.

An optogenetic gene expression system with rapid activation and deactivation kinetics.

Optogenetic gene expression systems can control transcription with spatial and temporal detail unequaled with traditional inducible promoter systems. However, current eukaryotic light-gated transcription systems are limited by toxicity, dynamic range or slow activation and deactivation. Here we present an optogenetic gene expression system that addresses these shortcomings and demonstrate its broad utility. Our approach uses an engineered version of EL222, a bacterial light-oxygen-voltage protein that binds DNA when illuminated with blue light. The system has a large (>100-fold) dynamic range of protein expression, rapid activation (<10 s) and deactivation kinetics (<50 s) and a highly linear response to light. With this system, we achieve light-gated transcription in several mammalian cell lines and intact zebrafish embryos with minimal basal gene activation and toxicity. Our approach provides a powerful new tool for optogenetic control of gene expression in space and time.

Two distinct functions for PI3-kinases in macropinocytosis.

Class-1 PI3-kinases are major regulators of the actin cytoskeleton, whose precise contributions to chemotaxis, phagocytosis and macropinocytosis remain unresolved. We used systematic genetic ablation to examine this question in growing Dictyostelium cells. Mass spectroscopy shows that a quintuple mutant lacking the entire genomic complement of class-1 PI3-kinases retains only 10% of wild-type PtdIns(3,4,5)P3 levels. Chemotaxis to folate and phagocytosis of bacteria proceed normally in the quintuple mutant but macropinocytosis is abolished. In this context PI3-kinases show specialized functions, only one of which is directly linked to gross PtdIns(3,4,5)P3 levels: macropinosomes originate in patches of PtdIns(3,4,5)P3, with associated F-actin-rich ruffles, both of which depend on PI3-kinase 1/2 (PI3K1/2) but not PI3K4, whereas conversion of ruffles into vesicles requires PI3K4. A biosensor derived from the Ras-binding domain of PI3K1 suggests that Ras is activated throughout vesicle formation. Binding assays show that RasG and RasS interact most strongly with PI3K1/2 and PI3K4, and single mutants of either Ras have severe macropinocytosis defects. Thus, the fundamental function of PI3-kinases in growing Dictyostelium cells is in macropinocytosis where they have two distinct functions, supported by at least two separate Ras proteins.