Spatially coordinated cell cycle activity and motility govern bifurcation of mammary branches.

Branching morphogenesis is an evolutionary solution to maximize epithelial function in a compact organ. It involves successive rounds of branch elongation and branch point formation to generate a tubular network. In all organs, branch points can form by tip splitting, but it is unclear how tip cells coordinate elongation and branching. Here, we addressed these questions in the embryonic mammary gland. Live imaging revealed that tips advance by directional cell migration and elongation relies upon differential cell motility that feeds a retrograde flow of lagging cells into the trailing duct, supported by tip proliferation. Tip bifurcation involved localized repression of cell cycle and cell motility at the branch point. Cells in the nascent daughter tips remained proliferative but changed their direction to elongate new branches. We also report the fundamental importance of epithelial cell contractility for mammary branching morphogenesis. The co-localization of cell motility, non-muscle myosin II, and ERK activities at the tip front suggests coordination/cooperation between these functions.

Myosin light chain kinase-driven myosin II turnover regulates actin cortex contractility during mitosis.

Force generation by the molecular motor myosin II (MII) at the actin cortex is a universal feature of animal cells. Despite its central role in driving cell shape changes, the mechanisms underlying MII regulation at the actin cortex remain incompletely understood. Here we show that myosin light chain kinase (MLCK) promotes MII turnover at the mitotic cortex. Inhibition of MLCK resulted in an alteration of the relative levels of phosphorylated regulatory light chain (RLC), with MLCK preferentially creating a short-lived pRLC species and Rho-associated kinase (ROCK) preferentially creating a stable ppRLC species during metaphase. Slower turnover of MII and altered RLC homeostasis on MLCK inhibition correlated with increased cortex tension, driving increased membrane bleb initiation and growth, but reduced bleb retraction during mitosis. Taken together, we show that ROCK and MLCK play distinct roles at the actin cortex during mitosis; ROCK activity is required for recruitment of MII to the cortex, while MLCK activity promotes MII turnover. Our findings support the growing evidence that MII turnover is an essential dynamic process influencing the mechanical output of the actin cortex.

Evolution of mechanical cooperativity among myosin II motors.

Myosin II is a biomolecular machine that is responsible for muscle contraction. Myosin II motors act cooperatively: during muscle contraction, multiple motors bind to a single actin filament and pull it against an external load, like people pulling on a rope in a tug-of-war. We model the dynamics of actomyosin filaments in order to study the evolution of motor-motor cooperativity. We find that filament backsliding-the distance an actin slides backward when a motor at the end of its cycle releases-is central to the speed and efficiency of muscle contraction. Our model predicts that this backsliding has been reduced through evolutionary adaptations to the motor's binding propensity, the strength of the motor's power stroke, and the force dependence of the motor's release from actin. These properties optimize the collective action of myosin II motors, which is not a simple sum of individual motor actions. The model also shows that these evolutionary variables can explain the speed-efficiency trade-off observed across different muscle tissues. This is an example of how evolution can tune the microscopic properties of individual proteins in order to optimize complex biological functions.

Myosin II and Arp2/3 cross-talk governs intracellular hydraulic pressure and lamellipodia formation.

Human fibroblasts can switch between lamellipodia-dependent and -independent migration mechanisms on two-dimensional surfaces and in three-dimensional (3D) matrices. RhoA GTPase activity governs the switch from low-pressure lamellipodia to high-pressure lobopodia in response to the physical structure of the 3D matrix. Inhibiting actomyosin contractility in these cells reduces intracellular pressure and reverts lobopodia to lamellipodial protrusions via an unknown mechanism. To test the hypothesis that high pressure physically prevents lamellipodia formation, we manipulated pressure by activating RhoA or changing the osmolarity of the extracellular environment and imaged cell protrusions. We find RhoA activity inhibits Rac1-mediated lamellipodia formation through two distinct pathways. First, RhoA boosts intracellular pressure by increasing actomyosin contractility and water influx but acts upstream of Rac1 to inhibit lamellipodia formation. Increasing osmotic pressure revealed a second RhoA pathway, which acts through nonmuscle myosin II (NMII) to disrupt lamellipodia downstream from Rac1 and elevate pressure. Interestingly, Arp2/3 inhibition triggered a NMII-dependent increase in intracellular pressure, along with lamellipodia disruption. Together, these results suggest that actomyosin contractility and water influx are coordinated to increase intracellular pressure, and RhoA signaling can inhibit lamellipodia formation via two distinct pathways in high-pressure cells.

Myosin II isoforms promote internalization of spatially distinct clathrin-independent endocytosis cargoes through modulation of cortical tension downstream of ROCK2.

Although the actomyosin cytoskeleton has been implicated in clathrin-mediated endocytosis, a clear requirement for actomyosin in clathrin-independent endocytosis (CIE) has not been demonstrated. We discovered that the Rho-associated kinase ROCK2 is required for CIE of MHCI and CD59 through promotion of myosin II activity. Myosin IIA promoted internalization of MHCI and myosin IIB drove CD59 uptake in both HeLa and polarized Caco2 intestinal epithelial cells. In Caco2 cells, myosin IIA localized to the basal cortex and apical brush border and mediated MHCI internalization from the basolateral domain, while myosin IIB localized at the basal cortex and apical cell-cell junctions and promoted CD59 uptake from the apical membrane. Atomic force microscopy demonstrated that myosin IIB mediated apical epithelial tension in Caco2 cells. Thus, specific cargoes are internalized by ROCK2-mediated activation of myosin II isoforms to mediate spatial regulation of CIE, possibly by modulation of local cortical tension.

A Myosin II-Based Nanomachine Devised for the Study of Ca2+-Dependent Mechanisms of Muscle Regulation.

The emergent properties of the array arrangement of the molecular motor myosin II in the sarcomere of the striated muscle, the generation of steady force and shortening, can be studied in vitro with a synthetic nanomachine made of an ensemble of eight heavy-meromyosin (HMM) fragments of myosin from rabbit psoas muscle, carried on a piezoelectric nanopositioner and brought to interact with a properly oriented actin filament attached via gelsolin (a Ca2+-regulated actin binding protein) to a bead trapped by dual laser optical tweezers. However, the application of the original version of the nanomachine to investigate the Ca2+-dependent regulation mechanisms of the other sarcomeric (regulatory or cytoskeleton) proteins, adding them one at a time, was prevented by the impossibility to preserve [Ca2+] as a free parameter. Here, the nanomachine is implemented by assembling the bead-attached actin filament with the Ca2+-insensitive gelsolin fragment TL40. The performance of the nanomachine is determined both in the absence and in the presence of Ca2+ (0.1 mM, the concentration required for actin attachment to the bead with gelsolin). The nanomachine exhibits a maximum power output of 5.4 aW, independently of [Ca2+], opening the possibility for future studies of the Ca2+-dependent function/dysfunction of regulatory and cytoskeletal proteins.

Nonmuscle myosin-2 contractility-dependent actin turnover limits the length of epithelial microvilli.

Brush border microvilli enable functions that are critical for epithelial homeostasis, including solute uptake and host defense. However, the mechanisms that regulate the assembly and morphology of these protrusions are poorly understood. The parallel actin bundles that support microvilli have their pointed-end rootlets anchored in a filamentous meshwork referred to as the "terminal web." Although classic electron microscopy studies revealed complex ultrastructure, the composition and function of the terminal web remain unclear. Here we identify nonmuscle myosin-2C (NM2C) as a component of the terminal web. NM2C is found in a dense, isotropic layer of puncta across the subapical domain, which transects the rootlets of microvillar actin bundles. Puncta are separated by ∼210 nm, the expected size of filaments formed by NM2C. In intestinal organoid cultures, the terminal web NM2C network is highly dynamic and exhibits continuous remodeling. Using pharmacological and genetic perturbations in cultured intestinal epithelial cells, we found that NM2C controls the length of growing microvilli by regulating actin turnover in a manner that requires a fully active motor domain. Our findings answer a decades-old question on the function of terminal web myosin and hold broad implications for understanding apical morphogenesis in diverse epithelial systems.

The Drosophila melanogaster Rab GAP RN-tre cross-talks with the Rho1 signaling pathway to regulate nonmuscle myosin II localization and function.

To identify novel regulators of nonmuscle myosin II (NMII) we performed an image-based RNA interference screen using stable Drosophila melanogaster S2 cells expressing the enhanced green fluorescent protein (EGFP)-tagged regulatory light chain (RLC) of NMII and mCherry-Actin. We identified the Rab-specific GTPase-activating protein (GAP) RN-tre as necessary for the assembly of NMII RLC into contractile actin networks. Depletion of RN-tre led to a punctate NMII phenotype, similar to what is observed following depletion of proteins in the Rho1 pathway. Depletion of RN-tre also led to a decrease in active Rho1 and a decrease in phosphomyosin-positive cells by immunostaining, while expression of constitutively active Rho or Rho-kinase (Rok) rescues the punctate phenotype. Functionally, RN-tre depletion led to an increase in actin retrograde flow rate and cellular contractility in S2 and S2R+ cells, respectively. Regulation of NMII by RN-tre is only partially dependent on its GAP activity as overexpression of constitutively active Rabs inactivated by RN-tre failed to alter NMII RLC localization, while a GAP-dead version of RN-tre partially restored phosphomyosin staining. Collectively, our results suggest that RN-tre plays an important regulatory role in NMII RLC distribution, phosphorylation, and function, likely through Rho1 signaling and putatively serving as a link between the secretion machinery and actomyosin contractility.

Unilateral Cleavage Furrows in Multinucleate Cells.

Multinucleate cells can be produced in Dictyostelium by electric pulse-induced fusion. In these cells, unilateral cleavage furrows are formed at spaces between areas that are controlled by aster microtubules. A peculiarity of unilateral cleavage furrows is their propensity to join laterally with other furrows into rings to form constrictions. This means cytokinesis is biphasic in multinucleate cells, the final abscission of daughter cells being independent of the initial direction of furrow progression. Myosin-II and the actin filament cross-linking protein cortexillin accumulate in unilateral furrows, as they do in the normal cleavage furrows of mononucleate cells. In a myosin-II-null background, multinucleate or mononucleate cells were produced by cultivation either in suspension or on an adhesive substrate. Myosin-II is not essential for cytokinesis either in mononucleate or in multinucleate cells but stabilizes and confines the position of the cleavage furrows. In fused wild-type cells, unilateral furrows ingress with an average velocity of 1.7 µm × min-1, with no appreciable decrease of velocity in the course of ingression. In multinucleate myosin-II-null cells, some of the furrows stop growing, thus leaving space for the extensive broadening of the few remaining furrows.

Physical Constraints and Forces Involved in Phagocytosis.

Phagocytosis is a specialized process that enables cellular ingestion and clearance of microbes, dead cells and tissue debris that are too large for other endocytic routes. As such, it is an essential component of tissue homeostasis and the innate immune response, and also provides a link to the adaptive immune response. However, ingestion of large particulate materials represents a monumental task for phagocytic cells. It requires profound reorganization of the cell morphology around the target in a controlled manner, which is limited by biophysical constraints. Experimental and theoretical studies have identified critical aspects associated with the interconnected biophysical properties of the receptors, the membrane, and the actin cytoskeleton that can determine the success of large particle internalization. In this review, we will discuss the major physical constraints involved in the formation of a phagosome. Focusing on two of the most-studied types of phagocytic receptors, the Fcγ receptors and the complement receptor 3 (αMß2 integrin), we will describe the complex molecular mechanisms employed by phagocytes to overcome these physical constraints.

Precise Tuning of Cortical Contractility Regulates Cell Shape during Cytokinesis.

The mechanical properties of the actin cortex regulate shape changes during cell division, cell migration, and tissue morphogenesis. We show that modulation of myosin II (MII) filament composition allows tuning of surface tension at the cortex to maintain cell shape during cytokinesis. Our results reveal that MIIA generates cortex tension, while MIIB acts as a stabilizing motor and its inclusion in MII hetero-filaments reduces cortex tension. Tension generation by MIIA drives faster cleavage furrow ingression and bleb formation. We also show distinct roles for the motor and tail domains of MIIB in maintaining cytokinetic fidelity. Maintenance of cortical stability by the motor domain of MIIB safeguards against shape instability-induced chromosome missegregation, while its tail domain mediates cortical localization at the terminal stages of cytokinesis to mediate cell abscission. Because most non-muscle contractile systems are cortical, this tuning mechanism will likely be applicable to numerous processes driven by myosin-II contractility.

Direct visualization of human myosin II force generation using DNA origami-based thick filaments.

The sarcomere, the minimal mechanical unit of muscle, is composed of myosins, which self-assemble into thick filaments that interact with actin-based thin filaments in a highly-structured lattice. This complex imposes a geometric restriction on myosin in force generation. However, how single myosins generate force within the restriction remains elusive and conventional synthetic filaments do not recapitulate the symmetric bipolar filaments in sarcomeres. Here we engineered thick filaments using DNA origami that incorporate human muscle myosin to directly visualize the motion of the heads during force generation in a restricted space. We found that when the head diffuses, it weakly interacts with actin filaments and then strongly binds preferentially to the forward region as a Brownian ratchet. Upon strong binding, the two-step lever-arm swing dominantly halts at the first step and occasionally reverses direction. Our results illustrate the usefulness of our DNA origami-based assay system to dissect the mechanistic details of motor proteins.

In vivo optochemical control of cell contractility at single-cell resolution.

The spatial and temporal dynamics of cell contractility plays a key role in tissue morphogenesis, wound healing, and cancer invasion. Here, we report a simple optochemical method to induce cell contractions in vivo during Drosophila morphogenesis at single-cell resolution. We employed the photolabile Ca2+ chelator o-nitrophenyl EGTA to induce bursts of intracellular free Ca2+ by laser photolysis in the epithelial tissue. Ca2+ bursts appear within seconds and are restricted to individual target cells. Cell contraction reliably followed within a minute, causing an approximately 50% drop in the cross-sectional area. Increased Ca2+ levels are reversible, and the target cells further participated in tissue morphogenesis. Depending on Rho kinase (ROCK) activity but not RhoGEF2, cell contractions are paralleled with non-muscle myosin II accumulation in the apico-medial cortex, indicating that Ca2+ bursts trigger non-muscle myosin II activation. Our approach can be, in principle, adapted to many experimental systems and species, as no specific genetic elements are required.

Pulling in new directions: Myosin 2, Piezo, and metabolism.

Myosin 2 plays a central role in numerous, fundamental, actin-based biological processes, including cell migration, cell division, and the adhesion of cells to substrates and other cells. Here, we highlight recent studies in which the forces created by actomyosin 2 have been shown to also impact tension-sensitive ion channels and cell metabolism.

Cytoskeletal polarization and cytokinetic signaling drives polar lobe formation in spiralian embryos.

In many spiralians, asymmetry in the first two cleavages is achieved through the formation of a polar lobe (PL), which transiently constricts to sequester vegetal cytoplasm into the CD and D blastomeres. While microtubules and actin filaments are required for polar lobe formation, little else is known regarding the structural and functional similarities with the contractile ring, or how the PL constriction is able to form perpendicular to the cleavage plane. Examination of scallop embryos revealed that while activated myosin II could be detected in both the cleavage furrow and early PL constriction, astral or central spindle microtubules were not observed associated with the PL neck until the constriction was nearly complete. Further, inhibition of Aurora B had no effect on polar lobe initiation, but blocked both contractile ring ingression and PL constriction beyond phase II. The cortex destined for PL sequestration was marked by enrichment of the Arp2/3 complex, which was first detected during meiosis and remained enriched at the vegetal pole through the first two cleavages. Inhibition of Arp2/3 affected PL formation and partitioning of cytoplasm into the two daughter cells, suggesting that Arp2/3 plays a functional role in defining the zone of cortex to be sequestered into the polar lobe. Together, these data offer for the first time a mechanism by which a cytoskeletal specialization defines the polar lobe in this atypical form of asymmetric cell division.

Coordination of protrusion dynamics within and between collectively migrating border cells by myosin II.

Collective cell migration is emerging as a major driver of embryonic development, organogenesis, tissue homeostasis, and tumor dissemination. In contrast to individually migrating cells, collectively migrating cells maintain cell-cell adhesions and coordinate direction-sensing as they move. While nonmuscle myosin II has been studied extensively in the context of cells migrating individually in vitro, its roles in cells migrating collectively in three-dimensional, native environments are not fully understood. Here we use genetics, Airyscan microscopy, live imaging, optogenetics, and Förster resonance energy transfer to probe the localization, dynamics, and functions of myosin II in migrating border cells of the Drosophila ovary. We find that myosin accumulates transiently at the base of protrusions, where it functions to retract them. E-cadherin and myosin colocalize at border cell-border cell contacts and cooperate to transmit directional information. A phosphomimetic form of myosin is sufficient to convert border cells to a round morphology and blebbing migration mode. Together these studies demonstrate that distinct and dynamic pools of myosin II regulate protrusion dynamics within and between collectively migrating cells and suggest a new model for the role of protrusions in collective direction sensing in vivo.

Physical and functional cell-matrix uncoupling in a developing tissue under tension.

Tissue mechanics play a crucial role in organ development. They rely on the properties of cells and the extracellular matrix (ECM), but the relative physical contribution of cells and ECM to morphogenesis is poorly understood. Here, we have analyzed the behavior of the peripodial epithelium (PE) of the Drosophila leg disc in the light of the dynamics of its cellular and ECM components. The PE undergoes successive changes during leg development, including elongation, opening and removal to free the leg. During elongation, we found that the ECM and cell layer are progressively uncoupled. Concomitantly, the tension, mainly borne by the ECM at first, builds up in the cell monolayer. Then, each layer of the peripodial epithelium is removed by an independent mechanism: while the ECM layer withdraws following local proteolysis, cellular monolayer withdrawal is independent of ECM degradation and is driven by myosin II-dependent contraction. These results reveal a surprising physical and functional cell-matrix uncoupling in a monolayer epithelium under tension during development.This article has an associated 'The people behind the papers' interview.

Efficient Front-Rear Coupling in Neutrophil Chemotaxis by Dynamic Myosin II Localization.

Efficient chemotaxis requires rapid coordination between different parts of the cell in response to changing directional cues. Here, we investigate the mechanism of front-rear coordination in chemotactic neutrophils. We find that changes in the protrusion rate at the cell front are instantaneously coupled to changes in retraction at the cell rear, while myosin II accumulation at the rear exhibits a reproducible 9-15-s lag. In turning cells, myosin II exhibits dynamic side-to-side relocalization at the cell rear in response to turning of the leading edge and facilitates efficient turning by rapidly re-orienting the rear. These manifestations of front-rear coupling can be explained by a simple quantitative model incorporating reversible actin-myosin interactions with a rearward-flowing actin network. Finally, the system can be tuned by the degree of myosin regulatory light chain (MRLC) phosphorylation, which appears to be set in an optimal range to balance persistence of movement and turning ability.

Hic-5 regulates Src-induced invadopodia rosette formation and organization.

Fibroblasts transformed by the proto-oncogene Src form individual invadopodia that can spontaneously self-organize into large matrix-degrading superstructures called rosettes. However, the mechanisms by which the invadopodia can spatiotemporally reorganize their architecture is not well understood. Here, we show that Hic-5, a close relative of the scaffold protein paxillin, is essential for the formation and organization of rosettes in active Src-transfected NIH3T3 fibroblasts and cancer-associated fibroblasts. Live cell imaging, combined with domain-mapping analysis of Hic-5, identified critical motifs as well as phosphorylation sites that are required for the formation and dynamics of rosettes. Using pharmacological inhibition and mutant expression, we show that FAK kinase activity, along with its proximity to and potential interaction with the LD2,3 motifs of Hic-5, is necessary for rosette formation. Invadopodia dynamics and their coalescence into rosettes were also dependent on Rac1, formin, and myosin II activity. Superresolution microscopy revealed the presence of formin FHOD1 and INF2-mediated unbranched radial F-actin fibers emanating from invadopodia and rosettes, which may facilitate rosette formation. Collectively, our data highlight a novel role for Hic-5 in orchestrating the organization of invadopodia into higher-order rosettes, which may promote the localized matrix degradation necessary for tumor cell invasion.

Multiple feedback mechanisms fine-tune Rho signaling to regulate morphogenetic outcomes.

Rho signaling is a conserved mechanism for generating forces through activation of contractile actomyosin. How this pathway can produce different cell morphologies is poorly understood. In the Drosophila embryonic epithelium, we investigate how Rho signaling controls force asymmetry to drive morphogenesis. We study a distinct morphogenetic process termed 'alignment'. This process results in striking columns of rectilinear cells connected by aligned cell-cell contacts. We found that this is driven by contractile actomyosin cables that elevate tension along aligning interfaces. Our data show that polarization of Rho effectors, Rok and Dia, directs formation of these cables. Constitutive activation of these effectors causes aligning cells to instead invaginate. This suggests that moderating Rho signaling is essential to producing the aligned geometry. Therefore, we tested for feedback that could fine-tune Rho signaling. We discovered that F-actin exerts negative feedback on multiple nodes in the pathway. Further, we present evidence that suggests that Rok in part mediates feedback from F-actin to Rho in a manner independent of Myo-II. Collectively, our work suggests that multiple feedback mechanisms regulate Rho signaling, which may account for diverse morphological outcomes.