Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis.

Lamellipodia are sheet-like, leading edge protrusions in firmly adherent cells that contain Arp2/3-generated dendritic actin networks. Although lamellipodia are widely believed to be critical for directional cell motility, this notion has not been rigorously tested. Using fibroblasts derived from Ink4a/Arf-deficient mice, we generated a stable line depleted of Arp2/3 complex that lacks lamellipodia. This line shows defective random cell motility and relies on a filopodia-based protrusion system. Utilizing a microfluidic gradient generation system, we tested the role of Arp2/3 complex and lamellipodia in directional cell migration. Surprisingly, Arp2/3-depleted cells respond normally to shallow gradients of PDGF, indicating that lamellipodia are not required for fibroblast chemotaxis. Conversely, these cells cannot respond to a surface-bound gradient of extracellular matrix (haptotaxis). Consistent with this finding, cells depleted of Arp2/3 fail to globally align focal adhesions, suggesting that one principle function of lamellipodia is to organize cell-matrix adhesions in a spatially coherent manner.

Cells lay their own tracks - optogenetic Cdc42 activation stimulates fibronectin deposition supporting directed migration.

Rho GTPase family members are known regulators of directed migration and therefore play key roles in processes including development, the immune response and cancer metastasis. However, their individual contributions to these processes are complex. Here, we modify the activity of the two Rho GTPase family members Rac and Cdc42 by optogenetically recruiting specific guanine nucleotide exchange factor (GEF) DH or PH domains to defined regions of the cell membrane. We find that the localized activation of both GTPases produces lamellipodia in cells plated on a fibronectin substrate. By using a novel optotaxis assay, we show that biased activation can drive directional migration. Interestingly, in the absence of exogenous fibronectin, Rac activation is insufficient to produce stable lamellipodia or directional migration whereas Cdc42 activation is sufficient for these processes. We find that a remarkably small amount of fibronectin (<10 puncta per protrusion) is necessary to support stable GTPase-driven lamellipodia formation. Cdc42 bypasses the need for exogenous fibronectin by stimulating cellular fibronectin deposition under the newly formed lamellipodia.This article has an associated First Person interview with the first author of the paper.

Lamellipodia are crucial for haptotactic sensing and response.

Haptotaxis is the process by which cells respond to gradients of substrate-bound cues, such as extracellular matrix proteins (ECM); however, the cellular mechanism of this response remains poorly understood and has mainly been studied by comparing cell behavior on uniform ECMs with different concentrations of components. To study haptotaxis in response to gradients, we utilized microfluidic chambers to generate gradients of the ECM protein fibronectin, and imaged the cell migration response. Lamellipodia are fan-shaped protrusions that are common in migrating cells. Here, we define a new function for lamellipodia and the cellular mechanism required for haptotaxis - differential actin and lamellipodial protrusion dynamics lead to biased cell migration. Modest differences in lamellipodial dynamics occurring over time periods of seconds to minutes are summed over hours to produce differential whole cell movement towards higher concentrations of fibronectin. We identify a specific subset of lamellipodia regulators as being crucial for haptotaxis. Numerous studies have linked components of this pathway to cancer metastasis and, consistent with this, we find that expression of the oncogenic Rac1 P29S mutation abrogates haptotaxis. Finally, we show that haptotaxis also operates through this pathway in 3D environments.

Emergent spatiotemporal dynamics of the actomyosin network in the presence of chemical gradients.

We used particle-based computer simulations to study the emergent properties of the actomyosin cytoskeleton. Our model accounted for biophysical interactions between filamentous actin and non-muscle myosin II and was motivated by recent experiments demonstrating that spatial regulation of myosin activity is required for fibroblasts responding to spatial gradients of platelet derived growth factor (PDGF) to undergo chemotaxis. Our simulations revealed the spontaneous formation of actin asters, consistent with the punctate actin structures observed in chemotacting fibroblasts. We performed a systematic analysis of model parameters to identify biochemical steps in myosin activity that significantly affect aster formation and performed simulations in which model parameter values vary spatially to investigate how the model responds to chemical gradients. Interestingly, spatial variations in motor stiffness generated time-dependent behavior of the actomyosin network, in which actin asters continued to spontaneously form and dissociate in different regions of the gradient. Our results should serve as a guide for future experimental investigations.

Self-sorting of nonmuscle myosins IIA and IIB polarizes the cytoskeleton and modulates cell motility.

Nonmuscle myosin II (NMII) is uniquely responsible for cell contractility and thus defines multiple aspects of cell behavior. To generate contraction, NMII molecules polymerize into bipolar minifilaments. Different NMII paralogs are often coexpressed in cells and can copolymerize, suggesting that they may cooperate to facilitate cell motility. However, whether such cooperation exists and how it may work remain unknown. We show that copolymerization of NMIIA and NMIIB followed by their differential turnover leads to self-sorting of NMIIA and NMIIB along the front-rear axis, thus producing a polarized actin-NMII cytoskeleton. Stress fibers newly formed near the leading edge are enriched in NMIIA, but over time, they become progressively enriched with NMIIB because of faster NMIIA turnover. In combination with retrograde flow, this process results in posterior accumulation of more stable NMIIB-rich stress fibers, thus strengthening cell polarity. By copolymerizing with NMIIB, NMIIA accelerates the intrinsically slow NMIIB dynamics, thus increasing cell motility and traction and enabling chemotaxis.

Arp2/3 Complex Is Required for Macrophage Integrin Functions but Is Dispensable for FcR Phagocytosis and In Vivo Motility.

The Arp2/3 complex nucleates branched actin, forming networks involved in lamellipodial protrusion, phagocytosis, and cell adhesion. We derived primary bone marrow macrophages lacking Arp2/3 complex (Arpc2-/-) and directly tested its role in macrophage functions. Despite protrusion and actin assembly defects, Arpc2-/- macrophages competently phagocytose via FcR and chemotax toward CSF and CX3CL1. However, CR3 phagocytosis and fibronectin haptotaxis, both integrin-dependent processes, are disrupted. Integrin-responsive actin assembly and αM/ß2 integrin localization are compromised in Arpc2-/- cells. Using an in vivo system to observe endogenous monocytes migrating toward full-thickness ear wounds we found that Arpc2-/- monocytes maintain cell speeds and directionality similar to control. Our work reveals that the Arp2/3 complex is not a general requirement for phagocytosis or chemotaxis but is a critical driver of integrin-dependent processes. We demonstrate further that cells lacking Arp2/3 complex function in vivo remain capable of executing important physiological responses that require rapid directional motility.

Quantitative analysis of B-lymphocyte migration directed by CXCL13.

B-lymphocyte migration, directed by chemokine gradients, is essential for homing to sites of antigen presentation. B cells move rapidly, exhibiting amoeboid morphology like other leukocytes, yet quantitative studies addressing B-cell migration are currently lacking relative to neutrophils, macrophages, and T cells. Here, we used total internal reflection fluorescence (TIRF) microscopy to characterize the changes in shape (morphodynamics) of primary, murine B cells as they migrated on surfaces with adsorbed chemokine, CXCL13, and the adhesive ligand, ICAM-1. B cells exhibited frequent, spontaneous dilation and shrinking events at the sides of the leading membrane edge, a phenomenon that was predictive of turning versus directional persistence. To characterize directed B-cell migration, a microfluidic device was implemented to generate gradients of adsorbed CXCL13 gradients. Haptotaxis assays revealed a modest yet consistently positive bias of the cell's persistent random walk behavior towards CXCL13 gradients. Quantification of tactic fidelity showed that bias is optimized by steeper gradients without excessive midpoint density of adsorbed chemokine. Under these conditions, B-cell migration is more persistent when the direction of migration is better aligned with the gradient.

Tumor Cell-Driven Extracellular Matrix Remodeling Drives Haptotaxis during Metastatic Progression.

UNLABELLED: Fibronectin (FN) is a major component of the tumor microenvironment, but its role in promoting metastasis is incompletely understood. Here, we show that FN gradients elicit directional movement of breast cancer cells, in vitro and in vivo Haptotaxis on FN gradients requires direct interaction between α5ß1 integrin and MENA, an actin regulator, and involves increases in focal complex signaling and tumor cell-mediated extracellular matrix (ECM) remodeling. Compared with MENA, higher levels of the prometastatic MENA(INV) isoform associate with α5, which enables 3-D haptotaxis of tumor cells toward the high FN concentrations typically present in perivascular space and in the periphery of breast tumor tissue. MENA(INV) and FN levels were correlated in two breast cancer cohorts, and high levels of MENA(INV) were significantly associated with increased tumor recurrence as well as decreased patient survival. Our results identify a novel tumor cell-intrinsic mechanism that promotes metastasis through ECM remodeling and ECM-guided directional migration. SIGNIFICANCE: Here, we provide new insight into how tumor cell:ECM interactions generate signals and structures that promote directed tumor cell migration, a critical component of metastasis. Our results identify a tumor cell-intrinsic mechanism driven by the actin regulatory protein MENA that promotes ECM remodeling and haptotaxis along FN gradients. Cancer Discov; 6(5); 516-31. ©2016 AACR.See related commentary by Santiago-Medina and Yang, p. 474This article is highlighted in the In This Issue feature, p. 461.

GMFß controls branched actin content and lamellipodial retraction in fibroblasts.

The lamellipodium is an important structure for cell migration containing branched actin nucleated via the Arp2/3 complex. The formation of branched actin is relatively well studied, but less is known about its disassembly and how this influences migration. GMF is implicated in both Arp2/3 debranching and inhibition of Arp2/3 activation. Modulation of GMFß, a ubiquitous GMF isoform, by depletion or overexpression resulted in changes in lamellipodial dynamics, branched actin content, and migration. Acute pharmacological inhibition of Arp2/3 by CK-666, coupled to quantitative live-cell imaging of the complex, showed that depletion of GMFß decreased the rate of branched actin disassembly. These data, along with mutagenesis studies, suggest that debranching (not inhibition of Arp2/3 activation) is a primary activity of GMFß in vivo. Furthermore, depletion or overexpression of GMFß disrupted the ability of cells to directionally migrate to a gradient of fibronectin (haptotaxis). These data suggest that debranching by GMFß plays an important role in branched actin regulation, lamellipodial dynamics, and directional migration.

F-actin bundles direct the initiation and orientation of lamellipodia through adhesion-based signaling.

Mesenchymal cells such as fibroblasts are weakly polarized and reorient directionality by a lamellipodial branching mechanism that is stabilized by phosphoinositide 3-kinase (PI3K) signaling. However, the mechanisms by which new lamellipodia are initiated and directed are unknown. Using total internal reflection fluorescence microscopy to monitor cytoskeletal and signaling dynamics in migrating cells, we show that peripheral F-actin bundles/filopodia containing fascin-1 serve as templates for formation and orientation of lamellipodia. Accordingly, modulation of fascin-1 expression tunes cell shape, quantified as the number of morphological extensions. Ratiometric imaging reveals that F-actin bundles/filopodia play both structural and signaling roles, as they prime the activation of PI3K signaling mediated by integrins and focal adhesion kinase. Depletion of fascin-1 ablated fibroblast haptotaxis on fibronectin but not platelet-derived growth factor chemotaxis. Based on these findings, we conceptualize haptotactic sensing as an exploration, with F-actin bundles directing and lamellipodia propagating the process and with signaling mediated by adhesions playing the role of integrator.

Mesenchymal chemotaxis requires selective inactivation of myosin II at the leading edge via a noncanonical PLCγ/PKCα pathway.

Chemotaxis, migration toward soluble chemical cues, is critical for processes such as wound healing and immune surveillance and is exhibited by various cell types, from rapidly migrating leukocytes to slow-moving mesenchymal cells. To study mesenchymal chemotaxis, we observed cell migration in microfluidic chambers that generate stable gradients of platelet-derived growth factor (PDGF). Surprisingly, we found that pathways implicated in amoeboid chemotaxis, such as PI3K and mammalian target of rapamycin signaling, are dispensable for PDGF chemotaxis. Instead, we find that local inactivation of Myosin IIA, through a noncanonical Ser1/2 phosphorylation of the regulatory light chain, is essential. This site is phosphorylated by PKCα, which is activated by an intracellular gradient of diacylglycerol generated by PLCγ. Using a combination of live imaging and gradients of activators/inhibitors in the microfluidic chambers, we demonstrate that this signaling pathway and subsequent inhibition of Myosin II activity at the leading edge are required for mesenchymal chemotaxis.

LKB1 loss in melanoma disrupts directional migration toward extracellular matrix cues.

Somatic inactivation of the serine/threonine kinase gene STK11/LKB1/PAR-4 occurs in a variety of cancers, including ∼10% of melanoma. However, how the loss of LKB1 activity facilitates melanoma invasion and metastasis remains poorly understood. In LKB1-null cells derived from an autochthonous murine model of melanoma with activated Kras and Lkb1 loss and matched reconstituted controls, we have investigated the mechanism by which LKB1 loss increases melanoma invasive motility. Using a microfluidic gradient chamber system and time-lapse microscopy, in this paper, we uncover a new function for LKB1 as a directional migration sensor of gradients of extracellular matrix (haptotaxis) but not soluble growth factor cues (chemotaxis). Systematic perturbation of known LKB1 effectors demonstrated that this response does not require canonical adenosine monophosphate-activated protein kinase (AMPK) activity but instead requires the activity of the AMPK-related microtubule affinity-regulating kinase (MARK)/PAR-1 family kinases. Inhibition of the LKB1-MARK pathway facilitated invasive motility, suggesting that loss of the ability to sense inhibitory matrix cues may promote melanoma invasion.

Loss of Arp2/3 induces an NF-κB-dependent, nonautonomous effect on chemotactic signaling.

Arp2/3-branched actin is critical for cytoskeletal dynamics and cell migration. However, perturbations and diseases affecting this network have phenotypes that cannot be fully explained by cell-autonomous effects. In this paper, we report nonautonomous effects of Arp2/3 depletion. We show that, upon Arp2/3 depletion, the expression of numerous genes encoding secreted factors, including chemokines, growth factors, and matrix metalloproteases, was increased, a signature resembling the senescence-associated secretory phenotype. These factors affected epidermal growth factor chemotaxis in a nonautonomous way, resolving the recent contradictions about the role of Arp2/3 in chemotaxis. We demonstrate that these genes were activated by nuclear factor κB via a CCM2­MEKK3 pathway that has been implicated in hyperosmotic stress signaling. Consistent with this, Arp2/3-depleted cells showed misregulation of volume control and reduced actin in the submembranous cortex. The defects in osmotic signaling in the Arp2/3-depleted cells can be rescued by hypoosmotic treatment. Thus, perturbations of Arp2/3 have nonautonomous effects that should be considered when evaluating experimental manipulations and diseases affecting the Arp2/3-actin cytoskeleton.