Sreeja B. Asokan

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.

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 α5β1 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.

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

During cell migration, F-actin bundles/filopodia serve as templates for formation and orientation of lamellipodia and prime their stabilization by adhesion-based PI3K signaling.

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.

GMFβ controls branched actin content and lamellipodial retraction in fibroblasts

The primary activity of GMFβ in vivo is actin branch disassembly (and not inhibition of Arp2/3 activation), and this activity plays an important role in lamellipodial dynamics and directional migration toward ECM cues.

Lamellipodia are crucial for haptotactic sensing and response

ABSTRACT 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. Highlighted Article: Haptotaxis (directed migration) on a substrate-bound gradient is perhaps the least-well understood form of directed migration. We show that differential lamellipodial dynamics are crucial for this process.

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.

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

ABSTRACT 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. Highlighted Article: Through an optogenetic approach, differences in the contributions of Rac and Cdc42 to directed migration are analyzed. Cdc42 can stabilize protrusions and directed migration via fibronectin secretion while Rac cannot.

Lysophosphatidic acid provokes fibroblast chemotaxis through combinatorial regulation of myosin II

Lysophophatidic acid (LPA), a biologically active phospholipid that is ubiquitously present in tissues and organs, provokes cellular responses such as proliferation, apoptosis, differentiation and migration via activation of G-protein coupled receptors. These receptors activate a broad range of intracellular signaling cascades to mediate these responses. Using microfluidic chambers that generate and maintain stable gradients, we observed that chemotaxis of fibroblasts to LPA has higher directional fidelity than chemotaxis provoked by the receptor tyrosine kinase (RTK) ligand platelet-derived growth factor (PDGF). Unlike fast moving amoeboid cells, mesenchymal cells such as fibroblasts do not require PI3K for chemotaxis to a GPCR ligand. In addition, the Arp2/3 complex is not required for fibroblast GPCR-based chemotaxis in either 2D or 3D environments. Our data indicate that combinatorial regulation of myosin II involving global activation by RhoA/ROCK and local inhibition of myosin II at the leading edge by PKC results in highly efficient chemotaxis of fibroblasts to LPA. Based on these observations, we develop a simple mathematical model to explain how dual regulation of myosin II is responsible for enhanced chemotaxis in LPA gradients relative to PDGF. Using pharmacological approaches, we test predictions of this model and modulate the fidelity of LPA and PDGF chemotaxis.

Abstract PR15: Haptotaxis and direct remodeling of the extracellular matrix by tumor cells is important for metastasis

The tissue microenvironment, composed of stromal cells and extracellular matrix (ECM), is known to contribute to tumor progression, by providing both the structure and signals that promote tumor cell proliferation, survival and invasion. Fibronectin and collagen are major components of the tumor microenvironment, however, how tumor cells gain the ability to respond to these substrate-bound cues and how ECM sensing contributes to promoting metastasis remain poorly understood. Here, using a combination of in vitro and in vivo imaging, we show that gradients of FN can promote directional cell motility of breast cancer cells, a process dependent upon the actin regulatory Mena. Expression of the pro-metastatic isoform Mena INV allows cells to haptotax towards very high concentrations of FN, found near blood vessels and tumor periphery. Haptotaxis requires the direct interaction between α5β1 integrin and Mena/ Mena INV and is driven by outside-in signaling at focal complexes, crosstalk between α5β1 and EGFR, and inside-out tumor cell dependent ECM remodeling. These findings are clinically relevant as patients with high levels of Mena INV protein and FN have increased recurrence and decreased survival in two breast cancer cohorts. Our results identify a novel tumor cell-intrinsic mechanism that promotes ECM remodeling and directed migration, ultimately affecting metastasis. Citation Format: Madeleine J. Oudin, Oliver Jonas, Tatiana Kosciuk, Liliane Broye, Jeff Wyckoff, Joelle Klazen, John Lamar, Sreeja Asokan, Charlie Whittaker, Robert Langer, Michael Cima, Kari Wisinski, Richard Hynes, Douglas Lauffenburger, Patricia Keely, James Bear, Frank Gertler. Haptotaxis and direct remodeling of the extracellular matrix by tumor cells is important for metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr PR15.