Vera DesMarais

Cofilin takes the lead

Cofilin has emerged as a key regulator of actin dynamics at the leading edge of motile cells. Through its actin-severing activity, it creates new actin barbed ends for polymerization and also depolymerizes old actin filaments. Its function is tightly regulated in the cell. Spatially, its activity is restricted by other actin-binding proteins, such as tropomyosin, which compete for accessibility of actin filament populations in different regions of the cell. At the molecular level, it is regulated by phosphorylation, pH and phosphatidylinositol (4,5)-bisphosphate binding downstream of signaling cascades. In addition, it also appears to be regulated by interactions with 14-3-3ζ and cyclase-associated protein. In vivo, cofilin acts synergistically with the Arp2/3 complex to amplify local actin polymerization responses upon cell stimulation, which gives it a central role in setting the direction of motility in crawling cells.

Cortactin regulates cofilin and N-WASp activities to control the stages of invadopodium assembly and maturation

Invadopodia are matrix-degrading membrane protrusions in invasive carcinoma cells. The mechanisms regulating invadopodium assembly and maturation are not understood. We have dissected the stages of invadopodium assembly and maturation and show that invadopodia use cortactin phosphorylation as a master switch during these processes. In particular, cortactin phosphorylation was found to regulate cofilin and Arp2/3 complex–dependent actin polymerization. Cortactin directly binds cofilin and inhibits its severing activity. Cortactin phosphorylation is required to release this inhibition so cofilin can sever actin filaments to create barbed ends at invadopodia to support Arp2/3-dependent actin polymerization. After barbed end formation, cortactin is dephosphorylated, which blocks cofilin severing activity thereby stabilizing invadopodia. These findings identify novel mechanisms for actin polymerization in the invadopodia of metastatic carcinoma cells and define four distinct stages of invadopodium assembly and maturation consisting of invadopodium precursor formation, actin polymerization, stabilization, and matrix degradation.

Phospholipase C and cofilin are required for carcinoma cell directionality in response to EGF stimulation

The epidermal growth factor (EGF)–induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.

EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells

Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP2; however, this is mainly based on in vitro–binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP2 in living cells. We show that EGF induces a rapid loss of PIP2 through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation. Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF.

Spatial regulation of actin dynamics: a tropomyosin-free, actin-rich compartment at the leading edge

Rapid polymerization of a network of short, branched actin filaments takes place at the leading edge of migrating cells, a compartment enriched in activators of actin polymerization such as the Arp2/3 complex and cofilin. Actin filaments elsewhere in the cell are long and unbranched. Results reported here show that the presence or absence of tropomyosin in these different actin-containing regions helps establish functionally distinct actin-containing compartments in the cell. Tropomyosin, an inhibitor of the Arp2/3 complex and cofilin function, was localized in relation to actin filaments, the Arp2/3 complex, and free barbed ends of actin filaments in MTLn3 cells, which rapidly extend flat lamellipodia following EGF stimulation. All tropomyosin isoforms examined using indirect immunofluorescence were relatively absent from the dynamic leading edge compartment, but did colocalize with actin structures deeper in the lamellipodium and in stress fibers. An in vitro light microscopy assay revealed that tropomyosin protects actin filaments from cofilin severing. The results suggest that tropomyosin-free actin filaments under the membrane can participate in rapid, dynamic processes that depend on interactions between the activities of the Arp2/3 complex and ADF/cofilin that tropomyosin inhibits elsewhere in the cell.

Spatial and Temporal Control of Cofilin Activity Is Required for Directional Sensing during Chemotaxis

BACKGROUND Previous work has led to the hypothesis that cofilin severing, as regulated by PLC, is involved in chemotactic sensing. We have tested this hypothesis by investigating whether activation of endogenous cofilin is spatially and temporally linked to sensing an EGF point source in carcinoma cells. RESULTS We demonstrate that inhibition of endogenous cofilin activity with either siRNA or overexpression of LIMK suppresses directional sensing in carcinoma cells. LIMK siRNA knockdown, which suppresses cofilin phosphorylation, and microinjection of S3C cofilin, a cofilin mutant that is constitutively active and not phosphorylated by LIMK, also inhibits directional sensing and chemotaxis. These results indicate that phosphorylation of cofilin by LIMK, in addition to cofilin activity, is required for chemotaxis. Cofilin activity concentrates rapidly at the newly formed leading edge facing the gradient, whereas cofilin phosphorylation increases throughout the cell. Quantification of these results indicates that the amplification of asymmetric actin polymerization required for protrusion toward the EGF gradient occurs at the level of cofilin but not at the level of PLC activation by EGFR. CONCLUSIONS These results indicate that local activation of cofilin by PLC and its global inactivation by LIMK phosphorylation combine to generate the local asymmetry of actin polymerization required for chemotaxis.

Synergistic interaction between the Arp2/3 complex and cofilin drives stimulated lamellipod extension.

Both the Arp2/3 complex and cofilin are believed to be important for the generation of protrusive force at the leading edge; however, their relative contributions have not been explored in vivo. Our results with living cells show that cofilin enters the leading edge immediately before the start of lamellipod extension, slightly earlier than Arp2/3, which begins to be recruited slightly later as the lamellipod is extended. Blocking either the Arp2/3 complex or cofilin function in cells results in failure to extend broad lamellipods and inhibits free barbed ends, suggesting that neither factor on its own can support actin polymerization-mediated protrusion in response to growth factor stimulation. High-resolution analysis of the actin network at the leading edge supports the idea that both the severing activity of cofilin and the specific branching activity of the Arp2/3 complex are essential for lamellipod protrusion. These results are the first to document the relative contributions of cofilin and Arp2/3 complex in vivo and indicate that cofilin begins to initiate the generation of free barbed ends that act in synergy with the Arp2/3 complex to create a large burst in nucleation activity.

WASP family members and formin proteins coordinate regulation of cell protrusions in carcinoma cells

We examined the role of the actin nucleation promoters neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE2 in cell protrusion in response to epidermal growth factor (EGF), a key regulator in carcinoma cell invasion. We found that WAVE2 knockdown (KD) suppresses lamellipod formation and increases filopod formation, whereas N-WASP KD has no effect. However, simultaneous KD of both proteins results in the formation of large jagged protrusions with lamellar properties and increased filopod formation. This suggests that another actin nucleation activity is at work in carcinoma cells in response to EGF. A mammalian Diaphanous–related formin, mDia1, localizes at the jagged protrusions in double KD cells. Constitutively active mDia1 recapitulated the phenotype, whereas inhibition of mDia1 blocked the formation of these protrusions. Increased RhoA activity, which stimulates mDia1 nucleation, was observed in the N-WASP/WAVE2 KD cells and was shown to be required for the N-WASP/WAVE2 KD phenotype. These data show that coordinate regulation between the WASP family and mDia proteins controls the balance between lamellar and lamellipodial protrusion activity.

N-WASP and cortactin are involved in invadopodium-dependent chemotaxis to EGF in breast tumor cells

Metastatic mammary carcinoma cells, which have previously been observed to form mature, matrix degrading invadopodia on a thick ECM matrix, are able to form invadopodia with similar characteristics on glass without previously applied matrix. They form in response to epidermal growth factor (EGF), and contain the usual invadopodium core proteins N-WASP, Arp2/3, cortactin, cofilin, and F-actin. The study of invadopodia on glass allows for higher resolution analysis including the use of total internal reflection microscopy and analysis of their relationship to other cell motility events, in particular, lamellipodium extension and chemotaxis toward an EGF gradient. Invadopodium formation on glass requires N-WASP and cortactin but not microtubules. In a gradient of EGF more invadopodia form on the side of the cells facing the source of EGF. In addition, depletion of N-WASP or cortactin, which blocks invadopodium fromation, inhibits chemotaxis of cells towards EGF. This appears to be a localized defect in chemotaxis since depletion of N-WASP or cortactin via siRNA had no effect on lamellipodium protrusion or barbed end generation at the lamellipodiums leading edge. Since chemotaxis to EGF by breast tumor cells is involved in metastasis, inhibiting N-WASP activity in breast tumor cells might prevent metastasis of tumor cells while not affecting chemotaxis-dependent innate immunity which depends on WASp function in macrophages.