Lamellipodial Actin Research Articles

The murine Nck SH2/SH3 adaptors are important for the development of mesoderm-derived embryonic structures and for regulating the cellular actin network.

Mammalian Nck1 and Nck2 are closely related adaptor proteins that possess three SH3 domains, followed by an SH2 domain, and are implicated in coupling phosphotyrosine signals to polypeptides that regulate the actin cytoskeleton. However, the in vivo functions of Nck1 and Nck2 have not been defined. We have mutated the murine Nck1 and Nck2 genes and incorporated beta-galactosidase reporters into the mutant loci. In mouse embryos, the two Nck genes have broad and overlapping expression patterns. They are functionally redundant in the sense that mice deficient for either Nck1 or Nck2 are viable, whereas inactivation of both Nck1 and Nck2 results in profound defects in mesoderm-derived notochord and embryonic lethality at embryonic day 9.5. Fibroblast cell lines derived from Nck1(-/-) Nck2(-/-) embryos have defects in cell motility and in the organization of the lamellipodial actin network. These data suggest that the Nck SH2/SH3 adaptors have important functions in the development of mesodermal structures during embryogenesis, potentially linked to a role in cell movement and cytoskeletal organization.

Regulation of leading edge microtubule and actin dynamics downstream of Rac1.

Actin in migrating cells is regulated by Rho GTPases. However, Rho proteins might also affect microtubules (MTs). Here, we used time-lapse microscopy of PtK1 cells to examine MT regulation downstream of Rac1. In these cells, “pioneer” MTs growing into leading-edge protrusions exhibited a decreased catastrophe frequency and an increased time in growth as compared with MTs further from the leading edge. Constitutively active Rac1(Q61L) promoted pioneer behavior in most MTs, whereas dominant-negative Rac1(T17N) eliminated pioneer MTs, indicating that Rac1 is a regulator of MT dynamics in vivo. Rac1(Q61L) also enhanced MT turnover through stimulation of MT retrograde flow and breakage. Inhibition of p21-activated kinases (Paks), downstream effectors of Rac1, inhibited Rac1(Q61L)-induced MT growth and retrograde flow. In addition, Rac1(Q61L) promoted lamellipodial actin polymerization and Pak-dependent retrograde flow. Together, these results indicate coordinated regulation of the two cytoskeletal systems in the leading edge of migrating cells.

Pointed-end capping by tropomodulin3 negatively regulates endothelial cell motility.

Actin filament pointed-end dynamics are thought to play a critical role in cell motility, yet regulation of this process remains poorly understood. We describe here a previously uncharacterized tropomodulin (Tmod) isoform, Tmod3, which is widely expressed in human tissues and is present in human microvascular endothelial cells (HMEC-1). Tmod3 is present in sufficient quantity to cap pointed ends of actin filaments, localizes to actin filament structures in HMEC-1 cells, and appears enriched in leading edge ruffles and lamellipodia. Transient overexpression of GFP–Tmod3 leads to a depolarized cell morphology and decreased cell motility. A fivefold increase in Tmod3 results in an equivalent decrease in free pointed ends in the cells. Unexpectedly, a decrease in the relative amounts of F-actin, free barbed ends, and actin-related protein 2/3 (Arp2/3) complex in lamellipodia are also observed. Conversely, decreased expression of Tmod3 by RNA interference leads to faster average cell migration, along with increases in free pointed and barbed ends in lamellipodial actin filaments. These data collectively demonstrate that capping of actin filament pointed ends by Tmod3 inhibits cell migration and reveal a novel control mechanism for regulation of actin filaments in lamellipodia.

Selective roles of protein kinase C isoforms on cell motility of GT1 immortalized hypothalamic neurones.

Recently, we demonstrated that activation of the protein kinase C (PKC) signalling pathway promoted morphological differentiation of GT1 hypothalamic neurones via an increase in beta-catenin, a cell-cell adhesion molecule, indicating a possible involvement of PKC in cellular motility. In this study, we explored the differential roles of PKC isoforms in GT1 cell migration. First, we transiently transfected GT1 cells with enhanced green fluorescence protein (EGFP)-tagged actin to monitor the dynamic rearrangement of filamentous-actin (F-actin) in living cells. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, markedly promoted lamellipodia formation, while safingol (a PKC alpha-selective inhibitor) blocked the TPA-induced lamellipodial actin structure. Both wound-healing and Boyden migration assays showed that TPA treatment promoted neuronal migration of GT1 cells; however, cotreatment of TPA with safingol or rottlerin (a PKC delta-selective inhibitor) clearly blocked this TPA effect, indicating that both PKC alpha and PKC delta may be positive regulators of neuronal migration. By contrast, PKC gamma-EGFP-expressing GT1 cells exhibited decreased cellular motility and weak staining for actin stress fibres, suggesting that PKC gamma may act as a negative mediator of cell migration in these neurones. Among the PKC downstream signal molecules, p130Cas, a mediator of cell migration, and its kinase, focal adhesion kinase (FAK), increased following TPA treatment; phosphorylation of p130Cas was induced in a PKC alpha-dependent manner. Together, these results demonstrate that PKC alpha promotes GT1 neuronal migration by activating focal adhesion complex proteins such as p130Cas and FAK.

Urge to merge erects filopodia

Crawling cells often produce both broad, flat lamellipodia and long, thin filopodia. Once formed, both structures appear to use an actin-treadmilling mechanism to extend themselves. During treadmilling, lamellipodia continuously nucleate new filaments, but filopodia continue to use the same filaments. So how does a cell form a filopodium in the first place? Svitkina et al., page 409, address this question with a detailed kinetic and structural analysis, presenting a novel model that should help to guide future work in the field. Figure Fascin (green) helps to bundle actin filaments into filopodia. Using GFP-tagged versions of the Arp2/3, VASP, and fascin proteins and innovative electron microscopy techniques, the authors examined the molecular dynamics of filopodium initiation in mouse melanoma cells. Rather than forming a distinct nucleation complex, Arp2/3 seems to lay the groundwork for filopodium birth by producing a normal lamellipodial actin array. Conical structures, termed Λ-precursors, then rise up from this array. VASP gradually coalesces from a broad distribution along the lamellipodial edge into defined spots at the tips of the Λ-precursors. Fascin then abruptly appears at the Λ-precursor vertex before filopodia grow from these structures. The data suggest that, in a normal dendritic actin network in the lamellipodium, the elongating barbed ends of certain actin filaments bind to a set of molecules that protect them from capping and mediate their association with other elongating barbed ends. When these privileged filaments collide, they merge to form the conical Λ-precursors, and their tip complexes then recruit or activate the bundling protein fascin, allowing filament cross-linking and the growth of long, strong actin bundles characteristic of filopodia. This model also implies that microspikes, and retraction fibers—filopodium-like structures seen in some migrating cells—arise by a similar mechanism and are interconvertible with filopodia. In separate work, the authors have developed an in vitro system that mimics this convergent elongation process. They are now trying to identify proteins in the filopodial tip complex that regulate initiation. ▪

Association of cortactin with dynamic actin in lamellipodia and on endosomal vesicles.

We have used fluorescent protein tagging to study the localization and dynamics of the actin-binding protein cortactin in living NIH 3T3 fibroblast cells. Cortactin was localized to active lamellipodia and to small cytoplasmic spots. Time-lapse imaging revealed that these cortactin labeled structures were very dynamic. In the lamellipodia, cortactin labeled structures formed at the leading edge and then moved toward the cell center. Experiments with green fluorescent protein (GFP)-tagged actin showed that cortactin movement was coincident with the actin retrograde flow in the lamellipodia. Cytoplasmic cortactin spots also contained F-actin and were propelled by actin polymerization. Arp3, a component of the arp2/3 complex which is a key regulator of actin polymerization, co-localized with cortactin. Cytoplasmic cortactin-labeled spots were found to be associated with endosomal vesicles. Association was asymmetric and approximately half of the endosomes were associated with cortactin spots. Time-lapse imaging suggested that these cortactin and F-actin-containing spots propelled endosomes. Actin polymerization based propulsion may be a common mechanism for endomembrane trafficking in the same manner as used in the plasma membrane protrusions. As cortactin is known to interact with membrane-associated signaling proteins it could have a role in linking signaling complexes with dynamic actin on endosomes and in lamellipodia.

Association of cortactin with dynamic actin in lamellipodia and on endosomal vesicles

Association of cortactin with dynamic actin in lamellipodia and on endosomal vesicles

In growth cones of rat cerebral neurons and human neuroblastoma cells, activation of protein kinase C causes a shift from filopodial to lamellipodial actin dynamics

In cultures of rat cerebral neurons addition of the protein kinase C (PKC) activator 1,2-dioctanoyl-s,n-glycerol (diC8; 5μM) induces a transient elongation of filopodia which is followed by a striking enhancement of lamellar protrusions. After 30–40 min, lamellipodia are slowly retracted, filopodia reappear and become predominant again. The reappearance of filopodia is accelerated by addition of the potent PKC-inhibitor RO-31-8220 (2 μM). A similar transient promotion of lamellar protrusive activity is obtained in SH-SY5Y neuroblastoma cells upon stimulation by acetylcholine or diC8. Immunostainings showed that the new space created by extending actin-driven lamellipodia is rapidly entered by microtubules. Preincubation with or permanent presence of RO-31-8220 totally inhibits both filopodial and lamellipodial protrusive activity. The data suggest that both filopodial and lamellipodial protrusion require active PKC, however of different levels of activation.

Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity.

The organization and polarity of actin filaments in neuronal growth cones was studied with negative stain and freeze-etch EM using a permeabilization protocol that caused little detectable change in morphology when cultured nerve growth cones were observed by video-enhanced differential interference contrast microscopy. The lamellipodial actin cytoskeleton was composed of two distinct subpopulations: a population of 40-100-nm-wide filament bundles radiated from the leading edge, and a second population of branching short filaments filled the volume between the dorsal and ventral membrane surfaces. Together, the two populations formed the three-dimensional structural network seen within expanding lamellipodia. Interaction of the actin filaments with the ventral membrane surface occurred along the length of the filaments via membrane associated proteins. The long bundled filament population was primarily involved in these interactions. The filament tips of either population appeared to interact with the membrane only at the leading edge; this interaction was mediated by a globular Triton-insoluble material. Actin filament polarity was determined by decoration with myosin S1 or heavy meromyosin. Previous reports have suggested that the polarity of the actin filaments in motile cells is uniform, with the barbed ends toward the leading edge. We observed that the actin filament polarity within growth cone lamellipodia is not uniform; although the predominant orientation was with the barbed end toward the leading edge (47-56%), 22-25% of the filaments had the opposite orientation with their pointed ends toward the leading edge, and 19-31% ran parallel to the leading edge. The two actin filament populations display distinct polarity profiles: the longer filaments appear to be oriented predominantly with their barbed ends toward the leading edge, whereas the short filaments appear to be randomly oriented. The different length, organization and polarity of the two filament populations suggest that they differ in stability and function. The population of bundled long filaments, which appeared to be more ventrally located and in contact with membrane proteins, may be more stable than the population of short branched filaments. The location, organization, and polarity of the long bundled filaments suggest that they may be necessary for the expansion of lamellipodia and for the production of tension mediated by receptors to substrate adhesion molecules.

Roles of actin filaments and three second-messenger systems in short-term regulation of chick dorsal root ganglion neurite outgrowth.

In a previous study (J. Cell Biol. 109: 1229-1243, 1989), we reported that conditions which increased growth cone calcium levels and induced neurite retraction in cultured chick DRG neurons also resulted in an apparent loss of actin filaments in the growth cone periphery. We further showed that the actin-stabilizing drug phalloidin could block or reverse calcium-ionophore-induced neurite retraction, indicating that the behavioral changes were mediated, at least in part, by changes in actin filament stability. In this study, we have further characterized the calcium sensitivity of growth cone behavior to identify which features of calcium-induced behavioral effects can be attributed to effects on actin filaments alone, and to assess whether two other second-messenger systems, cAMP and protein kinase C, might influence neurite outgrowth by altering calcium levels or actin stability. The results indicated that growth cone behavior was highly sensitive to small changes in calcium concentrations. Neurite outgrowth was only observed in calcium-permeabilized cells when extracellular calcium concentrations were between 200 and 300 nM, and changes as small as 50 nM commonly produced detectable changes in behavior. Furthermore, low doses of cytochalasins mimicked all of the grossly observable features of growth cone responses to elevation of intracellular calcium, including the apparent preferential destruction of lamellipodial actin filaments and sparing of filopodial actin, suggesting that the behavioral effects of calcium elevation could be explained by loss of actin filaments alone. The effects of cAMP elevation and protein kinase C activation on growth cone behavior, ultrastructure, and fura2-AM-measured calcium levels indicated that the effects of cAMP manipulations could be partially explained by a cAMP-induced lowering of growth cone calcium levels and concomitant increased stabilization of actin filaments, but protein kinase C appeared to act through an independent mechanism.