The actin-binding protein UNC-115/abLIM controls formation of lamellipodia and filopodia and neuronal morphogenesis in Caenorhabditis elegans

The roles of actin-binding proteins in development and morphogenesis are not well understood. The actin-binding protein UNC-115 has been implicated in cytoskeletal signaling downstream of Rac in Caenorhabditis elegans axon pathfinding, but the cellular role of UNC-115 in this process remains undefined. Here we report that UNC-115 overactivity in C. elegans neurons promotes the formation of neurites and lamellipodial and filopodial extensions similar to those induced by activated Rac and normally found in C. elegans growth cones. We show that UNC-115 activity in neuronal morphogenesis is enhanced by two molecular mechanisms: when ectopically driven to the plasma membrane by the myristoylation sequence of c-Src, and by mutation of a putative serine phosphorylation site in the actin-binding domain of UNC-115. In support of the hypothesis that UNC-115 modulates actin cytoskeletal organization, we show that UNC-115 activity in serum-starved NIH 3T3 fibroblasts results in the formation of lamellipodia and filopodia. We conclude that UNC-115 is a novel regulator of the formation of lamellipodia and filopodia in neurons, possibly in the growth cone during axon pathfinding.     

The Arp2/3 activators WAVE and WASP have distinct genetic interactions with Rac GTPases in Caenorhabditis elegans axon guidance

In the developing nervous system, axons are guided to their targets by the growth cone. Lamellipodial and filopodial protrusions from the growth cone underlie motility and guidance. Many molecules that control lamellipodia and filopodia formation, actin organization, and axon guidance have been identified, but it remains unclear how these molecules act together to control these events. Experiments are described here that indicate that, in Caenorhabditis elegans, two WH2-domain-containing activators of the Arp2/3 complex, WVE-1/WAVE and WSP-1/WASP, act redundantly in axon guidance and that GEX-2/Sra-1 and GEX-3/Kette, molecules that control WAVE activity, might act in both pathways. WAVE activity is controlled by Rac GTPases, and data are presented here that suggest WVE-1/WAVE and CED-10/Rac act in parallel to a pathway containing WSP-1/WASP and MIG-2/RhoG. Furthermore, results here show that the CED-10/WVE-1 and MIG-2/WSP-1 pathways act in parallel to two other molecules known to control lamellipodia and filopodia and actin organization, UNC-115/abLIM and UNC-34/Enabled. These results indicate that at least three actin-modulating pathways act in parallel to control actin dynamics and lamellipodia and filopodia formation during axon guidance (WASP-WAVE, UNC-115/abLIM, and UNC-34/Enabled).     

The Rac GTP exchange factor TIAM-1 acts with CDC-42 and the guidance receptor UNC-40/DCC in neuronal protrusion and axon guidance

The mechanisms linking guidance receptors to cytoskeletal dynamics in the growth cone during axon extension remain mysterious. The Rho-family GTPases Rac and CDC-42 are key regulators of growth cone lamellipodia and filopodia formation, yet little is understood about how these molecules interact in growth cone outgrowth or how the activities of these molecules are regulated in distinct contexts. UNC-73/Trio is a well-characterized Rac GTP exchange factor in Caenorhabditis elegans axon pathfinding, yet UNC-73 does not control CED-10/Rac downstream of UNC-6/Netrin in attractive axon guidance. Here we show that C. elegans TIAM-1 is a Rac-specific GEF that links CDC-42 and Rac signaling in lamellipodia and filopodia formation downstream of UNC-40/DCC. We also show that TIAM-1 acts with UNC-40/DCC in axon guidance. Our results indicate that a CDC-42/TIAM-1/Rac GTPase signaling pathway drives lamellipodia and filopodia formation downstream of the UNC-40/DCC guidance receptor, a novel set of interactions between these molecules. Furthermore, we show that TIAM-1 acts with UNC-40/DCC in axon guidance, suggesting that TIAM-1 might regulate growth cone protrusion via Rac GTPases in response to UNC-40/DCC. Our results also suggest that Rac GTPase activity is controlled by different GEFs in distinct axon guidance contexts, explaining how Rac GTPases can specifically control multiple cellular functions.     

Interactions of UNC-34 Enabled with Rac GTPases and the NIK kinase MIG-15 in Caenorhabditis elegans axon pathfinding and neuronal migration

Many genes that affect axon pathfinding and cell migration have been identified. Mechanisms by which these genes and the molecules they encode interact with one another in pathways and networks to control developmental events are unclear. Rac GTPases, the cytoskeletal signaling molecule Enabled, and NIK kinase have all been implicated in regulating axon pathfinding and cell migration. Here we present evidence that, in Caenorhabditis elegans, three Rac GTPases, CED-10, RAC-2, and MIG-2, define three redundant pathways that each control axon pathfinding, and that the NIK kinase MIG-15 acts in each Rac pathway. Furthermore, we show that the Enabled molecule UNC-34 defines a fourth partially redundant pathway that acts in parallel to Rac/MIG-15 signaling in axon pathfinding. Enabled and the three Racs also act redundantly to mediate AQR and PQR neuronal cell migration. The Racs and UNC-34 Ena might all control the formation of actin-based protrusive structures (lamellipodia and filopodia) that mediate growth cone outgrowth and cell migration. MIG-15 does not act with the three Racs in execution of cell migration. Rather, MIG-15 affects direction of PQR neuronal migration, similar to UNC-40 and DPY-19, which control initial Q cell polarity, and Wnt signaling, which acts later to control Q cell-directed migration. MIG-2 Rac, which acts with CED-10 Rac, RAC-2 Rac, and UNC-34 Ena in axon pathfinding and cell migration, also acts with MIG-15 in PQR directional migration.     

The actin-binding protein UNC-115 is an effector of Rac signaling during axon pathfinding in C. elegans

Rac GTPases control cell shape by regulating downstream effectors that influence the actin cytoskeleton. UNC-115, a putative actin-binding protein similar to human abLIM/limatin, has previously been implicated in axon pathfinding. We have discovered the role of UNC-115 as a downstream cytoskeletal effector of Rac signaling in axon pathfinding. We show that unc-115 double mutants with ced-10 Rac, mig-2 Rac or unc-73 GEF but not with rac-2/3 Rac displayed synthetic axon pathfinding defects, and that loss of unc-115 function suppressed the formation of ectopic plasma membrane extensions induced by constitutively-active rac-2 in neurons. Furthermore, we show that UNC-115 can bind to actin filaments. Thus, UNC-115 is an actin-binding protein that acts downstream of Rac signaling in axon pathfinding.     

Arp2/3 is a negative regulator of growth cone translocation

Arp2/3 is an actin binding complex that is enriched in the peripheral lamellipodia of fibroblasts, where it forms a network of short, branched actin filaments, generating the protrusive force that extends lamellipodia and drives fibroblast motility. Although it has been assumed that Arp2/3 would play a similar role in growth cones, our studies indicate that Arp2/3 is enriched in the central, not the peripheral, region of growth cones and that the growth cone periphery contains few branched actin filaments. Arp2/3 inhibition in fibroblasts severely disrupts actin organization and membrane protrusion. In contrast, Arp2/3 inhibition in growth cones minimally affects actin organization and does not inhibit lamellipodia protrusion or de novo filopodia formation. Surprisingly, Arp2/3 inhibition significantly enhances axon elongation and causes defects in growth cone guidance. These results indicate that Arp2/3 is a negative regulator of growth cone translocation.     

SWAN-1, a Caenorhabditis elegans WD repeat protein of the AN11 family, is a negative regulator of Rac GTPase function

Rac GTPases are key regulators of cell shape and cytoskeletal organization. While some regulators of Rac activity are known, such as GTPase-activating proteins (GAPs) that repress Rac activity, other Rac regulators remain to be identified. The novel Caenorhabditis elegans WD-repeat protein SWAN-1 was identified in a yeast two-hybrid screen with the LIM domains of the Rac effector UNC-115/abLIM. SWAN-1 was found to also associate physically with Rac GTPases. The swan-1(ok267) loss-of-function mutation suppressed defects caused by the hypomorphic ced-10(n1993) allele and enhanced ectopic lamellipodia and filopodia formation induced by constitutively active Rac in C. elegans neurons. Furthermore, SWAN-1(+) transgenic expression suppressed the effects of overactive Rac, including ectopic lamellipodia and filopodia formation in C. elegans neurons, ectopic lamellipodia formation in cultured mammalian fibroblasts, and cell polarity and actin cytoskeleton defects in yeast. These studies indicate that SWAN-1 is an inhibitor of Rac GTPase function in cellular morphogenesis and cytoskeletal organization. While broadly conserved across species, SWAN-1 family members show no sequence similarity to previously known Rac inhibitors.     

The Role of Rac1 in the Growth Cone Dynamics and Force Generation of DRG Neurons

We used optical tweezers, video imaging, immunocytochemistry and a variety of inhibitors to analyze the role of Rac1 in the motility and force generation of lamellipodia and filopodia from developing growth cones of isolated Dorsal Root Ganglia neurons. When the activity of Rac1 was inhibited by the drug EHop-016, the period of lamellipodia protrusion/retraction cycles increased and the lamellipodia retrograde flow rate decreased; moreover, the axial force exerted by lamellipodia was reduced dramatically. Inhibition of Arp2/3 by a moderate amount of the drug CK-548 caused a transient retraction of lamellipodia followed by a complete recovery of their usual motility. This recovery was abolished by the concomitant inhibition of Rac1. The filopodia length increased upon inhibition of both Rac1 and Arp2/3, but the speed of filopodia protrusion increased when Rac1 was inhibited and decreased instead when Arp2/3 was inhibited. These results suggest that Rac1 acts as a switch that activates upon inhibition of Arp2/3. Rac1 also controls the filopodia dynamics necessary to explore the environment.     

Arp2/3 complex is important for filopodia formation, growth cone motility, and neuritogenesis in neuronal cells

A role of Arp2/3 complex in lamellipodia is well established, whereas its roles in filopodia formation remain obscure. We addressed this question in neuronal cells, in which motility is heavily based on filopodia, and we found that Arp2/3 complex is involved in generation of both lamellipodia and filopodia in growth cones, and in neuritogenesis, the processes thought to occur largely in Arp2/3 complex-independent manner. Depletion of Arp2/3 complex in primary neurons and neuroblastoma cells by small interfering RNA significantly decreased the F-actin contents and inhibited lamellipodial protrusion and retrograde flow in growth cones, but also initiation and dynamics of filopodia. Using electron microscopy, immunochemistry, and gene expression, we demonstrated the presence of the Arp2/3 complex-dependent dendritic network of actin filaments in growth cones, and we showed that individual actin filaments in filopodia originated at Arp2/3 complex-dependent branch points in lamellipodia, thus providing a mechanistic explanation of Arp2/3 complex functions during filopodia formation. Additionally, Arp2/3 complex depletion led to formation of multiple neurites, erratic pattern of neurite extension, and excessive formation of stress fibers and focal adhesions. Consistent with this phenotype, RhoA activity was increased in Arp2/3 complex-depleted cells, indicating that besides nucleating actin filaments, Arp2/3 complex may influence cell motility by altering Rho GTPase signaling.     

The Caenorhabditis elegans Eph receptor activates NCK and N-WASP, and inhibits Ena/VASP to regulate growth cone dynamics during axon guidance

The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.     

UNC-6/netrin and its receptors UNC-5 and UNC-40/DCC modulate growth cone protrusion in vivo in C. elegans

The UNC-6/netrin guidance cue functions in axon guidance in vertebrates and invertebrates, mediating attraction via UNC-40/DCC family receptors and repulsion via by UNC-5 family receptors. The growth cone reads guidance cues and extends lamellipodia and filopodia, actin-based structures that sense the extracellular environment and power the forward motion of the growth cone. We show that UNC-6/netrin, UNC-5 and UNC-40/DCC modulated the extent of growth cone protrusion that correlated with attraction versus repulsion. Loss-of-function unc-5 mutants displayed increased protrusion in repelled growth cones, whereas loss-of-function unc-6 or unc-40 mutants caused decreased protrusion. In contrast to previous studies, our work suggests that the severe guidance defects in unc-5 mutants may be due to latent UNC-40 attractive signaling that steers the growth cone back towards the ventral source of UNC-6. UNC-6/Netrin signaling also controlled polarity of growth cone protrusion and F-actin accumulation that correlated with attraction versus repulsion. However, filopodial dynamics were affected independently of polarity of protrusion, indicating that the extent versus polarity of protrusion are at least in part separate mechanisms. In summary, we show here that growth cone guidance in response to UNC-6/netrin involves a combination of polarized growth cone protrusion as well as a balance between stimulation and inhibition of growth cone (e.g. filopodial) protrusion.     

Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes

Cell migration is initiated by plasma membrane protrusions, in the form of lamellipodia and filopodia. The latter rod-like projections may exert sensory functions and are found in organisms as distant in evolution as mammals and amoeba such as Dictyostelium discoideum. In mammals, lamellipodia protrusion downstream of the small GTPase Rac1 requires a multimeric protein assembly, the WAVE-complex, which activates Arp2/3-mediated actin filament nucleation and actin network assembly. A current model of filopodia formation postulates that these structures arise from a dendritic network of lamellipodial actin filaments by selective elongation and bundling. Here, we have analyzed filopodia formation in mammalian cells abrogated in expression of essential components of the lamellipodial actin polymerization machinery. Cells depleted of the WAVE-complex component Nck-associated protein 1 (Nap1), and, in consequence, of lamellipodia, exhibited normal filopodia protrusion. Likewise, the Arp2/3-complex, which is essential for lamellipodia protrusion, is dispensable for filopodia formation. Moreover, genetic disruption of nap1 or the WAVE-orthologue suppressor of cAMP receptor (scar) in Dictyostelium was also ineffective in preventing filopodia protrusion. These data suggest that the molecular mechanism of filopodia formation is conserved throughout evolution from Dictyostelium to mammals and show that lamellipodia and filopodia formation are functionally separable.     

Rho family GTPases and neuronal growth cone remodelling: relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid.

Rho family GTPases have been assigned important roles in the formation of actin-based morphologies in nonneuronal cells. Here we show that microinjection of Cdc42Hs and Rac1 promoted formation of filopodia and lamellipodia in N1E-115 neuroblastoma growth cones and along neurites. These actin-containing structures were also induced by injection of Clostridium botulinum C3 exoenzyme, which abolishes RhoA-mediated functions such as neurite retraction. The C3 response was inhibited by coinjection with the dominant negative mutant Cdc42Hs(T17N), while the Cdc42Hs response could be competed by coinjection with RhoA. We also demonstrate that the neurotransmitter acetylcholine (ACh) can induce filopodia and lamellipodia on neuroblastoma growth cones via muscarinic ACh receptor activation, but only when applied in a concentration gradient. ACh-induced formation of filopodia and lamellipodia was inhibited by preinjection with the dominant negative mutants Cdc42Hs(T17N) and Rac1(T17N), respectively. Lysophosphatidic acid (LPA)-induced neurite retraction, which is mediated by RhoA, was inhibited by ACh, while C3 exoenzyme-mediated neurite outgrowth was inhibited by injection with Cdc42Hs(T17N) or Rac1(T17N). Together these results suggest that there is competition between the ACh- and LPA-induced morphological pathways mediated by Cdc42Hs and/or Rac1 and by RhoA, leading to either neurite development or collapse.     

Critical role of Ena/VASP proteins for filopodia formation in neurons and in function downstream of netrin-1

Ena/VASP proteins play important roles in axon outgrowth and guidance. Ena/VASP activity regulates the assembly and geometry of actin networks within fibroblast lamellipodia. In growth cones, Ena/VASP proteins are concentrated at filopodia tips, yet their role in growth cone responses to guidance signals has not been established. We found that Ena/VASP proteins play a pivotal role in formation and elongation of filopodia along neurite shafts and growth cone. Netrin-1-induced filopodia formation was dependent upon Ena/VASP function and directly correlated with Ena/VASP phosphorylation at a regulatory PKA site. Accordingly, Ena/VASP function was required for filopodial formation from the growth cone in response to global PKA activation. We propose that Ena/VASP proteins control filopodial dynamics in neurons by remodeling the actin network in response to guidance cues.     

Transducer of Cdc42-dependent actin assembly promotes epidermal growth factor-induced cell motility and invasiveness

Toca-1 (transducer of Cdc42-dependent actin assembly) interacts with the Cdc42·N-WASP and Abi1·Rac·WAVE F-actin branching pathways that function in lamellipodia formation and cell motility. However, the potential role of Toca-1 in these processes has not been reported. Here, we show that epidermal growth factor (EGF) induces Toca-1 localization to lamellipodia, where it co-localizes with F-actin and Arp2/3 complex in A431 epidermoid carcinoma cells. EGF also induces tyrosine phosphorylation of Toca-1 and interactions with N-WASP and Abi1. Stable knockdown of Toca-1 expression by RNA interference has no effect on cell growth, EGF receptor expression, or internalization. However, Toca-1 knockdown cells display defects in EGF-induced filopodia and lamellipodial protrusions compared with control cells. Further analyses reveal a role for Toca-1 in localization of Arp2/3 and Abi1 to lamellipodia. Toca-1 knockdown cells also display a significant defect in EGF-induced motility and invasiveness. Taken together, these results implicate Toca-1 in coordinating actin assembly within filopodia and lamellipodia to promote EGF-induced cell migration and invasion.     

The acidic regions of WASp and N-WASP can synergize with CDC42Hs and Rac1 to induce filopodia and lamellipodia

The acidic (A) region of WASp family proteins is thought to represent a high-affinity binding site for Arp2/3 complex without activating properties. Here we show that GST-fused WASp-A and N-WASP-A, but not a WASP-A/W500S mutant, several truncated WASp-A constructs or WAVE1-A can pull down Arp2/3 complex from cell lysates. Significantly, WASp-A and N-WASP-A synergistically trigger formation of filopodia or lamellipodia when coinjected with sub-effective concentrations of V12CDC42Hs or V12Rac1, respectively, into macrophages. The ability of WASp family A region constructs to induce this effect is closely correlated with their ability to bind Arp2/3 complex in vitro. These results imply that (i) Arp2/3 complex is critically involved in filopodia and lamellipodia formation in macrophages and (ii) acidic regions of WASp and N-WASP are not simply binding sites for Arp2/3 complex but can prime it for RhoGTPase-triggered signals leading to actin nucleation.     

Distribution and possible interactions of actin-associated proteins and cell adhesion molecules of nerve growth cones

Actin filaments and their interactions with cell surface molecules have key roles in tissue cell behaviour. Axonal pathfinding during embryogenesis, an especially complex cell behaviour, is based on the migration of nerve growth cones. We have used fluorescence immunocytochemistry to examine the distribution in growth cones, their filopodia and lamellipodia of several actin-associated proteins and nerve cell adhesion molecules. The leading margins of chick dorsal root ganglion nerve growth cones and their protrusions stain strongly for f-actin, filamin, alpha-actinin, myosin, tropomyosin, talin and vinculin. MAP2 is absent from DRG growth cones, and staining for spectrin fodrin extends into growth cones, but not along filopodia. Thus, organization of the leading margins of growth cones may strongly resemble the leading lamella of migrating fibroblasts. The adhesion-mediating molecules integrin, L1, N-CAM and A-CAM are all found on DRG neurites and growth cones. However, filopodia stain relatively more strongly for integrin and L1 than for A-CAM or N-CAM. In fact, the 180 X 10(3) Mr form of N-CAM may be absent from most of the length of filopodia. DRG neurones cultured in cytochalasin B display differences in immunofluorescence staining which further emphasize that these adhesion molecules interact differentially with the actin filament system of migrating growth cones. Several models for neuronal morphogenesis emphasize the importance of regulation of the expression of adhesion molecules. Our results support hypotheses that cellular distribution and transmembrane interactions are key elements in the functions of these adhesion molecules during axonal pathfinding.     

Rac1-dependent actin filament organization in growth cones is necessary for β1-integrin–mediated advance but not for growth on poly-D-lysine

The activity of filopodia and lamellipodia determines the advance, motility, adhesion, and sensory capacity of neuronal growth cones. The shape and dynamics of these highly motile structures originate from the continuous reorganization of the actin cytoskeleton in response to extracellular signals. The small GTPases, Rac1, Rho, and CDC42, regulate the organization of actin filament structures in nonneuronal cells; yet, their role in growth cone motility and neurite outgrowth is poorly understood. We investigated in vitro the function of Rac1 in neurite outgrowth and differentiation by introducing purified recombinant mutants of Rac1 into primary chick embryo motor neurons via trituration. Endogenous Rac1 was expressed in growth cone bodies as well as in the tips and shafts of filopodia, where it often colocalized with actin filament structures. The introduction of constitutively active Rac1 resulted in an increase in rhodamine–phalloidin staining, presumably from an accumulation of actin filaments in growth cones, while dominant negative Rac1 caused a decrease in rhodamine–phalloidin staining. Nevertheless, both Rac1 mutants retarded growth cone advance, and hence attenuated neurite outgrowth and inhibited differentiation of neurites into axons and dendrites on laminin and fibronectin. In contrast, on poly-D -lysine, neither Rac1 mutant affected growth cone advance, neurite outgrowth, or neurite differentiation despite inducing similar changes in the amount of rhodamine–phalloidin staining in growth cones. Our data demonstrate that Rac1 regulates actin filament organization in neuronal growth cones and is pivotal for β1 integrin–mediated growth cone advance, but not for growth on poly-D lysine. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 524–540, 1998     

Macondo crude oil from the Deepwater Horizon oil spill disrupts specific developmental processes during zebrafish embryogenesis

Background

During nerve growth, cytoplasmic vesicles add new membrane preferentially to the growth cone located at the distal tip of extending axons. Growth cone membrane is also retrieved locally, and asymmetric retrieval facilitates membrane remodeling during growth cone repulsion by a chemorepellent gradient. Moreover, growth inhibitory factors can stimulate bulk membrane retrieval and induce growth cone collapse. Despite these functional insights, the processes mediating local membrane remodeling during axon extension remain poorly defined.

Results

To investigate the spatial and temporal dynamics of membrane retrieval in actively extending growth cones, we have used a transient labeling and optical recording method that can resolve single vesicle events. Live-cell confocal imaging revealed rapid membrane retrieval by distinct endocytic modes based on spatial distribution in Xenopus spinal neuron growth cones. These modes include endocytic "hot-spots" triggered at the base of filopodia, at the lateral margins of lamellipodia, and along dorsal ridges of the growth cone. Additionally, waves of endocytosis were induced when individual filopodia detached from the substrate and fused with the growth cone dorsal surface or with other filopodia. Vesicle formation at sites of membrane remodeling by self-contact required F-actin polymerization. Moreover, bulk membrane retrieval by macroendocytosis correlated positively with the substrate-dependent rate of axon extension and required the function of Rho-family GTPases.

Conclusions

This study provides insight into the dynamic membrane remodeling processes essential for nerve growth by identifying several distinct modes of rapid membrane retrieval in the growth cone during axon extension. We found that endocytic membrane retrieval is intensified at specific subdomains and may drive the dynamic membrane ruffling and re-absorption of filopodia and lamellipodia in actively extending growth cones. The findings offer a platform for determining the molecular mechanisms of distinct endocytic processes that may remodel the surface distribution of receptors, ion channels and other membrane-associated proteins locally to drive growth cone extension and chemotactic guidance.