Erik A. Lundquist

The Netrin receptor UNC-40/DCC stimulates axon attraction and outgrowth through Enabled and, in parallel, rac and UNC-115/AbLIM

Netrins promote axon outgrowth and turning through DCC/UNC-40 receptors. To characterize Netrin signaling, we generated a gain-of-function UNC-40 molecule, MYR::UNC-40. MYR::UNC-40 causes axon guidance defects, excess axon branching, and excessive axon and cell body outgrowth. These defects are suppressed by loss-of-function mutations in ced-10 (a Rac GTPase), unc-34 (an Enabled homolog), and unc-115 (a putative actin binding protein). ced-10, unc-34, and unc-115 also function in endogenous unc-40 signaling. Our results indicate that Enabled functions in axonal attraction as well as axon repulsion. UNC-40 has two conserved cytoplasmic motifs that mediate distinct downstream pathways: CED-10, UNC-115, and the UNC-40 P2 motif act in one pathway, and UNC-34 and the UNC-40 P1 motif act in the other. Thus, UNC-40 might act as a scaffold to deliver several independent signals to the actin cytoskeleton.

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.

Rac proteins and the control of axon development

Rac GTPases and their effectors control cellular morphogenesis in a wide range of developmental contexts by regulating the structure and dynamics of the actin cytoskeleton. Although much is known about the biochemistry of Racs and Rac regulators, less is known about how Racs control cellular morphogenesis, including axon development, in vivo. Recent loss-of-function genetic studies using model organisms have shown that Racs and their effectors are required for multiple aspects of axon development, including axon outgrowth, axon guidance and axon branching. Interestingly, these studies have also revealed that Rac activity is required to prune spurious axons and branches. Analyses of Racs and their upstream and downstream effectors suggest that Rac signaling is complex. Different neurons utilize distinct combinations of upstream Rac regulators during axon development, possibly reflecting responses to different axon path-finding signals, and Racs use distinct downstream effectors to mediate different aspects of axon development, possibly reflecting differential regulation of the lamellipodial and filopodial growth-cone actin-cytoskeleton domains underlying axon developmental events.

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 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 Arp2/3 complex, UNC-115/abLIM, and UNC-34/Enabled regulate axon guidance and growth cone filopodia formation in Caenorhabditis elegans

BackgroundWhile many molecules involved in axon guidance have been identified, the cellular and molecular mechanisms by which these molecules regulate growth cone morphology during axon outgrowth remain to be elucidated. The actin cytoskeleton of the growth cone underlies the formation of lamellipodia and filopodia that control growth cone outgrowth and guidance. The role of the Arp2/3 complex in growth cone filopodia formation has been controversial, and other mechanisms of growth cone filopodia formation remain to be described.ResultsHere we show that mutations in genes encoding the Arp2/3 complex (arx genes) caused defects in axon guidance. Analysis of developing growth cones in vivo showed that arx mutants displayed defects in filopodia and reduced growth cone size. Time-lapse analysis of growth cones in living animals indicated that arx mutants affected the rate of growth cone filopodia formation but not filopodia stability or length. Two other actin modulatory proteins, UNC-115/abLIM and UNC-34/Enabled, that had been shown previously to affect axon guidance had overlapping roles with Arp2/3 in axon guidance and also affected the rate of filopodia initiation but not stability or length.ConclusionOur results indicate that the Arp2/3 complex is required cell-autonomously for axon guidance and growth cone filopodia initiation. Furthermore, they show that two other actin-binding proteins, UNC-115/abLIM and UNC-34/Enabled, also control growth cone filopodia formation, possibly in parallel to Arp2/3. These studies indicate that, in vivo, multiple actin modulatory pathways including the Arp2/3 complex contribute to growth cone filopodia formation during growth cone outgrowth.

Mutationally activated Rho GTPases in cancer

The Rho family of GTPases (members of the Ras superfamily) are best known for their roles in regulating cytoskeletal dynamics. It is also well established that misregulation of Rho proteins contributes to tumorigenesis and metastasis. Unlike Ras proteins, which are frequently mutated in cancer (around 30%), Rho proteins themselves are generally not found to be mutated in cancer. Rather, misregulation of Rho activity in cancer was thought to occur by overexpression of these proteins or by misregulation of molecules that control Rho activity, such as activation or overexpression of GEFs and inactivation or loss of GAPs or GDIs. Recent studies, enabled by next-generation tumor exome sequencing, report activating point mutations in Rho GTPases as driver mutations in melanoma, as well as breast, and head and neck cancers. The Rac1(P29L) mutation identified in these tumor studies was previously identified by our lab as an activating Rac mutation in C. elegans neuronal development, highlighting the conserved nature of this mutation. Furthermore, this finding supports the relevance of studying Rho GTPases in model organisms such as C. elegans to study the mechanisms that underlie carcinogenesis. This review will describe the recent findings that report activating Rho mutations in various cancer types, moving Rho GTPases from molecules misregulated in cancer to mutagenic targets that drive tumorigenesis.

The MIG-15 NIK kinase acts cell-autonomously in neuroblast polarization and migration in C. elegans

Cell migration is a fundamental process in animal development, including development of the nervous system. In C. elegans, the bilateral QR and QL neuroblasts undergo initial anterior and posterior polarizations and migrations before they divide to produce neurons. A subsequent Wnt signal from the posterior instructs QL descendants to continue their posterior migration. Nck-interacting kinases (NIK kinases) have been implicated in cell and nuclear migration as well as lamellipodia formation. Studies here show that the C. elegans MIG-15 NIK kinase controls multiple aspects of initial Q cell polarization, including the ability of the cells to polarize, to maintain polarity, and to migrate. These data suggest that MIG-15 acts independently of the Wnt signal that controls QL descendant posterior migration. Furthermore, MIG-15 affects the later migrations of neurons generated from Q cell division. Finally, a mosaic analysis indicates that MIG-15 acts cell-autonomously in Q descendant migration.

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.

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.