The Ror-family receptor tyrosine kinases (RTKs) play crucial roles in the development of various organs and tissues. In mammals, Ror2, a member of the Ror-family RTKs, has been shown to act as a receptor or coreceptor for Wnt5a to mediate noncanonical Wnt signaling. Ror2-and Wnt5a-deficient mice exhibit similar abnormalities during developmental morphogenesis, reflecting their defects in convergent extension movements and planar cell polarity STRUCTURE OF THE ROR-FAMILY RECEPTOR TYROSINE KINASES (RTKS)The Ror-family of RTKs are type I transmembrane protein tyrosine kinases characterized by their extracellular Frizzled-like cysteine-rich domains (CRDs) and membrane-proximal Kringle domains (Fig. 1)

Growth cone motility and morphology are based on actin-filament dynamics. Cofilin plays an essential role for the rapid turnover of actin filaments by severing and depolymerizing them. The activity of cofilin is repressed by phosphorylation at Ser3 by LIM kinase (LIMK, in which LIM is an acronym of the three gene products Lin-11, Isl-1, and Mec-3) and is reactivated by dephosphorylation by phosphatases, termed Slingshot (SSH). We investigated the roles of cofilin, LIMK, and SSH in the growth cone motility and morphology and neurite extension by expressing fluorescence protein-labeled cofilin, LIMK1, SSH1, or their mutants in chick dorsal root ganglion (DRG) neurons and then monitoring live images of growth cones by time-lapse video fluorescence microscopy. The expression of LIMK1 remarkably repressed growth cone motility and neurite extension, whereas the expression of SSH1 or a nonphosphorylatable S3A mutant of cofilin enhanced these events. The fan-like shape of growth cones was disorganized by the expression of any of these proteins. The repressive effects on growth cone behavior by LIMK1 expression were significantly rescued by the coexpression of S3A-cofilin or SSH1. These findings suggest that LIMK1 and SSH1 play critical roles in controlling growth cone motility and morphology and neurite extension by regulating the activity of cofilin and may be involved in signaling pathways that regulate stimulus-induced growth cone guidance. Using various mutants of cofilin, we also obtained evidence that the actin-filament-severing activity of cofilin is critical for growth cone motility and neurite extension.

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The regulation of actin cytoskeleton dynamics plays a fundamental role in cell shape change, motility, and migration in response to stimuli. In neurons, actin filaments accumulate at the distal tip of the growth cone in the growing neurite, and actin filament dynamics and reorganization are essential for controlling growth cone motility and morphology and determining the direction and speed of neurite extension (1-4). Cofilin and its closely related protein, actin depolymerizing factor (ADF), 2 are key mediators of actin filament dynamics that act by stimulating the depolymerization and severing of actin filaments (5). The activities of cofilin and ADF are inhibited by phosphorylation at Ser-3 by LIM kinases (LIMKs, composed of LIMK1 and LIMK2) (6, 7) and TES kinases (TESKs, composed of TESK1 and TESK2) (8); the inactive Ser-3-phosphorylated cofilin and ADF (P-cofilin/P-ADF) are reactivated by dephosphorylation by Slingshot (SSH) family protein phosphatases (SSH1, SSH2, and SSH3) (9, 10) and chronophin (a haloacid dehalogenase) (11). Because cofilin/ADF and their upstream regulators, LIMKs and SSHs, are abundant in neuronal growth cones (12-15), these proteins have been implicated in the control of neurite extension and guidance through regulating actin filament dynamics.Neurotrophins are known to regulate neurite outgrowth and guidance (2). Rat pheochromocytoma PC12 cells have been often used as a model system to investigate nerve growth factor (NGF)-induced neurite outgrowth. NGF induces cofilin/ADF dephosphorylation in PC12 cells (16), and overexpression of cofilin/ADF or SSH1 enhances neurite extension from PC12 cells and primary cultured neurons, such as chick dorsal root ganglion (DRG) or rat cortical neurons (13,17). In contrast, overexpression of LIMK1 in neurons suppresses growth cone motility and extension (13). In addition, the growth cone collapse induced by semaphorin-3A (a repulsive guidance molecule) or Nogo-66 (a myelin-associated inhibitor of axon regeneration) requires transient activation of LIMK1 and cofilin phosphorylation in chick DRG neurons (18,19). These results suggest that LIMK1 acts as a negative regulator of neurite outgrowth by inhibiting cofilin/ADF activity. However, recent studies have suggested that LIMK1 has a seemingly opposite function on neurite outgrowth: neurite extension from hippocampal neurons was enhanced by LIMK1 expression and suppressed by blockade of LIMK1 activation (14, 20 -22). Thus, further studies are required to understand the role of LIMK1 in neurite extension.In this study, we examined the roles of cofilin/ADF and its phosphoregulation in neurite extension by knocking down the expression of cofilin/ADF, LIMK1/LIMK2, and SSH1/SSH2 using small interfering RNAs (siRNAs). Knockdown of cofilin/ ADF markedly blocked NGF-induced neurite extension of PC12 cells, and knockdown of SSH1/SSH2 suppressed NGFinduced cofilin/ADF dephosphorylation and neurite extension, indicating that SSH1/SSH2-mediated cofilin/ADF dephosphorylation is crucial for neurite extension. LIMK1...

We conclude that the work equivalent of W' is not affected by power variations during exhausting cycle ergometry, at least in the P range of 100-134% of CP.