During development, cells undergo dynamic morphological changes by rearrangements of the cytoskeleton including microtubules. However, molecular mechanisms underlying the microtubule remodeling between orientated and disoriented formations are almost unknown. Here we found that novel subtypes of collapsin response mediator proteins (CRMP-As) and the originals (CRMP-Bs), which occur from the alternative usage of different first coding exons, are involved in this conversion of microtubule patterns. Overexpression of CRMP2A and CRMP2B in chick embryonic fibroblasts induced orientated and disoriented patterns of microtubules, respectively. Moreover, sequential overexpression of another subtype overcame the effect of the former expression of the countersubtype. Overexpression experiments in cultured chick retinae showed that CRMP2B promoted axon branching and suppressed axon elongation of ganglion cells, while CRMP2A blocked these effects when co-overexpressed. Our findings suggest that the opposing activities of CRMP2A and CRMP2B contribute to the cellular morphogenesis including neuronal axonogenesis through remodeling of microtubule organization.

The CRMP (collapsin response mediator protein) family is thought to play key roles in growth cone guidance during neural development. The four members (CRMP1-4) identified to date have been demonstrated to form hetero-multimeric structures through mutual associations. In this study, we cloned a novel member of this family, which we call CRMP5, by the yeast twohybrid method. This protein shares relatively low amino acid identity with the other CRMP members (49 -50%) and also with dihydropyrimidinase (51%), whereas CRMP1-4 exhibit higher identity with each other (68 -75%), suggesting that CRMP5 might be categorized into a third subfamily. The mouse CRMP5 gene was located at chromosome 5 B1. Northern blot and in situ hybridization analyses indicated that CRMP5 is expressed throughout the nervous system similarly to the other members (especially CRMP1 and CRMP4) with the expression peak in the first postnatal week. Association experiments using the yeast two-hybrid method and coimmunoprecipitation showed that CRMP5 interacts with dihydropyrimidinase and all the CRMPs including itself, except for CRMP1, although the expression profile almost overlaps with that of CRMP1 during development. These results suggest that CRMP complexes in the developing nervous system are classifiable into two populations that contain either CRMP1 or CRMP5. This indicates that different complexes may have distinct functions in shaping the neural networks.

Slit regulates migration of not only neurons, but also nonneuronal cells, such as leukocytes and cancer cells. Slit effect on cancer cell migration has not been well-characterized. In this study, we used several different assays to examine Slit effect on breast cancer cell migration in vitro. We show that ubiquitin-specific protease 33 (USP33)/VDU1, originally identified as a von Hippel-Lindau tumor suppressor (VHL) protein-interacting deubiquitinating enzyme, binds to the Robo1 receptor, and that USP33 is required for Slit responsiveness in breast cancer cells. Slit induces redistribution of Robo1 from intracellular compartments to the plasma membrane in a USP33-dependent manner. Slit impairs directional migration of breast cancer cells without affecting their migration speed. This inhibitory effect is Robo-mediated and USP33-dependent. These data uncover a previously unknown function of USP33 and reveal a new player in Slit-Robo signaling in cancer cell migration.cell migration and motility ͉ metastasis ͉ Slit-Robo signaling C ell migration is a fundamental process critical for not only embryonic development but also homeostasis in adult animals. A number of molecular cues guide axons and migrating neurons (1-4). Recent studies suggest that molecular mechanisms modulating migration of cells in different tissues/organs are conserved. For example, guidance cues, receptors, and the intracellular signaling pathways for neuronal migration are also used for cells outside of the nervous system, ranging from immune cells, myoblasts, and endothelial cells to tumor cells (4-7). The Slit gene was first identified in Drosophila, and subsequent studies indicated that secreted proteins of the Slit family and their receptors of the Roundabout (Robo) family play important roles in neuronal guidance (8-13). The Slit genes are frequently inactivated in cancer (14-19).Accumulating evidence supports that chemokines and their receptors play important roles in tumorigenesis and cancer metastasis, including chemotactic invasion and migration of cancer cells (20,21). In some cases, cancer cells show increased expression of chemokine receptors not expressed in normal nontumor cells, providing a plausible explanation for distant metastasis to organs that secrete corresponding chemokine ligands (22). Studies from our group and other groups have shown that Slit suppresses chemokine-directed chemotaxis of leukocytes and breast cancer cells (23-25) and inhibits medulloblastoma cell invasion (26). These observations suggest a potential therapeutic strategy in controlling aberrant cell migration during cancer metastasis. Cellular and molecular mechanisms underlying Slit signaling in cancer cells remain to be elucidated.To dissect the Slit-Robo signaling pathway, we carried out yeast two-hybrid screens by using the intracellular domain of Robo1 as bait (27). From such screening, we have identified ubiquitin-specific protease 33 (USP33)/von Hippel-Lindau tumor suppressor protein (pVHL)-interacting deubiquitinating enzyme 1 (VDU1) as a protein ...

Commissural axons cross the ventral midline of the neural tube in a Slit-dependent manner. The underlying molecular mechanisms remain to be elucidated. Here we report that the deubiquitinating enzyme USP33 interacts with the Robo1 receptor. USP33 is essential for midline crossing by commissural axons and for their response to Slit. Our results reveal a previously unknown role of USP33 in vertebrate commissural axon guidance and in Slit signaling.

Collapsin response mediator proteins (CRMPs) have been implicated in signaling of axonal guidance, including semaphorins. We have previously identified a unique member of this gene family, CRMP-associated molecule CRAM (CRMP-5), which is phylogenetically divergent from the other four CRMPs. In this study, we have examined the distribution and function of CRAM in developing neurons. Immunohistochemical analysis showed accumulation of CRAM in the filopodia of growth cones. Experiments using cytochalasin D indicated that filopodial localization of CRAM was independent of filamentous actin. Overexpression of CRAM in neuronal cells significantly promoted filopodial growth and led to the formation of supernumerary growth cones, which acquired resistance to semaphorin-3A stimulation. Finally, knockdown of CRAM by using RNA interference blocked filopodial formation and revealed an aberrant morphology of growth cones. We propose that CRAM regulates filopodial dynamics and growth cone development, thereby restricting the response of growth cone to repulsive guidance cues.

Intracellular vesicular transport is important for photoreceptor function and maintenance. However, the mechanism underlying photoreceptor degeneration in response to vesicular transport defects is unknown. Here, we report that photoreceptors undergo apoptosis in a zebrafish β-soluble N-ethylmaleimide-sensitive factor attachment protein (β-SNAP) mutant. β-SNAP cooperates with N-ethylmaleimide-sensitive factor to recycle the SNAP receptor (SNARE), a key component of the membrane fusion machinery, by disassembling the cis-SNARE complex generated in the vesicular fusion process. We found that photoreceptor apoptosis in the β-SNAP mutant was dependent on the BH3-only protein BNip1. BNip1 functions as a component of the syntaxin-18 SNARE complex and regulates retrograde transport from the Golgi to the endoplasmic reticulum. Failure to disassemble the syntaxin-18 cis-SNARE complex caused BNip1-dependent apoptosis. These data suggest that the syntaxin-18 cis-SNARE complex functions as an alarm factor that monitors vesicular fusion competence and that BNip1 transforms vesicular fusion defects into photoreceptor apoptosis.

A switch in the response of commissural axons to the repellent Slit is crucial for ensuring that they cross the ventral midline only once. However, the underlying mechanisms remain to be elucidated. We have found that both endocytosis and recycling of Robo1 receptor are crucial for modulating Slit sensitivity in vertebrate commissural axons. Robo1 endocytosis and its recycling back to the cell surface maintained the stability of axonal Robo1 during Slit stimulation. We identified Arf6 guanosine triphosphatase and its activators, cytohesins, as previously unknown components in Slit-Robo1 signalling in vertebrate commissural neurons. Slit-Robo1 signalling activated Arf6. The Arf6-deficient mice exhibited marked defects in commissural axon midline crossing. Our data showed that a Robo1 endocytosis-triggered and Arf6-mediated positive-feedback strengthens the Slit response in commissural axons upon their midline crossing. Furthermore, the cytohesin-Arf6 pathways modulated this self-enhancement of the Slit response before and after midline crossing, resulting in a switch that reinforced robust regulation of axon midline crossing. Our study provides insights into endocytic trafficking-mediated mechanisms for spatiotemporally controlled axonal responses and uncovers new players in the midline switch in Slit responsiveness of commissural axons.

To understand the molecular basis of topographic retinotectal projection, an overall view of the asymmetrically expressed molecules in the developing retina is needed. We performed a large-scale screening using restriction landmark cDNA scanning (RLCS) in the embryonic day 8 (E8) chick retina. RLCS is a cDNA display system, in which a large number of cDNA species are displayed as two-dimensional spots with intensities reflecting their expression levels as mRNA. We searched for spots that gave different signal intensities between the nasal and temporal retinas or between the dorsal and ventral retinas, and detected about 200 spots that were preferential on one side in the retina. The asymmetric expression of each gene was verified by Northern blotting and in situ hybridization. By subsequent analyses using molecular cloning, DNA sequencing, and database searching, 33 asymmetric molecules along the nasotemporal (N-T) axis and 20 along the dorsoventral (D-V) axis were identified. These included transcription factors, secretory factors, transmembrane proteins, and intracellular proteins with various putative functions. Their expression profiles revealed by in situ hybridization are highly diverse and individual. Moreover, many of them begin to be expressed in the retina from the early developmental stages, suggesting that they are implicated in the establishment and maintenance of regional specificity in the developing retina. The molecular repertoire revealed by this work will provide candidates for future studies to elucidate the molecular mechanisms of topographic retinotectal map formation.