L1 interaction with ankyrin regulates mediolateral topography in the retinocollicular projection
ABSTRACT Dynamic modulation of adhesion provided by anchorage of axonal receptors with the cytoskeleton contributes to attractant or repellent responses that guide axons to topographic targets in the brain. The neural cell adhesion molecule L1 engages the spectrin-actin cytoskeleton through reversible linkage of its cytoplasmic domain to ankyrin. To investigate a role for L1 association with the cytoskeleton in topographic guidance of retinal axons to the superior colliculus, a novel mouse strain was generated by genetic knock-in that expresses an L1 point mutation (Tyr1229His) abolishing ankyrin binding. Axon tracing revealed a striking mistargeting of mutant ganglion cell axons from the ventral retina, which express high levels of ephrinB receptors, to abnormally lateral sites in the contralateral superior colliculus, where they formed multiple ectopic arborizations. These axons were compromised in extending interstitial branches in the medial direction, a normal response to the high medial to low lateral SC gradient of ephrinB1. Furthermore, ventral but not dorsal L1(Y1229H) retinal cells were impaired for ephrinB1-stimulated adhesion through beta1 integrins in culture. The retinocollicular phenotype of the L1(Tyr1229His) mutant provides the first evidence that L1 regulates topographic mapping of retinal axons through adhesion mediated by linkage to the actin cytoskeleton and functional interaction with the ephrinB/EphB targeting system.
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ABSTRACT: Background Members of the Semaphorin 3 family (Sema3s) influence the development of the central nervous system, and some are implicated in regulating aspects of visual system development. However, we lack information about the timing of expression of the Sema3s with respect to different developmental epochs in the mammalian visual system. In this time-course study in the rat, we document for the first time changes in the expression of RNAs for the majority of Class 3 Semaphorins (Sema3s) and their receptor components during the development of the rat retina and superior colliculus (SC).ResultsDuring retinal development, transcript levels changed for all of the Sema3s examined, as well as Nrp2, Plxna2, Plxna3, and Plxna4a. In the SC there were also changes in transcript levels for all Sema3s tested, as well as Nrp1, Nrp2, Plxna1, Plxna2, Plxna3, and Plxna4a. These changes correlate with well-established epochs, and our data suggest that the Sema3s could influence retinal ganglion cell (RGC) apoptosis, patterning and connectivity in the maturing retina and SC, and perhaps guidance of RGC and cortical axons in the SC. Functionally we found that SEMA3A, SEMA3C, SEMA3E, and SEMA3F proteins collapsed purified postnatal day 1 RGC growth cones in vitro. Significantly this is a developmental stage when RGCs are growing into and within the SC and are exposed to Sema3 ligands.Conclusion These new data describing the overall temporal regulation of Sema3 expression in the rat retina and SC provide a platform for further work characterising the functional impact of these proteins on the development and maturation of mammalian visual pathways.BMC Developmental Biology 07/2014; 14(1):34. DOI:10.1186/s12861-014-0034-9 · 2.75 Impact Factor
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ABSTRACT: NrCAM (Neuron-glial related cell adhesion molecule), a member of the L1 family of cell adhesion molecules, reversibly binds ankyrin and regulates axon growth, but it has not been studied for a role in retinotopic mapping. During development of retino-collicular topography, NrCAM was expressed uniformly in retinal ganglion cells (RGCs) along both mediolateral and anteroposterior retinal axes, and was localized on RGC axons within the optic tract and superior colliculus (SC). Anterograde tracing of RGC axons in NrCAM null mutant mice at P10, when the map resembles its mature form, revealed laterally displaced ectopic termination zones (eTZs) of axons from the temporal retina, indicating defective mediolateral topography, which is governed by ephrinB/EphBs. Axon tracing at P2 revealed that interstitial branch orientation of ventral-temporal RGC axons in NrCAM null mice was compromised in the medial direction, likely accounting for displacement of eTZs. A similar retinocollicular targeting defect in EphB mutant mice suggested that NrCAM and EphB interact to regulate mediolateral retino-collicular targeting. We found that EphB2 tyrosine kinase but not an EphB2 kinase dead mutant, phosphorylated NrCAM at a conserved tyrosine residue in the FIGQY ankyrin binding motif, perturbing ankyrin recruitment in NrCAM transfected HEK293 cells. Furthermore, the phosphorylation of NrCAM at FIGQY in SC was decreased in EphB1/3 and EphB1/2/3 null mice compared to WT, while phospho-FIGQY of NrCAM in SC was increased in EphB2 constitutively active (F620D/F620D) mice. These results demonstrate that NrCAM contributes to mediolateral retinocollicular axon targeting by regulating RGC branch orientation through a likely mechanism in which ephrinB/EphB phosphorylates NrCAM to modulate linkage to the actin cytoskeleton.PLoS ONE 09/2013; 8(9):e73000. DOI:10.1371/journal.pone.0073000 · 3.53 Impact Factor
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ABSTRACT: Ribonucleoprotein (RNP) complexes are widespread in nature and play crucial roles in gene regulation, RNA processing, and translation. Novel technologies, such as CRISPR-mediated genome engineering, stress the potential of RNP complexes to carry out complex tasks in molecular biology. Here we report a bottom-up approach for the programmable self-assembly of RNP complexes. The building blocks for RNP complex formation are RNAs and Pumilio proteins that can bind to RNA sequence-specifically. Correct RNP assembly triggers protein complementation of a tripartite GFP, thereby resulting in up to 25-fold increased fluorescence, and is strictly dependent on the correct RNA sequences. Our results indicate that Pumilio and guide RNAs are suitable building blocks for the correct self-assembly of RNP complexes consisting of up to six different components. Self-assembling RNP complexes might prove useful for complex biotechnological applications in RNA sensing, imaging, or processing.Chemistry - An Asian Journal 08/2014; 9(8). DOI:10.1002/asia.201402220 · 3.94 Impact Factor