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The centrosomal E3 ubiquitin ligase FBXO31-SCF regulates neuronal morphogenesis and migration.

Vadhvani M, Schwedhelm-Domeyer N, Mukherjee C, Stegmüller J - PLoS ONE (2013)

Bottom Line: In addition, we identified the polarity protein Par6c as a novel interaction partner and substrate targeted for proteasomal degradation in the control of axon but not dendrite growth.Finally, we ascribe a role for FBXO31 in dendrite growth and neuronal migration in the developing cerebellar cortex.Taken together, we uncovered the centrosomal E3 ligase FBXO31-SCF as a novel regulator of neuronal development.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany.

ABSTRACT
Neuronal development requires proper migration, polarization and establishment of axons and dendrites. Growing evidence identifies the ubiquitin proteasome system (UPS) with its numerous components as an important regulator of various aspects of neuronal development. F-box proteins are interchangeable subunits of the Cullin-1 based E3 ubiquitin ligase, but only a few family members have been studied. Here, we report that the centrosomal E3 ligase FBXO31-SCF (Skp1/Cullin-1/F-box protein) regulates neuronal morphogenesis and axonal identity. In addition, we identified the polarity protein Par6c as a novel interaction partner and substrate targeted for proteasomal degradation in the control of axon but not dendrite growth. Finally, we ascribe a role for FBXO31 in dendrite growth and neuronal migration in the developing cerebellar cortex. Taken together, we uncovered the centrosomal E3 ligase FBXO31-SCF as a novel regulator of neuronal development.

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FBXO31 regulates dendrite growth and neuronal migration in the developing cerebellum. A.HEK 293T cell lysates transfected with mycFBXO31 along with control or bi-cistronic FBXO31 RNAi #1/CMV-GFP plasmids were probed with α-myc antibody. 14-3-3ß served as a loading control. B. Snapshots of 3D-reconstructed cerebellar granule neurons from rat pups electroporated with control plasmid or with FBXO31 RNAi #1/CMV-GFP bi-cistronic plasmid at P4 and analyzed at P9. Arrows indicate dendrites and arrowheads indicate axons of granule neurons. Scale bar equals 50 µm. C. Histogram showing dendrite length measurements for control or FBXO31 knockdown neurons. A total of 84 neurons were analyzed for dendrite length measurements (n = 3, mean±SEM, unpaired t-test, ***p<0.001). D. Coronal sections of rat pup cerebellum electroporated as described in Figure 4B. IGL = internal granular layer, ML = molecular layer, EGL = external granular layer. Scale bar equals 50 µm. E. Histogram showing percentage of migrated neurons in EGL, ML or IGL. A total of 3637 neurons were analyzed. (n = 3, mean±SEM, two-way ANOVA ***p<0.001, n.s. = not significant). F. Histogram showing distance of granule neuron cell bodies from the pial surface. A total of 681 neurons were analyzed. (n = 3, mean±SEM, two-way ANOVA ***p<0.001).
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pone-0057530-g006: FBXO31 regulates dendrite growth and neuronal migration in the developing cerebellum. A.HEK 293T cell lysates transfected with mycFBXO31 along with control or bi-cistronic FBXO31 RNAi #1/CMV-GFP plasmids were probed with α-myc antibody. 14-3-3ß served as a loading control. B. Snapshots of 3D-reconstructed cerebellar granule neurons from rat pups electroporated with control plasmid or with FBXO31 RNAi #1/CMV-GFP bi-cistronic plasmid at P4 and analyzed at P9. Arrows indicate dendrites and arrowheads indicate axons of granule neurons. Scale bar equals 50 µm. C. Histogram showing dendrite length measurements for control or FBXO31 knockdown neurons. A total of 84 neurons were analyzed for dendrite length measurements (n = 3, mean±SEM, unpaired t-test, ***p<0.001). D. Coronal sections of rat pup cerebellum electroporated as described in Figure 4B. IGL = internal granular layer, ML = molecular layer, EGL = external granular layer. Scale bar equals 50 µm. E. Histogram showing percentage of migrated neurons in EGL, ML or IGL. A total of 3637 neurons were analyzed. (n = 3, mean±SEM, two-way ANOVA ***p<0.001, n.s. = not significant). F. Histogram showing distance of granule neuron cell bodies from the pial surface. A total of 681 neurons were analyzed. (n = 3, mean±SEM, two-way ANOVA ***p<0.001).

Mentions: Finally, we explored the role of FBXO31 in the context of the developing cerebellum. We generated a bicistronic plasmid encoding the small hairpin RNA, we have previously used in cultured neurons, and a GFP cassette. We verified the efficient knockdown of FBXO31 by this bicistronic plasmid in heterologous cells (Figure 6A). We then electroporated the cerebella of P4 rat pups and isolated the tissue 5 days later. In coronal sections of P9 cerebella, we assessed dendrite growth of the GFP-positive neurons in the internal granular layer and found a reduction in total dendritic length in FBXO31 knockdown condition as compared to control (Figure 6B, 6C). Due to technical limitations, it is not feasible to measure axons as they fasciculate in the molecular layer into untraceable fibers. Strikingly, we also found that while more than 80% of transfected control neurons descend rapidly into the internal granular layer, nearly 50% of FBXO31 RNAi neurons fail to migrate and remain in the external granule layer/molecular layer (Figure 6D, 6E). We further quantified the migration defect and measured the distance of transfected neurons from the pial surface and found that the migration of FBXO31 knockdown neurons is markedly stalled (Figure6F). These results indicate that FBXO31 promotes dendritic morphogenesis and migration of granule neurons in the developing cerebellum.


The centrosomal E3 ubiquitin ligase FBXO31-SCF regulates neuronal morphogenesis and migration.

Vadhvani M, Schwedhelm-Domeyer N, Mukherjee C, Stegmüller J - PLoS ONE (2013)

FBXO31 regulates dendrite growth and neuronal migration in the developing cerebellum. A.HEK 293T cell lysates transfected with mycFBXO31 along with control or bi-cistronic FBXO31 RNAi #1/CMV-GFP plasmids were probed with α-myc antibody. 14-3-3ß served as a loading control. B. Snapshots of 3D-reconstructed cerebellar granule neurons from rat pups electroporated with control plasmid or with FBXO31 RNAi #1/CMV-GFP bi-cistronic plasmid at P4 and analyzed at P9. Arrows indicate dendrites and arrowheads indicate axons of granule neurons. Scale bar equals 50 µm. C. Histogram showing dendrite length measurements for control or FBXO31 knockdown neurons. A total of 84 neurons were analyzed for dendrite length measurements (n = 3, mean±SEM, unpaired t-test, ***p<0.001). D. Coronal sections of rat pup cerebellum electroporated as described in Figure 4B. IGL = internal granular layer, ML = molecular layer, EGL = external granular layer. Scale bar equals 50 µm. E. Histogram showing percentage of migrated neurons in EGL, ML or IGL. A total of 3637 neurons were analyzed. (n = 3, mean±SEM, two-way ANOVA ***p<0.001, n.s. = not significant). F. Histogram showing distance of granule neuron cell bodies from the pial surface. A total of 681 neurons were analyzed. (n = 3, mean±SEM, two-way ANOVA ***p<0.001).
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Related In: Results  -  Collection

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pone-0057530-g006: FBXO31 regulates dendrite growth and neuronal migration in the developing cerebellum. A.HEK 293T cell lysates transfected with mycFBXO31 along with control or bi-cistronic FBXO31 RNAi #1/CMV-GFP plasmids were probed with α-myc antibody. 14-3-3ß served as a loading control. B. Snapshots of 3D-reconstructed cerebellar granule neurons from rat pups electroporated with control plasmid or with FBXO31 RNAi #1/CMV-GFP bi-cistronic plasmid at P4 and analyzed at P9. Arrows indicate dendrites and arrowheads indicate axons of granule neurons. Scale bar equals 50 µm. C. Histogram showing dendrite length measurements for control or FBXO31 knockdown neurons. A total of 84 neurons were analyzed for dendrite length measurements (n = 3, mean±SEM, unpaired t-test, ***p<0.001). D. Coronal sections of rat pup cerebellum electroporated as described in Figure 4B. IGL = internal granular layer, ML = molecular layer, EGL = external granular layer. Scale bar equals 50 µm. E. Histogram showing percentage of migrated neurons in EGL, ML or IGL. A total of 3637 neurons were analyzed. (n = 3, mean±SEM, two-way ANOVA ***p<0.001, n.s. = not significant). F. Histogram showing distance of granule neuron cell bodies from the pial surface. A total of 681 neurons were analyzed. (n = 3, mean±SEM, two-way ANOVA ***p<0.001).
Mentions: Finally, we explored the role of FBXO31 in the context of the developing cerebellum. We generated a bicistronic plasmid encoding the small hairpin RNA, we have previously used in cultured neurons, and a GFP cassette. We verified the efficient knockdown of FBXO31 by this bicistronic plasmid in heterologous cells (Figure 6A). We then electroporated the cerebella of P4 rat pups and isolated the tissue 5 days later. In coronal sections of P9 cerebella, we assessed dendrite growth of the GFP-positive neurons in the internal granular layer and found a reduction in total dendritic length in FBXO31 knockdown condition as compared to control (Figure 6B, 6C). Due to technical limitations, it is not feasible to measure axons as they fasciculate in the molecular layer into untraceable fibers. Strikingly, we also found that while more than 80% of transfected control neurons descend rapidly into the internal granular layer, nearly 50% of FBXO31 RNAi neurons fail to migrate and remain in the external granule layer/molecular layer (Figure 6D, 6E). We further quantified the migration defect and measured the distance of transfected neurons from the pial surface and found that the migration of FBXO31 knockdown neurons is markedly stalled (Figure6F). These results indicate that FBXO31 promotes dendritic morphogenesis and migration of granule neurons in the developing cerebellum.

Bottom Line: In addition, we identified the polarity protein Par6c as a novel interaction partner and substrate targeted for proteasomal degradation in the control of axon but not dendrite growth.Finally, we ascribe a role for FBXO31 in dendrite growth and neuronal migration in the developing cerebellar cortex.Taken together, we uncovered the centrosomal E3 ligase FBXO31-SCF as a novel regulator of neuronal development.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany.

ABSTRACT
Neuronal development requires proper migration, polarization and establishment of axons and dendrites. Growing evidence identifies the ubiquitin proteasome system (UPS) with its numerous components as an important regulator of various aspects of neuronal development. F-box proteins are interchangeable subunits of the Cullin-1 based E3 ubiquitin ligase, but only a few family members have been studied. Here, we report that the centrosomal E3 ligase FBXO31-SCF (Skp1/Cullin-1/F-box protein) regulates neuronal morphogenesis and axonal identity. In addition, we identified the polarity protein Par6c as a novel interaction partner and substrate targeted for proteasomal degradation in the control of axon but not dendrite growth. Finally, we ascribe a role for FBXO31 in dendrite growth and neuronal migration in the developing cerebellar cortex. Taken together, we uncovered the centrosomal E3 ligase FBXO31-SCF as a novel regulator of neuronal development.

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