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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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Par6c acts downstream of FBXO31-SCF in the control of axon growth. A.Representative images of cerebellar granule neurons transfected with control plasmid or FBXO31 RNAi #1 plasmid or Par6c RNAi plasmid or both FBXO31 RNAi #1 and Par6c RNAi plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 4. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. B. Quantification of longest process length of granule neurons shown in A (N = 3, n = 439, mean±SEM, one-way ANOVA, ***p<0.001). C. Quantification of total dendrite lengths of granule neurons shown in A (N = 3, n = 318, mean±SEM, one-way ANOVA, ***p<0.001, n.s. = not significant). D. Quantification of longest process length of granule neurons transfected with control plasmid or FBXO31 WT or Par6c WT or both FBXO31 WT and Par6c WT plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 3. (N = 3, n = 387, mean±SEM, one-way ANOVA, ***p<0.001, n.s. = not significant). E. Quantification of total dendrite length of granule neurons transfected with control plasmid or FBXO31 WT or Par6c WT or both FBXO31 WT and Par6c WT plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 3. (N = 3, n = 351, mean±SEM, one-way ANOVA, ***p<0.001, n.s. = not significant).
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pone-0057530-g005: Par6c acts downstream of FBXO31-SCF in the control of axon growth. A.Representative images of cerebellar granule neurons transfected with control plasmid or FBXO31 RNAi #1 plasmid or Par6c RNAi plasmid or both FBXO31 RNAi #1 and Par6c RNAi plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 4. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. B. Quantification of longest process length of granule neurons shown in A (N = 3, n = 439, mean±SEM, one-way ANOVA, ***p<0.001). C. Quantification of total dendrite lengths of granule neurons shown in A (N = 3, n = 318, mean±SEM, one-way ANOVA, ***p<0.001, n.s. = not significant). D. Quantification of longest process length of granule neurons transfected with control plasmid or FBXO31 WT or Par6c WT or both FBXO31 WT and Par6c WT plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 3. (N = 3, n = 387, mean±SEM, one-way ANOVA, ***p<0.001, n.s. = not significant). E. Quantification of total dendrite length of granule neurons transfected with control plasmid or FBXO31 WT or Par6c WT or both FBXO31 WT and Par6c WT plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 3. (N = 3, n = 351, mean±SEM, one-way ANOVA, ***p<0.001, n.s. = not significant).

Mentions: Since we identified Par6c as a target of FBXO31-SCF and a regulator of longest process growth, we reasoned that Par6c acts as a downstream component in the FBXO31-SCF pathway of neuronal morphogenesis. To establish such a cascade, we carried out epistasis analysis and transfected neurons with the FBXO31 RNAi or the Par6c RNAi plasmid or both and measured longest process length and dendritic lengths. While we found that FBXO31 RNAi reduces longest process and dendrite length and Par6c RNAi stimulates longest process growth, FBXO31/Par6c double knockdown results in enhanced growth longest processes and short dendrites. These results demonstrate that although the Par6c RNAi phenotype is dominant over FBXO31 RNAi phenotype regarding the longest process, the dendritic FBXO31 phenotype prevails (Figure 5A–C). In an analogous epistasis experiment, we overexpressed FBOX31 and Par6c individually or together and found that while FBXO31 increases and Par6c decreases longest process length, expression of Par6c and FBXO31 together results in reduced axonal length but has no effect on dendrites (Figure 5D, 5E). These data support the conclusion that Par6c acts downstream of FBXO31-SCF in longest process growth control, but not in dendrite growth.


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

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

Par6c acts downstream of FBXO31-SCF in the control of axon growth. A.Representative images of cerebellar granule neurons transfected with control plasmid or FBXO31 RNAi #1 plasmid or Par6c RNAi plasmid or both FBXO31 RNAi #1 and Par6c RNAi plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 4. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. B. Quantification of longest process length of granule neurons shown in A (N = 3, n = 439, mean±SEM, one-way ANOVA, ***p<0.001). C. Quantification of total dendrite lengths of granule neurons shown in A (N = 3, n = 318, mean±SEM, one-way ANOVA, ***p<0.001, n.s. = not significant). D. Quantification of longest process length of granule neurons transfected with control plasmid or FBXO31 WT or Par6c WT or both FBXO31 WT and Par6c WT plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 3. (N = 3, n = 387, mean±SEM, one-way ANOVA, ***p<0.001, n.s. = not significant). E. Quantification of total dendrite length of granule neurons transfected with control plasmid or FBXO31 WT or Par6c WT or both FBXO31 WT and Par6c WT plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 3. (N = 3, n = 351, mean±SEM, one-way ANOVA, ***p<0.001, n.s. = not significant).
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pone-0057530-g005: Par6c acts downstream of FBXO31-SCF in the control of axon growth. A.Representative images of cerebellar granule neurons transfected with control plasmid or FBXO31 RNAi #1 plasmid or Par6c RNAi plasmid or both FBXO31 RNAi #1 and Par6c RNAi plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 4. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. B. Quantification of longest process length of granule neurons shown in A (N = 3, n = 439, mean±SEM, one-way ANOVA, ***p<0.001). C. Quantification of total dendrite lengths of granule neurons shown in A (N = 3, n = 318, mean±SEM, one-way ANOVA, ***p<0.001, n.s. = not significant). D. Quantification of longest process length of granule neurons transfected with control plasmid or FBXO31 WT or Par6c WT or both FBXO31 WT and Par6c WT plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 3. (N = 3, n = 387, mean±SEM, one-way ANOVA, ***p<0.001, n.s. = not significant). E. Quantification of total dendrite length of granule neurons transfected with control plasmid or FBXO31 WT or Par6c WT or both FBXO31 WT and Par6c WT plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 3. (N = 3, n = 351, mean±SEM, one-way ANOVA, ***p<0.001, n.s. = not significant).
Mentions: Since we identified Par6c as a target of FBXO31-SCF and a regulator of longest process growth, we reasoned that Par6c acts as a downstream component in the FBXO31-SCF pathway of neuronal morphogenesis. To establish such a cascade, we carried out epistasis analysis and transfected neurons with the FBXO31 RNAi or the Par6c RNAi plasmid or both and measured longest process length and dendritic lengths. While we found that FBXO31 RNAi reduces longest process and dendrite length and Par6c RNAi stimulates longest process growth, FBXO31/Par6c double knockdown results in enhanced growth longest processes and short dendrites. These results demonstrate that although the Par6c RNAi phenotype is dominant over FBXO31 RNAi phenotype regarding the longest process, the dendritic FBXO31 phenotype prevails (Figure 5A–C). In an analogous epistasis experiment, we overexpressed FBOX31 and Par6c individually or together and found that while FBXO31 increases and Par6c decreases longest process length, expression of Par6c and FBXO31 together results in reduced axonal length but has no effect on dendrites (Figure 5D, 5E). These data support the conclusion that Par6c acts downstream of FBXO31-SCF in longest process growth control, but not in dendrite growth.

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.

Show MeSH