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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 as an axon growth suppressor. A.Representative images of granule neurons transfected with control vector or plasmid encoding mycPar6c WT together with the GFP plasmid at DIV 0 and analyzed at DIV 3. Arrowheads indicate granule neurons cell bodies. Scale bar represents 50 µm. B. Quantification of longest process length of granule neurons shown in A. (N = 3, n = 160, mean±SEM, unpaired t-test, *p<0.05). C. Quantification of percentage of non-polarized granule neurons shown in A. (N = 3, n = 226, mean±SEM, unpaired t-test, *p<0.05). D. and E. Quantification of 2nd longest (D) and 3rd longest process length (E) of granule neurons shown in A. (N = 3, n = 160, mean±SEM, unpaired t-test, *p<0.05). F. HEK 293T cell lysates transfected with mycPar6c WT or mycPar6c-Res plasmids together with control or Par6c RNAi plasmids were immunoblotted with α-myc antibody. 14-3-3ß served as a loading control. G. Representative images of granule neurons transfected with control vector or Par6c RNAi or Par6c RNAi and Par6c-Res together with the GFP plasmid at DIV 0 and analyzed at DIV 4. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. H. Quantification of longest process length of granule neurons transfected with control vector or Par6c RNAi plasmid or both Par6c RNAi plasmid and mycPar6c-Res plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 4 (N = 3, n = 309, mean±SEM, one-way ANOVA, ***p<0.001) I. Quantification of total dendrite lengths of granule neurons transfected with control vector or Par6c RNAi plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 4 (N = 3, n = 255, mean±SEM, one-way ANOVA, n.s. = not significant). J. Quantification of percentage of non-polarized granule neurons transfected with control vector or Par6c RNAi plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 4. (N = 3, n = 313, mean±SEM, one-way ANOVA, n.s. = not significant).
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pone-0057530-g004: Par6c acts as an axon growth suppressor. A.Representative images of granule neurons transfected with control vector or plasmid encoding mycPar6c WT together with the GFP plasmid at DIV 0 and analyzed at DIV 3. Arrowheads indicate granule neurons cell bodies. Scale bar represents 50 µm. B. Quantification of longest process length of granule neurons shown in A. (N = 3, n = 160, mean±SEM, unpaired t-test, *p<0.05). C. Quantification of percentage of non-polarized granule neurons shown in A. (N = 3, n = 226, mean±SEM, unpaired t-test, *p<0.05). D. and E. Quantification of 2nd longest (D) and 3rd longest process length (E) of granule neurons shown in A. (N = 3, n = 160, mean±SEM, unpaired t-test, *p<0.05). F. HEK 293T cell lysates transfected with mycPar6c WT or mycPar6c-Res plasmids together with control or Par6c RNAi plasmids were immunoblotted with α-myc antibody. 14-3-3ß served as a loading control. G. Representative images of granule neurons transfected with control vector or Par6c RNAi or Par6c RNAi and Par6c-Res together with the GFP plasmid at DIV 0 and analyzed at DIV 4. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. H. Quantification of longest process length of granule neurons transfected with control vector or Par6c RNAi plasmid or both Par6c RNAi plasmid and mycPar6c-Res plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 4 (N = 3, n = 309, mean±SEM, one-way ANOVA, ***p<0.001) I. Quantification of total dendrite lengths of granule neurons transfected with control vector or Par6c RNAi plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 4 (N = 3, n = 255, mean±SEM, one-way ANOVA, n.s. = not significant). J. Quantification of percentage of non-polarized granule neurons transfected with control vector or Par6c RNAi plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 4. (N = 3, n = 313, mean±SEM, one-way ANOVA, n.s. = not significant).

Mentions: We then examined if Par6c has in addition to its role in polarity, neurite growth-regulating functions. Since Par6c levels are regulated by FBXO31-SCF, we reasoned that overexpression of Par6c may phenocopy the FBXO31 RNAi phenotype. To acknowledge that both FBXO31 and Par6c affect axonal identity and polarity, we refer to the axon as longest process. Indeed, we found that Par6c gain-of-function results in reduced longest process growth and thus a shift towards a larger number of non-polarized neurons (Figure 4A–C). The lengths of the second and third longest processes however are left unaffected by Par6c overexpression. (Figure 4D, 4E). To investigate a possible role of the only other known substrate of FBXO31 in neuronal morphogenesis [18], we tested if Cyclin D1 overexpression has any effects on process length or polarity. However, we found that neither is the case (Figure S6A–D) Subsequently, we investigated loss-of-function of Par6c in neurons and generated a Par6c RNAi plasmid using the targeting region described by Zhang and Macara [32]. The Par6c RNAi plasmid was validated in heterologous cells using immunoblotting (Figure 4F). We then transfected neurons with control vector or Par6c RNAi plasmid and found that Par6c knockdown enhances longest process growth but has neither an effect on dendrites nor on the number of non-polarized neurons (Figure 4G–J). To ensure the specificity of the Par6c knockdown phenotype, we generated a Par6c rescue expression plasmid ( = Par6c-Res) by introducing silent mutations in the targeting region that renders Par6c insensitive to RNAi. The sustained expression of Par6c-Res under Par6c knockdown was confirmed in heterologous cells (Figure 4F). We then transfected neurons with either control vectors, the Par6c RNAi plasmid and control vector or the Par6c RNAi plasmid together with the Par6c-Res plasmid and found that the latter rescues the Par6c RNAi phenotype and reduces longest process length to baseline levels of control neurons (Figure 4G, 4H), but has no significant effects on dendrite length or polarity (Figure 4I, 4J). These experiments indicate that Par6c acts as a suppressor of longest process 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 as an axon growth suppressor. A.Representative images of granule neurons transfected with control vector or plasmid encoding mycPar6c WT together with the GFP plasmid at DIV 0 and analyzed at DIV 3. Arrowheads indicate granule neurons cell bodies. Scale bar represents 50 µm. B. Quantification of longest process length of granule neurons shown in A. (N = 3, n = 160, mean±SEM, unpaired t-test, *p<0.05). C. Quantification of percentage of non-polarized granule neurons shown in A. (N = 3, n = 226, mean±SEM, unpaired t-test, *p<0.05). D. and E. Quantification of 2nd longest (D) and 3rd longest process length (E) of granule neurons shown in A. (N = 3, n = 160, mean±SEM, unpaired t-test, *p<0.05). F. HEK 293T cell lysates transfected with mycPar6c WT or mycPar6c-Res plasmids together with control or Par6c RNAi plasmids were immunoblotted with α-myc antibody. 14-3-3ß served as a loading control. G. Representative images of granule neurons transfected with control vector or Par6c RNAi or Par6c RNAi and Par6c-Res together with the GFP plasmid at DIV 0 and analyzed at DIV 4. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. H. Quantification of longest process length of granule neurons transfected with control vector or Par6c RNAi plasmid or both Par6c RNAi plasmid and mycPar6c-Res plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 4 (N = 3, n = 309, mean±SEM, one-way ANOVA, ***p<0.001) I. Quantification of total dendrite lengths of granule neurons transfected with control vector or Par6c RNAi plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 4 (N = 3, n = 255, mean±SEM, one-way ANOVA, n.s. = not significant). J. Quantification of percentage of non-polarized granule neurons transfected with control vector or Par6c RNAi plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 4. (N = 3, n = 313, mean±SEM, one-way ANOVA, n.s. = not significant).
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pone-0057530-g004: Par6c acts as an axon growth suppressor. A.Representative images of granule neurons transfected with control vector or plasmid encoding mycPar6c WT together with the GFP plasmid at DIV 0 and analyzed at DIV 3. Arrowheads indicate granule neurons cell bodies. Scale bar represents 50 µm. B. Quantification of longest process length of granule neurons shown in A. (N = 3, n = 160, mean±SEM, unpaired t-test, *p<0.05). C. Quantification of percentage of non-polarized granule neurons shown in A. (N = 3, n = 226, mean±SEM, unpaired t-test, *p<0.05). D. and E. Quantification of 2nd longest (D) and 3rd longest process length (E) of granule neurons shown in A. (N = 3, n = 160, mean±SEM, unpaired t-test, *p<0.05). F. HEK 293T cell lysates transfected with mycPar6c WT or mycPar6c-Res plasmids together with control or Par6c RNAi plasmids were immunoblotted with α-myc antibody. 14-3-3ß served as a loading control. G. Representative images of granule neurons transfected with control vector or Par6c RNAi or Par6c RNAi and Par6c-Res together with the GFP plasmid at DIV 0 and analyzed at DIV 4. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. H. Quantification of longest process length of granule neurons transfected with control vector or Par6c RNAi plasmid or both Par6c RNAi plasmid and mycPar6c-Res plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 4 (N = 3, n = 309, mean±SEM, one-way ANOVA, ***p<0.001) I. Quantification of total dendrite lengths of granule neurons transfected with control vector or Par6c RNAi plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 4 (N = 3, n = 255, mean±SEM, one-way ANOVA, n.s. = not significant). J. Quantification of percentage of non-polarized granule neurons transfected with control vector or Par6c RNAi plasmid together with GFP plasmid at DIV 0 and analyzed at DIV 4. (N = 3, n = 313, mean±SEM, one-way ANOVA, n.s. = not significant).
Mentions: We then examined if Par6c has in addition to its role in polarity, neurite growth-regulating functions. Since Par6c levels are regulated by FBXO31-SCF, we reasoned that overexpression of Par6c may phenocopy the FBXO31 RNAi phenotype. To acknowledge that both FBXO31 and Par6c affect axonal identity and polarity, we refer to the axon as longest process. Indeed, we found that Par6c gain-of-function results in reduced longest process growth and thus a shift towards a larger number of non-polarized neurons (Figure 4A–C). The lengths of the second and third longest processes however are left unaffected by Par6c overexpression. (Figure 4D, 4E). To investigate a possible role of the only other known substrate of FBXO31 in neuronal morphogenesis [18], we tested if Cyclin D1 overexpression has any effects on process length or polarity. However, we found that neither is the case (Figure S6A–D) Subsequently, we investigated loss-of-function of Par6c in neurons and generated a Par6c RNAi plasmid using the targeting region described by Zhang and Macara [32]. The Par6c RNAi plasmid was validated in heterologous cells using immunoblotting (Figure 4F). We then transfected neurons with control vector or Par6c RNAi plasmid and found that Par6c knockdown enhances longest process growth but has neither an effect on dendrites nor on the number of non-polarized neurons (Figure 4G–J). To ensure the specificity of the Par6c knockdown phenotype, we generated a Par6c rescue expression plasmid ( = Par6c-Res) by introducing silent mutations in the targeting region that renders Par6c insensitive to RNAi. The sustained expression of Par6c-Res under Par6c knockdown was confirmed in heterologous cells (Figure 4F). We then transfected neurons with either control vectors, the Par6c RNAi plasmid and control vector or the Par6c RNAi plasmid together with the Par6c-Res plasmid and found that the latter rescues the Par6c RNAi phenotype and reduces longest process length to baseline levels of control neurons (Figure 4G, 4H), but has no significant effects on dendrite length or polarity (Figure 4I, 4J). These experiments indicate that Par6c acts as a suppressor of longest process 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