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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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Centrosomal FBXO31 promotes axon and dendrite growth in neurons. A.Cultured cerebellar granule neurons and hippocampal neurons were fixed using methanol followed by immunostaining with α-FBXO31 and α-γtubulin antibodies. The cells were counterstained with the DNA dye bisbenzimide Hoechst 33258. Arrows indicate centrosomes. Scale bar equals 5 µm. B. Cell lysates of HEK 293T cells transfected with indicated plasmids were probed with α-myc antibody. 14-3- ß served as a loading control. C. Representative images of cerebellar granule neurons transfected with empty control vectors, FXO31 RNAi #1 plasmid or FBXO31 RNAi #1 together with mycFBXO31-Res at DIV 0 and analyzed at DIV 4. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. D. Quantification of longest process lengths of granule neurons shown in C (N = 3, n = 296, mean±SEM, one-way ANOVA *p<0.05, ***p<0.001). E. Quantification of total dendrite lengths of granule neurons shown in C (N = 3, n = 291, mean±SEM, one-way ANOVA, *p<0.05, ***p<0.001). F. Representative images of cultured hippocampal neurons transfected with control vector or FBXO31 RNAi #1 plasmids at DIV 1 and analyzed at DIV 5. Arrowheads indicate hippocampal neuron cell bodies. Scale bar equals 50 µm. G. Quantification of longest process lengths of hippocampal neurons shown in F (N = 3, n = 190, mean±SEM, unpaired t-test, ***p<0.001). H. Quantification of total dendrite lengths of hippocampal neurons shown in F (N = 3, n = 184, mean±SEM, unpaired t-test, **p<0.01). I. Representative images of cultured cortical neurons transfected with control vector or FBXO31 RNAi #1 plasmids at DIV 1 and analyzed at DIV 5. Arrowheads indicate cortical neuron cell bodies. Scale bar equals 50 µm. J. Quantification of longest process lengths of cortical neurons shown in I (N = 3, n = 164, mean±SEM, unpaired t-test, ***p<0.001). K. Quantification of total dendrite lengths of cortical neurons shown in I (N = 3, n = 147, mean±SEM, unpaired t-test, ***p<0.001). L. Representative images of cerebellar granule neurons transfected with empty control vector, mycFBXO31 wild type (WT) plasmid or mycFBXO31 ΔF mutant plasmid at DIV 0 and analyzed at DIV 3. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. M. Quantification of longest process lengths of granule neurons shown in L (N = 3, n = 381, mean±SEM, one-way ANOVA ***p<0.001). N. Quantification of total dendrite lengths of granule neurons shown in L (N = 3, n = 341, mean±SEM, one-way ANOVA, ***p<0.001).
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pone-0057530-g001: Centrosomal FBXO31 promotes axon and dendrite growth in neurons. A.Cultured cerebellar granule neurons and hippocampal neurons were fixed using methanol followed by immunostaining with α-FBXO31 and α-γtubulin antibodies. The cells were counterstained with the DNA dye bisbenzimide Hoechst 33258. Arrows indicate centrosomes. Scale bar equals 5 µm. B. Cell lysates of HEK 293T cells transfected with indicated plasmids were probed with α-myc antibody. 14-3- ß served as a loading control. C. Representative images of cerebellar granule neurons transfected with empty control vectors, FXO31 RNAi #1 plasmid or FBXO31 RNAi #1 together with mycFBXO31-Res at DIV 0 and analyzed at DIV 4. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. D. Quantification of longest process lengths of granule neurons shown in C (N = 3, n = 296, mean±SEM, one-way ANOVA *p<0.05, ***p<0.001). E. Quantification of total dendrite lengths of granule neurons shown in C (N = 3, n = 291, mean±SEM, one-way ANOVA, *p<0.05, ***p<0.001). F. Representative images of cultured hippocampal neurons transfected with control vector or FBXO31 RNAi #1 plasmids at DIV 1 and analyzed at DIV 5. Arrowheads indicate hippocampal neuron cell bodies. Scale bar equals 50 µm. G. Quantification of longest process lengths of hippocampal neurons shown in F (N = 3, n = 190, mean±SEM, unpaired t-test, ***p<0.001). H. Quantification of total dendrite lengths of hippocampal neurons shown in F (N = 3, n = 184, mean±SEM, unpaired t-test, **p<0.01). I. Representative images of cultured cortical neurons transfected with control vector or FBXO31 RNAi #1 plasmids at DIV 1 and analyzed at DIV 5. Arrowheads indicate cortical neuron cell bodies. Scale bar equals 50 µm. J. Quantification of longest process lengths of cortical neurons shown in I (N = 3, n = 164, mean±SEM, unpaired t-test, ***p<0.001). K. Quantification of total dendrite lengths of cortical neurons shown in I (N = 3, n = 147, mean±SEM, unpaired t-test, ***p<0.001). L. Representative images of cerebellar granule neurons transfected with empty control vector, mycFBXO31 wild type (WT) plasmid or mycFBXO31 ΔF mutant plasmid at DIV 0 and analyzed at DIV 3. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. M. Quantification of longest process lengths of granule neurons shown in L (N = 3, n = 381, mean±SEM, one-way ANOVA ***p<0.001). N. Quantification of total dendrite lengths of granule neurons shown in L (N = 3, n = 341, mean±SEM, one-way ANOVA, ***p<0.001).

Mentions: We then determined the localization of FBXO31 in neurons and discovered that FBXO31 localizes to the centrosome in both cerebellar granule neurons and in hippocampal neurons (Figure 1A). Co-staining with the centrosomal protein -tubulin supported FBXO31’s centrosomal localization (Figure 1A). In addition, we confirmed the specificity of the immunostaining in neurons by preincubation of the antibody with recombinant FBXO31 protein (Figure S1D). Interestingly, a previous study described the SCF complex subunit Skp1 and Cullin-1 as components of the centrosomal material [19], which prompted us to determine if the binding to Skp1 or Cullin-1 is required to recruit FBXO31 to the centrosome. An FBXO31 mutant lacking the F-box domain, which mediates the binding to Skp1 and Cullin-1 (Figure S2) [17], [20], still localizes to the centrosome. Further mapping analysis revealed that a stretch in the N-terminal region of FBXO31 is required for its recruitment to the centrosome (Figure S3).


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

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

Centrosomal FBXO31 promotes axon and dendrite growth in neurons. A.Cultured cerebellar granule neurons and hippocampal neurons were fixed using methanol followed by immunostaining with α-FBXO31 and α-γtubulin antibodies. The cells were counterstained with the DNA dye bisbenzimide Hoechst 33258. Arrows indicate centrosomes. Scale bar equals 5 µm. B. Cell lysates of HEK 293T cells transfected with indicated plasmids were probed with α-myc antibody. 14-3- ß served as a loading control. C. Representative images of cerebellar granule neurons transfected with empty control vectors, FXO31 RNAi #1 plasmid or FBXO31 RNAi #1 together with mycFBXO31-Res at DIV 0 and analyzed at DIV 4. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. D. Quantification of longest process lengths of granule neurons shown in C (N = 3, n = 296, mean±SEM, one-way ANOVA *p<0.05, ***p<0.001). E. Quantification of total dendrite lengths of granule neurons shown in C (N = 3, n = 291, mean±SEM, one-way ANOVA, *p<0.05, ***p<0.001). F. Representative images of cultured hippocampal neurons transfected with control vector or FBXO31 RNAi #1 plasmids at DIV 1 and analyzed at DIV 5. Arrowheads indicate hippocampal neuron cell bodies. Scale bar equals 50 µm. G. Quantification of longest process lengths of hippocampal neurons shown in F (N = 3, n = 190, mean±SEM, unpaired t-test, ***p<0.001). H. Quantification of total dendrite lengths of hippocampal neurons shown in F (N = 3, n = 184, mean±SEM, unpaired t-test, **p<0.01). I. Representative images of cultured cortical neurons transfected with control vector or FBXO31 RNAi #1 plasmids at DIV 1 and analyzed at DIV 5. Arrowheads indicate cortical neuron cell bodies. Scale bar equals 50 µm. J. Quantification of longest process lengths of cortical neurons shown in I (N = 3, n = 164, mean±SEM, unpaired t-test, ***p<0.001). K. Quantification of total dendrite lengths of cortical neurons shown in I (N = 3, n = 147, mean±SEM, unpaired t-test, ***p<0.001). L. Representative images of cerebellar granule neurons transfected with empty control vector, mycFBXO31 wild type (WT) plasmid or mycFBXO31 ΔF mutant plasmid at DIV 0 and analyzed at DIV 3. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. M. Quantification of longest process lengths of granule neurons shown in L (N = 3, n = 381, mean±SEM, one-way ANOVA ***p<0.001). N. Quantification of total dendrite lengths of granule neurons shown in L (N = 3, n = 341, mean±SEM, one-way ANOVA, ***p<0.001).
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pone-0057530-g001: Centrosomal FBXO31 promotes axon and dendrite growth in neurons. A.Cultured cerebellar granule neurons and hippocampal neurons were fixed using methanol followed by immunostaining with α-FBXO31 and α-γtubulin antibodies. The cells were counterstained with the DNA dye bisbenzimide Hoechst 33258. Arrows indicate centrosomes. Scale bar equals 5 µm. B. Cell lysates of HEK 293T cells transfected with indicated plasmids were probed with α-myc antibody. 14-3- ß served as a loading control. C. Representative images of cerebellar granule neurons transfected with empty control vectors, FXO31 RNAi #1 plasmid or FBXO31 RNAi #1 together with mycFBXO31-Res at DIV 0 and analyzed at DIV 4. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. D. Quantification of longest process lengths of granule neurons shown in C (N = 3, n = 296, mean±SEM, one-way ANOVA *p<0.05, ***p<0.001). E. Quantification of total dendrite lengths of granule neurons shown in C (N = 3, n = 291, mean±SEM, one-way ANOVA, *p<0.05, ***p<0.001). F. Representative images of cultured hippocampal neurons transfected with control vector or FBXO31 RNAi #1 plasmids at DIV 1 and analyzed at DIV 5. Arrowheads indicate hippocampal neuron cell bodies. Scale bar equals 50 µm. G. Quantification of longest process lengths of hippocampal neurons shown in F (N = 3, n = 190, mean±SEM, unpaired t-test, ***p<0.001). H. Quantification of total dendrite lengths of hippocampal neurons shown in F (N = 3, n = 184, mean±SEM, unpaired t-test, **p<0.01). I. Representative images of cultured cortical neurons transfected with control vector or FBXO31 RNAi #1 plasmids at DIV 1 and analyzed at DIV 5. Arrowheads indicate cortical neuron cell bodies. Scale bar equals 50 µm. J. Quantification of longest process lengths of cortical neurons shown in I (N = 3, n = 164, mean±SEM, unpaired t-test, ***p<0.001). K. Quantification of total dendrite lengths of cortical neurons shown in I (N = 3, n = 147, mean±SEM, unpaired t-test, ***p<0.001). L. Representative images of cerebellar granule neurons transfected with empty control vector, mycFBXO31 wild type (WT) plasmid or mycFBXO31 ΔF mutant plasmid at DIV 0 and analyzed at DIV 3. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. M. Quantification of longest process lengths of granule neurons shown in L (N = 3, n = 381, mean±SEM, one-way ANOVA ***p<0.001). N. Quantification of total dendrite lengths of granule neurons shown in L (N = 3, n = 341, mean±SEM, one-way ANOVA, ***p<0.001).
Mentions: We then determined the localization of FBXO31 in neurons and discovered that FBXO31 localizes to the centrosome in both cerebellar granule neurons and in hippocampal neurons (Figure 1A). Co-staining with the centrosomal protein -tubulin supported FBXO31’s centrosomal localization (Figure 1A). In addition, we confirmed the specificity of the immunostaining in neurons by preincubation of the antibody with recombinant FBXO31 protein (Figure S1D). Interestingly, a previous study described the SCF complex subunit Skp1 and Cullin-1 as components of the centrosomal material [19], which prompted us to determine if the binding to Skp1 or Cullin-1 is required to recruit FBXO31 to the centrosome. An FBXO31 mutant lacking the F-box domain, which mediates the binding to Skp1 and Cullin-1 (Figure S2) [17], [20], still localizes to the centrosome. Further mapping analysis revealed that a stretch in the N-terminal region of FBXO31 is required for its recruitment to the centrosome (Figure S3).

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
Related in: MedlinePlus