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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 axonal identity in neurons. A.Representative images of cerebellar granule neurons transfected with control vector or mycFBXO31 WT plasmid at DIV 0 and analyzed at DIV 3. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. B. Quantification of percentage of non-polarized granule neurons shown in A. (N = 3, n = 256, mean±SEM, unpaired t-test, **p<0.01). C. Representative images of cultured hippocampal neurons transfected at DIV 1 with control vector, plasmids encoding mycFBXO31 WT or mycFBXO31 ΔF together with the GFP plasmid and immunostained at DIV 7 with α-GFP and α-AnkG antibodies and counterstained with Hoechst. Arrows indicate axon initial segment. Scale bar equals 10 µm. D. Quantification of number of axons in C. A total of 169 cells were analyzed (N = 3, mean±SEM, two-way ANOVA ***p<0.001). E. Representative images of cultured hippocampal neurons from E18 rat embryos transfected with control vector or FBXO31 RNAi#1/CMVGFP plasmid at DIV 1 and immunostained at DIV 6 with α-GFP and α-AnkG antibody and counterstained with Hoechst. Arrows indicate axon initial segment. Scale bar equals 10 µm. F. Quantification of number of axons in E. A total of 121 cells were analyzed (N = 3, mean±SEM, two-way ANOVA ***p<0.001).
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pone-0057530-g002: FBXO31 regulates axonal identity in neurons. A.Representative images of cerebellar granule neurons transfected with control vector or mycFBXO31 WT plasmid at DIV 0 and analyzed at DIV 3. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. B. Quantification of percentage of non-polarized granule neurons shown in A. (N = 3, n = 256, mean±SEM, unpaired t-test, **p<0.01). C. Representative images of cultured hippocampal neurons transfected at DIV 1 with control vector, plasmids encoding mycFBXO31 WT or mycFBXO31 ΔF together with the GFP plasmid and immunostained at DIV 7 with α-GFP and α-AnkG antibodies and counterstained with Hoechst. Arrows indicate axon initial segment. Scale bar equals 10 µm. D. Quantification of number of axons in C. A total of 169 cells were analyzed (N = 3, mean±SEM, two-way ANOVA ***p<0.001). E. Representative images of cultured hippocampal neurons from E18 rat embryos transfected with control vector or FBXO31 RNAi#1/CMVGFP plasmid at DIV 1 and immunostained at DIV 6 with α-GFP and α-AnkG antibody and counterstained with Hoechst. Arrows indicate axon initial segment. Scale bar equals 10 µm. F. Quantification of number of axons in E. A total of 121 cells were analyzed (N = 3, mean±SEM, two-way ANOVA ***p<0.001).

Mentions: Aside from the enhanced of neurite growth, we noticed that a larger number of FBXO31-overexpressing granule neurons appeared non-polarized as compared to control neurons. We carried out morphological analysis and defined a neuron as polarized when the longest process was at least twice as long as the second longest. When we quantified the number of non-polarized neurons, we found that while 18% of control neurons appear non-polarized, 38% of FBXO31-overexpressing neurons display a non-polarized morphology (Figure 2A, 2B). We then used molecular markers to examine a possible defect. Axons harbor several unique features including the axon initial segment (AIS), which is characterized by the presence of AnkyrinG. AnkyrinG is responsible for the organization of the AIS and the maintenance of neuronal polarity [21], [22]. We subjected control vector-, wild type FBXO31- and FBXO31 ΔF-expressing hippocampal neurons to immunocytochemistry and found that a significantly larger number of wild type FBXO31-expressing neurons harbor two or more AnkG-positive processes while control or FBXO31 ΔF neurons display mostly one AnkG-positive axon (Figure 2C, 2D). Conversely, when we triggered FBXO31 knockdown, a large percentage of longest processes failed to display the axonal marker AnkyrinG (Figure 2E, 2F). These data suggest that FBXO31 controls not only neuronal morphogenesis but also axonal identity.


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 axonal identity in neurons. A.Representative images of cerebellar granule neurons transfected with control vector or mycFBXO31 WT plasmid at DIV 0 and analyzed at DIV 3. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. B. Quantification of percentage of non-polarized granule neurons shown in A. (N = 3, n = 256, mean±SEM, unpaired t-test, **p<0.01). C. Representative images of cultured hippocampal neurons transfected at DIV 1 with control vector, plasmids encoding mycFBXO31 WT or mycFBXO31 ΔF together with the GFP plasmid and immunostained at DIV 7 with α-GFP and α-AnkG antibodies and counterstained with Hoechst. Arrows indicate axon initial segment. Scale bar equals 10 µm. D. Quantification of number of axons in C. A total of 169 cells were analyzed (N = 3, mean±SEM, two-way ANOVA ***p<0.001). E. Representative images of cultured hippocampal neurons from E18 rat embryos transfected with control vector or FBXO31 RNAi#1/CMVGFP plasmid at DIV 1 and immunostained at DIV 6 with α-GFP and α-AnkG antibody and counterstained with Hoechst. Arrows indicate axon initial segment. Scale bar equals 10 µm. F. Quantification of number of axons in E. A total of 121 cells were analyzed (N = 3, mean±SEM, two-way ANOVA ***p<0.001).
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pone-0057530-g002: FBXO31 regulates axonal identity in neurons. A.Representative images of cerebellar granule neurons transfected with control vector or mycFBXO31 WT plasmid at DIV 0 and analyzed at DIV 3. Arrowheads indicate granule neuron cell bodies. Scale bar equals 50 µm. B. Quantification of percentage of non-polarized granule neurons shown in A. (N = 3, n = 256, mean±SEM, unpaired t-test, **p<0.01). C. Representative images of cultured hippocampal neurons transfected at DIV 1 with control vector, plasmids encoding mycFBXO31 WT or mycFBXO31 ΔF together with the GFP plasmid and immunostained at DIV 7 with α-GFP and α-AnkG antibodies and counterstained with Hoechst. Arrows indicate axon initial segment. Scale bar equals 10 µm. D. Quantification of number of axons in C. A total of 169 cells were analyzed (N = 3, mean±SEM, two-way ANOVA ***p<0.001). E. Representative images of cultured hippocampal neurons from E18 rat embryos transfected with control vector or FBXO31 RNAi#1/CMVGFP plasmid at DIV 1 and immunostained at DIV 6 with α-GFP and α-AnkG antibody and counterstained with Hoechst. Arrows indicate axon initial segment. Scale bar equals 10 µm. F. Quantification of number of axons in E. A total of 121 cells were analyzed (N = 3, mean±SEM, two-way ANOVA ***p<0.001).
Mentions: Aside from the enhanced of neurite growth, we noticed that a larger number of FBXO31-overexpressing granule neurons appeared non-polarized as compared to control neurons. We carried out morphological analysis and defined a neuron as polarized when the longest process was at least twice as long as the second longest. When we quantified the number of non-polarized neurons, we found that while 18% of control neurons appear non-polarized, 38% of FBXO31-overexpressing neurons display a non-polarized morphology (Figure 2A, 2B). We then used molecular markers to examine a possible defect. Axons harbor several unique features including the axon initial segment (AIS), which is characterized by the presence of AnkyrinG. AnkyrinG is responsible for the organization of the AIS and the maintenance of neuronal polarity [21], [22]. We subjected control vector-, wild type FBXO31- and FBXO31 ΔF-expressing hippocampal neurons to immunocytochemistry and found that a significantly larger number of wild type FBXO31-expressing neurons harbor two or more AnkG-positive processes while control or FBXO31 ΔF neurons display mostly one AnkG-positive axon (Figure 2C, 2D). Conversely, when we triggered FBXO31 knockdown, a large percentage of longest processes failed to display the axonal marker AnkyrinG (Figure 2E, 2F). These data suggest that FBXO31 controls not only neuronal morphogenesis but also axonal identity.

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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