The Centrosomal E3 Ubiquitin Ligase FBXO31-SCF Regulates Neuronal Morphogenesis and Migration

<div><p>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.</p> </div

Par6c acts as an axon growth suppressor. A.

<p>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>B.</b> Quantification of longest process length of granule neurons shown in A. (N = 3, n = 160, mean±SEM, unpaired t-test, *p<0.05). <b>C.</b> Quantification of percentage of non-polarized granule neurons shown in A. (N = 3, n = 226, mean±SEM, unpaired t-test, *p<0.05). <b>D. and E.</b> Quantification of 2^nd longest (D) and 3^rd longest process length (E) of granule neurons shown in A. (N = 3, n = 160, mean±SEM, unpaired t-test, *p<0.05). <b>F.</b> 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. <b>G.</b> 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. <b>H.</b> 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) <b>I.</b> 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). <b>J.</b> 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).</p

FBXO31-SCF targets Par6c to proteasomal degradation. A.

<p>Transfected HEK 293T cells lysates were subjected to immunoprecipitation with α-Flag antibody and immunoblotted with α-myc antibody. <b>B.</b> Transfected HEK 293T cells lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with α-Flag antibody. <b>C.</b> Schematic showing various Par6c deletion mutants and their interaction with FBXO31. <b>D.</b> Granule neurons were transfected with Par6c plasmid at DIV0 and treated with DMSO or lactacystin for 10 hours prior to lysis at DIV 3. Lysates were immunoblotted with α-myc antibody. 14-3-3ß served as a loading control. <b>E.</b> and <b>F.</b> HEK 293T cells (E) and granule neurons (F) were transfected with mycPar6c plasmid together with FBXO31 RNAi plasmids or respective control vectors as indicated. Cell lysates were immunoblotted with α-myc and α-Flag antibodies. 14-3-3ß served as a loading control. <b>G.</b> Cerebellar granule neurons were transfected with mycPar6c plasmid together with FBXO31 RNAi #1 plasmid or respective control vector at DIV 2. Centrosomal purification was performed at DIV 6 using sucrose density gradient centrifugation. The fractions were probed with α-myc antibody. γ-tubulin served as a positive control for centrosomal protein. The histogram shows Par6c levels relative to γ-tubulin at the centrosome (N = 3, mean±SEM, unpaired t-test, *p<0.05). <b>H.</b> HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT or ΔF plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with α-ubiquitin antibody. <b>I.</b> HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with K48-specific α-ubiquitin antibody. <b>J.</b> HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with anti-myc antibody and immunoblotted with K63-specific anti-ubiquitin antibody.</p

FBXO31 regulates dendrite growth and neuronal migration in the developing cerebellum. A.

<p>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>B.</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. <b>C.</b> 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). <b>D.</b> Coronal sections of rat pup cerebellum electroporated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057530#pone-0057530-g004" target="_blank">Figure 4B</a>. IGL = internal granular layer, ML = molecular layer, EGL = external granular layer. Scale bar equals 50 µm. <b>E.</b> 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). <b>F.</b> 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).</p

FBXO31 regulates axonal identity in neurons. A.

<p>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>B.</b> Quantification of percentage of non-polarized granule neurons shown in A. (N = 3, n = 256, mean±SEM, unpaired t-test, **p<0.01). <b>C.</b> 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. <b>D.</b> Quantification of number of axons in C. A total of 169 cells were analyzed (N = 3, mean±SEM, two-way ANOVA ***p<0.001). <b>E.</b> 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. <b>F.</b> Quantification of number of axons in E. A total of 121 cells were analyzed (N = 3, mean±SEM, two-way ANOVA ***p<0.001).</p

Centrosomal FBXO31 promotes axon and dendrite growth in neurons. A.

<p>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>B.</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. <b>C.</b> 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. <b>D.</b> 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). <b>E.</b> 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). <b>F.</b> 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. <b>G.</b> Quantification of longest process lengths of hippocampal neurons shown in F (N = 3, n = 190, mean±SEM, unpaired t-test, ***p<0.001). <b>H.</b> Quantification of total dendrite lengths of hippocampal neurons shown in F (N = 3, n = 184, mean±SEM, unpaired t-test, **p<0.01). <b>I.</b> 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. <b>J.</b> Quantification of longest process lengths of cortical neurons shown in I (N = 3, n = 164, mean±SEM, unpaired t-test, ***p<0.001). <b>K.</b> Quantification of total dendrite lengths of cortical neurons shown in I (N = 3, n = 147, mean±SEM, unpaired t-test, ***p<0.001). <b>L.</b> 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. <b>M.</b> Quantification of longest process lengths of granule neurons shown in L (N = 3, n = 381, mean±SEM, one-way ANOVA ***p<0.001). <b>N.</b> Quantification of total dendrite lengths of granule neurons shown in L (N = 3, n = 341, mean±SEM, one-way ANOVA, ***p<0.001).</p

Par6c acts downstream of FBXO31-SCF in the control of axon growth. A.

<p>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>B.</b> Quantification of longest process length of granule neurons shown in A (N = 3, n = 439, mean±SEM, one-way ANOVA, ***p<0.001). <b>C.</b> 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). <b>D.</b> 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). <b>E.</b> 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).</p