10 research outputs found
The role of E3 ubiquitin ligase FBXO31-SCF in neuronal morphogenesis
Neuronale Entwicklung wird durch das
Zusammenspiel von extrinsischen Signalen und intrinsischen Faktoren
koordiniert. Diese extrinsischen Signale regulieren das Zytoskelett
des Neurons über mehrere intrazelluläre Signalwege, welche während
der neuronalen Morphogenese essentiell sind. Neuere
Forschungsergebnisse zeigen das Ubiquitin-Proteasom System (UPS)
als einen entscheidenden zell-intrinsischen Regulator von
neuronaler Entwicklung. Die Skp-Cullin1-F-box Protein (SCF)
E3-Ubiquitin Ligase und insbesondere die F-box Proteine als
Substrat-rekrutierende Adaptor-Untereinheit wurden als essentielle
Modulatoren von diversen Aspekten neuronaler Entwicklung
identifiziert, z.B. Vorläuferzellen-Vermehrung, Zellwanderung,
Axonen- und Dendritenwachstum und Synaptogenese. In der
vorliegenden Studie habe ich das im Gehirn angereicherte,
zentrosomale F-box Protein FBXO31-SCF als einen neuen Regulator
neuronaler Morphogenese sowohl in vitro als auch im Cerebellum
identifiziert. Meine Studie zeigt nicht nur, dass FBXO31-SCF als
Regulator axonaler Identität agiert, sondern auch dass FBXO31-SCF
Axonen- und Dendritenwachstum in Neuronen fördert. Um
mechanistische Einsichten in die FBXO31-regulierten Phänotypen zu
bekommen, habe ich das Polaritätsprotein Par6c als einen neuen
Interaktionspartner und bona fide Substrat von FBXO31
herausgefunden. Weitere Analysen zeigten, dass FBXO31-SCF upstream
vom Polaritätkomplexprotein Par6c in der Regulation von Axonen-,
aber nicht Dendritenwachstum in Neuronen agiert. Zusammengefasst
gibt meine Studie systematische Einsicht in FBXO31-regulierte
Ereignisse in sich entwickelnden Neuronen und zeigt dadurch die
E3-Ubiquitin Ligase FBXO31-SCF als einen Schlüssel-Regulator von
neuronaler Entwicklung.Neuronal development is coordinated by the
interplay of extrinsic cues and intrinsic factors. These extrinsic
cues act through multiple intracellular signaling pathways to
regulate the cytoskeleton machinery of the neuron that is essential
during neuronal morphogenesis. Recent evidence identifies the
ubiquitin proteasome system (UPS) as a crucial cell-intrinsic
regulator of neuronal development. The Skp1-Cullin1-F-box protein
(SCF) E3 ubiquitin ligase and in particular the
substrate-recruiting adaptor subunit F-box proteins have emerged as
essential modulators of diverse aspects of neuronal development
including progenitor proliferation, migration, axon and dendrite
growth and synaptogenesis. In this study, I identified the
brain-enriched centrosomal F-box protein FBXO31-SCF as a novel
regulator of neuronal morphogenesis both in vitro and in the
developing cerebellum. While my study identifies FBXO31-SCF as a
regulator of axonal identity, I also found that FBXO31-SCF promotes
of axon and dendrite growth in neurons. To gain mechanistic insight
into the FBXO31-regulated phenotypes, I uncovered the polarity
protein Par6c as a novel interaction partner and a bona de
substrate of FBXO31. Further analysis revealed that FBXO31-SCF acts
upstream of polarity complex protein Par6c to regulate axon growth
but not dendrite growth in neurons. Taken together, my study gives
a systematic insight into FBXO31-regulated events in developing
neurons and thus introduces the E3 ubiquitin ligase FBXO31-SCF as a
key regulator of neuronal development
Genetic Manipulation of Cerebellar Granule Neurons <em>In Vitro</em> and <em>In Vivo</em> to Study Neuronal Morphology and Migration
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<sup>nd</sup> longest (D) and 3<sup>rd</sup> 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
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
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