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An OBSL1-Cul7Fbxw8 ubiquitin ligase signaling mechanism regulates Golgi morphology and dendrite patterning.

Litterman N, Ikeuchi Y, Gallardo G, O'Connell BC, Sowa ME, Gygi SP, Harper JW, Bonni A - PLoS Biol. (2011)

Bottom Line: Accordingly, OBSL1 is required for the morphogenesis of the Golgi apparatus and the elaboration of dendrites.Finally, we identify the Golgi protein Grasp65 as a novel and physiologically relevant substrate of Cul7(Fbxw8) in the control of Golgi and dendrite morphogenesis in neurons.Collectively, these findings define a novel OBSL1-regulated Cul7(Fbxw8) ubiquitin signaling mechanism that orchestrates the morphogenesis of the Golgi apparatus and patterning of dendrites, with fundamental implications for our understanding of brain development.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
The elaboration of dendrites in neurons requires secretory trafficking through the Golgi apparatus, but the mechanisms that govern Golgi function in neuronal morphogenesis in the brain have remained largely unexplored. Here, we report that the E3 ubiquitin ligase Cul7(Fbxw8) localizes to the Golgi complex in mammalian brain neurons. Inhibition of Cul7(Fbxw8) by independent approaches including Fbxw8 knockdown reveals that Cul7(Fbxw8) is selectively required for the growth and elaboration of dendrites but not axons in primary neurons and in the developing rat cerebellum in vivo. Inhibition of Cul7(Fbxw8) also dramatically impairs the morphology of the Golgi complex, leading to deficient secretory trafficking in neurons. Using an immunoprecipitation/mass spectrometry screening approach, we also uncover the cytoskeletal adaptor protein OBSL1 as a critical regulator of Cul7(Fbxw8) in Golgi morphogenesis and dendrite elaboration. OBSL1 forms a physical complex with the scaffold protein Cul7 and thereby localizes Cul7 at the Golgi apparatus. Accordingly, OBSL1 is required for the morphogenesis of the Golgi apparatus and the elaboration of dendrites. Finally, we identify the Golgi protein Grasp65 as a novel and physiologically relevant substrate of Cul7(Fbxw8) in the control of Golgi and dendrite morphogenesis in neurons. Collectively, these findings define a novel OBSL1-regulated Cul7(Fbxw8) ubiquitin signaling mechanism that orchestrates the morphogenesis of the Golgi apparatus and patterning of dendrites, with fundamental implications for our understanding of brain development.

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Fbxw8 is localized at the Golgi apparatus in neurons.(A) Lysates of embryonic brain and placenta were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (B) Lysates of cerebellum from rat pups from P6 to adult and of primary P6 granule neurons cultured DIV1 to DIV9 were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (C) Lysates of granule neurons were subjected to subcellular fractionation and immunoblotted with the Fbxw8, 14-3-3β, and SnoN antibodies, the latter two to mark cytoplasmic (C) and nuclear (N) fractions, respectively. (D) Granule neurons were subjected to immunocytochemistry with the Fbxw8 antibody together with the TGN38, GM130, Hsp60, or PDI antibody. DNA dye bisbenzimide (Hoechst 33258) was used to stain the nucleus. Arrows indicate localization of Fbxw8 at the Golgi apparatus. Scale bar = 10 µm. (E) Lysates of 293T cells transfected with the expression plasmids encoding Flag-Fbxw8 and GFP together with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid were immunoblotted with the Flag and GFP antibodies. (F) Lysates of granule neurons transfected with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (G) Granule neurons transfected at DIV2 with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid together with an expression plasmid encoding farnesylated GFP to label membranes were fixed at DIV5 and were subjected to immunocytochemistry using the GFP and Fbxw8 antibodies. Dotted lines represent tracing of transfected cells. Arrows indicate Golgi-localized Fbxw8 in transfected neurons. Scale bar = 10 µm. Fbxw8 knockdown reduced almost completely Fbxw8 immunofluorescence in neurons.
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pbio-1001060-g001: Fbxw8 is localized at the Golgi apparatus in neurons.(A) Lysates of embryonic brain and placenta were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (B) Lysates of cerebellum from rat pups from P6 to adult and of primary P6 granule neurons cultured DIV1 to DIV9 were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (C) Lysates of granule neurons were subjected to subcellular fractionation and immunoblotted with the Fbxw8, 14-3-3β, and SnoN antibodies, the latter two to mark cytoplasmic (C) and nuclear (N) fractions, respectively. (D) Granule neurons were subjected to immunocytochemistry with the Fbxw8 antibody together with the TGN38, GM130, Hsp60, or PDI antibody. DNA dye bisbenzimide (Hoechst 33258) was used to stain the nucleus. Arrows indicate localization of Fbxw8 at the Golgi apparatus. Scale bar = 10 µm. (E) Lysates of 293T cells transfected with the expression plasmids encoding Flag-Fbxw8 and GFP together with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid were immunoblotted with the Flag and GFP antibodies. (F) Lysates of granule neurons transfected with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (G) Granule neurons transfected at DIV2 with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid together with an expression plasmid encoding farnesylated GFP to label membranes were fixed at DIV5 and were subjected to immunocytochemistry using the GFP and Fbxw8 antibodies. Dotted lines represent tracing of transfected cells. Arrows indicate Golgi-localized Fbxw8 in transfected neurons. Scale bar = 10 µm. Fbxw8 knockdown reduced almost completely Fbxw8 immunofluorescence in neurons.

Mentions: Among F-box proteins, Fbxw8 uniquely assembles with the scaffold protein Cul7 [21],[22], and unlike Cul1, which may associate with all F-box proteins, Cul7 associates only with Fbxw8 [23],[24]. Cul7Fbxw8 is highly expressed in placenta [21]–[23]. We found that Fbxw8 was also highly expressed in the developing rat brain (Figure 1A). In the rat cerebellum, Fbxw8 was abundantly expressed in the first two postnatal weeks, and its levels decreased thereafter (Figure 1B). Consistent with these results, Fbxw8 was highly expressed in primary granule neurons isolated from postnatal day 6 (P6) rat pups and cultured for 1 to 9 d in vitro, with expression decreasing over time with neuron maturation (Figure 1B). These results reveal that Fbxw8 is expressed in neurons with a temporal profile that coincides with the period of dendrite development.


An OBSL1-Cul7Fbxw8 ubiquitin ligase signaling mechanism regulates Golgi morphology and dendrite patterning.

Litterman N, Ikeuchi Y, Gallardo G, O'Connell BC, Sowa ME, Gygi SP, Harper JW, Bonni A - PLoS Biol. (2011)

Fbxw8 is localized at the Golgi apparatus in neurons.(A) Lysates of embryonic brain and placenta were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (B) Lysates of cerebellum from rat pups from P6 to adult and of primary P6 granule neurons cultured DIV1 to DIV9 were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (C) Lysates of granule neurons were subjected to subcellular fractionation and immunoblotted with the Fbxw8, 14-3-3β, and SnoN antibodies, the latter two to mark cytoplasmic (C) and nuclear (N) fractions, respectively. (D) Granule neurons were subjected to immunocytochemistry with the Fbxw8 antibody together with the TGN38, GM130, Hsp60, or PDI antibody. DNA dye bisbenzimide (Hoechst 33258) was used to stain the nucleus. Arrows indicate localization of Fbxw8 at the Golgi apparatus. Scale bar = 10 µm. (E) Lysates of 293T cells transfected with the expression plasmids encoding Flag-Fbxw8 and GFP together with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid were immunoblotted with the Flag and GFP antibodies. (F) Lysates of granule neurons transfected with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (G) Granule neurons transfected at DIV2 with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid together with an expression plasmid encoding farnesylated GFP to label membranes were fixed at DIV5 and were subjected to immunocytochemistry using the GFP and Fbxw8 antibodies. Dotted lines represent tracing of transfected cells. Arrows indicate Golgi-localized Fbxw8 in transfected neurons. Scale bar = 10 µm. Fbxw8 knockdown reduced almost completely Fbxw8 immunofluorescence in neurons.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3091842&req=5

pbio-1001060-g001: Fbxw8 is localized at the Golgi apparatus in neurons.(A) Lysates of embryonic brain and placenta were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (B) Lysates of cerebellum from rat pups from P6 to adult and of primary P6 granule neurons cultured DIV1 to DIV9 were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (C) Lysates of granule neurons were subjected to subcellular fractionation and immunoblotted with the Fbxw8, 14-3-3β, and SnoN antibodies, the latter two to mark cytoplasmic (C) and nuclear (N) fractions, respectively. (D) Granule neurons were subjected to immunocytochemistry with the Fbxw8 antibody together with the TGN38, GM130, Hsp60, or PDI antibody. DNA dye bisbenzimide (Hoechst 33258) was used to stain the nucleus. Arrows indicate localization of Fbxw8 at the Golgi apparatus. Scale bar = 10 µm. (E) Lysates of 293T cells transfected with the expression plasmids encoding Flag-Fbxw8 and GFP together with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid were immunoblotted with the Flag and GFP antibodies. (F) Lysates of granule neurons transfected with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid were immunoblotted with the Fbxw8 and Erk1/2 antibodies. (G) Granule neurons transfected at DIV2 with the U6/fbxw8-1, U6/fbxw8-2, or control U6 RNAi plasmid together with an expression plasmid encoding farnesylated GFP to label membranes were fixed at DIV5 and were subjected to immunocytochemistry using the GFP and Fbxw8 antibodies. Dotted lines represent tracing of transfected cells. Arrows indicate Golgi-localized Fbxw8 in transfected neurons. Scale bar = 10 µm. Fbxw8 knockdown reduced almost completely Fbxw8 immunofluorescence in neurons.
Mentions: Among F-box proteins, Fbxw8 uniquely assembles with the scaffold protein Cul7 [21],[22], and unlike Cul1, which may associate with all F-box proteins, Cul7 associates only with Fbxw8 [23],[24]. Cul7Fbxw8 is highly expressed in placenta [21]–[23]. We found that Fbxw8 was also highly expressed in the developing rat brain (Figure 1A). In the rat cerebellum, Fbxw8 was abundantly expressed in the first two postnatal weeks, and its levels decreased thereafter (Figure 1B). Consistent with these results, Fbxw8 was highly expressed in primary granule neurons isolated from postnatal day 6 (P6) rat pups and cultured for 1 to 9 d in vitro, with expression decreasing over time with neuron maturation (Figure 1B). These results reveal that Fbxw8 is expressed in neurons with a temporal profile that coincides with the period of dendrite development.

Bottom Line: Accordingly, OBSL1 is required for the morphogenesis of the Golgi apparatus and the elaboration of dendrites.Finally, we identify the Golgi protein Grasp65 as a novel and physiologically relevant substrate of Cul7(Fbxw8) in the control of Golgi and dendrite morphogenesis in neurons.Collectively, these findings define a novel OBSL1-regulated Cul7(Fbxw8) ubiquitin signaling mechanism that orchestrates the morphogenesis of the Golgi apparatus and patterning of dendrites, with fundamental implications for our understanding of brain development.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
The elaboration of dendrites in neurons requires secretory trafficking through the Golgi apparatus, but the mechanisms that govern Golgi function in neuronal morphogenesis in the brain have remained largely unexplored. Here, we report that the E3 ubiquitin ligase Cul7(Fbxw8) localizes to the Golgi complex in mammalian brain neurons. Inhibition of Cul7(Fbxw8) by independent approaches including Fbxw8 knockdown reveals that Cul7(Fbxw8) is selectively required for the growth and elaboration of dendrites but not axons in primary neurons and in the developing rat cerebellum in vivo. Inhibition of Cul7(Fbxw8) also dramatically impairs the morphology of the Golgi complex, leading to deficient secretory trafficking in neurons. Using an immunoprecipitation/mass spectrometry screening approach, we also uncover the cytoskeletal adaptor protein OBSL1 as a critical regulator of Cul7(Fbxw8) in Golgi morphogenesis and dendrite elaboration. OBSL1 forms a physical complex with the scaffold protein Cul7 and thereby localizes Cul7 at the Golgi apparatus. Accordingly, OBSL1 is required for the morphogenesis of the Golgi apparatus and the elaboration of dendrites. Finally, we identify the Golgi protein Grasp65 as a novel and physiologically relevant substrate of Cul7(Fbxw8) in the control of Golgi and dendrite morphogenesis in neurons. Collectively, these findings define a novel OBSL1-regulated Cul7(Fbxw8) ubiquitin signaling mechanism that orchestrates the morphogenesis of the Golgi apparatus and patterning of dendrites, with fundamental implications for our understanding of brain development.

Show MeSH