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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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The cytoskeletal adaptor protein OBSL1 forms a physical complex with the scaffold protein Cul7.(A) Lysates of 293T cells harboring an inducible HA-Fbxw8 lentivirus were immunoprecipitated with the HA antibody and subjected to proteomic analysis using LC-MS/MS. CompPASS was utilized to interrogate datasets and assign the DN and Z scoring metrics. Proteins with a DN score greater than 1 and a Z score greater than 3.5 were considered HCIPs. We confirmed that endogenous Fbxw8 and endogenous Cul7 form a complex in cells (Figure S6G). (B) Lysates of 293T cells transfected with expression plasmids encoding V5-OBSL1 and Cul7-HA or the control vectors were immunoprecipitated (IP) with the V5 or HA antibodies. Immunoprecipitates and lysates were immunoblotted (IB) with the V5 and HA antibodies. (C) Domain map of full-length (FL) Cul7 protein and deletion mutant proteins. Cul7 consists of a large N-terminal domain unique among the cullin family that contains a CPH domain, a DOC domain, a cullin domain, and an extreme C-terminal region that contains a neddylation motif. (D) Lysates of 293T cells transfected with expression plasmids encoding V5-OBSL1 and full-length Cul7-HA, deletion mutants, or the control vector were immunoprecipitated with the HA antibody. Immunoprecipitates and lysates were immmunoblotted with the HA and V5 antibodies. (E) Granule neurons were transfected with the expression plasmids encoding V5-OBSL1 and the Golgi marker, GFP-GT, and subjected to immunocytochemistry with the V5 and GFP antibodies. Arrow denotes co-localization of OBSL1 and GFP-GT. Scale bar = 5 µm. (F) Granule neurons were transfected with the expression plasmids encoding Cul7-HA or deletion mutants and the Golgi marker, GFP-GT, and subjected to immunocytochemistry with the HA and GFP antibodies. Arrows denote the Golgi apparatus as labeled by GFP-GT. Scale bar = 5 µm.
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pbio-1001060-g005: The cytoskeletal adaptor protein OBSL1 forms a physical complex with the scaffold protein Cul7.(A) Lysates of 293T cells harboring an inducible HA-Fbxw8 lentivirus were immunoprecipitated with the HA antibody and subjected to proteomic analysis using LC-MS/MS. CompPASS was utilized to interrogate datasets and assign the DN and Z scoring metrics. Proteins with a DN score greater than 1 and a Z score greater than 3.5 were considered HCIPs. We confirmed that endogenous Fbxw8 and endogenous Cul7 form a complex in cells (Figure S6G). (B) Lysates of 293T cells transfected with expression plasmids encoding V5-OBSL1 and Cul7-HA or the control vectors were immunoprecipitated (IP) with the V5 or HA antibodies. Immunoprecipitates and lysates were immunoblotted (IB) with the V5 and HA antibodies. (C) Domain map of full-length (FL) Cul7 protein and deletion mutant proteins. Cul7 consists of a large N-terminal domain unique among the cullin family that contains a CPH domain, a DOC domain, a cullin domain, and an extreme C-terminal region that contains a neddylation motif. (D) Lysates of 293T cells transfected with expression plasmids encoding V5-OBSL1 and full-length Cul7-HA, deletion mutants, or the control vector were immunoprecipitated with the HA antibody. Immunoprecipitates and lysates were immmunoblotted with the HA and V5 antibodies. (E) Granule neurons were transfected with the expression plasmids encoding V5-OBSL1 and the Golgi marker, GFP-GT, and subjected to immunocytochemistry with the V5 and GFP antibodies. Arrow denotes co-localization of OBSL1 and GFP-GT. Scale bar = 5 µm. (F) Granule neurons were transfected with the expression plasmids encoding Cul7-HA or deletion mutants and the Golgi marker, GFP-GT, and subjected to immunocytochemistry with the HA and GFP antibodies. Arrows denote the Golgi apparatus as labeled by GFP-GT. Scale bar = 5 µm.

Mentions: Having identified an essential role for the ubiquitin ligase Cul7Fbxw8 ubiquitin signaling pathway in Golgi morphogenesis and dendrite patterning in neurons, we next addressed the major question of how the function of neuronal Cul7Fbxw8 is controlled. To identify novel regulators of Cul7Fbxw8 in an unbiased manner, we utilized an IP/mass spec screening approach [31]. We immunopurified Fbxw8 complexes from 293T cells that were infected with an inducible HA-Fbxw8 lentivirus. Purified Fbxw8 complexes were next subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses to identify Fbxw8-associated proteins. We interrogated datasets using the platform Comparative Proteomics Analysis Software Suite (CompPASS) to assign scoring metrics, DN and Z scores, for parallel mass spectral studies. The DN score is similar to the conventional Z score, but it also incorporates the frequency of the observed interactor, its abundance, and the reproducibility of the interaction [31]. Proteins with a DN score greater than 1 and a Z score greater than 3.5 were considered high-confidence candidate-interacting proteins (HCIP) (Figure 5A). Proteins reproducibly identified as HCIPs in our analyses included those established to interact with Fbxw8, including Cul7 and Skp1A, validating the IP/mass spec approach. We also identified five subunits of the CCT (chaperonin containing TCP1) complex in association with Fbxw8. The CCT complex has been implicated in the proper folding of WD40 domain proteins [32], suggesting that Fbxw8 may also employ CCT for folding.


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)

The cytoskeletal adaptor protein OBSL1 forms a physical complex with the scaffold protein Cul7.(A) Lysates of 293T cells harboring an inducible HA-Fbxw8 lentivirus were immunoprecipitated with the HA antibody and subjected to proteomic analysis using LC-MS/MS. CompPASS was utilized to interrogate datasets and assign the DN and Z scoring metrics. Proteins with a DN score greater than 1 and a Z score greater than 3.5 were considered HCIPs. We confirmed that endogenous Fbxw8 and endogenous Cul7 form a complex in cells (Figure S6G). (B) Lysates of 293T cells transfected with expression plasmids encoding V5-OBSL1 and Cul7-HA or the control vectors were immunoprecipitated (IP) with the V5 or HA antibodies. Immunoprecipitates and lysates were immunoblotted (IB) with the V5 and HA antibodies. (C) Domain map of full-length (FL) Cul7 protein and deletion mutant proteins. Cul7 consists of a large N-terminal domain unique among the cullin family that contains a CPH domain, a DOC domain, a cullin domain, and an extreme C-terminal region that contains a neddylation motif. (D) Lysates of 293T cells transfected with expression plasmids encoding V5-OBSL1 and full-length Cul7-HA, deletion mutants, or the control vector were immunoprecipitated with the HA antibody. Immunoprecipitates and lysates were immmunoblotted with the HA and V5 antibodies. (E) Granule neurons were transfected with the expression plasmids encoding V5-OBSL1 and the Golgi marker, GFP-GT, and subjected to immunocytochemistry with the V5 and GFP antibodies. Arrow denotes co-localization of OBSL1 and GFP-GT. Scale bar = 5 µm. (F) Granule neurons were transfected with the expression plasmids encoding Cul7-HA or deletion mutants and the Golgi marker, GFP-GT, and subjected to immunocytochemistry with the HA and GFP antibodies. Arrows denote the Golgi apparatus as labeled by GFP-GT. Scale bar = 5 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3091842&req=5

pbio-1001060-g005: The cytoskeletal adaptor protein OBSL1 forms a physical complex with the scaffold protein Cul7.(A) Lysates of 293T cells harboring an inducible HA-Fbxw8 lentivirus were immunoprecipitated with the HA antibody and subjected to proteomic analysis using LC-MS/MS. CompPASS was utilized to interrogate datasets and assign the DN and Z scoring metrics. Proteins with a DN score greater than 1 and a Z score greater than 3.5 were considered HCIPs. We confirmed that endogenous Fbxw8 and endogenous Cul7 form a complex in cells (Figure S6G). (B) Lysates of 293T cells transfected with expression plasmids encoding V5-OBSL1 and Cul7-HA or the control vectors were immunoprecipitated (IP) with the V5 or HA antibodies. Immunoprecipitates and lysates were immunoblotted (IB) with the V5 and HA antibodies. (C) Domain map of full-length (FL) Cul7 protein and deletion mutant proteins. Cul7 consists of a large N-terminal domain unique among the cullin family that contains a CPH domain, a DOC domain, a cullin domain, and an extreme C-terminal region that contains a neddylation motif. (D) Lysates of 293T cells transfected with expression plasmids encoding V5-OBSL1 and full-length Cul7-HA, deletion mutants, or the control vector were immunoprecipitated with the HA antibody. Immunoprecipitates and lysates were immmunoblotted with the HA and V5 antibodies. (E) Granule neurons were transfected with the expression plasmids encoding V5-OBSL1 and the Golgi marker, GFP-GT, and subjected to immunocytochemistry with the V5 and GFP antibodies. Arrow denotes co-localization of OBSL1 and GFP-GT. Scale bar = 5 µm. (F) Granule neurons were transfected with the expression plasmids encoding Cul7-HA or deletion mutants and the Golgi marker, GFP-GT, and subjected to immunocytochemistry with the HA and GFP antibodies. Arrows denote the Golgi apparatus as labeled by GFP-GT. Scale bar = 5 µm.
Mentions: Having identified an essential role for the ubiquitin ligase Cul7Fbxw8 ubiquitin signaling pathway in Golgi morphogenesis and dendrite patterning in neurons, we next addressed the major question of how the function of neuronal Cul7Fbxw8 is controlled. To identify novel regulators of Cul7Fbxw8 in an unbiased manner, we utilized an IP/mass spec screening approach [31]. We immunopurified Fbxw8 complexes from 293T cells that were infected with an inducible HA-Fbxw8 lentivirus. Purified Fbxw8 complexes were next subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses to identify Fbxw8-associated proteins. We interrogated datasets using the platform Comparative Proteomics Analysis Software Suite (CompPASS) to assign scoring metrics, DN and Z scores, for parallel mass spectral studies. The DN score is similar to the conventional Z score, but it also incorporates the frequency of the observed interactor, its abundance, and the reproducibility of the interaction [31]. Proteins with a DN score greater than 1 and a Z score greater than 3.5 were considered high-confidence candidate-interacting proteins (HCIP) (Figure 5A). Proteins reproducibly identified as HCIPs in our analyses included those established to interact with Fbxw8, including Cul7 and Skp1A, validating the IP/mass spec approach. We also identified five subunits of the CCT (chaperonin containing TCP1) complex in association with Fbxw8. The CCT complex has been implicated in the proper folding of WD40 domain proteins [32], suggesting that Fbxw8 may also employ CCT for folding.

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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