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The role of Rab6a and phosphorylation of non-muscle myosin IIA tailpiece in alcohol-induced Golgi disorganization.

ABSTRACT: Abnormalities in the Golgi apparatus function are important to the development of alcoholic liver injury. We recently reported that Golgi disorganization in ethanol (EtOH)-treated hepatocytes is caused by impaired dimerization of the largest Golgi matrix protein, giantin. However, little is known about the mechanism which forces fragmentation. Here, in both HepG2 cells overexpressing alcohol dehydrogenase and in rat hepatocytes, we found that EtOH administration reduces the complex between giantin and Rab6a GTPase and results in the S1943 phosphorylation of non-muscle Myosin IIA (NMIIA) heavy chain, thus facilitating NMIIA association with Golgi enzymes, as detected by biochemical approaches and 3D Structured Illumination Microscopy. We revealed that NMIIA-P-S1943 competes with giantin for the Rab6a dimer, which was converted to monomer after Golgi fragmentation. Therefore, Rab6a plays a dual role in the Golgi, serving as master regulator of Golgi organization and disorganization, and that NMIIA and giantin engage in a "tug-of-war". However, the inhibition of F-actin and downregulation of NMIIA or overexpression of NMHC-IIA?tailpiece, as well the overexpression of dominant negative Rab6a(T27N), preserved a compact Golgi phenotype. Thus, the actomyosin complex forces EtOH-induced Golgi disorganization, and the targeting of NMIIA-P-S1943 may be important for preventing the damaging effects of alcohol metabolism on the cell.

SUBMITTER: Petrosyan A

PROVIDER: S-EPMC4989220 | biostudies-other | 2016 Aug

REPOSITORIES: biostudies-other

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The role of Rab6a and phosphorylation of non-muscle myosin IIA tailpiece in alcohol-induced Golgi disorganization.

Petrosyan Armen A Casey Carol A CA Cheng Pi-Wan PW

Scientific reports 20160818

Abnormalities in the Golgi apparatus function are important to the development of alcoholic liver injury. We recently reported that Golgi disorganization in ethanol (EtOH)-treated hepatocytes is caused by impaired dimerization of the largest Golgi matrix protein, giantin. However, little is known about the mechanism which forces fragmentation. Here, in both HepG2 cells overexpressing alcohol dehydrogenase and in rat hepatocytes, we found that EtOH administration reduces the complex between giant ...[more]

PMID: 27535804

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Project description:It is known that ethanol (EtOH) and its metabolites have a negative effect on protein glycosylation. The fragmentation of the Golgi apparatus induced by alteration of the structure of largest Golgi matrix protein, giantin, is the major consequence of damaging effects of EtOH-metabolism on the Golgi; however, the link between this and abnormal glycosylation remains unknown. Because previously we have shown that Golgi morphology dictates glycosylation, we examined the effect EtOH administration has on function of Golgi residential enzymes involved in N-glycosylation.HepG2 cells transfected with mouse ADH1 (VA-13 cells) were treated with 35 mM EtOH for 72 hours. Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8 weeks. Characterization of Golgi-associated mannosyl (?-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (MGAT1), ?-1,2-mannosidase (Man-I), and ?-mannosidase II (Man-II) were performed in VA-13 cells and rat hepatocytes followed by three-dimensional structured illumination microscopy (3D SIM).First, we detected that EtOH administration results in the loss of sialylated N-glycans on asialoglycoprotein receptor; however, the high-mannose-type N-glycans are increased. Further analysis by 3D SIM revealed that EtOH treatment despite Golgi disorganization does not change cis-Golgi localization for Man-I, but does induce medial-to-cis relocation of MGAT1 and Man-II. Using different approaches, including electron microscopy, we revealed that EtOH treatment results in dysfunction of ADP-ribosylation factor 1 (Arf1) GTPase followed by a deficiency in COPI vesicles at the Golgi. Silencing beta-COP or expression of GDP-bound mutant Arf1(T31N) mimics the EtOH effect on retaining MGAT1 and Man-II at the cis-Golgi, suggesting that (i) EtOH specifically blocks activation of Arf1, and (ii) EtOH alters the proper localization of Golgi enzymes through impairment of COPI. Importantly, the level of MGAT1 was reduced, because likely MGAT1, contrary to Man-I and Man-II, is giantin sensitive.Thus, we provide the mechanism by which EtOH-induced Golgi remodeling may significantly modify formation of N-glycans.

Project description:BackgroundGlioblastoma (GBM) is the most aggressive form of brain tumor and has vascular-rich features. The S100A4/non-muscle myosin IIA (NMIIA) axis contributes to aggressive phenotypes in a variety of human malignancies, but little is known about its involvement in GBM tumorigenesis. Herein, we examined the role of the S100A4/NMIIA axis during tumor progression and vasculogenesis in GBM.MethodsWe performed immunohistochemistry for S100A4, NMIIA, and two hypoxic markers, hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase 9 (CA9), in samples from 94 GBM cases. The functional impact of S100A4 knockdown and hypoxia were also assessed using a GBM cell line.ResultsIn clinical GBM samples, overexpression of S100A4 and NMIIA was observed in both non-pseudopalisading (Ps) and Ps (-associated) perinecrotic lesions, consistent with stabilization of HIF-1α and CA9. CD34(+) microvascular densities (MVDs) and the interaction of S100A4 and NMIIA were significantly higher in non-Ps perinecrotic lesions compared to those in Ps perinecrotic areas. In non-Ps perinecrotic lesions, S100A4(+)/HIF-1α(-) GBM cells were recruited to the surface of preexisting host vessels in the vascular-rich areas. Elevated vascular endothelial growth factor A (VEGFA) mRNA expression was found in S100A4(+)/HIF-1α(+) GBM cells adjacent to the vascular-rich areas. In addition, GBM patients with high S100A4 protein expression had significantly worse OS and PFS than did patients with low S100A4 expression. Knockdown of S100A4 in the GBM cell line KS-1 decreased migration capability, concomitant with decreased Slug expression; the opposite effects were elicited by blebbistatin-dependent inhibition of NMIIA.ConclusionS100A4(+)/HIF-1α(-) GBM cells are recruited to (and migrate along) preexisting vessels through inhibition of NMIIA activity. This is likely stimulated by extracellular VEGF that is released by S100A4(+)/HIF-1α(+) tumor cells in non-Ps perinecrotic lesions. In turn, these events engender tumor progression via acceleration of pro-tumorigenic vascular functions. Video abstract.

Dataset Information

The role of Rab6a and phosphorylation of non-muscle myosin IIA tailpiece in alcohol-induced Golgi disorganization.

Publications

The role of Rab6a and phosphorylation of non-muscle myosin IIA tailpiece in alcohol-induced Golgi disorganization.

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