Dataset Information

Rab10 GTPase regulates ER dynamics and morphology.

ABSTRACT: We have identified Rab10 as an ER-specific Rab GTPase that regulates ER structure and dynamics. We show that Rab10 localizes to the ER and to dynamic ER-associated structures that track along microtubules and mark the position of new ER tubule growth. Rab10 depletion or expression of a Rab10 GDP-locked mutant alters ER morphology, resulting in fewer ER tubules. We demonstrate that this defect is due to a reduced ability of dynamic ER tubules to grow out and successfully fuse with adjacent ER. Consistent with this function, Rab10 partitions to dynamic ER-associated domains found at the leading edge of almost half of all dynamic ER tubules. Interestingly, this Rab10 domain is highly enriched with at least two ER enzymes that regulate phospholipid synthesis, phosphatidylinositol synthase (PIS) and CEPT1. Both the formation and function of this Rab10/PIS/CEPT1 dynamic domain are inhibited by expression of a GDP-locked Rab10 mutant. Together, these data demonstrate that Rab10 regulates ER dynamics and further suggest that these dynamics could be coupled to phospholipid synthesis.

SUBMITTER: English AR

PROVIDER: S-EPMC3582403 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Rab10 GTPase regulates ER dynamics and morphology.

English Amber R AR Voeltz Gia K GK

Nature cell biology 20121223 2

We have identified Rab10 as an ER-specific Rab GTPase that regulates ER structure and dynamics. We show that Rab10 localizes to the ER and to dynamic ER-associated structures that track along microtubules and mark the position of new ER tubule growth. Rab10 depletion or expression of a Rab10 GDP-locked mutant alters ER morphology, resulting in fewer ER tubules. We demonstrate that this defect is due to a reduced ability of dynamic ER tubules to grow out and successfully fuse with adjacent ER. Co ...[more]

PMID: 23263280

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Project description:mRNA trafficking and local protein translation are associated with protrusive cellular domains, such as neuronal growth cones, and deregulated control of protein translation is associated with tumor malignancy. We show here that activated RhoA, but not Rac1, is enriched in pseudopodia of MSV-MDCK-INV tumor cells and that Rho, Rho kinase (ROCK) and myosin II regulate the microtubule-independent targeting of RNA to these tumor cell domains. ROCK inhibition does not affect pseudopodial actin turnover but significantly reduces the dynamics of pseudopodial RNA turnover. Gene array analysis shows that 7.3% of the total genes analyzed exhibited a greater than 1.6-fold difference between the pseudopod and cell body fractions. Of these, only 13.2% (261 genes) are enriched in pseudopodia, suggesting that only a limited number of total cellular mRNAs are enriched in tumor cell protrusions. Comparison of the tumor pseudopod mRNA cohort and a cohort of mRNAs enriched in neuronal processes identified tumor pseudopod-specific signaling networks that were defined by expression of M-Ras and the Shp2 protein phosphatase. Pseudopod expression of M-Ras and Shp2 mRNA were diminished by ROCK inhibition linking pseudopodial Rho/ROCK activation to the localized expression of specific mRNAs. Pseudopodial enrichment for mRNAs involved in protein translation and signaling suggests that local mRNA translation regulates pseudopodial expression of less stable signaling molecules as well as the cellular machinery to translate these mRNAs. mRNA trafficking and local protein translation are associated with protrusive cellular domains, such as neuronal growth cones, and deregulated control of protein translation is associated with tumor malignancy. We show here that activated RhoA, but not Rac1, is enriched in pseudopodia of MSV-MDCK-INV tumor cells and that Rho, Rho kinase (ROCK) and myosin II regulate the microtubule-independent targeting of RNA to these tumor cell domains. ROCK inhibition does not affect pseudopodial actin turnover but significantly reduces the dynamics of pseudopodial RNA turnover. Gene array analysis shows that 7.3% of the total genes analyzed exhibited a greater than 1.6-fold difference between the pseudopod and cell body fractions. Of these, only 13.2% (261 genes) are enriched in pseudopodia, suggesting that only a limited number of total cellular mRNAs are enriched in tumor cell protrusions. Comparison of the tumor pseudopod mRNA cohort and a cohort of mRNAs enriched in neuronal processes identified tumor pseudopod-specific signaling networks that were defined by expression of M-Ras and the Shp2 protein phosphatase. Pseudopod expression of M-Ras and Shp2 mRNA were diminished by ROCK inhibition linking pseudopodial Rho/ROCK activation to the localized expression of specific mRNAs. Pseudopodial enrichment for mRNAs involved in protein translation and signaling suggests that local mRNA translation regulates pseudopodial expression of less stable signaling molecules as well as the cellular machinery to translate these mRNAs. Pseudopodial Rho/ROCK activation may impact on tumor cell migration and metastasis by stimulating the pseudopodial translocation of mRNAs and thereby regulating the expression of local signaling cascades. Keywords: tumor cells, protrusive cellular domains, pseudopodia vs cell body, RNA trafficking Pseudopod purification- Pseudopodia purification was performed as previously described (Jia et al, J. Biol. Chem. 280, 30564-73, 2005). Briefly, 107 MSV-MDCK-INV cells were plated on 100 mm 1um pore filters mounted between two 3-3.5â custom-made washers (Boulons Plus, Anjou, QC) in a 100 mm Falcon Petri dish and the exterior sealed with agarose to prevent cell leakage to the bottom of the filter. After 24 hours culture, the filter was washed 4 times with cold PBS containing 0.1 mM Ca2+ and 1 mM Mg2+ and the top (cell bodies) and bottom (pseudopodia) of the filter scraped with a glass cover slip in ice-cold lysis buffer (20 mM Tris-HCl pH 7.6, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA containing freshly added 2 mM DTT, 0.5 mM PMSF, 1 mM sodium vanadate, 2.5 mM sodium fluoride and 1uM leupeptin). Affymetrix gene array- GeneChips were processed at the London Regional Genomics Centre (Robarts Research Institute, London, Ontario, Canada; http://www.lrge.ca). Six Affymetrix canine arrays (3 pseudopodia, 3 cell body) were scanned with the Affymetrix GeneChip Scanner 3000 and signal intensities for genes generated with GCOS1.2 (Affymetrix Inc., Santa Clara, CA) using default values for statistical expression algorithm parameters, a target signal of 150 for all probe sets and a normalization value of 1. To determine significant differences in gene expression levels between pseudopodia and cell body samples, 1 way-ANOVA was performed using Partek Pro (St. Louis, MO). Significantly modulated genes were defined as those with absolute log2 fold change greater than 0.7. Genes were summarized together using hierarchical clustering by Z score normalized data crossing all 6 samples.

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Rab10 GTPase regulates ER dynamics and morphology.

Publications

Rab10 GTPase regulates ER dynamics and morphology.

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