scholarly journals The Small GTP-binding Protein Rho Regulates Cortical Activities in Cultured Cells during Division

1999 ◽  
Vol 144 (2) ◽  
pp. 305-313 ◽  
Author(s):  
Christopher B. O'Connell ◽  
Sally P. Wheatley ◽  
Sohail Ahmed ◽  
Yu-li Wang
Keyword(s):  
Epithelial Cells ◽  
Actin Filaments ◽  
Cultured Cells ◽  
Binding Protein ◽  
Rat Kidney ◽  
Myosin Ii ◽  
Metaphase Plate ◽  
Cortical Actin ◽  
Gtp Binding Protein ◽  
Gtp Binding

We have investigated the role of the small GTP-binding protein Rho in cytokinesis by microinjecting an inhibitor, C3 ribosyltransferase, into cultured cells. Microinjection of C3 into prometaphase or metaphase normal rat kidney epithelial cells induced immediate and global cortical movement of actin toward the metaphase plate, without an apparent effect on the mitotic spindle. During anaphase, concentrated cortical actin filaments migrated with separating chromosomes, leaving no apparent concentration of actin filaments along the equator. Myosin II in injected epithelial cells showed a diffuse distribution throughout cell division. All treated, well-adherent cells underwent cleavage-like activities and most of them divided successfully. However, cytokinesis became abnormal, generating irregular ingressions and ectopic cleavage sites even when mitosis was blocked with nocodazole. The effects of C3 appeared to be dependent on cell adhesion; less adherent 3T3 fibroblasts exhibited irregular cortical ingression only when cells started to increase attachment during respreading, but managed to complete cytokinesis. Poorly adherent HeLa cells showed neither ectopic cleavage nor completion of cytokinesis. Our results indicate that Rho does not simply activate actin–myosin II interactions during cytokinesis, but regulates the spatial pattern of cortical activities and completion of cytokinesis possibly through modulating the mechanical strength of the cortex.

Localization of ral, a small Mr GTP-binding protein, to collecting duct cells in bovine and rat kidney

1991 ◽  
Vol 261 (6) ◽  
pp. F1063-F1070
Author(s):  
A. Gupta ◽  
B. Bastani ◽  
P. Chardin ◽  
K. A. Hruska
Keyword(s):  
Gene Product ◽  
Plasma Membranes ◽  
Binding Protein ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Gtp Binding Protein ◽  
Bovine Kidney ◽  
Gtp Binding ◽  
Collecting Ducts

Plasma membranes from bovine kidney cortex were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Blotting with [alpha-32P]GTP and [35S]GTP gamma S demonstrated specific binding to three and six distinct protein bands, respectively, in the 20,000- to 29,000-Mr range. This indicated the presence of small Mr GTP binding proteins (smg) in bovine kidney cortex. Only one smg with 28,000 Mr was labeled with hydrolysis-resistant GTP photoaffinity probe p3-(4-azidoanilido)-p1-5GTP (AAGTP). The major smg in platelet membranes that binds GTP on nitrocellulose blots has been identified as ral-Mr 29,000. With the use of an antiserum against the ral A gene product, one of the smg with Mr of 29,000 present in bovine renal cortical plasma membranes was identified as ral. Ral was absent from glomerular homogenate, suggesting that it is localized to the tubular segments of the nephron. Ral was detected only in the particulate fraction and not the cytosol. Further subcellular localization of ral was investigated by immunohistochemical staining. Anti-ral antibody immunostained the apical and basolateral membranes of cells in the cortical and medullary collecting ducts in a speckled pattern in the bovine kidney. In the rat kidney, however, uniform linear staining of cortical and medullary collecting ducts predominantly localized to the apical membrane was observed. To date, no function has been assigned to ral. Localization of the ral gene product to the collecting duct suggests a specific functional role for this GTP-binding protein.

The small GTP-binding protein, Rab6p, is associated with both Golgi and post-Golgi synaptophysin-containing membranes during synaptogenesis of hypothalamic neurons in culture

1993 ◽  
Vol 105 (4) ◽  
pp. 935-947 ◽  
Author(s):  
A. Tixier-Vidal ◽  
A. Barret ◽  
R. Picart ◽  
V. Mayau ◽  
D. Vogt ◽  
...  
Keyword(s):  
Synaptic Vesicles ◽  
Laser Scanning ◽  
Synapse Formation ◽  
Binding Protein ◽  
Rat Kidney ◽  
Integral Membrane Protein ◽  
Laser Scanning Confocal Microscopy ◽  
Gtp Binding Protein ◽  
Scanning Confocal Microscopy ◽  
Gtp Binding

We have recently localized a small GTP-binding protein (Rab6p) thought to be involved in vesicular membrane transport, to the medial and trans-cisternae of the Golgi apparatus in NRK (normal rat kidney) cells. Here, we have localized and quantified Rab6p during the development in culture of embryonic neurons, up to synapse formation, and compared its subcellular distribution and level of expression to that of synaptophysin, a major integral membrane protein of small synaptic vesicles. Using immunocytochemistry (laser scanning confocal microscopy, immunoelectron microscopy), fractionation and immunoisolation methods, we show that during the early phase of synaptogenesis, Rab6p is associated with synaptophysin-containing membranes of a trans-Golgi subcompartment, post-Golgi vesicles and small synaptic vesicles or their precursors. Concomitantly, Rab6p undergoes translocation from cytosol to membranes and its level of expression increases. However, at late stages, the association of Rab6p to small synaptic vesicles sharply decreases and its level of expression plateaus. These findings suggest a role for Rab6p in the post-Golgi transport of synaptophysin, at an early step of the biogenesis of small synaptic vesicles.

Secretion from permeabilised mast cells is enhanced by addition of gelsolin: contrasting effects of endogenous gelsolin

1995 ◽  
Vol 108 (2) ◽  
pp. 657-666 ◽  
Author(s):  
Y.S. Borovikov ◽  
J.C. Norman ◽  
L.S. Price ◽  
A. Weeds ◽  
A. Koffer
Keyword(s):  
Mast Cells ◽  
Resting State ◽  
Actin Filaments ◽  
Cortical Actin ◽  
Gtp Binding Protein ◽  
Independent Manner ◽  
Calcium Dependent ◽  
Gtp Binding ◽  
Gamma S ◽  
Rat Mast Cells

Permeabilised rat mast cells were exposed to gelsolin and its N-terminal half (S1-3), proteins that sever actin filaments in a calcium-dependent and independent manner, respectively. Gelsolin and S1-3 induced a decrease in cellular F-actin content and an increase in the extent of the secretory response. The calcium sensitivities of both these effects were consistent with the differential calcium requirements of the two proteins. Segment 1 (S1), which binds G-actin and caps filaments but does not sever them, did not show these effects. Thus, secretion of mast cells is promoted as a consequence of the severing activity of exogenous gelsolin or S1-3. Most of the endogenous gelsolin remained within permeabilised, washed mast cells and its distribution in resting state was predominantly cortical. Addition of calcium in the absence of MgATP did not reduce the F-actin content; by contrast, calcium with MgATP induced F-actin loss that was unaffected by the presence of anti-gelsolin. Because this antibody inhibits the severing activity of gelsolin, these results indicate that in permeabilised mast cells the severing activity of the remaining endogenous gelsolin is not involved in cortical actin filaments disassembly. Upon exposure to GTP-gamma-S in the absence of calcium, the content of cortical gelsolin was reduced. This parallels our previous observation of a GTP-gamma-S induced reduction of cortical actin filaments followed by their relocation to the cell's interior (Norman et al. (1994) J. Cell Biol. 126, 1005–1015) and suggests that actin redistribution may be a consequence of dissociation of gelsolin caps brought about by activation of a GTP-binding protein.

scholarly journals The identification and characterization of a new GTP-binding protein (Gbp45) involved in cell proliferation and death related to mitochondrial function

2008 ◽  
Vol 13 (4) ◽  
Author(s):  
Yukimi Kira ◽  
Manabu Nishikawa
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Mitochondrial Function ◽  
Cultured Cells ◽  
Binding Protein ◽  
Immunoblot Analysis ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Identification And Characterization

AbstractWe describe the identification and characterization of a GTP-binding protein with a molecular weight of 45 kD (Gbp45). Gbp45 cDNA was found to overlap with a hypothetical human protein, PTD004, the sequence of which was previously deposited in the databases. The gene for PTD004 was recently found to be one of the ATPases, hOLA1 (human Obg-like ATPase 1). The Gbp45 gene encodes a protein of 396 amino acid residues. Immunocytochemical analysis and examination with GFP-tagged protein revealed that Gbp45 is primarily located in the cytosolic compartment. Immunoblot analysis showed that the Gbp45 protein is strongly expressed in the neuronal tissues and pancreas. T43N and T56N mutations resulted in a loss of Gbp45’s ability to bind to GTP and a loss of GTPase activity. In cultured cells, the transfection of wild-type Gbp45 accelerated cell proliferation, though T43N and T56N mutations induced cell death. Down-regulating Gbp45 expression decreased the cell proliferation rate and increased the rate of cell death induced by the inhibition of mitochondrial electron transport. These findings indicate that Gbp45 plays important roles in cell proliferation and death related to mitochondrial function.

scholarly journals rho, a Small GTP-Binding Protein, Is Essential for Shigella Invasion of Epithelial Cells

1997 ◽  
Vol 185 (2) ◽  
pp. 281-292 ◽  
Author(s):  
Masahisa Watarai ◽  
Yoichi Kamata ◽  
Shunji Kozaki ◽  
Chihiro Sasakawa
Keyword(s):  
Epithelial Cells ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Shigella Flexneri ◽  
Chinese Hamster ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Causative Agents ◽  
Invasive Capacity

Shigella, the causative agents of bacillary dysentery, are capable of invading mammalian cells that are not normally phagocytic. Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with α5β1 integrin. We report here that Shigella invasion of epithelial cells requires rho activity, a ras-related GTP-binding protein. The invasive capacity of Shigella flexneri for Chinese hamster ovary (CHO) cells and other epithelial cells were greatly reduced when treated with Clostridium botulinum exoenzyme C3 transferase. Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA. Attachment of S. flexneri to CHO cells can elicit tyrosine phosphorylation of pp125FAK and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase. Our results indicate that cellular signal transduction regulated by rho is essential for Shigella invasion of epithelial cells.

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