scholarly journals The GTPase Rab4 Interacts with Chlamydia trachomatis Inclusion Membrane Protein CT229

2006 ◽  
Vol 74 (9) ◽  
pp. 5362-5373 ◽  
Author(s):  
K. A. Rzomp ◽  
A. R. Moorhead ◽  
M. A. Scidmore
Keyword(s):  
Chlamydia Trachomatis ◽  
Hela Cells ◽  
Intracellular Trafficking ◽  
Specific Interaction ◽  
Dominant Negative ◽  
Rab Gtpases ◽  
Wild Type ◽  
Inclusion Membrane ◽  
Chlamydial Inclusion ◽  
Constitutively Active

ABSTRACT Chlamydiae, which are obligate intracellular bacteria, replicate in a nonlysosomal vacuole, termed an inclusion. Although neither the host nor the chlamydial proteins that mediate the intracellular trafficking of the inclusion have been clearly identified, several enhanced green fluorescent protein (GFP)-tagged Rab GTPases, including Rab4A, are recruited to chlamydial inclusions. GFP-Rab4A associates with inclusions in a species-independent fashion by 2 h postinfection by mechanisms that have not yet been elucidated. To test whether chlamydial inclusion membrane proteins (Incs) recruit Rab4 to the inclusion, we screened a collection of chlamydial Incs for their ability to interact with Rab4A by using a yeast two-hybrid assay. From our analysis, we identified a specific interaction between Rab4A and Chlamydia trachomatis Inc CT229, which is expressed during the initial stages of infection. CT229 interacts with only wild-type Rab4A and the constitutively active GTPase-deficient Rab4AQ67L but not with the dominant-negative GDP-restricted Rab4AS22N mutant. To confirm the interaction between CT229 and Rab4A, we demonstrated that DsRed-CT229 colocalized with GFP-Rab4A in HeLa cells and more importantly wild-type and constitutively active GFP-Rab4A colocalized with CT229 at the inclusion membrane in C. trachomatis serovar L2-infected HeLa cells. Taken together, these data suggest that CT229 interacts with and recruits Rab4A to the inclusion membrane and therefore may play a role in regulating the intracellular trafficking or fusogenicity of the chlamydial inclusion.

Role of Rab Proteins In Endocytosis and Trafficking of Factor VIIa and Endothelial Cell Protein C Receptor In Endothelial Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1145-1145
Author(s):  
Ramesh C Nayak ◽  
Shiva Keshava ◽  
Usha Pendurthi ◽  
L. Vijaya Mohan Rao
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Dominant Negative ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Wild Type ◽  
Dominant Negative Form ◽  
Negative Form ◽  
Constitutively Active

Abstract Abstract 1145 Recent studies from our laboratory and others showed that endothelial cell protein C receptor (EPCR), the cellular receptor for protein C and activated protein C (APC), also serves as a receptor for factor VII (FVII) and activated factor VII (FVIIa). At present, the physiological importance of FVII/FVIIa binding to EPCR is largely unknown, but this interaction may play a role in the clearance or transport of FVII/FVIIa from circulation to tissues. Our recent studies showed that FVIIa (or APC) binding to EPCR promoted the endocytosis of EPCR via dynamin and caveolar-dependent pathways, and the endocytosed receptor-ligand complexes were accumulated in the recycling compartment (REC) before being targeted back to the cell surface (Blood 2009;114:1974-1986). Rab GTPases (Rab 4, Rab 5, Rab 7 and Rab 11 etc.), which localize to specific endosomal structures, have been shown to play crucial roles in the endocytic and exocytic pathways of receptor or receptor/ligand complexes. The role of these Ras-like small GTPases is unknown in endocytosis and trafficking of EPCR and EPCR/FVIIa complexes. The present study was undertaken in order to investigate the role of different Rab GTPases (Rab 4A, Rab 5 and Rab11) in the intracellular trafficking of EPCR and internalized FVIIa in endothelial cells. For this, we examined the effect of expressing wild-type (wt) or mutant Rab proteins on the intracellular distribution of FVIIa in human umbilical vein endothelial cells (HUVEC). The wild-type, constitutively active and dominant negative mutants of Rab 4A, Rab 5 and Rab 11 were cloned in adenoviral shuttle vector pacAd5 K-N pA CMV and the recombinant adenoviruses expressing these Rab GTPase variants were generated in human embryonic kidney (HEK) cells. HUVEC were infected with recombinant adenoviruses encoding for the wild-type, active or dominant negative mutant of Rab 4A, Rab 5 and Rab 11 (25 moi/cell). After culturing the cells for 24 h, they were incubated with recombinant FVIIa conjugated with Alexa fluor 488 fluorescent dye (AF488-FVIIa) for 1 h at 37°C. The intracellular distribution of FVIIa was analyzed by monitoring the fluorescence of AF488-FVIIa by confocal microscopy. The intracellular distribution of EPCR and Rab proteins was evaluated by confocal microscopy after immunofluorescence staining. Expression of Rab 4A wt or constitutively active Rab 4A (Q67L) forms led to accumulation of AF488-FVIIa within the Rab 4A positive early/sorting endosomes, whereas FVIIa accumulation in the REC was inhibited. In cells expressing Rab 4A dominant negative form (S22N), FVIIa was trafficked normally and accumulated in the REC. Rab 4A is known to regulate fusion of early and sorting endosomes, as well as recycling of the internalized receptor or receptor/ligand complexes from early/sorting endosomes back to the cell surface. Increased accumulation of FVIIa in early/sorting endosomes but a decrease in REC in HUVEC transduced to express wt and constitutively active Rab 4A, suggests that Rab 4A plays a role in the transport of internalized FVIIa and FVIIa-EPCR complexes from sorting endosomes back to the cell surface. HUVEC expressing Rab 5 wt or active mutant (Q79L) showed larger endosomal structures beneath the plasma membrane where EPCR and FVIIa were accumulated; very little FVIIa entered the REC. The trafficking of internalized FVIIa remained unaffected in HUVEC expressing Rab 5A dominant negative form (S34N). As Rab 5 is known to induce receptor internalization and fusion between early endosomes, the large endosomal structures containing AF488-FVIIa found in HUVEC expressing wt or constitutively active form but not in cells expressing the dominant negative form suggests that Rab 5 facilitates internalization of FVIIa-EPCR complexes. In contrast to the data obtained in HUVEC expressing Rab 4A and Rab 5, the intracellular trafficking of AF488-FVIIa remained unaffected in HUVEC expressing either wt or constitutively active Rab 11 mutant. Rab 11 dominant negative mutant (S34N) prevented the entry of AF488-FVIIa into REC. The observation that the dominant negative form of Rab 11 inhibits the entry of internalized FVIIa to the REC indicates that the activation of Rab 11 by GTP is required for the transport of FVIIa from sorting endosomes toward the recycling compartment. Overall our present data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR and internalized FVIIa in endothelial cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium

2007 ◽  
Vol 176 (3) ◽  
pp. 263-268 ◽  
Author(s):  
Adam C. Smith ◽  
Won Do Heo ◽  
Virginie Braun ◽  
Xiuju Jiang ◽  
Chloe Macrae ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Dominant Negative ◽  
Rab Gtpases ◽  
Wild Type ◽  
Intracellular Growth ◽  
Non Invasive ◽  
Serovar Typhimurium ◽  
Guanosine Triphosphatase ◽  
Kinetics Of

Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome–lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth.

H-Ras Is Involved in the Inside-out Signaling Pathway of Interleukin-3–Induced Integrin Activation

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1540-1548 ◽  
Author(s):  
Hirohiko Shibayama ◽  
Naoyuki Anzai ◽  
Stephen E. Braun ◽  
Seiji Fukuda ◽  
Charlie Mantel ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Dominant Negative ◽  
The Other ◽  
Wild Type ◽  
Integrin Activation ◽  
Mapk Kinase ◽  
Interleukin 3 ◽  
Other Hand ◽  
Inside Out ◽  
Constitutively Active

Abstract The proto-oncogene product, p21ras, has been implicated in the cellular mechanism of adhesion, although its precise role has been controversial. Numerous cytokines and growth-factors activate Ras, which is an important component of their growth-promoting signaling pathways. On the other hand, the role of Ras in cytokine-induced adhesion has not been elucidated. We therefore investigated the function of H-Ras in the inside-out signaling pathway of interleukin-3 (IL-3)–induced integrin activation in the murine Baf3 cell line after transfection of cells with either constitutively active, dominant-negative, or wild-type H-Ras cDNAs. Adhesion of Baf3 cells to fibronectin was induced by IL-3 in a dose-dependent manner via very late antigen-4 (VLA-4; 4β1 integrins) and VLA-5 (5β1 integrins) activation. On the other hand, IL-4 did not induce the adhesion of Baf3 cells to fibronectin, although IL-4 did stimulate the cell proliferation of Baf3 cells. Constitutively active H-Ras–transfected Baf3 cells adhered to fibronectin without IL-3 stimulation through VLA-4 and VLA-5, whereas dominant-negative H-Ras–transfected Baf3 cells showed significantly less adhesion induced by IL-3 compared with wild-type and constitutively active H-Ras–transfected Baf3 cells. Anti-β1 integrin antibody (clone; 9EG7), which is known to change integrin conformation and activate integrins, induced the adhesion of dominant-negative H-Ras–transfected Baf3 cells as much as the other types of H-Ras–transfected Baf3 cells. 8-Br-cAMP, Dibutyryl-cAMP, Ras-Raf-1 pathway inhibitors, and PD98059, a MAPK kinase inhibitor, suppressed proliferation and phosphorylation of MAPK detected by Western blotting with anti–phospho-MAPK antibody, but not adhesion of any type of H-Ras–transfected Baf3 cells, whereas U-73122, a phospholipase C (PLC) inhibitor, suppressed adhesion of these cells completely. These data indicate that H-Ras and PLC, but not Raf-1, MAPK kinase, or the MAPK pathway, are involved in the inside-out signaling pathway of IL-3–induced VLA-4 and VLA-5 activation in Baf3 cells.

scholarly journals Different consequences of cataract-associated mutations at adjacent positions in the first extracellular boundary of connexin50

2011 ◽  
Vol 300 (5) ◽  
pp. C1055-C1064 ◽  
Author(s):  
Jun-Jie Tong ◽  
Peter J. Minogue ◽  
Wenji Guo ◽  
Tung-Ling Chen ◽  
Eric C. Beyer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Hela Cells ◽  
Xenopus Oocytes ◽  
Dominant Negative ◽  
Wild Type ◽  
Gap Junction Channels ◽  
Dominant Negative Inhibitor ◽  
Intercellular Channels ◽  
Gap Junctional

Gap junction channels, which are made of connexins, are critical for intercellular communication, a function that may be disrupted in a variety of diseases. We studied the consequences of two cataract-associated mutations at adjacent positions at the first extracellular boundary in human connexin50 (Cx50), W45S and G46V. Both of these mutants formed gap junctional plaques when they were expressed in HeLa cells, suggesting that they trafficked to the plasma membrane properly. However, their functional properties differed. Dual two-microelectrode voltage-clamp studies showed that W45S did not form functional intercellular channels in paired Xenopus oocytes or hemichannel currents in single oocytes. When W45S was coexpressed with wild-type Cx50, the mutant acted as a dominant negative inhibitor of wild-type function. In contrast, G46V formed both functional gap junctional channels and hemichannels. G46V exhibited greatly enhanced currents compared with wild-type Cx50 in the presence of physiological calcium concentrations. This increase in hemichannel activity persisted when G46V was coexpressed with wild-type lens connexins, consistent with a dominant gain of hemichannel function for G46V. These data suggest that although these two mutations are in adjacent amino acids, they have very different effects on connexin function and cause disease by different mechanisms: W45S inhibits gap junctional channel function; G46V reduces cell viability by forming open hemichannels.

scholarly journals Vav2 Activates Rac1, Cdc42, and RhoA Downstream from Growth Factor Receptors but Not β1 Integrins

2000 ◽  
Vol 20 (19) ◽  
pp. 7160-7169 ◽  
Author(s):  
Betty P. Liu ◽  
Keith Burridge
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Phosphorylation ◽  
G Proteins ◽  
Growth Factor Receptors ◽  
Dominant Negative ◽  
Wild Type ◽  
Epidermal Growth ◽  
Rho Family ◽  
Constitutively Active

ABSTRACT The Rho family of GTPases plays a major role in the organization of the actin cytoskeleton. These G proteins are activated by guanine nucleotide exchange factors that stimulate the exchange of bound GDP for GTP. In their GTP-bound state, these G proteins interact with downstream effectors. Vav2 is an exchange factor for Rho family GTPases. It is a ubiquitously expressed homologue of Vav1, and like Vav1, it has previously been shown to be activated by tyrosine phosphorylation. Because Vav1 becomes tyrosine phosphorylated and activated following integrin engagement in hematopoietic cells, we investigated the tyrosine phosphorylation of Vav2 in response to integrin-mediated adhesion in fibroblasts and epithelial cells. However, no tyrosine phosphorylation of Vav2 was detected in response to integrin engagement. In contrast, treating cells with either epidermal growth factor or platelet-derived growth factor stimulated tyrosine phosphorylation of Vav2. We have examined the effects of overexpressing either wild-type or amino-terminally truncated (constitutively active) forms of Vav2 as fusion proteins with green fluorescent protein. Overexpression of either wild-type or constitutively active Vav2 resulted in prominent membrane ruffles and enhanced stress fibers. These cells revealed elevated rates of cell migration that were inhibited by expression of dominant negative forms of Rac1 and Cdc42. Using a binding assay to measure the activity of Rac1, Cdc42, and RhoA, we found that overexpression of Vav2 resulted in increased activity of each of these G proteins. Expression of a carboxy-terminal fragment of Vav2 decreased the elevation of Rac1 activity induced by epidermal growth factor, consistent with Vav2 mediating activation of Rac1 downstream from growth factor receptors.

scholarly journals Chlamydia trachomatis IncA Is Localized to the Inclusion Membrane and Is Recognized by Antisera from Infected Humans and Primates

1998 ◽  
Vol 66 (12) ◽  
pp. 6017-6021 ◽  
Author(s):  
John P. Bannantine ◽  
Walter E. Stamm ◽  
Robert J. Suchland ◽  
Daniel D. Rockey
Keyword(s):  
Chlamydia Trachomatis ◽  
Immunoblot Analysis ◽  
Chlamydia Psittaci ◽  
Inclusion Membrane ◽  
Chlamydial Inclusion

ABSTRACT Chlamydia psittaci produces a collection of proteins, termed IncA, IncB, and IncC, that are localized to the chlamydial inclusion membrane. In this report we demonstrate that IncA is also produced by Chlamydia trachomatis. C. trachomatis IncA is structurally similar to C. psittaci IncA and is also localized to the inclusion membrane. Immunoblot analysis demonstrated that sera from C. trachomatis-infected patients and from experimentally infected monkeys both recognized C. trachomatis IncA.

scholarly journals Cytoplasmic accumulation of cdc25B phosphatase in mitosis triggers centrosomal microtubule nucleation in HeLa cells

1996 ◽  
Vol 109 (5) ◽  
pp. 1081-1093 ◽  
Author(s):  
B.G. Gabrielli ◽  
C.P. De Souza ◽  
I.D. Tonks ◽  
J.M. Clark ◽  
N.K. Hayward ◽  
...  
Keyword(s):  
Functional Status ◽  
Hela Cells ◽  
Mitotic Spindle ◽  
Dominant Negative ◽  
Cyclin B ◽  
Wild Type ◽  
Microtubule Nucleation ◽  
Cytoplasmic Accumulation ◽  
Low Levels ◽  
Essential Prerequisite

The formation of the mitotic spindle is an essential prerequisite for successful mitosis. The dramatic changes in the level of microtubule (Mt) nucleation at the centrosomes and Mt dynamics that occur in prophase are presumed to be initiated through the activity of cdc2/cyclin B. Here we present data that the cdc25B isoform functions to activate the cytoplasmic pool of cdc2/cyclin B responsible for these events. In contrast to cdc25C, cdc25B is present at low levels in HeLa cells during interphase, but sharply increases in prophase, when cdc25B accumulation in the cytoplasm correlates with prophase spindle formation. Overexpression of wild type and dominant negative mutants of cdc25B and cdc25C shows that prophase Mt nucleation is a consequence of cytoplasmic cdc25B activity, and that cdc25C regulates nuclear G2/M events. Our data also suggest that the functional status of the centrosome can regulate nuclear mitotic events.

scholarly journals Receptor-Mediated Moloney Murine Leukemia Virus Entry Can Occur Independently of the Clathrin-Coated-Pit-Mediated Endocytic Pathway

1999 ◽  
Vol 73 (7) ◽  
pp. 5994-6005 ◽  
Author(s):  
Sunyoung Lee ◽  
Yi Zhao ◽  
W. French Anderson
Keyword(s):  
Hela Cells ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Moloney Murine Leukemia Virus ◽  
Wild Type ◽  
293 Cells ◽  
Immunofluorescence Labeling ◽  
Murine Leukemia

ABSTRACT To investigate receptor-mediated Moloney murine leukemia virus (MoMuLV) entry, the green fluorescent protein (GFP)-tagged ecotropic receptor designated murine cationic amino acid transporter (MCAT-1) (MCAT-1-GFP) was constructed and expressed in 293 cells (293/MCAT-1-GFP). 293/MCAT-1-GFP cells displayed green fluorescence primarily at the cell membrane and supported wild-type levels of MoMuLV vector binding and transduction. Using immunofluorescence labeling and confocal microscopy, it was demonstrated that the surface envelope protein (SU) gp70 of MoMuLV virions began to appear inside cells 5 min after virus binding and was colocalized with MCAT-1-GFP. However, clathrin was not colocalized with MCAT-1-GFP, suggesting that MoMuLV entry, mediated by MCAT-1, does not involve clathrin. Double immunofluorescence labeling of SU and clathrin in 293 cells expressing untagged receptor (293/MCAT-1) gave the same results, i.e., SU and clathrin did not colocalize. In addition, we examined the transduction ability of MoMuLV vector on HeLa cells overexpressing the dominant-negative GTPase mutant of dynamin (K44A). HeLa cells overexpressing mutant dynamin have a severe block in endocytosis by the clathrin-coated-pit pathway. No significant titer difference was observed when MoMuLV vector was tranduced into HeLa cells overexpressing either wild-type or mutant dynamin, while the transduction ability of vesicular stomatitis virus glycoprotein pseudotyped vector into HeLa cells overexpressing mutant dynamin was decreased significantly. Taken together, these data suggest that MoMuLV entry does not occur through the clathrin-coated-pit-mediated endocytic pathway.

scholarly journals Connexin 32 mutations from X-linked Charcot-Marie-Tooth disease patients: functional defects and dominant negative effects.

1996 ◽  
Vol 7 (6) ◽  
pp. 907-916 ◽  
Author(s):  
Y Omori ◽  
M Mesnil ◽  
H Yamasaki
Keyword(s):  
Hela Cells ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Base Substitution ◽  
Cell Contact ◽  
Wild Type ◽  
Gap Junctional Intercellular Communication ◽  
Charcot Marie Tooth ◽  
Charcot Marie Tooth Disease ◽  
Tooth Disease

We have characterized the function of connexin (Cx) 32 gene mutations found in X-linked dominant Charcot-Marie-Tooth disease with respect to their ability to form functional gap junctions among themselves and to inactivate wild-type Cx32 by a dominant negative mechanism. We prepared four types of Cx32 mutant cDNAs and transfected them into HeLa cells, which do not show detectable levels of gap junctional intercellular communication (GJIC), nor expression of any connexins examined. Cells transfected with the wild-type Cx32 gene, but not those transfected with three different base substitution mutations (i.e. Cys 60 to Phe, Val 139 to Met, and Arg 215 to Trp), restored GJIC. Unexpectedly, in cells transfected with a nonsense mutant at codon 220, there was also restored GJIC. When we double-transfected these mutant constructs into the HeLa cells that had already been transfected with the wild-type Cx32 gene and thus were GJIC proficient, three base substitution mutants inhibited GJIC, suggesting that these three mutants can eliminate the function of wild-type Cx32 in a dominant negative manner. The nonsense mutation at codon 220 did not show such a dominant negative effect. Since both mutant and wild-type Cx32 mRNAs were detected, but only poor Cx32 protein expression at cell-cell contact areas was observed in the double transfectants, it is suggested that certain mutants form nonfunctional chimeric connexons with wild-type connexins, which are not properly inserted into the cytoplasmic membrane.

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