The rho family of GTP-binding proteins regulates actin filament organization. In unpolarized mammalian cells, rho proteins regulate the assembly of actin-containing stress fibers at the cell-matrix interface. Polarized epithelial cells, in contrast, are tall and cylindrical with well developed intercellular tight junctions that permit them to behave as biologic barriers. We report that rho regulates filamentous actin organization preferentially in the apical pole of polarized intestinal epithelial cells and, in so doing, influences the organization and permeability of the associated apical tight junctions. Thus, barrier function, which is an essential characteristic of columnar epithelia, is regulated by rho.
The GTPase RhoA is a major regulator of the assembly of actin stress fibers and the contractility of the actomyosin cytoskeleton. The epidermal cell differentiation inhibitor (EDIN) and EDIN-like ADP-ribosyltransferases of Staphylococcus aureus catalyze the inactivation of RhoA, producing actin cable disruption. We report that purified recombinant EDIN and EDIN-producing S. aureus provoke large transcellular tunnels in endothelial cells that we have named macroapertures (MAs). These structures open transiently, followed by the appearance of actin-containing membrane waves extending over the aperture. Disruption of actin cables, either directly or indirectly, through rhoA RNAi knockdown also triggers the formation of MAs. Intoxication of endothelial monolayers by EDIN produces a loss of barrier function and provides direct access of the endothelium basement membrane to S. aureus.
Background Motility is an important component of Salmonella enterica serovar Typhimurium (ST) pathogenesis allowing the bacteria to move into appropriate niches, across the mucus layer and invade the intestinal epithelium. In vitro, flagellum-associated motility is closely related to the invasive properties of ST. The probiotic yeast Saccharomyces boulardii BIOCODEX (S.b-B) is widely prescribed for the prophylaxis and treatment of diarrheal diseases caused by bacteria or antibiotics. In case of Salmonella infection, S.b-B has been shown to decrease ST invasion of T84 colon cell line. The present study was designed to investigate the impact of S.b-B on ST motility. Methodology/Principal Findings Experiments were performed on human colonic T84 cells infected by the Salmonella strain 1344 alone or in the presence of S.b-B. The motility of Salmonella was recorded by time-lapse video microscopy. Next, a manual tracking was performed to analyze bacteria dynamics (MTrackJ plugin, NIH image J software). This revealed that the speed of bacterial movement was modified in the presence of S.b-B. The median curvilinear velocity (CLV) of Salmonella incubated alone with T84 decreased from 43.3 µm/sec to 31.2 µm/sec in the presence of S.b-B. Measurement of track linearity (TL) showed similar trends: S.b-B decreased by 15% the number of bacteria with linear tract (LT) and increased by 22% the number of bacteria with rotator tract (RT). Correlation between ST motility and invasion was further established by studying a non-motile flagella-deficient ST strain. Indeed this strain that moved with a CLV of 0.5 µm/sec, presented a majority of RT and a significant decrease in invasion properties. Importantly, we show that S.b-B modified the motility of the pathogenic strain SL1344 and significantly decreased invasion of T84 cells by this strain. Conclusions This study reveals that S.b-B modifies Salmonella's motility and trajectory which may account for the modification of Salmonella's invasion.
The proteins of the RAF family (A-RAF, B-RAF, and C-RAF) are serine/threonine kinases that play important roles in development, mature cell regulation, and cancer. Although it is widely held that their localization on membranes is an important aspect of their function, there are few data that address this aspect of their mode of action. Here, we report that each member of the RAF family exhibits a specific distribution at the level of cellular membranes and that C-RAF is the only isoform that directly targets mitochondria. We found that the RAF kinases exhibit intrinsic differences in terms of mitochondrial affinity and that C-RAF is the only isoform that binds this organelle efficiently. This affinity is conferred by the C-RAF amino-terminal domain and does not depend on the presence of RAS GTPases on the surface of mitochondria. Finally, we analyzed the consequences of C-RAF activation on mitochondria and observed that this event dramatically changes their morphology and their subcellular distribution. Our observations indicate that: (i) RAF kinases exhibit different localizations at the level of cellular membranes; (ii) C-RAF is the only isoform that directly binds mitochondria; and (iii) through its functional coupling with MEK, C-RAF regulates the shape and the cellular distribution of mitochondria. The proteins of the RAF family (A-RAF, B-RAF, and C-RAF) are serine/threonine kinases that play important roles in development, mature cell regulation, and cancer. Although it is widely held that their localization on membranes is an important aspect of their function, there are few data that address this aspect of their mode of action. Here, we report that each member of the RAF family exhibits a specific distribution at the level of cellular membranes and that C-RAF is the only isoform that directly targets mitochondria. We found that the RAF kinases exhibit intrinsic differences in terms of mitochondrial affinity and that C-RAF is the only isoform that binds this organelle efficiently. This affinity is conferred by the C-RAF amino-terminal domain and does not depend on the presence of RAS GTPases on the surface of mitochondria. Finally, we analyzed the consequences of C-RAF activation on mitochondria and observed that this event dramatically changes their morphology and their subcellular distribution. Our observations indicate that: (i) RAF kinases exhibit different localizations at the level of cellular membranes; (ii) C-RAF is the only isoform that directly binds mitochondria; and (iii) through its functional coupling with MEK, C-RAF regulates the shape and the cellular distribution of mitochondria. RAF kinases are a family of Ser/Thr kinases (A-RAF, B-RAF, and C-RAF) that are found upstream of the highly conserved mitogen-activated protein kinase (MAPK) 3The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; HUVEC, human umbilical vein endothelial cell; LT, lethal toxin; PA, protective antigen; LF, lethal factor; MEF, mouse embryonic fibroblasts; PMA, phorbol 12-myristate 13-acetate; 4-OHT, 4-hydroxytamoxifene; WT, wild type; HEK, human embryonic kidney. signaling module, MEK-ERK. As such, they link ERK activation to different growth signals (1Wellbrock C. Kasarides M. Marais R. Nat. Rev. Mol. Cell Biol. 2004; 5: 875-885Crossref PubMed Scopus (939) Google Scholar). Mutations that confer a constitutively active status to the protein B-RAF are among the most commonly detected genetic abnormalities in human carcinogenesis (1Wellbrock C. Kasarides M. Marais R. Nat. Rev. Mol. Cell Biol. 2004; 5: 875-885Crossref PubMed Scopus (939) Google Scholar). The variety of defects observed upon the knock-out of each of the RAF genes indicate that they play both overlapping and distinct roles in mouse development. However, it is unclear to which extent this can be attributed to their tissue-specific expression pattern and/or to specific roles that these kinases might exert at the cellular level (2Hindley A. Kolch W. J. Cell Sci. 2002; 115: 1575-1581Crossref PubMed Google Scholar, 3Pelkmans L. Zerial M. Nature. 2005; 436: 128-133Crossref PubMed Scopus (288) Google Scholar, 4Pelkmans L. Fava E. Grabner H. Hannus M. Habermann B. Krausz E. Zerial M. Nature. 2005; 436: 78-86Crossref PubMed Scopus (529) Google Scholar). A distinctive property of the RAF kinases is their ability to interact with cellular membranes. This localization imparts distinct properties to RAF kinases in terms of signaling; the small GTPases of the RAS family, which are membrane-anchored proteins, link RAF kinases to activated membrane receptors (5Plowman S.J. Hancock J.F. Biochim. Biophys. Acta. 2005; 1746: 274-283Crossref PubMed Scopus (96) Google Scholar). Compared with the cytosol, cellular membranes also contain a reduced content in phosphatases, thereby increasing the functional output of C-RAF activation when this kinase is located on membranes (6Harding A. Tian T. Westbury E. Frische E. Hancock J.F. Curr. Biol. 2005; 15: 869-873Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). RAF kinases interact not only with the plasma membrane; C-RAF is also present on the surface of mitochondria (7Wang H.G. Rapp U.R. Reed J.C. Cell. 1996; 87: 629-638Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 8Wiese S. Pei G. Karch C. Troppmair J. Holtmann B. Rapp U.R. Sendtner M. Nat. Neurosci. 2001; 4: 137-142Crossref PubMed Scopus (95) Google Scholar, 9Alavi A. Hood J.D. Frausto R. Stupack D.G. Cheresh D.A. Science. 2003; 301: 94-96Crossref PubMed Scopus (297) Google Scholar, 10Jin S. Zhuo Y. Guo W. Field J. J. Biol. Chem. 2005; 280: 24698-24705Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 11Goetz R. Wiese S. Takayama S. Camarero G.C. Rossoll W. Schweizer U. Troppmair J. Jablonka S. Holtmann B. Reed J.C. Rapp U.R. Sendtner M. Nat. Neurosci. 2005; 8: 1169-1178Crossref PubMed Scopus (102) Google Scholar, 12Galmiche A. Fueller J. Biochim. Biophys. Acta. 2007; 1773: 1256-1262Crossref PubMed Scopus (16) Google Scholar). Although the molecular mechanisms and the consequences of this localization are far from being uncovered, all reports to date mention that C-RAF exerts anti-apoptotic and pro-oncogenic effects at this level (9Alavi A. Hood J.D. Frausto R. Stupack D.G. Cheresh D.A. Science. 2003; 301: 94-96Crossref PubMed Scopus (297) Google Scholar, 10Jin S. Zhuo Y. Guo W. Field J. J. Biol. Chem. 2005; 280: 24698-24705Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The existence of target proteins for RAF kinases on the surface of mitochondria, such as the protein BAD, a "BH3-only" member of the Bcl2 family (7Wang H.G. Rapp U.R. Reed J.C. Cell. 1996; 87: 629-638Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 13She Q.B. Solit D.B. Ye Q. O'Reilly K.E. Lobo J. Rosen N. Cancer Cell. 2005; 8: 287-297Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar), suggests that these kinases could control directly some important aspects of the physiology of these membrane organelles. Despite the potentially important consequences of RAF localization to cell membranes, many important questions remain to be addressed. First, do all RAF isoforms exhibit identical or distinct membrane tropism? Considering the interaction of RAF kinases with mitochondria, it remains unclear whether C-RAF is the only isoform that is present at this level. What are the structural determinants of RAF involved in mitochondrial interaction, and what type of molecules are recognized? How is the interaction of RAF kinases with mitochondria regulated? What are the consequences in terms of mitochondrial physiology? Here, we have used different experimental strategies to address these questions. Our findings reveal the specificity of RAF interaction with cellular membrane organelles and shed light on their mode of action at this level. Cell Culture and Reagents—The cell lines used in this study were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 2 mm l-glutamine, and antibiotics (penicillin-streptomycin), with the exception of HUVECs (PromoCell), which were grown in an endothelial basal growth medium supplemented with 20% fetal bovine serum, 20 ng/ml basic fibroblast growth factor, 10 ng/ml epidermal growth factor (Invitrogen), and 1 μg/ml heparin (Sigma). Mouse embryonic fibroblasts (MEF) immortalized with the SV40 large T antigen (clones K2 (C-RAF−/-) and K9 (C-RAF+/+)) were obtained from C. Pritchard (University of Leicester, United Kingdom) (14Hüser M. Luckett J. Chiloeches A. Mercer K. Iwobi M. Giblett S. Sun X.M. Brown J. Marais R. Pritchard C. EMBO J. 2001; 20: 1940-1951Crossref PubMed Scopus (278) Google Scholar). Mice of the FVB strain used in these studies were kept and anaesthetized according to protocols approved by the animal care and use committee at the University of Würzburg. The primers and strategy used for the construction of all RAF expression vectors, as well as the antibodies used in this study, are described in the online supplemental material. Rabbit reticulocyte lysates were synthesized and radiolabeled with [35S]Cys/Met (Redivue-Cys-Met Mix, GE Healthcare) using the TnT T7 quick-coupled transcription-translation system (Promega) according to the manufacturer's instructions. Standard protocols were used for immunoblotting and enhanced chemiluminescence detection of the proteins (ECL kit from Pierce). Immunofluorescence—Immunofluorescence labeling was performed according to conventional protocols. For experiments presented in Figs. 1 and 4, cells were transfected with vectors encoding untagged versions of each RAF isoform according to standard procedures (Lipofectamine 2000, Invitrogen). After overnight expression, we performed a prepermeabilization step; the cells were treated with digitonin (50 μg/ml) in a buffer containing 10 mm KCl, 1.5 mm MgCl2, 1 mm EGTA, 1 mm EDTA, 250 mm sucrose, 20 mm Hepes-KOH, pH 7.4, with protease inhibitors for 4 min on ice. The cells were subsequently fixed and processed for immunofluorescence. Pictures were acquired on a TCS SP2 confocal microscope (Leica) equipped with a 63× HCX PL APO, NA = 1.40 objective (Leica), under oil immersion. Following acquisition, the images were combined using Photoshop software (Adobe).FIGURE 4Role of mitochondrial surface proteins and of the small GTPases of the RAS family as interaction partners for C-RAF. A, mitochondrial surface proteins are required for C-RAF binding. Purified mitochondria were treated with trypsin. To monitor the selective removal of mitochondrial surface proteins by this protocol, mitochondria were analyzed by immunoblot for their content of proteins of the mitochondrial surface (Bcl-XL) or matrix (manganese-superoxide dismutase (Mn-SOD)). Trypsinized mitochondria exhibit a drastically reduced ability to bind C-RAF (*, p < 0.01 compared with control (Ctrl)). B, EDTA treatment. Mitochondria were pretreated on ice with 20 mm EDTA and used later for a binding assay with C-RAF. ns, indicates no significant difference compared with control. C, mitochondrial interaction properties of the mutant C-RAF R89L. A mutant C-RAF R89L, which abolishes the interaction of C-RAF with RAS GTPases, was compared with C-RAF WT for its ability to bind mitochondria in vitro.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Videomicroscopy—HUVECs were electroporated with the mitochondrial fluorescent marker DsRed-mito (Clontech). Cells were filmed for 2 h under constant conditions of 5% CO2 and 37 °C and observed by UV-lamp using an Axiovert 200 microscope equipped with shutter-controlled illumination (Carl Zeiss) and a cooled digital charge-coupled device camera (Roper Scientific) using a ×40 lens. Images were processed using MetaMorph 2.0 image analysis software (Universal Imaging) and QuickTime pro 7 software (Apple Computers). Mitochondrial Purification—Mitochondria were purified from different sources according to a protocol detailed in the supplemental "Materials and Methods." The analysis of different markers revealed that this preparation was devoid of plasma membrane (E-cadherin), nuclear (histone H3), and cytoskeletal markers (tubulin) but contained membranes from the endoplasmic reticulum (Grp98) and endosomes (Rab7) (supplemental Fig. 1A). The mitochondria retained their functionality (polarity and ability to import intrinsic preproteins) after isolation (data not shown). In Vitro Assay for Mitochondrial Binding and Import—For each mitochondrial binding reaction, we mixed 50 μg of mitochondrial proteins with 10 μl of reticulocyte lysate in a binding buffer consisting of 55 mm mannitol, 18 mm sucrose, 80 mm KOAc, 8 mm MgCl2, 1 mm dithiothreitol, 20 mm Hepes-KOH, pH 7.4, and 0.5 mg/ml bovine serum albumin. Unless stated otherwise, the samples were incubated for 15 min on ice. After a brief centrifugation, the mitochondrial pellets were rinsed, reisolated, and resuspended in sample buffer. Following separation on SDS-PAGE, radioactivity was detected with the autoradiography enhancer En3hance (PerkinElmer Life Sciences) with ECL films (Amersham Biosciences). The results were quantified using NIH ImageJ software and presented as the percentage of input bound. To test their ability to import RAF kinases, mitochondria were incubated with RAF kinases in the same buffer with 5 mm succinate, 1 mm ATP, and 0.08 mm ADP. The import reactions were performed at 30 °C for 20 min. Following the reisolation of mitochondria, proteinase K was applied on ice for 10 min (10 μg/ml final concentration). Proteolysis was stopped with addition of phenylmethylsulfonyl fluoride, and the samples were analyzed by autoradiography as described previously. Mitochondrial Treatments—For trypsinization, to remove proteins from the surface of mitochondria, preparations of this purified organelles were incubated with 20 μg/ml trypsin for 15 min on ice in storage buffer. Proteolysis was stopped with the addition of 500 μg/ml soybean tryptic inhibitor, and mitochondria were reisolated and used for binding assays. For EDTA treatment, mitochondria were pretreated for 5 min with 20 mm EDTA and later incubated with lysates, again in the presence of EDTA. Electron Microscopy—Cells grown on coverslips were fixed for 45 min with 2.5% glutaraldehyde (50 mm cacodylate, pH 7.2, 50 mm KCl, 2.5 mm MgCl2) at room temperature, fixed for 2 h at 4 °C with 2% OsO4 buffered with 50 mm cacodylate (pH 7.2), washed with H2O, and incubated overnight at 4 °C with 0.5% uranyl acetate. The cells were dehydrated, embedded in Epon 812, and ultrathin sectioned. The sections were analyzed with a Zeiss EM900 (Carl Zeiss). Anthrax Lethal Toxin (LT) Purification and Use on Cells—The two components of LT, protective antigen (PA) and lethal factor (LF), were produced and purified separately as recombinant proteins in Escherichia coli. The genes encoding LF and PA were PCR-amplified from genomic DNA of Bacillus anthracis strain Sterne (a gift from Patrice Boquet, Nice, France) and cloned into pQE30 (Qiagen) and pET22b (Novagen) expression plasmids. Both proteins were obtained in a His6-tagged form and purified by immobilized metal affinity chromatography (IMAC, Chelating Fast Flow, Amersham Biosciences). PA was further purified on a mono-Q column (Amersham Biosciences). Following their purification, the proteins were concentrated and dialyzed in 25 mm Tris-HCl, pH.7.4, 250 mm NaCl. PA and LF were simultaneously applied on cells at final concentrations of 3 and 1 μg/ml respectively, in normal culture medium. Statistics—Statistical significance was determined using a one-tailed paired Student's t test. RAF Kinases Exhibit Distinct Localizations on Cellular Membrane Organelles—Although the literature contains multiple fragmentary reports on the ability of RAF kinases to interact with cellular membranes, no comparison has yet been performed among all isoforms. To analyze simultaneously the membrane localization of all isoforms of RAF kinases, we devised a strategy intended to overcome the following technical obstacles: (i) RAF kinases exhibit non-overlapping tissue-specific expression patterns and are generally expressed at low endogenous levels; (ii) RAF kinases tend to heterodimerize under growth conditions (15Weber C.K. Slupsky J.R. Kalmes H.A. Rapp U.R. Cancer Res. 2001; 61: 3595-3598PubMed Google Scholar, 16Garnett M.J. Rana S. Paterson H. Barford D. Marais R. Mol. Cell. 2005; 20: 963-969Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 17Rushworth L.K. Hindley A.D. O'Neill E. Kolch W. Mol. Cell. Biol. 2006; 26: 2262-2272Crossref PubMed Scopus (311) Google Scholar), rendering the analysis of individual isoform behavior difficult; (iii) each RAF kinase is present predominantly as a cytoplasmic pool. To compare directly the membrane localization of each RAF isoform, we overexpressed them and applied conditions of serum starvation. Then, immediately before the coverslips were fixed and processed, we performed a mild permeabilization in order to eliminate the cytosolic pool of the kinases. Using this strategy in HeLa cells, we noticed that each RAF kinase exhibited distinct membrane targeting properties (Fig. 1). C-RAF almost completely colocalized with mitochondria (Fig. 1, A-C). Although we also detected a strong immunoreactivity for C-RAF in the nuclei of HeLa cells, this immunoreactivity was not attributable to the presence of the full length C-RAF but rather to a short form corresponding to the carboxyl terminus of this kinase (supplemental Fig. 1B). Only C-RAF was detected at the level of mitochondria; neither A-RAF (Fig. 1, D-F), B-RAF (Fig. 1, G-I), nor the kinase KSR (kinase suppressor of RAS, a kinase closely related to the RAFs) (data not shown) colocalized with this organelle. Although A-RAF fluorescence highlighted intracellular tubules and vesicles that we identified as endosomes, and KSR was present on the Golgi apparatus (data not shown), B-RAF did not associate with intracellular membranes of permeabilized cells. To obtain direct evidence that members of the RAF kinase family exhibit distinct subcellular localization, we coexpressed C-RAF and A-RAF and analyzed simultaneously the localization of both kinases using again our pre-permeabilization procedure (Fig. 1, J-L). We noticed a complete lack of overlap between A-RAF and C-RAF, thereby confirming at the single cell level that these kinases target different intracellular membranes. Immunoblots of purified mitochondria confirmed that, at endogenous expression levels, C-RAF is the only isoform present in significant amounts on the mitochondria of serum-starved cells (Fig. 2A). Collectively, these findings revealed the existence of distinct membrane localizations of the RAF kinases and the preferential localization of C-RAF to mitochondria. C-RAF Efficiently Binds to Purified Mitochondria in Vitro—To gain more insight into the interaction of RAF kinases with mitochondria, we decided to analyze this interaction in vitro. RAF kinases were produced and radiolabeled with [35S]Cys/Met by in vitro transcription/translation in reticulocyte lysates (as initially reported by Stancato et al. (18Stancato L.F. Chow Y.H. Hutchison K.A. Perdew G.H. Jove R. Pratt W.B. J. Biol. Chem. 1993; 268: 21711-21716Abstract Full Text PDF PubMed Google Scholar), and mixed with purified mitochondria obtained from human embryonic kidney (HEK) 293 cells. Autoradiographic analysis revealed that the RAF isoforms exhibit different abilities to associate with mitochondria (Fig. 2B). Although C-RAF associated with mitochondria in a fast and efficient fashion, the two other isoforms exhibited a significantly reduced binding (A-RAF) or behaved like the kinase MEK, which was included as a negative control in this assay (B-RAF) (Fig. 2B). The binding efficiency of C-RAF, measured as a percentage of the input bound at 15 min, was more than 2-fold higher than A-RAF and more than 5-fold higher than B-RAF (Fig. 2C). At this stage, a contamination of our mitochondrial preparations with endosomal membranes, detected with the markers Rab7 (supplemental Fig. 1A) and EEA1 (data not shown), constituted the most likely explanation for the residual binding observed with A-RAF using mitochondria from cultured cells. We extended our analysis to mitochondria obtained from a normal tissue, in this case from mouse liver (Fig. 2D). Again, we noticed a clear difference between C-RAF and the two other isoforms; C-RAF bound almost 5-fold more efficiently to mitochondria than B-RAF and A-RAF (Fig. 2D). We verified the specificity of our binding assay by replacing mitochondria with purified microsomal membranes (Fig. 2E) and detected no binding of C-RAF to microsomes, suggesting that the experiments presented indeed reflected C-RAF-specific interaction with mitochondria. Because the possibility that one of the RAF kinase isoforms (A-RAF) might be partially present inside mitochondria had been proposed by Yuryev et al. (19Yuryev A. Ono M. Goff S.A. Macaluso F. Wennogle L.P. Mol. Cell. Biol. 2000; 20: 4870-4878Crossref PubMed Scopus (60) Google Scholar), we decided to test whether RAF kinases would be imported into this organelle. We tested this possibility by performing proteolytic treatments of mitochondria that had been incubated with RAF kinases (Fig. 2F). We had observed previously that RAF kinases produced in reticulocyte lysates are sensitive to proteinase K (data not shown). Although we observed that the iron-sulfur protein Rieske was processed and imported inside mitochondria in a membrane potential-dependent fashion (abrogated by valinomycin), none of the RAF kinases demonstrated resistance to externally added protease upon incubation with mitochondria (Fig. 2F). Therefore, RAF kinases were not imported inside mitochondria in vitro. At this point of the work, we concluded that C-RAF exhibits a specific capacity to bind to the surface of mitochondria. These results strongly confirmed our initial observation that RAF kinases have different tropisms for membrane organelles and encouraged us to use our acellular system further to learn more about C-RAF interaction with mitochondria. Mitochondrial Affinity Is an Intrinsic Property of C-RAF and the C-RAF Mitochondrial Binding Domain Localizes to Its Amino Terminus—Our observations that RAF kinases exhibit different affinities for mitochondria in acellular conditions suggested that the differences of subcellular distribution we had initially observed were intrinsic properties of the RAF kinases rather than the product of a cellular regulation that would have applied to one of the RAF isoforms. To examine the possibility that the activation of these kinases, for example obtained through the introduction of an oncogenic mutation, might interfere with their recruitment to mitochondria, we compared C-RAF and B-RAF wild type with their oncogenic versions, C-RAF DDED (mutated on residues 338, 341, 491, and 494) (20Chong H. Lee J. Guan K.L. EMBO J. 2001; 20: 3716-3727Crossref PubMed Scopus (196) Google Scholar) and B-RAF V600E. We found that these mutations did not alter the mitochondrial binding properties of either C-RAF or B-RAF (supplemental Fig. 2A). We also focused our attention on the role of Ser-338, a residue in which phosphorylation had previously been proposed to constitute a mitochondrial targeting signal (9Alavi A. Hood J.D. Frausto R. Stupack D.G. Cheresh D.A. Science. 2003; 301: 94-96Crossref PubMed Scopus (297) Google Scholar, 10Jin S. Zhuo Y. Guo W. Field J. J. Biol. Chem. 2005; 280: 24698-24705Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). A non-phosphorylatable mutant in which this residue was replaced with Ala was compared with C-RAF WT; we found that C-RAF S338A exhibits an affinity for mitochondria that is comparable to C-RAF WT (supplemental Fig. 2B). Reciprocally, phosphomimetic mutants with an Asp residue introduced on Ser at positions 338 and 339 did not increase the recruitment of C-RAF to mitochondria (data not shown). These findings indicated that the phosphorylation of Ser-338 is neither necessary nor sufficient for the mitochondrial targeting of C-RAF. We concluded that the conformational changes that accompany the activation of RAF kinases do not change their affinity for mitochondria. To address directly the possibility that C-RAF affinity for mitochondria might be an intrinsic property of this isoform, we decided to identify the sequence determinants that account for it. We constructed a set of vectors allowing the expression of different parts of C-RAF, as well as some chimeras between C-RAF and B-RAF (Fig. 3). Each protein was synthesized and radiolabeled in vitro in reticulocyte lysates and incubated with purified mitochondria as described above (Fig. 3). Interestingly, all chimeras in which the first 100 amino-terminal residues of B-RAF were substituted with corresponding residues from C-RAF showed an increased recruitment to mitochondria (Fig. 3). These results indicated that the amino-terminal regulatory region of RAF kinases determines their mitochondrial affinity, an interesting point considering that this region concentrates almost all sequence divergences between the isoforms of RAF. To determine directly which part of the C-RAF kinase binds mitochondria, two constructs covering the carboxyl terminus of C-RAF (residues 324-648) or its amino terminus (residues 1-323) were compared in this assay. This analysis revealed that the amino-terminal domain of C-RAF binds efficiently to mitochondria, in contrast to its carboxyl terminus (Fig. 3). We concluded that C-RAF amino terminus confers its mitochondrial interaction properties on the entire C-RAF kinase. Protein Binding Partner(s) Other Than RAS GTPases Support C-RAF Recruitment on Mitochondria—RAF kinases, and in particular C-RAF, can interact in vitro both with proteins and also with lipid mixtures (21Hekman M. Hamm H. Villar A.V. Bader B. Kuhlmann J. Nickel J. Rapp U.R. J. Biol. Chem. 2002; 277: 24090-24102Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). To determine which of these molecules C-RAF would recognize, we treated purified mitochondria with trypsin (Fig. 4A). This procedure, which removed the proteins of the mitochondrial outer membrane (Bcl-XL) but left intact intrinsic mitochondrial proteins (the matrix protein Mn-SOD), almost completely abolished the recruitment of C-RAF (Fig. 4A). Therefore, we concluded that C-RAF interacts with proteins of the mitochondrial surface. Small GTPases of the RAS family constitute a prime candidate as binding partners for RAF kinases on the surface of mitochondria. To test their contribution to the recruitment of C-RAF, we used the following approaches: (i) we tested the effect of Mg2+ chelation by EDTA, a procedure that triggers the transition of small GTPases to an inactive, nucleotide-free form (Fig. 4B); and (ii) we compared the binding of C-RAF WT with the mutant R89L, a mutation that abrogates the binding of C-RAF to activated RAS (22Bondeva T. Balla A. Varnai P. Balla T. Mol. Biol. Cell. 2002; 13: 2323-2333Crossref PubMed Scopus (68) Google Scholar) (Fig. 4C). Clearly, neither of these two procedures abrogated the binding of C-RAF to mitochondria (Fig. 4, B and C). We concluded that, under the conditions of our assay, protein binding partners other than RAS GTPases account for most of the binding of C-RAF to mitochondria. To confirm this observation in intact cells, we tested the intracellular distribution of the mutant R89L of C-RAF with the protocol that we had used for the experiments presented in Fig. 1. We found that C-RAF R89L exhibited the same mitochondrial localization as C-RAF WT (supplemental Fig. 3). We concluded that the mitochondrial recruitment of C-RAF is, at least in this cell system, independent of RAS GTPases. Active C-RAF Specifically Changes Mitochondrial Subcellular Distribution in a MEK-dependent Fashion—To analyze the consequences of C-RAF activation on mitochondria, we decided to use an inducible system, allowing a kinetic analysis of the consequences of C-RAF activation. We used a cell line stably expressing low levels of a fusion protein between an activated form of C-RAF (C-RAF-BXB) and the hormone-binding domain of the estrogen receptor (23Kerkhoff E. Rapp U.R. Mol. Cell. Biol. 1997; 17: 2576-2586Crossref PubMed Scopus (152) Google Scholar). This oncogenic version of C-RAF, which consists of an internal deletion of its Ras-binding domain (between residues 65 and 304) still bound mitochondria with high affinity in vitro and in intact cells (data not shown). In this system, C-RAF activation was achieved through the application of the estrogen receptor agonist 4-hydroxytamoxifene (4-OHT, 1 μm). Using this system, we observed that C-RAF activation causes an intense remodeling of this organelle, characterized by a change from long, filamentous to short, spherical mitochondria clustered around the nucleus. The activation of C-RAF was sufficient to produce this mitochondrial remodeling, because it could be observed in serum-free conditions (Fig. 5, A-D). Transmission electron microscopy confirmed these observations (Fig. 5, E and F); although mitochondria from control cells were visible as longitudinal as well as cross-sections, we observed spherical elements only in cells in which C-RAF had been activated for 12 h. Despite this intense change in shape and distribution, the inner architecture of mitochondria remained normal (Fig. 5, E and F). Mitochondrial remodeling occurred progressively upon 4-OHT addition and was systematically noticed after 12 h of treatment
Directional cell migration is crucially dependent on the spatiotemporal control of intracellular signalling events. These events regulate polarized actin dynamics, resulting in protrusion at the front of the cell and contraction at the rear. The actin cytoskeleton is regulated through signalling by Rho-like GTPases, such as RhoA, which stimulates myosin-based contractility, and CDC42 and Rac1, which promote actin polymerization and protrusion. Here, we show that Rac1 binds the adapter protein caveolin-1 (Cav1) and that Rac1 activity promotes Cav1 accumulation at Rac1-positive peripheral adhesions. Using Cav1-deficient mouse fibroblasts and depletion of Cav1 expression in human epithelial and endothelial cells mediated by small interfering RNA and short hairpin RNA, we show that loss of Cav1 induces an increase in Rac1 protein and its activated, GTP-bound form. Cav1 controls Rac1 protein levels by regulating ubiquitylation and degradation of activated Rac1 in an adhesion-dependent fashion. Finally, we show that Rac1 ubiquitylation is not required for effector binding, but regulates the dynamics of Rac1 at the periphery of the cell. These data extend the canonical model of Rac1 inactivation and uncover Cav1-regulated polyubiquitylation as an additional mechanism to control Rac1 signalling. PMID: 20460433
It remains a challenge to decode the molecular basis of the long-term actin cytoskeleton rearrangements that are governed by the reprogramming of gene expression. Bacillus anthracis lethal toxin (LT) inhibits mitogen-activated protein kinase (MAPK) signaling, thereby modulating gene expression, with major consequences for actin cytoskeleton organization and the loss of endothelial barrier function. Using a laser ablation approach, we characterized the contractile and tensile mechanical properties of LT-induced stress fibers. These actin cables resist pulling forces that are transmitted at cell-matrix interfaces and at cell-cell discontinuous adherens junctions. We report that treating the cells with trichostatin A (TSA), a broad range inhibitor of histone deacetylases (HDACs), or with MS-275, which targets HDAC1, 2 and 3, induces stress fibers. LT decreased the cellular levels of HDAC1, 2 and 3 and reduced the global HDAC activity in the nucleus. Both the LT and TSA treatments induced Rnd3 expression, which is required for the LT-mediated induction of actin stress fibers. Furthermore, we reveal that treating the LT-intoxicated cells with garcinol, an inhibitor of histone acetyl-transferases (HATs), disrupts the stress fibers and limits the monolayer barrier dysfunctions. These data demonstrate the importance of modulating the flux of protein acetylation in order to control actin cytoskeleton organization and the endothelial cell monolayer barrier.
