Cap G, a Gelsolin Family Protein Modulating Protective Effects of Unidirectional Shear Stress

Abstract

Atherosclerosis is a progressive and complex pathophysiological process occurring in large arteries. Although it is of multifactorial origin, the disease develops at preferential sites along the vasculature in regions experiencing specific hemodynamic conditions that are predisposed to endothelial dysfunction. The exact mechanisms allowing endothelial cells to discriminate between plaque-free and plaque-prone flows remain to be explored. To investigate such mechanisms, we performed a proteomic analysis on endothelial cells exposed in vitro to these two-flow patterns. A few spots on the two-dimensional gel had an intensity that was differentially regulated by plaque-free versus plaque-prone flows. One of them was further investigated and identified as macrophage-capping protein (Cap G), a member of the gelsolin protein superfamily. A 2-fold increase of Cap G protein and a 5-fold increase of Cap G mRNA were observed in cells exposed to a plaque-free flow as compared with static cultures. This increase was not observed in cells exposed to plaque-prone flow. Plaque-free flow induced a corresponding increase in nuclear and cytoskeletal-associated Cap G. Finally, overexpression of Cap G in transfection assays increased the motility potential of endothelial cells. These observations together with the known functions of Cap G suggest that Cap G may contribute to the protective effect exerted by plaque-free flow on endothelial cells. On the contrary, in cells exposed to a plaque-prone flow, no induction of Cap G expression could be observed. Atherosclerosis is a progressive and complex pathophysiological process occurring in large arteries. Although it is of multifactorial origin, the disease develops at preferential sites along the vasculature in regions experiencing specific hemodynamic conditions that are predisposed to endothelial dysfunction. The exact mechanisms allowing endothelial cells to discriminate between plaque-free and plaque-prone flows remain to be explored. To investigate such mechanisms, we performed a proteomic analysis on endothelial cells exposed in vitro to these two-flow patterns. A few spots on the two-dimensional gel had an intensity that was differentially regulated by plaque-free versus plaque-prone flows. One of them was further investigated and identified as macrophage-capping protein (Cap G), a member of the gelsolin protein superfamily. A 2-fold increase of Cap G protein and a 5-fold increase of Cap G mRNA were observed in cells exposed to a plaque-free flow as compared with static cultures. This increase was not observed in cells exposed to plaque-prone flow. Plaque-free flow induced a corresponding increase in nuclear and cytoskeletal-associated Cap G. Finally, overexpression of Cap G in transfection assays increased the motility potential of endothelial cells. These observations together with the known functions of Cap G suggest that Cap G may contribute to the protective effect exerted by plaque-free flow on endothelial cells. On the contrary, in cells exposed to a plaque-prone flow, no induction of Cap G expression could be observed. The endothelium is a semiselective barrier between the blood and the vessel wall. Endothelial cells (ECs) 1The abbreviations used are: EC, endothelial cell; BAECs, bovine aorta endothelial cells; Cap G, macrophage-capping protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MS, mass spectrometry; eGFP, enhanced green fluorescent protein; PIPES, 1,4-piperazinediethanesulfonic acid; ANOVA, analysis of variance; IRES, internal ribosome entry site; EST, expressed sequence tag.1The abbreviations used are: EC, endothelial cell; BAECs, bovine aorta endothelial cells; Cap G, macrophage-capping protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MS, mass spectrometry; eGFP, enhanced green fluorescent protein; PIPES, 1,4-piperazinediethanesulfonic acid; ANOVA, analysis of variance; IRES, internal ribosome entry site; EST, expressed sequence tag. are continuously exposed to mechanical forces generated by blood flow (1Davies P.F. Physiol. Rev. 1995; 75: 519-560Crossref PubMed Scopus (2285) Google Scholar). The disturbance of endothelial homeostasis can lead to endothelial dysfunction, triggering several pathological processes including atherosclerosis. Hemodynamic forces have been shown to play a preponderant role in regulating cell function and structure in the endothelium. Among these forces, shear stress represents a major stimulus. Localization of atherosclerotic plaques is correlated with regions characterized by a flow with low mean shear stress values and a cyclic reversal flow direction (named also oscillatory flow) (2Ku D.N. Giddens D.P. Zarins C.K. Glagov S. Arteriosclerosis. 1985; 5: 293-302Crossref PubMed Google Scholar, 3Glagov S. Zarins C. Giddens D.P. Ku D.N. Arch. Pathol. Lab. Med. 1988; 112: 1018-1031PubMed Google Scholar, 4Davies P.F. Shi C. Depaola N. Helmke B.P. Polacek D.C. Ann. N. Y. Acad. Sci. 2001; 947: 7-17Crossref PubMed Scopus (96) Google Scholar). This contrasts with plaque-free sections where ECs are exposed to unidirectional shear stress. However, little is known regarding the mechanism by which this particular environment might predispose the endothelium to damage. Interestingly, the expression of several genes has been shown to be differentially regulated by unidirectional and oscillatory flow. Unidirectional shear stress increases expression of proteins involved in adaptive process to reduce flow-induced endothelium damage as shown by the up-regulation of endothelial nitric-oxide synthase gene expression (5Ziegler T. Silacci P. Harrison V.J. Hayoz D. Hypertension. 1998; 32: 351-355Crossref PubMed Scopus (208) Google Scholar, 6Ziegler T. Bouzourene K. Harrison V.J. Brunner H.R. Hayoz D. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 686-692Crossref PubMed Scopus (282) Google Scholar, 7Silacci P. Formentin K. Bouzourene K. Daniel F. Brunner H.R. Hayoz D. Nitric Oxide. 2000; 4: 47-56Crossref PubMed Scopus (51) Google Scholar). In contrast, oscillatory shear stress induces expression of genes implied in the development of plaques such as the vascular cell adhesion molecule-1 or endothelin-1 growth factor (6Ziegler T. Bouzourene K. Harrison V.J. Brunner H.R. Hayoz D. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 686-692Crossref PubMed Scopus (282) Google Scholar, 8Chappell D.C. Varner S.E. Nerem R.M. Medford R.M. Alexander R.W. Circ. Res. 1998; 82: 532-539Crossref PubMed Scopus (464) Google Scholar). Endothelial morphology is also influenced by the prevailing hemodynamic conditions. Indeed, unidirectional but not oscillatory shear stress induces an elongation and an alignment of ECs in flow direction through actin assembly and reorganization (6Ziegler T. Bouzourene K. Harrison V.J. Brunner H.R. Hayoz D. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 686-692Crossref PubMed Scopus (282) Google Scholar, 9Malek A.M. Izumo S. J. Cell Sci. 1996; 109: 713-726Crossref PubMed Google Scholar). This elongation process is evident 12–24 h after flow exposure and is reversible (10Cucina A. Sterpetti A.V. Pupelis G. Fragale A. Lepidi S. Cavallaro A. Giustiniani Q. Santoro D'Angelo L. Eur. J. Vasc. Endovasc. Surg. 1995; 9: 86-92Abstract Full Text PDF PubMed Scopus (52) Google Scholar). Mechanisms triggering this process involve integrin molecules activation, transient RhoA GTPase inhibition (11Li S. Chen B.P. Azuma N. Hu Y.L. Wu S.Z. Sumpio B.E. Shyy J.Y. Chien S. J. Clin. Invest. 1999; 103: 1141-1150Crossref PubMed Scopus (173) Google Scholar, 12Tzima E. del Pozo M.A. Shattil S.J. Chien S. Schwartz M.A. EMBO J. 2001; 20: 4639-4647Crossref PubMed Scopus (438) Google Scholar), intracellular Ca2+ mobilization, and p38 mitogen-activating protein kinase activation (9Malek A.M. Izumo S. J. Cell Sci. 1996; 109: 713-726Crossref PubMed Google Scholar, 13Azuma N. Akasaka N. Kito H. Ikeda M. Gahtan V. Sasajima T. Sumpio B.E. Am. J. Physiol. 2001; 280: H189-H197Crossref PubMed Google Scholar). Modulation of these signaling molecules is necessary to induce a complex cytoskeletal reorganization that includes formation of actin-stress fibers and that is necessary for cellular morphology changes. Therefore, mechanotransduction mechanisms leading to cytoskeletal dynamic changes are strongly suggested to imply a large number of actin-binding proteins. Among these binding proteins, the gelsolin family can control many aspects of actin organization by severing filament ends and nucleating actin assembly (14Yin H.L. Bioessays. 1987; 7: 176-179Crossref PubMed Scopus (173) Google Scholar). Gelsolin is the founding member of this family that now contains six members: gelsolin, villin, adseverin, macrophage-capping protein (Cap G), advillin, and supervillin (15Sun H.Q. Yamamoto M. Mejillano M. Yin H.L. J. Biol. Chem. 1999; 274: 33179-33182Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). In contrast to gelsolin, Cap G binds and caps actin filament but does not sever them (16Yu F.X. Johnston P.A. Sudhof T.C. Yin H.L. Science. 1990; 250: 1413-1415Crossref PubMed Scopus (167) Google Scholar, 17Yu F.X. Zhou D.M. Yin H.L. J. Biol. Chem. 1991; 266: 19269-19275Abstract Full Text PDF PubMed Google Scholar). Its binding to actin is reversible by reducing Ca2+ concentration (18Kwiatkowski D.J. Curr. Opin. Cell Biol. 1999; 11: 103-108Crossref PubMed Scopus (325) Google Scholar). Indeed, Cap G rapidly dissociates from the barbed end of actin when Ca2+ concentration becomes submicromolar (14Yin H.L. Bioessays. 1987; 7: 176-179Crossref PubMed Scopus (173) Google Scholar). Cap G and gelsolin also differentiate themselves by their cellular distribution. Whereas gelsolin is exclusively present in the cytoplasm, Cap G has been found in the nuclei (19Onoda K. Yu F.X. Yin H.L. Cell. Motil. Cytoskeleton. 1993; 26: 227-238Crossref PubMed Scopus (40) Google Scholar). Moreover, phosphorylated Cap G is preferentially associated with nuclear location (20Onoda K. Yin H.L. J. Biol. Chem. 1993; 268: 4106-4112Abstract Full Text PDF PubMed Google Scholar). Studies of gelsolin knock-out cells suggest that this protein regulates morphology of stress fibers, facilitates the wound healing responses, and enhances chemotaxis (18Kwiatkowski D.J. Curr. Opin. Cell Biol. 1999; 11: 103-108Crossref PubMed Scopus (325) Google Scholar, 21Cunningham C.C. Stossel T.P. Kwiatkowski D.J. Science. 1991; 251: 1233-1236Crossref PubMed Scopus (256) Google Scholar). Understanding the mechanisms responsible for the selective sensitivity of ECs to different hemodynamic environments could allow us to get new insights into the development of atherogenesis. Taking advantage of our unique perfusion system combining shear stress, hoop stretch, and pressure (22Zhao S. Suciu A. Ziegler T. Moore Jr., J.E. Burki E. Meister J.J. Brunner H.R. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1781-1786Crossref PubMed Scopus (168) Google Scholar) in association with a proteomic analysis, we identify Cap G as a novel differentially regulated gene. Indeed, Cap G gene expression is induced in ECs exposed to plaque-free flow, whereas a plaque-prone flow (oscillatory flow) fails to induce such expression. We also demonstrated that unidirectional flow, but not oscillatory flow, increases the association of Cap G with the cytoskeleton and Cap G nuclear location. Moreover, overexpression of Cap G enhances EC-migrating capacities. Taken together, these observations suggest a key role of Cap G in the signaling pathway triggered by unidirectional flow and also in the increased wound healing facility of plaque-free regions. Bovine aorta endothelial cells (BAECs) were isolated from aorta as described previously (6Ziegler T. Bouzourene K. Harrison V.J. Brunner H.R. Hayoz D. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 686-692Crossref PubMed Scopus (282) Google Scholar). Cells were grown in low glucose Dulbecco's modified Eagle's medium (Amimed) supplemented with 10% fetal calf serum (Amimed), 4 mmol/liter GLUTAMAX (Invitrogen), 10 mg/ml streptomycin, and 100 IU/ml penicillin (Invitrogen). Cells were used between passages 3 and 5. The system has been described in more detail elsewhere (6Ziegler T. Bouzourene K. Harrison V.J. Brunner H.R. Hayoz D. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 686-692Crossref PubMed Scopus (282) Google Scholar, 22Zhao S. Suciu A. Ziegler T. Moore Jr., J.E. Burki E. Meister J.J. Brunner H.R. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1781-1786Crossref PubMed Scopus (168) Google Scholar). It is composed of compliant tubes coated with 20 μg/ml bovine fibronectin and with were cells a were in a perfusion composed of a medium and a The is by a function to a flow medium is composed of medium supplemented with to increase the to The is by a 10% to a of and were at a of in a Cells were exposed to unidirectional flow 3 or or to oscillatory flow 3 mean was to 100 the end of the cells were by and further were in CHAPS, 100 of proteins were on a two-dimensional was performed were in gel and further on an A of was to were on a were and a Full were in for and were used to the The number was isolated from a gel with of protein In this proteins were and in The was from the gel and to to be further characterized by mass and and of Bovine Cap G Cap G has been identified through in of for was by cells as a of The were and The was performed with a The was into the as in the different were for The and sequence were the The bovine Cap G protein was with the Cap G from and the and in mmol/liter mmol/liter mmol/liter protein were to 10 of proteins were in a 10% and were used to Cap G and protein by was performed in 20 for h at 4 was or to and a system were in and with in for 3 We the cells with a specific for Cap G in 10% of cells were in the of a in 10% Cells were for and green and was from cells of were on to a A and with the Cap G were in mmol/liter mmol/liter for at and in for at were an and to the was performed as described by and Biol. 1999; 112: Google Scholar). Cells were mmol/liter 100 mmol/liter 10 mmol/liter PIPES, for 10 was at for 10 at 4 and was were in mmol/liter 100 mmol/liter 10 mmol/liter PIPES, for at 4 The was by at for 10 at 4 was in mmol/liter PIPES, mmol/liter at for 10 cytoskeletal was in 10 mmol/liter of the were supplemented with and Cell G was into into was performed A of 3 of for of was Cells were h after was by after transfection with Cell was a modified cells were the and to for h through cells were and and for are expressed as analysis was performed between was performed values were Cap G in ECs to a Plaque-free investigate effect of plaque-free versus plaque-prone flow on protein we performed two-dimensional gel on protein of ECs exposed to these flow patterns. two-dimensional for and 3 were This analysis us to protein spots Among the of spots and were shown to be differentially regulated by unidirectional and oscillatory flow We to further investigate the of number corresponding to an protein with an of and an mass of This was on the of intensity in cells exposed to unidirectional flow as compared with cells exposed to oscillatory flow and on the of this protein present in of cells in static that was for further corresponding to has been identified by mass to of from a with Cap G. To the two-dimensional gel G were used in Cap G increases in exposed to unidirectional shear stress for h Cap G protein was induced in cells exposed to unidirectional shear stress, whereas oscillatory flow to induce expression of Cap G protein induction by unidirectional shear stress a induction after 4 h of flow exposure 3 and a after h To Cap G gene expression also at the mRNA cells were exposed to unidirectional or oscillatory flow for h and Cap G were by a in cells exposed to flow for h were compared with that observed in static conditions. shear stress induced a increase in Cap G whereas of unidirectional flow 3 a increase in Cap G mRNA expression as compared with static conditions A and it was the at the protein oscillatory flow to induce an increase in Cap G mRNA and of Bovine Cap G the Cap G sequence as we performed a analysis of in and of the bovine Among these were and for to a sequence of and The sequence has been in as To that this mRNA is expressed in BAECs, we the and A of was by from cells The isolated was into different were The were to that by The an from to of a with a mass of analysis in protein as gelsolin gelsolin gelsolin gelsolin To the sequence of the bovine Cap G we have the from mass in our The by two-dimensional gel were at the of the protein and of bovine Cap G protein with from and is shown in Bovine Cap G protein a of to Cap G and and Cap G and proteins. Unidirectional Cap G of ECs the of Cap G in of the but in the nuclear nuclear and cytoskeletal To investigate unidirectional shear stress influenced cellular Cap G association to the cells were exposed to different flow conditions to a Cap G increased rapidly and was after h of flow exposure This association was in and was present after h of unidirectional flow Moreover, h of exposure to oscillatory shear stress to induce Cap G cytoskeletal association Interestingly, Cap G in the nuclear also increased in cells exposed to unidirectional shear stress for it was the for the cytoskeletal-associated Cap G, no increase was observed in cells exposed for h to an oscillatory shear stress and Cap G to static at of the in cells exposed to unidirectional and oscillatory shear stress Cap G To the role of Cap G in cells were with a a the expression of Cap G and and with the the as a control The used us to cells not of Cap G in cells was demonstrated by Cap G was shown to be in cells with Cap G expression whereas was expressed also in cells with control was used to protein in the different These were also by To motility cells were in the and motility was h as the of cell through The number of cells is increased when cells have been previously with Cap as compared with cells and to cells with the expression Moreover, when were with the Cap G and of the cells green further the that of the cells Cap G were plaques preferentially where vasculature is exposed to a particular flow corresponding to low mean shear stress values and a cyclic reversal of flow This flow is in contrast to that observed in regions which is characterized by a unidirectional and of shear stress (2Ku D.N. Giddens D.P. Zarins C.K. Glagov S. Arteriosclerosis. 1985; 5: 293-302Crossref PubMed Google Scholar, 3Glagov S. Zarins C. Giddens D.P. Ku D.N. Arch. Pathol. Lab. Med. 1988; 112: 1018-1031PubMed Google Scholar, 4Davies P.F. Shi C. Depaola N. Helmke B.P. Polacek D.C. Ann. N. Y. Acad. Sci. 2001; 947: 7-17Crossref PubMed Scopus (96) Google Scholar, Jr., J.E. Ku D.N. Zarins C.K. Glagov S. J. PubMed Scopus Google Scholar, Ku D.N. J. 1996; PubMed Scopus Google Scholar). the of the hemodynamic forces on ECs has at the of cell morphology as as at the of gene expression (1Davies P.F. Physiol. Rev. 1995; 75: 519-560Crossref PubMed Scopus (2285) Google Scholar, 4Davies P.F. Shi C. Depaola N. Helmke B.P. Polacek D.C. Ann. N. Y. Acad. Sci. 2001; 947: 7-17Crossref PubMed Scopus (96) Google Scholar). Moreover, it has been demonstrated that different hemodynamic conditions in in vitro induce effect on in the of endothelial nitric-oxide vascular cell adhesion and growth factor gene expression (5Ziegler T. Silacci P. Harrison V.J. Hayoz D. Hypertension. 1998; 32: 351-355Crossref PubMed Scopus (208) Google Scholar, 6Ziegler T. Bouzourene K. Harrison V.J. Brunner H.R. Hayoz D. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 686-692Crossref PubMed Scopus (282) Google Scholar, 8Chappell D.C. Varner S.E. Nerem R.M. Medford R.M. Alexander R.W. Circ. Res. 1998; 82: 532-539Crossref PubMed Scopus (464) Google Scholar, V.J. Ziegler T. Bouzourene K. Suciu A. Silacci P. Hayoz D. J. 1998; PubMed Scopus Google Scholar). of these suggest that unidirectional flow, of plaque-free a effect on endothelial cells. On the contrary, oscillatory flow at plaque-prone to the preferential development of atherosclerotic plaques to the of a protective effect or the activation of a this no are on the mechanisms allowing ECs to discriminate between a plaque-free and a plaque-prone flow. To investigate of these mechanisms, we the protein in cells exposed to hemodynamic conditions plaque-free and plaque-prone flows. We used a perfusion system combining shear stress, hoop stretch, and pressure in to hemodynamic conditions to (22Zhao S. Suciu A. Ziegler T. Moore Jr., J.E. Burki E. Meister J.J. Brunner H.R. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1781-1786Crossref PubMed Scopus (168) Google Scholar). The two-dimensional gel analysis several protein spots with intensity differentially regulated by unidirectional versus oscillatory flows One of these further characterized by mass and a with macrophage-capping protein or Cap G which is a member of the family (15Sun H.Q. Yamamoto M. Mejillano M. Yin H.L. J. Biol. Chem. 1999; 274: 33179-33182Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). of Cap G expression by unidirectional shear stress was by and analysis and of mRNA was more that observed for Cap G This may be by an Cap G This is by the induction of Cap G protein observed Indeed, induction observed in the cytoskeletal with that observed in the nuclear a induction of 5-fold is in cells exposed to unidirectional shear stress This is more with the induction observed at the mRNA and of bovine Cap G sequence the structure of Cap G A with and a of and of cellular in that Cap G is associated with the and the (19Onoda K. Yu F.X. Yin H.L. Cell. Motil. Cytoskeleton. 1993; 26: 227-238Crossref PubMed Scopus (40) Google Scholar). to the of the gelsolin Cap G, which is a actin-binding has a of actin (16Yu F.X. Johnston P.A. Sudhof T.C. Yin H.L. Science. 1990; 250: 1413-1415Crossref PubMed Scopus (167) Google Scholar) and in the of the in Cap G in cells exposed to unidirectional flow is and can be observed after h of flow exposure This in Cap G is with a role of Cap G in the of cytoskeletal reorganization induced by unidirectional shear stress (9Malek A.M. Izumo S. J. Cell Sci. 1996; 109: 713-726Crossref PubMed Google Scholar), leading to cell alignment in flow Moreover, flow-induced cytoskeletal reorganization as as cellular alignment in the direction of flow are processes to Cap G actin binding (9Malek A.M. Izumo S. J. Cell Sci. 1996; 109: 713-726Crossref PubMed Google Scholar, D. C. Science. 2001; PubMed Scopus Google Scholar, F. D. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. Biol. PubMed Scopus Google Scholar) and further an of Cap G in these In oscillatory flow, which fails to changes in ECs (6Ziegler T. Bouzourene K. Harrison V.J. Brunner H.R. Hayoz D. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 686-692Crossref PubMed Scopus (282) Google Scholar), also fails to induce such an increase in Cap G this However, the exact mechanisms by which proteins of the family may on flow-induced cytoskeletal reorganization are not Unidirectional flow also increases of Cap G associated with the Cap G increase is observed after of exposure to unidirectional shear stress. location of Cap G as as the role of this increase in nuclear Cap G by unidirectional flow to be but Yin H.L. Chen 1998; PubMed Scopus Google Scholar, J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, Science. 2000; PubMed Scopus Google Scholar) suggest the of a nuclear signaling This together with the known of Cap G to and to Yin H.L. Chen 1998; PubMed Scopus Google Scholar), is with the of Cap G in the of this signaling pathway in cells exposed to flow. Moreover, Cap G could on gene by binding to as be suggested by the of a EMBO J. 1991; PubMed Scopus Google Scholar). The of this is that plaque-prone flow to induce an increase in Cap G the of severing (16Yu F.X. Johnston P.A. Sudhof T.C. Yin H.L. Science. 1990; 250: 1413-1415Crossref PubMed Scopus (167) Google Scholar), Cap G overexpression increases the motility of cells H.Q. K. D.C. Yin H.L. J. Cell Biol. 1995; PubMed Scopus Google Scholar). Moreover, Cap G on function Kwiatkowski D.J. J. Cell Biol. 2001; PubMed Scopus Google Scholar). To Cap G on the motility of we have with a Cap G and of Cap G protein in these cells was by and by The number of as is increased in Cap G and the increase in the number of cells to the number of that the observed are not to to transfection but from Cap G overexpression This that in of the vasculature exposed to a plaque-free flow, the process may be in to increased Cap G gene expression. Indeed, the process has been demonstrated to be by unidirectional flow S. Am. J. Pathol. 1996; Google the mechanisms remain In we that Cap G is a to to the sensitivity to plaque-free versus plaque-prone flows. Moreover, on the observations as as on the different known of Cap G, this protein may at several including cytoskeletal gene and of the signaling be necessary to the of Cap G processes and to the role of nuclear Cap G.