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• W2890544989 abstract "•A FRET-based biosensor (named MSS) is developed to study cell membrane tension•MSS is a beneficial tool to visualize the distribution of membrane tension•Membrane tension is inhomogeneous in response to shear stress•Membrane tension does not display polar distribution during mechanotransduction Cell membrane is the first medium from where a cell senses and responds to external stress stimuli. Exploring the tension changes in cell membrane will help us to understand intracellular force transmission. Here, a biosensor (named MSS) based on fluorescence resonance energy transfer is developed to visualize cell membrane tension. Validity of the biosensor is first verified for the detection of cell membrane tension. Results show a shear stress-induced heterogeneous distribution of membrane tension with the biosensor, which is strengthened by the disruption of microfilaments or enhancement of membrane fluidity, but weakened by the reduction of membrane fluidity or disruption of microtubules. These findings suggest that the MSS biosensor is a beneficial tool to visualize the changes and distribution of cell membrane tension. Besides, cell membrane tension does not display obvious polar distribution, indicating that cellular polarity changes do not first occur on the cell membrane during mechanical transmission. Cell membrane is the first medium from where a cell senses and responds to external stress stimuli. Exploring the tension changes in cell membrane will help us to understand intracellular force transmission. Here, a biosensor (named MSS) based on fluorescence resonance energy transfer is developed to visualize cell membrane tension. Validity of the biosensor is first verified for the detection of cell membrane tension. Results show a shear stress-induced heterogeneous distribution of membrane tension with the biosensor, which is strengthened by the disruption of microfilaments or enhancement of membrane fluidity, but weakened by the reduction of membrane fluidity or disruption of microtubules. These findings suggest that the MSS biosensor is a beneficial tool to visualize the changes and distribution of cell membrane tension. Besides, cell membrane tension does not display obvious polar distribution, indicating that cellular polarity changes do not first occur on the cell membrane during mechanical transmission. It is well observed that external mechanical stimulus can be transmitted or transduced into intracellular biological signal to regulate cell alignment (Goldfinger et al., 2008Goldfinger L.E. Tzima E. Stockton R. Kiosses W.B. Kinbara K. Tkachenko E. Gutierrez E. Groisman A. Nguyen P. Chien S. et al.Localized alpha4 integrin phosphorylation directs shear stress-induced endothelial cell alignment.Circ. Res. 2008; 103: 177-185Crossref PubMed Scopus (41) Google Scholar), deformation (Pfafferott et al., 1985Pfafferott C. Nash G.B. Meiselman H.J. Red blood cell deformation in shear flow. effects of internal and external phase viscosity and of in vivo aging.Biophys. J. 1985; 47: 695-704Abstract Full Text PDF PubMed Scopus (64) Google Scholar), differentiation (Altman et al., 2002Altman G.H. Horan R.L. Martin I. Farhadi J. Stark P.R. Volloch V. Richmond J.C. Vunjak-Novakovic G. Kaplan D.L. Cell differentiation by mechanical stress.FASEB J. 2002; 16: 270-272Crossref PubMed Scopus (565) Google Scholar), and some other cellular functions (Liu et al., 2010Liu B. Kim T.J. Wang Y. Live cell imaging of mechanotransduction.J. R. Soc. Interface. 2010; 7 Suppl 3: S365-S375Crossref PubMed Scopus (24) Google Scholar). However, the information about detailed signal pathway is limited. At present, it is extensively believed that mechanical stress signal acts on the cell membrane, which activates membrane proteins such as sensitive ion channels, G-protein-coupled receptors, and focal adhesions kinases (Boycott et al., 2013Boycott H.E. Barbier C.S. Eichel C.A. Costa K.D. Martins R.P. Louault F. Dilanian G. Coulombe A. Hatem S.N. Balse E. Shear stress triggers insertion of voltage-gated potassium channels from intracellular compartments in atrial myocytes.Proc. Natl. Acad. Sci. USA. 2013; 110: E3955-E3964Crossref PubMed Scopus (56) Google Scholar, Chachisvilis et al., 2006Chachisvilis M. Zhang Y.L. Frangos J.A. G protein-coupled receptors sense fluid shear stress in endothelial cells.Proc. Natl. Acad. Sci. USA. 2006; 103: 15463-15468Crossref PubMed Scopus (361) Google Scholar, Liu et al., 2014Liu B. Lu S. Hu Y.L. Liao X. Ouyang M. Wang Y. RhoA and membrane fluidity mediates the spatially polarized Src/FAK activation in response to shear stress.Sci. Rep. 2014; 4: 7008Crossref PubMed Scopus (31) Google Scholar). Consequently, they further activate intracellular signaling chain and regulate gene expression as well as protein synthesis. This could explain the major phenomena of mechanotransduction. However, it has been reported that a rapid (<300 ms) activation of Rac was induced by the stress at cell periphery but there was no Rac activation within 30 s when it was stimulated by platelet-derived growth factor (Poh et al., 2009Poh Y.C. Na S. Chowdhury F. Ouyang M. Wang Y. Wang N. Rapid activation of Rac GTPase in living cells by force is independent of Src.PLoS One. 2009; 4: e7886Crossref PubMed Scopus (65) Google Scholar). Besides, cells display a lot of polar changes upon shear stress stimuli including Cdc42-dependent polarization of microtubule system (microtubule-organizing center) (Tzima et al., 2003Tzima E. Kiosses W.B. del Pozo M.A. Schwartz M.A. Localized cdc42 activation, detected using a novel assay, mediates microtubule organizing center positioning in endothelial cells in response to fluid shear stress.J. Biol. Chem. 2003; 278: 31020-31023Crossref PubMed Scopus (153) Google Scholar, McCue et al., 2006McCue S. Dajnowiec D. Xu F. Zhang M. Jackson M.R. Langille B.L. Shear stress regulates forward and reverse planar cell polarity of vascular endothelium in vivo and in vitro.Circ. Res. 2006; 98: 939-946Crossref PubMed Scopus (90) Google Scholar) and different activation levels of Src or focal adhesion kinase at upstream and downstream sites (Liu et al., 2014Liu B. Lu S. Hu Y.L. Liao X. Ouyang M. Wang Y. RhoA and membrane fluidity mediates the spatially polarized Src/FAK activation in response to shear stress.Sci. Rep. 2014; 4: 7008Crossref PubMed Scopus (31) Google Scholar). These cellular polarized changes may result from the asymmetric distribution of stress during transmission via cell membrane, cytoskeleton, or other signal proteins. As a barrier of the whole cell, cell membrane is the first medium of mechanotransduction to sense extracellular stress. The induced alteration of membrane tension is likely to be an association between cellular polarized changes and stress distribution. In some neural cells, membrane tension also takes part in regulating vesicle trafficking and driving fusion pore expansion (Apodaca, 2002Apodaca G. Modulation of membrane traffic by mechanical stimuli.Am. J. Physiol. Renal Physiol. 2002; 282: F179-F190Crossref PubMed Google Scholar, Kozlov and Chernomordik, 2015Kozlov M.M. Chernomordik L.V. Membrane tension and membrane fusion.Curr. Opin. Struct. Biol. 2015; 33: 61-67Crossref PubMed Scopus (84) Google Scholar). Studies have reported that high membrane tension activates exocytosis, whereas low membrane tension activates endocytosis (Gauthier et al., 2009Gauthier N.C. Rossier O.M. Mathur A. Hone J.C. Sheetz M.P. Plasma membrane area increases with spread area by exocytosis of a GPI-anchored protein compartment.Mol. Biol. Cell. 2009; 20: 3261-3272Crossref PubMed Scopus (94) Google Scholar). In addition, tension of cell membrane has been shown to regulate many cell behaviors, including cell motility (Gauthier et al., 2011Gauthier N.C. Fardin M.A. Roca-Cusachs P. Sheetz M.P. Temporary increase in plasma membrane tension coordinates the activation of exocytosis and contraction during cell spreading.Proc. Natl. Acad. Sci. USA. 2011; 108: 14467-14472Crossref PubMed Scopus (250) Google Scholar), polarization (Houk et al., 2012Houk A.R. Jilkine A. Mejean C.O. Boltyanskiy R. Dufresne E.R. Angenent S.B. Altschuler S.J. Wu L.F. Weiner O.D. Membrane tension maintains cell polarity by confining signals to the leading edge during neutrophil migration.Cell. 2012; 148: 175-188Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar), spreading (Raucher and Sheetz, 2000Raucher D. Sheetz M.P. Cell spreading and lamellipodial extension rate is regulated by membrane tension.J. Cell Biol. 2000; 148: 127-136Crossref PubMed Scopus (355) Google Scholar), and membrane repair (Togo et al., 2000Togo T. Krasieva T.B. Steinhardt R.A. A decrease in membrane tension precedes successful cell-membrane repair.Mol. Biol. Cell. 2000; 11: 4339-4346Crossref PubMed Scopus (143) Google Scholar). However, how the membrane tension changes in response to external stress remains unclear due to the lack of appropriate tension sensors. Membrane tension is composed of in-plane tension in the lipid bilayer and tension from membrane-cytoskeleton adhesion (Keren, 2011Keren K. Membrane tension leads the way.Proc. Natl. Acad. Sci. USA. 2011; 108: 14379-14380Crossref PubMed Scopus (37) Google Scholar). In recent years, some methods have been developed gradually to measure membrane-associated tension. One way is by using micropipette aspiration, which sucks the cellular surface into a micropipette to form a hemispheric protrusion and thus calculates the tension by taking advantage of Laplace's law (Hochmuth, 2000Hochmuth R.M. Micropipette aspiration of living cells.J. Biomech. 2000; 33: 15-22Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar). This technique supplies information about the overall tension of the cell, so it is not appropriate for measuring membrane tension only (Tinevez et al., 2009Tinevez J.Y. Schulze U. Salbreux G. Roensch J. Joanny J.F. Paluch E. Role of cortical tension in bleb growth.Proc. Natl. Acad. Sci. USA. 2009; 106: 18581-18586Crossref PubMed Scopus (393) Google Scholar). Tether pulling is another way to measure membrane tension by pulling membrane tethers from the plasma membrane with the help of optical or magnetic tweezers (Diz-Munoz et al., 2013Diz-Munoz A. Fletcher D.A. Weiner O.D. Use the force: membrane tension as an organizer of cell shape and motility.Trends Cell Biol. 2013; 23: 47-53Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). The tether force measured by this approach is contributed by in-plane tension, a component of membrane tension, and cytoskeleton attachment to some extent (Lafaurie-Janvore et al., 2013Lafaurie-Janvore J. Maiuri P. Wang I. Pinot M. Manneville J.B. Betz T. Balland M. Piel M. ESCRT-III assembly and cytokinetic abscission are induced by tension release in the intercellular bridge.Science. 2013; 339: 1625-1629Crossref PubMed Scopus (118) Google Scholar, Dai and Sheetz, 1999Dai J. Sheetz M.P. Membrane tether formation from blebbing cells.Biophys. J. 1999; 77: 3363-3370Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). Recently, a new technology based on fluorescence resonance energy transfer (FRET) has been developed to evaluate tension across proteins in focal adhesions that gives a complex distribution of tension within individual focal adhesion (Grashoff et al., 2010Grashoff C. Hoffman B.D. Brenner M.D. Zhou R. Parsons M. Yang M.T. McLean M.A. Sligar S.G. Chen C.S. Ha T. et al.Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics.Nature. 2010; 466: 263-266Crossref PubMed Scopus (1037) Google Scholar, Morimatsu et al., 2013Morimatsu M. Mekhdjian A.H. Adhikari A.S. Dunn A.R. Molecular tension sensors report forces generated by single integrin molecules in living cells.Nano Lett. 2013; 13: 3985-3989Crossref PubMed Scopus (155) Google Scholar). Such a method could provide the spatial and temporal dynamics of tension visually in living cells in an invasive way. In the current project, a membrane-bound FRET-based tension sensor (named MSS) is constructed to visualize the dynamics of membrane tension in HeLa cells under applied laminar shear stress of 0.5 (low shear stress), 2 (intermediate shear stress), and 4 Pa (high shear stress) (Mitchell and King, 2013Mitchell M.J. King M.R. Computational and experimental models of cancer cell response to fluid shear stress.Front. Oncol. 2013; 3: 44Crossref PubMed Scopus (147) Google Scholar). Cell membrane tension is found to be positively related to cell membrane fluidity but not to the magnitude of shear stress. In addition, shear stress-induced membrane tension depends on the cooperation between cytoskeleton components, although it is mainly maintained by microtubules. MSS is a membrane-bound tension sensor, including a tension sensor module and two anchoring proteins, which are linked with lipid molecules in lipid raft and non-lipid raft regions through Lyn and K-Ras kinases, respectively. The tension sensor module comprises an elastic spider silk protein inserted between two fluorescence proteins, enhanced cyan fluorescence protein (ECFP) and yellow fluorescent protein for energy transfer (YPet) (Figure 1A). With this sensor, tension changes can be transformed into FRET efficiency changes of these two fluorescence proteins. The control probe, KMSS, is a head-less mutant whose amino terminal disassociates in the cytoplasm, whereas only the carboxyl terminal anchors on the membrane (Figure 1B). Therefore, no tension could be applied to the KMSS mutant. The FRET ratio of HeLa cells expressing MSS shows no changes over time under static condition (Video S1), demonstrating the stability of the sensor in a living cell. To further check these tension sensors' effects, the osmotic pressure of culture medium is changed artificially. HeLa cells expressing KMSS are exposed to hypertonic solution (0.05 g/mL, sucrose) or hypotonic solution (adding 3 times volume of H2O into culture medium) separately, but the FRET efficiency remains almost unchanged from the baseline (p > 0.05; Figures 1C and 1D), indicating that KMSS is inert to the changes of membrane tension. In contrast, the FRET ratio of HeLa cells expressing MSS achieves a clear higher level in hypertonic solution, whereas it decreases significantly from the baseline in hypotonic solution after 15 min (p < 0.05; Figure 1E), demonstrating that MSS is significantly affected and that it is sensitive to the changes of membrane tension induced by osmotic pressure. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI1NWRlOTNlMWFmMTQyMDVlZDI2OTAzZjcyMzk3NmU1NyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc5MDEyNzgxfQ.W9-BcdIIjbsP7JK36tyG-XncD_pi2ObOewahRJ3ovBfn_258Y7RxbA1VdG5Hr079sTNK8WlsXCiWS7jOFTp2WqXYWbGsORL5QZmS1HoYvG1MNllpZgtvwrf9HB4mBmFoaMIG_fucaThP3-B7d2xis4Xo6mVDhqEbEeFDyAM6xfc8REnNn3PHPQljneYPA0ZVIpeFSRKE57byphHjs_hbct-6jMFl8izMzX8QtIwF5HgsmUQuNwykbNKu4Gk_1IfNsoup_QMol8kkxuT0GNlbr0YDpeVPlqzufjWAE-dZNzfGGbaJjOp05RiqV6V2IsemMDQ3k_KHz6yEt1JguG5VEg Download .mp4 (0.23 MB) Help with .mp4 files Video S1. The MSS Biosensor in HeLa Cells without Performing Shear Stress and without Any Cytoskeletal Inhibitors, Related to Figure 1 Furthermore, when the cells expressing MSS are exposed to hypertonic solution (0.025 g/mL, sucrose) after a hypotonic stimulus (adding 2 times volume of H2O into culture medium) for 10 min, the FRET ratio reverses immediately, even though it decreases slowly in the beginning (Figure S1A; Video S2). On the contrary, the FRET ratio increases from the start but later decreases slowly when the cells expressing MSS are exposed to hypertonic solution (0.05 g/mL, sucrose) for 10 min first and then to hypotonic stimulus (adding the same volume of H2O into the culture medium) (Figure S1B). These results suggest that the response of this biosensor to the changes in membrane tension is reversible, whereby it will react quickly if the membrane tension changes in the opposite direction. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJhN2U3ODU2OGRhOWVmMjFkZTYzM2I1NjNjYjNmN2M1YSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc5MDEyNzgxfQ.ph6rCBuvxE1nRstqYEo5hf2nZnm4jCXrYg_L9s7OLuU-n-ZHXXfKnoAHSt_lU4XeZahSbR4yIbfo9AkgLa37P8noPx8g-RCRBZxoivudfF-VYdiXhiJih3nW6fGSyd3fgOOlAKZjnAOwYVgPgAguzz78e4r-sbsTQ4BsKO1xBDqaL0hwcYMhO1RIoL0zRVR8J3vJYc1GZtvtCmL685KjLTHXn3I91m9PPrysy0YSr651TDhQe2PeY9Pq0gaO42IdzOwrQ95wyt3XcZREPmZnegzC5JCDmDYrkIVmTXe5mAu_54uN_PLuxQaZXu8Q3A5jp6OKqKhdruUVggtiZ2jIkw Download .mp4 (0.37 MB) Help with .mp4 files Video S2. The MSS Biosensor in HeLa Cells after Exposure to Hypotonic Solution First and Then to Hypertonic Solution 10 min Later, Related to Figure 1 In addition, the FRET ratio of MSS increases linearly when cells are exposed to 0.025, 0.5, and 0.075 g/mL sucrose solution (Figure S1C). On the contrary, the ratio decreases linearly after reducing the osmotic pressure of culture medium by adding 1, 2, 3, or 4 times volume of H2O separately (Figure S1D). Results indicate a linear relationship between the response of MSS biosensor and the changes in membrane tension induced by osmotic pressure in current experiments. Therefore, MSS could be applied to visualize the changes and distribution of membrane tension with good sensitivity, reversibility, and linearity. To check the effects of different shear stresses on cell membrane tension, HeLa cells expressing MSS are exposed to shear stress of 0.5 (MSS-0.5 group), 2 (MSS-2 group), and 4 Pa (MSS-4 group) for 15 min. The group of HeLa cells transfected with KMSS, with shear stress of 2 Pa, serves as control. Results show that the FRET ratio of MSS decreases obviously in response to shear tress, whereas that of the control group remains unchanged basically (Figures 2A and 2B ). In addition, when the cells are exposed to shear stress, the FRET ratio of MSS decreases sharply within 30 s and then decreases slowly with time whatever be the magnitude of shear stress. Strangely, these are coincident changes of FRET ratio under shear stress of 0.5 and 4 Pa, both of which are lower than the change of FRET ratio under stress of 2 Pa (Figures 2B and 2C), suggesting that membrane tension is not positively related to the magnitude of shear stress. Further analyses show that cells display a heterogeneous distribution of FRET ratio with the lowest in the middle part, whereas the highest in upstream and downstream parts initially. This distribution manner remains unchanged over time and is not affected by low or high shear stress (Figures 3A and 3B ; Videos S3, S4, and S5). By fitting the FRET ratio in different regions of a cell at 0 and 15 min with Extreme equation (Tables S1 and S2), it is found that after its exposure to shear stress, the minimal FRET ratio at 15 min (named min value) decreases, and that 2 Pa induces a significantly larger min value than the other two groups (p < 0.05; Figure 3C). However, the position of min value shows a tendency of moving toward downstream as the shear stress ranges from 0.5 to 4 Pa (Figure S2). Furthermore, upstream variation range (UVR) and downstream variation range (DVR) become smaller under shear stress application, especially for 0.5 and 4 Pa, although there is no significant difference between UVR and DVR in spite of the magnitude of shear stress (Figure 3D). These results suggest that shear stress can enlarge membrane tension but plays only a gentle role in its uneven distribution. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJhYjdmNDI2M2EwNGU2OTZkYzRjZjM0ZWRkNzFlMzc3ZiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc5MDEyNzgxfQ.aT-TPmE1fxPCKHjzHELOusxfbWLIpyYzucvlhinS_SYr42YRgGZ6FGY2iY3Civ9XTGktlJHKFHcEgvCUKmQVDAjVfOeAmx3Eue_evE_-gGms1VKX0vuiTGq1__oL5ZrRywYqw1SxhKaU0DlCn-E1vWtOppS1OjQFl88EVZ0l7pgPVR7xaIMBwkIiinjrvxgorSsao37vmEANOItOpZKuK8nKlTkRWjFeHVKmlFufbztiC7lOLkjzVA-8K6sC0wJeBQiSjpbPoICvf21__cmkG2svYSMj77lkLJK7HcNmKt1TsiKRuadCQNTjW8jRonQ6xirbosv1E3Mr8LrA2btXwg Download .mp4 (0.26 MB) Help with .mp4 files Video S3. The MSS Biosensor in HeLa Cells under Shear Stress of 0.5 Pa, Related to Figure 2 eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI5Y2EwZmFlZTk2NTU2ZGFjYTkyZWJmNDM4ZDcwNWIxNiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc5MDEyNzgxfQ.VZMMJrpc9AWytuUr4rnqD0pCM2786OWr9Skevxb4UNjYWVDZpVl-cUTu-azzSP6p3IHPAf-2fomv-C4bsM_iS1oXCMocwNjcvNQ5SlKReyz2Yx7zidB0_1C2uhjOVXJXb8DuC6qF67r-G-6h5E05XvE5n9qeb3utqe_f41nckviHlE3i6eYnsedg2VE5mdrhclBiyTQsFhVHq13nDDMKg8WdXmvvlXgDbtVpuGvkT2zRQEy2kRIRCHm3FpCqEWqEqAOXI7up2IXJPsxNWU3rZib1SFKQxvk9OiRcDyRjxgubOwXUkYvT2HBhqg3XNVQ6PhC12C2WFWhyZs0s1C1XeQ Download .mp4 (0.21 MB) Help with .mp4 files Video S4. The MSS Biosensor in HeLa Cells under Shear Stress of 2 Pa, Related to Figure 2 eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIwMzY2ZDZiMTZjOGYyNWMzMzhhNDQwNzMyMTEzMzdhYiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc5MDEyNzgxfQ.ET_T4PMN7Tll9aeboUAqaw4_DjCCWe5Ldc_wdB1gP6b5LLbPcLo0QNYYaZcNUkGiujouNn1mKEq418_PBLZax1j2NPTeNhhfi678vt0CMGo_kw3yVhp3F1yfTpMoMum0yoH_dwx6hj2i2pSQEVvpeowSBLl76bKi36ECbNE1NljGeYPRYQA9ogAf7WYwD7UWetfuEtUKA9zfItb4Qr4NJrIQmN0f9dr6z_DorlDEWNfhN_x2p37RID9F7Qe1BEYTDqEhhEPrn3a2CJLMO8htQ00DGEQTWD6EDrlOFEOzH70zemdu9uzhRc06Zc7jGsTNdN5JeiUW0xV1MR1iHgfneg Download .mp4 (0.25 MB) Help with .mp4 files Video S5. The MSS Biosensor in HeLa Cells under Shear Stress of 4 Pa, Related to Figure 2 Cell membrane fluidity is reported to display polarity upon shear stress application (Butler et al., 2002Butler P.J. Tsou T.C. Li J.Y. Usami S. Chien S. Rate sensitivity of shear-induced changes in the lateral diffusion of endothelial cell membrane lipids: a role for membrane perturbation in shear-induced MAPK activation.FASEB J. 2002; 16: 216-218Crossref PubMed Scopus (79) Google Scholar). To check whether the cell membrane fluidity participates in the regulation of membrane tension induced by shear stress, HeLa cells expressing MSS are pretreated with 0.1 mmol/L of cholesterol (Cho) for 3 hr or 45 mmol/L benzyl alcohol (BA) for 15 min to reduce or enhance the fluidity of cell membrane, respectively (Butler et al., 2002Butler P.J. Tsou T.C. Li J.Y. Usami S. Chien S. Rate sensitivity of shear-induced changes in the lateral diffusion of endothelial cell membrane lipids: a role for membrane perturbation in shear-induced MAPK activation.FASEB J. 2002; 16: 216-218Crossref PubMed Scopus (79) Google Scholar). Cells transfected with MSS plasmid under shear stress of 2 Pa, namely, the MSS-2 group, serves as a control group. Upon the stimulus of 2 Pa, BA treatment decreases the FRET ratio sharply, whereas Cho decreases it slowly, although both treatments show a decreasing trend in the FRET ratio (Figures 4A and 4B ; Videos S6 and S7). At 15 min, BA treatment leads to a significant lower FRET ratio but Cho leads to a significant higher FRET ratio compared with the control group (p < 0.05; Figure 4C). These results demonstrate that cell membrane fluidity affects the alteration of membrane tension induced by shear stress, and higher membrane tension is associated with higher cell membrane fluidity. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJiNTQ5YjAwMDNjOGIxOWFkYTA3ZDViNzBkNTU3ZGUyYiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc5MDEyNzgxfQ.Lt3E3EGQW70WnIe_D8SU6OKJ15LnXqMkbQcc2MnHK88jLePJxebnMTFuzrhTQ3_ZJp0Jrc9JB4In3Tj0K3of__qL9qTQZSIB_P7rOhhzGi0Jr42vtKKlcnEJEYlfc_UoFfPq1CqOAzQB75z2bqrJkeCUwovGJytofhYymBR9lVG4zVF-zBiaQFkf-KywXxxOCOvjPM-bg2uaiO917q5x7oS_vzNN-G9TqZOc7YQdf5mGG-66TIVo8q_yJxFTO85MqIZA9ngvclSKdCFB_mEx7shOrzRDcJTvZQ4ZHiIB8x0lkebKjcQkyC77In383c0rmUuhzZquXEr3CuAP_D-VWg Download .mp4 (0.28 MB) Help with .mp4 files Video S6. The MSS Biosensor in HeLa Cells Pretreated for 15 min with 45 mmol/L Benzyl Alcohol under Shear Stress of 2 Pa, Related to Figure 4 eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJjNDA5NzFlNDlmMjgyMDI0NTJmNTczYjU3MTU4Y2NjOSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc5MDEyNzgxfQ.RnQzCfxtTtqH9N0GNYR00Lxi5XukgVHWuOS7Yc-1Pcb9xF67MeX6h7749_3Dh-zYfK8Ib_w3QDbizHj2kTR_lFU517HcakiJ6ez-ryXyb4lIvvGFup_WwFQ3U4Mbb0rK37GmtGvlzNN2gRlw0hYlM8iATB5GjpRmySW9aifuZKbEpwpi04Qf1Qi5VbD-Kl_a1j44_-r6ifHkiVOxUGimIbTKzfOJ1foYORUh7i203fEsyZeHS-9Gw0UoMVTccxvHFpmJmd2TFuQmz4z7PPtBhSzo-Ty4CEig1yUpFRmCTHVIbtTlKb3_u98qPjIObl2ugB__7KGeqp9snu0i-5xPLA Download .mp4 (0.24 MB) Help with .mp4 files Video S7. The MSS Biosensor in HeLa Cells Pretreated for 3 hr with 0.1 mmol/L Cholesterol under Shear Stress of 2 Pa, Related to Figure 4 Initially, FRET ratio is low in the whole cell after BA or Cho applications, especially under Cho treatment. After exposure to shear stress, the ratio decreases in all regions of the cell (Figure 5A). However, the smallest ratio (at 15 min) is still located in the middle part of the cell (Figure 5B). From the fitting curve, it can be obtained that BA treatment significantly decreases the min value, whereas Cho plays an opposite role (p < 0.05; Figure 5C; Tables S1 and S2), but neither of the above treatments changes the position of the min value (Figure S2). Besides, either UVR or DVR is drastically decreased by BA treatment but not by Cho. UVR has no significant difference with DVR whether under BA or Cho application (Figure 5D). These results indicate that cell membrane fluidity plays a significant role in adjusting the distribution of membrane tension induced by shear stress. They also suggest that enhanced cell membrane fluidity can help to homogenize cellular membrane tension. Studies show that cytoskeleton binds with cell membrane directly and takes part in the process of sensing external stress and mediating mechanical force transduction (Choi and Helmke, 2008Choi C.K. Helmke B.P. Short-term shear stress induces rapid actin dynamics in living endothelial cells.Mol. Cell. Biomech. 2008; 5: 247-258PubMed Google Scholar, Davies, 2009Davies P.F. Hemodynamic shear stress and the endothelium in cardiovascular pathophysiology.Nat. Clin. Pract. Cardiovasc. Med. 2009; 6: 16-26Crossref PubMed Scopus (812) Google Scholar, Alenghat and Ingber, 2002Alenghat F.J. Ingber D.E. Mechanotransduction: all signals point to cytoskeleton, matrix, and integrins.Sci. STKE. 2002; 2002: pe6Crossref PubMed Scopus (412) Google Scholar, Raucher et al., 2000Raucher D. Stauffer T. Chen W. Shen K. Guo S. York J.D. Sheetz M.P. Meyer T. Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion.Cell. 2000; 100: 221-228Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar). Therefore, it is tested whether cytoskeleton plays any role in the regulation of membrane tension induced by shear stress. HeLa cells expressing MSS are pretreated with 1 μg/mL of cytochalasin D (CytoD) for 30 min or 5 μg/mL of nocodazole (Noco) for 15 min to destroy actin filaments or microtubules, respectively (Kim et al., 2012Kim M. Song K. Jin E.J. Sonn J. Staurosporine and cytochalasin D induce chondrogenesis by regulation of actin dynamics in different way.Exp. Mol. Med. 2012; 44: 521-528Crossref PubMed Scopus (15) Google Scholar, Zaal et al., 2011Zaal K.J. Reid E. Mousavi K. Zhang T. Mehta A. Bugnard E. Sartorelli V. Ralston E. Who needs microtubules? myogenic reorganization of MTOC, Golgi complex and ER exit sites persists despite lack of normal microtubule tracks.PLoS One. 2011; 6: e29057Crossref PubMed Scopus (24) Google Scholar). It is observed that CytoD decreases FRET ratio significantly, whereas Noco strangely increases the ratio to a higher level compared with the control group (MSS-2 group) (p < 0.05; Figure 6; Videos S8 and S9). When HeLa cells expressing MSS are pretreated with 1 μg/mL of myosin light-chain kinase (MLCK) inhibitor, ML7, for 1 hr to suppress the contraction but keep the structure of microfilament intact (Liu et al., 2011Liu B. Lu S. Zheng S. Jiang Z. Wang Y. Two distinct phases of calcium signalling under flow.Cardiovasc. Res. 2011; 91: 124-133Crossref PubMed Scopus (37) Google Scholar), the FRET ratio decreases obviously but is slightly higher than that obtained from CytoD treatment, although the tendency is similar to that of CytoD (p < 0.05; Figure 6; Video S10). These results indicate that both microfilaments and microtubules are necessary for maintaining cellular membrane tension. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIzNGM5Y2FmZTQ4ZDE0ZTgyYTkzNjc1ZWI2NTNiZDBmOCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc5MDEyNzgxfQ.IM-T_CWi7u6kBevqimoweUiyWDafxhljdfkvg1G_VxK0Lcy2rR3_zRQN_GJFqQjcUqF9M0ojmAI5bbqXV-xczKF26rT4OB2ccapXXDREv57U5moqUT5lTkdT64o8HG9C8wpbB48agHT7q5_NEHkReEuZtcr4evibFPXbbhOv-A6jQD2ay42Xs62581MpS9nEEFtihscGi1UZOnP3SVCnoJ1Azr_w6oBTql2V1fh2dQcWSSNP9LfX9kTMo9doY4Qta3xv9o98d7GucfQ5zFl3fhCEnmAeOGsYQ8lY6XxqIGEI2yo8vuW4RN4A82UMRI1CYn3vBO9dsarXVa3Uog" @default.
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• W2890544989 date "2018-09-01" @default.
• W2890544989 modified "2023-10-15" @default.
• W2890544989 title "A Membrane-Bound Biosensor Visualizes Shear Stress-Induced Inhomogeneous Alteration of Cell Membrane Tension" @default.
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• W2890544989 doi "https://doi.org/10.1016/j.isci.2018.09.002" @default.
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