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RhoA and membrane fluidity mediates the spatially polarized Src/FAK activation in response to shear stress.

Liu B, Lu S, Hu YL, Liao X, Ouyang M, Wang Y - Sci Rep (2014)

Bottom Line: Further experiments revealed that HSS decreased RhoA activity, with a constitutively active RhoA mutant inhibiting while a negative RhoA mutant enhancing the HSS-induced Src polarity.Further results indicate that HSS stimulates FAK activation with a spatial polarity similar to Src.The inhibition of Src by PP1, as well as the perturbation of RhoA activity and membrane fluidity, can block this HSS-induced FAK polarity.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biomedical Engineering, Dalian University of Technology, Dalian, Liaoning Province, 116024, P. R. China [2] Department of Bioengineering and the Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

ABSTRACT
While Src plays crucial roles in shear stress-induced cellular processes, little is known on the spatiotemporal pattern of high shear stress (HSS)-induced Src activation. HSS (65 dyn/cm(2)) was applied on bovine aortic endothelial cells to visualize the dynamic Src activation at subcellular levels utilizing a membrane-targeted Src biosensor (Kras-Src) based on fluorescence resonance energy transfer (FRET). A polarized Src activation was observed with higher activity at the side facing the flow, which was enhanced by a cytochalasin D-mediated disruption of actin filaments but inhibited by a benzyl alcohol-mediated enhancement of membrane fluidity. Further experiments revealed that HSS decreased RhoA activity, with a constitutively active RhoA mutant inhibiting while a negative RhoA mutant enhancing the HSS-induced Src polarity. Cytochalasin D can restore the polarity in cells expressing the active RhoA mutant. Further results indicate that HSS stimulates FAK activation with a spatial polarity similar to Src. The inhibition of Src by PP1, as well as the perturbation of RhoA activity and membrane fluidity, can block this HSS-induced FAK polarity. These results indicate that the HSS-induced Src and subsequently FAK polarity depends on the coordination between intracellular tension distribution regulated by RhoA, its related actin structures and the plasma membrane fluidity.

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RhoA mediates the HSS-induced Src polarity, but not overall activation.(A)The averaged time courses of representative YFP/CFP emission ratios of the RhoA FRET biosensor to represent the RhoA activity in different cell areas under HSS application (n = 17). (B) Bar graphs (mean ± S.E.M.) represent the averaged values of YFP/CFP emission ratios measuring the RhoA activity in the upstream and the downstream sides of BAECs pretreated with or without 50 μmol/l of PP1 before 30 min of HSS application (n = 14). (C) The representative YFP/CFP emission ratio images of (left panels) the RhoA FRET biosensors in BAECs pretreated with or without PP1, or (right panels) pretreated with 4 μmol/l of LPA (N = 7), Kras-Src FRET biosensors co-transfected with V14-RhoA (n = 11) or N19-RhoA (n = 11), or CytoD in V14-RhoA co-expressing BAECs (n = 4) as indicated. (D–E): Bar graphs (mean ± S.E.M.) represent the averaged values of YFP/CFP emission ratios measuring (D) the overall Src activation or (E) the Src polarity in BEACs with different pretreatments as indicated. * represents a statistically significant P < 0.05 when comparing to the control group; # represents a statistically significant P < 0.05 when comparing to the V14-RhoA co-transfected group.
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f3: RhoA mediates the HSS-induced Src polarity, but not overall activation.(A)The averaged time courses of representative YFP/CFP emission ratios of the RhoA FRET biosensor to represent the RhoA activity in different cell areas under HSS application (n = 17). (B) Bar graphs (mean ± S.E.M.) represent the averaged values of YFP/CFP emission ratios measuring the RhoA activity in the upstream and the downstream sides of BAECs pretreated with or without 50 μmol/l of PP1 before 30 min of HSS application (n = 14). (C) The representative YFP/CFP emission ratio images of (left panels) the RhoA FRET biosensors in BAECs pretreated with or without PP1, or (right panels) pretreated with 4 μmol/l of LPA (N = 7), Kras-Src FRET biosensors co-transfected with V14-RhoA (n = 11) or N19-RhoA (n = 11), or CytoD in V14-RhoA co-expressing BAECs (n = 4) as indicated. (D–E): Bar graphs (mean ± S.E.M.) represent the averaged values of YFP/CFP emission ratios measuring (D) the overall Src activation or (E) the Src polarity in BEACs with different pretreatments as indicated. * represents a statistically significant P < 0.05 when comparing to the control group; # represents a statistically significant P < 0.05 when comparing to the V14-RhoA co-transfected group.

Mentions: RhoA has been shown to play critical roles in regulating actin cytoskeleton and the translocation/activation of Src3738. It has also been shown that RhoA activity changed prior to the activation of several other signaling molecules induced by shear stress39. We hence examined the effects of HSS on RhoA activity and its role in regulating the Src polarity. BAECs expressing a RhoA FRET biosensor were exposed to HSS. A decrease of RhoA activity can be clearly observed upon HSS application (Fig. 3A–C, Movie 4). This HSS-induced RhoA decrease was not suppressed by 50 μM PP1 pretreatment (Fig. 3B–C), indicating that RhoA may indeed act upstream to Src activation under flow. Consistently, an active RhoA mutant (V14-RhoA) blocked and a negative mutant (N19-RhoA) enhanced the HSS-induced Src polarity (Fig. 3C and 3E, Movie 5). The HSS-induced Src polarity was also blocked by pretreatment for 1 hr with 4 μM lysophosphatidic acid (LPA), a RhoA activator (Fig. 3C–E). Interestingly, neither V14-RhoA nor N19-RhoA affected the overall HSS-induced Src activation level (Fig. 3C–D). These results indicate that HSS may regulate the RhoA activity to control Src polarity, but not overall level of Src activation. Because RhoA has been shown to regulate the stress fibers formation, it may regulate the HSS-induced Src activation via actin stress fiber formation. Indeed, more F-actin was observed when the actin-GFP was co-transfected with V14-RhoA into BAECs (Supplemental Fig. 2), which is also consistent with previous reports4041.


RhoA and membrane fluidity mediates the spatially polarized Src/FAK activation in response to shear stress.

Liu B, Lu S, Hu YL, Liao X, Ouyang M, Wang Y - Sci Rep (2014)

RhoA mediates the HSS-induced Src polarity, but not overall activation.(A)The averaged time courses of representative YFP/CFP emission ratios of the RhoA FRET biosensor to represent the RhoA activity in different cell areas under HSS application (n = 17). (B) Bar graphs (mean ± S.E.M.) represent the averaged values of YFP/CFP emission ratios measuring the RhoA activity in the upstream and the downstream sides of BAECs pretreated with or without 50 μmol/l of PP1 before 30 min of HSS application (n = 14). (C) The representative YFP/CFP emission ratio images of (left panels) the RhoA FRET biosensors in BAECs pretreated with or without PP1, or (right panels) pretreated with 4 μmol/l of LPA (N = 7), Kras-Src FRET biosensors co-transfected with V14-RhoA (n = 11) or N19-RhoA (n = 11), or CytoD in V14-RhoA co-expressing BAECs (n = 4) as indicated. (D–E): Bar graphs (mean ± S.E.M.) represent the averaged values of YFP/CFP emission ratios measuring (D) the overall Src activation or (E) the Src polarity in BEACs with different pretreatments as indicated. * represents a statistically significant P < 0.05 when comparing to the control group; # represents a statistically significant P < 0.05 when comparing to the V14-RhoA co-transfected group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4228346&req=5

f3: RhoA mediates the HSS-induced Src polarity, but not overall activation.(A)The averaged time courses of representative YFP/CFP emission ratios of the RhoA FRET biosensor to represent the RhoA activity in different cell areas under HSS application (n = 17). (B) Bar graphs (mean ± S.E.M.) represent the averaged values of YFP/CFP emission ratios measuring the RhoA activity in the upstream and the downstream sides of BAECs pretreated with or without 50 μmol/l of PP1 before 30 min of HSS application (n = 14). (C) The representative YFP/CFP emission ratio images of (left panels) the RhoA FRET biosensors in BAECs pretreated with or without PP1, or (right panels) pretreated with 4 μmol/l of LPA (N = 7), Kras-Src FRET biosensors co-transfected with V14-RhoA (n = 11) or N19-RhoA (n = 11), or CytoD in V14-RhoA co-expressing BAECs (n = 4) as indicated. (D–E): Bar graphs (mean ± S.E.M.) represent the averaged values of YFP/CFP emission ratios measuring (D) the overall Src activation or (E) the Src polarity in BEACs with different pretreatments as indicated. * represents a statistically significant P < 0.05 when comparing to the control group; # represents a statistically significant P < 0.05 when comparing to the V14-RhoA co-transfected group.
Mentions: RhoA has been shown to play critical roles in regulating actin cytoskeleton and the translocation/activation of Src3738. It has also been shown that RhoA activity changed prior to the activation of several other signaling molecules induced by shear stress39. We hence examined the effects of HSS on RhoA activity and its role in regulating the Src polarity. BAECs expressing a RhoA FRET biosensor were exposed to HSS. A decrease of RhoA activity can be clearly observed upon HSS application (Fig. 3A–C, Movie 4). This HSS-induced RhoA decrease was not suppressed by 50 μM PP1 pretreatment (Fig. 3B–C), indicating that RhoA may indeed act upstream to Src activation under flow. Consistently, an active RhoA mutant (V14-RhoA) blocked and a negative mutant (N19-RhoA) enhanced the HSS-induced Src polarity (Fig. 3C and 3E, Movie 5). The HSS-induced Src polarity was also blocked by pretreatment for 1 hr with 4 μM lysophosphatidic acid (LPA), a RhoA activator (Fig. 3C–E). Interestingly, neither V14-RhoA nor N19-RhoA affected the overall HSS-induced Src activation level (Fig. 3C–D). These results indicate that HSS may regulate the RhoA activity to control Src polarity, but not overall level of Src activation. Because RhoA has been shown to regulate the stress fibers formation, it may regulate the HSS-induced Src activation via actin stress fiber formation. Indeed, more F-actin was observed when the actin-GFP was co-transfected with V14-RhoA into BAECs (Supplemental Fig. 2), which is also consistent with previous reports4041.

Bottom Line: Further experiments revealed that HSS decreased RhoA activity, with a constitutively active RhoA mutant inhibiting while a negative RhoA mutant enhancing the HSS-induced Src polarity.Further results indicate that HSS stimulates FAK activation with a spatial polarity similar to Src.The inhibition of Src by PP1, as well as the perturbation of RhoA activity and membrane fluidity, can block this HSS-induced FAK polarity.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biomedical Engineering, Dalian University of Technology, Dalian, Liaoning Province, 116024, P. R. China [2] Department of Bioengineering and the Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

ABSTRACT
While Src plays crucial roles in shear stress-induced cellular processes, little is known on the spatiotemporal pattern of high shear stress (HSS)-induced Src activation. HSS (65 dyn/cm(2)) was applied on bovine aortic endothelial cells to visualize the dynamic Src activation at subcellular levels utilizing a membrane-targeted Src biosensor (Kras-Src) based on fluorescence resonance energy transfer (FRET). A polarized Src activation was observed with higher activity at the side facing the flow, which was enhanced by a cytochalasin D-mediated disruption of actin filaments but inhibited by a benzyl alcohol-mediated enhancement of membrane fluidity. Further experiments revealed that HSS decreased RhoA activity, with a constitutively active RhoA mutant inhibiting while a negative RhoA mutant enhancing the HSS-induced Src polarity. Cytochalasin D can restore the polarity in cells expressing the active RhoA mutant. Further results indicate that HSS stimulates FAK activation with a spatial polarity similar to Src. The inhibition of Src by PP1, as well as the perturbation of RhoA activity and membrane fluidity, can block this HSS-induced FAK polarity. These results indicate that the HSS-induced Src and subsequently FAK polarity depends on the coordination between intracellular tension distribution regulated by RhoA, its related actin structures and the plasma membrane fluidity.

Show MeSH
Related in: MedlinePlus