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Focal adhesion kinase (pp125FAK) phosphorylation is linked to force generation in F9 cells
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Journal o f Biomechanics 2006, Vol. 39 (Suppl 1)
5159 Th, 09:00-09:15 (P38) Focal adhesion kinase (pp125 FAK) p h o s p h o r y l a t i o n is linked to force generation in F9 cells A.H. Klemm, D.P. Zitterbart, W.H. Goldmann. Friedrich-Alexander-University of Erlangen-Nuremberg, Center for Medical Physics and Technology, Biophysics Group, Erlangen, Germany Mechanical forces, both intra- and extracellular, activate signaling events through integrins. It is known that an intact actin cytoskeleton (CSK) and focal adhesion complex (FAC) are prerequisites for integrin-mediated activation and subsequent downstream signaling events that control cell mechanical functions, but the molecular details are still elusive. Recent reports suggested that the focal adhesion kinase (pp125 FAK) and in particular its phosphorylation site at residue p-Tyr397 may play an essential role in this process, pp125 FAK binds to several focal adhesion proteins, including talin, alpha-actinin, and paxillin. In addition, pp125 FAK has been shown to be necessary for cell spreading on the extracellular matrix (ECM) and cytoskeletal stiffening. Here we measured the time course of pp125 FAK phosphorylation and traction force generation during the process of cell adhesion and spreading on ECM. Wild type mouse F9 embryonic carcinoma cells were plated onto fibrinogencoated dishes, harvested, and lysed at 10, 30, 60, and 240 minutes after plating to determine the phosphorylation of pp125 FAK by SDS-Page and Western blotting. In a parallel experiment, we used the method of Fourier Transform Traction Microscopy (Butler et al. Am. J. Physiol. Cell Physiol. 282: C595C605, 2002) to measure the tractions that these cells generate during adhesion and spreading onto collagen-coated polyacrylamide gels (Young's modulus 3000Pa). We found a time-dependent increase of pp125 FAK phosphorylation that reached a plateau value within -20 minutes of adhesion and spreading. Cell tractions expressed as strain energy exhibited an almost identical time course and reached a plateau value of -0.3 [fJ] within 20 minutes. These results demonstrate that pp125 FAK and force generation during cell adhesion and spreading are tightly linked. 5349 Th, 09:15-09:30 (P38) A model for the role o f integrins in flow induced mechanotransduction in osteocytes Y. Wang 1, M. Schaffler 2, S. Weinbaum 1. 1Department of Biomedical Engineering, CUNY Graduate Center and the City College of New York, NY, USA, 2Department of Orthopedics, Mount Sinai School of Medicine, New York, NY, USA Models for flow induced strain amplification in osteocytes have been proposed by You at el. (2001, J Biomech. 34, 1375-86) and Han et al. (2004, PNAS, 101, 16689-94). While both models elegantly predict how whole tissue strains lead to greatly amplified flow induced strains on the central actin filament bundle and the cell process membrane, the underlying molecular-level mechanotransduction mechanism, especially the transmembrane machinery, is still poorly understood. Integrins are ubiquitous transmembrane cell adhesion proteins that couple the cytoskeleton to extracellular matrix (ECM) proteins, and are involved in signaling between cells and their ECM. McNamara et al (2006, ORS abstract) have reported that, in regions of curvature along the canaliculi, collagen fibrils underlying the inner wall intermittently protrude into the pericellular space and attach periodically to the cell process membrane. This intermittent attachment appears to be integrin-based. In the current paper, we shall develop a new structural model to describe these local regions of curvature with fixed integrin attachment sites and an asymmetric loading of tethering elements along the cell process membrane. We predict the magnitude of such integrin attachments and their loading rate as a function of frequency. Our model predicts that fluid induced drag forces on the tethering elements will give rise to hoop strains of >0.5% for tissue-level strains of >1000 microstrain at 1 Hz and >250 microstrain at frequencies >10 Hz. The magnitude of the tension on the integrin attachment is lower than that required for disruption, but is sufficient to trigger integrin associated signaling pathways as reported by Li et al. (2003, J Biophys. 84, 1252-62). Therefore, we propose that these integrin attachments between the osteocyte process and the canalicular wall function as the primary transmembrane machinery in the strain amplification theory for the initiation of intracellular signaling. 6110 Th, 09:30-09:45 (P38) Mechanotransduction: models how extracellular matrix proteins and integrins convert force into biochemical signals V. Vogel. Laboratory for Biologically Oriented Materials, Department of Materials, ETH Zurich, Switzerland Cells can sense and transduce a broad range of mechanical forces into distinct sets of biochemical signals that ultimately regulate cellular processes, including adhesion, proliferation, differentiation, and apoptosis. Deciphering at the nanoscale the design principles by which sensory elements are integrated into structural protein motifs whose conformations can be switched
Oral Presentations mechanically is crucial to understand the process of transduction of force into biochemical signals that are then integrated to regulate mechanoresponsive pathways. While the major focus has been on membrane proteins such as ion channels, integrins, and associated cytoplasmic complexes as mechanosensory units, a multimodular design of tandem repeats of various structural motifs is ubiquitously found among extracellular matrix proteins, as well as cell adhesion molecules, and among many intracellular players that physically link transmembrane proteins to the contractile cytoskeleton. Considering the lack of experimental techniques to determine high resolution structures of partially unraveled proteins, powerful new insights are provided by steered molecular dynamics (SMD)in which force is applied to known crystal structures of proteins after equilibrating them in a box of explicit water molecules. SMD derived high resolution structural hypotheses will be discussed how force might alter protein function. In case of fibronectin, we furthermore applied fluorescence resonance energy transfer combined with other biochemical assays, to investigate how cells can locally stretch and partially unfold fibronectin and how matrix unfolding relates to a strain-dependent exposure of cryptic sites. Using SMD, we then asked what role mechanical forces might play in the process of integrin "activation". Fibronectin's 10 th type III module (fnllll0)was thereby docked to the ~J,v153 integrin head piece. 4349 Th, 11:00-11:15 (P41) The glycocalyx as a mechanosensor for fluid shear stress '~ Yao, A. Rabodzey, C.F. Dewey Jr.. Hatsopoulos Microfluids Lab, Massachusetts Institute of Technology, Cambridge, USA Flow-induced mechanotransduction in the endothelial cells has been studied over the years with a major focus on focal adhesions. Recent studies [1] have brought in a new perspective that the glycocalyx, a structure decorating the luminal surface of vascular endothelium, may play an important role in the mechanotransduction. Our studies have shown that by modifying the amount of the glycocalyx, both short term and long term shear responses are affected significantly. It is well established that after 24 hr of laminar flow, endothelial cells align in the direction of flow [2] and their proliferation is suppressed [3]. We report here that by removing the glycocalyx using a specific enzyme heparinase III, endothelial cells no long align under flow after 24 hrs, and they proliferate as if there were no flow present. In addition, recent results obtained in our group have shown that wild-type endothelial cells rapidly respond to flow by decreasing migration speed by 50% [4] and increasing the amount of VECadherin in junction. These responses are not observed in the cells treated with heparinase II1. Interestingly, we found that heparan sulfate proteoglycans (a major component of glycocalyx) redistribute after 24 hr of flow application from a uniform surface profile to a distinct pattern with more molecules detected close to regions above the cell-cell junction and less detected above the nucleus. In summary, we conclude that the presence of the glycocalyx is necessary for the endothelial cells to respond to fluid shear, and the glycocalyx itself is affected by the flow. We suggest that the redistribution of the glycocalyx can serve as a cell adaptive mechanism by reducing the shear gradient the cell experiences. References [1] Thi M.M., et al. Proc Natl Acad Sci USA 2004; 101(47): 16483-8. [2] Dewey Jr. C.F., et al. J Biomech Eng. 1981; 103: 177-85. [3] Levesque M.J., et al. Biomaterials 1990; 11(9): 702-7. [4] Osborn E.A., et al. Am J Physiol Cell Physiol. 2006; 290(2): C444-52.
6948 Th, 11:15-11:30 (P41) The dynamics o f extracellular mechanotransduction in bronchial epithelium N. Kojic 1, P. So 1, D.J. Tschumperlin 2. 1Harvard-MIT Division of Health Sciences and Technology, Cambridge, USA, 2physiology Program, Harvard School of Public Health, Boston, USA Mechanical stress activates the epidermal growth factor receptor (EGFR) in airway epithelial cells both in culture and in constricted mouse airways. The in vitro response requires an active autocrine ligand-receptor loop localized to the basolateral surfaces of the epithelium. We have shown previously that transcellular compressive stress shrinks the lateral intercellular space (LIS) separating epithelial cells, and hypothesized that constitutively-present EGFR autocrine signaling in a collapsing LIS is sufficient to account for mechanotransduction. To evaluate the dynamics of the proposed mechanism we compared high temporal resolution biochemical measurements of EGFR activation with high speed imaging of LIS geometry during compression of cultured human bronchial epithelial cells. We used ELISAs to quantify total and phosphorylated EGFR during the first 20 minutes of compressive stress (30cmH20). Our results demonstrated a rapid change in EGFR phosphorylation peaking at a 3.4±0.2-fold increase 3 minutes after the onset of compressive stress, returning to near baseline (1.5±0.3-fold) by 20 minutes. Using a two-photon fastscan imaging technique we found that compressive stress rapidly reduced LIS
Journal o f Biomechanics 2006, Vol. 39 (Suppl 1)
5159 Th, 09:00-09:15 (P38) Focal adhesion kinase (pp125 FAK) p h o s p h o r y l a t i o n is linked to force generation in F9 cells A.H. Klemm, D.P. Zitterbart, W.H. Goldmann. Friedrich-Alexander-University of Erlangen-Nuremberg, Center for Medical Physics and Technology, Biophysics Group, Erlangen, Germany Mechanical forces, both intra- and extracellular, activate signaling events through integrins. It is known that an intact actin cytoskeleton (CSK) and focal adhesion complex (FAC) are prerequisites for integrin-mediated activation and subsequent downstream signaling events that control cell mechanical functions, but the molecular details are still elusive. Recent reports suggested that the focal adhesion kinase (pp125 FAK) and in particular its phosphorylation site at residue p-Tyr397 may play an essential role in this process, pp125 FAK binds to several focal adhesion proteins, including talin, alpha-actinin, and paxillin. In addition, pp125 FAK has been shown to be necessary for cell spreading on the extracellular matrix (ECM) and cytoskeletal stiffening. Here we measured the time course of pp125 FAK phosphorylation and traction force generation during the process of cell adhesion and spreading on ECM. Wild type mouse F9 embryonic carcinoma cells were plated onto fibrinogencoated dishes, harvested, and lysed at 10, 30, 60, and 240 minutes after plating to determine the phosphorylation of pp125 FAK by SDS-Page and Western blotting. In a parallel experiment, we used the method of Fourier Transform Traction Microscopy (Butler et al. Am. J. Physiol. Cell Physiol. 282: C595C605, 2002) to measure the tractions that these cells generate during adhesion and spreading onto collagen-coated polyacrylamide gels (Young's modulus 3000Pa). We found a time-dependent increase of pp125 FAK phosphorylation that reached a plateau value within -20 minutes of adhesion and spreading. Cell tractions expressed as strain energy exhibited an almost identical time course and reached a plateau value of -0.3 [fJ] within 20 minutes. These results demonstrate that pp125 FAK and force generation during cell adhesion and spreading are tightly linked. 5349 Th, 09:15-09:30 (P38) A model for the role o f integrins in flow induced mechanotransduction in osteocytes Y. Wang 1, M. Schaffler 2, S. Weinbaum 1. 1Department of Biomedical Engineering, CUNY Graduate Center and the City College of New York, NY, USA, 2Department of Orthopedics, Mount Sinai School of Medicine, New York, NY, USA Models for flow induced strain amplification in osteocytes have been proposed by You at el. (2001, J Biomech. 34, 1375-86) and Han et al. (2004, PNAS, 101, 16689-94). While both models elegantly predict how whole tissue strains lead to greatly amplified flow induced strains on the central actin filament bundle and the cell process membrane, the underlying molecular-level mechanotransduction mechanism, especially the transmembrane machinery, is still poorly understood. Integrins are ubiquitous transmembrane cell adhesion proteins that couple the cytoskeleton to extracellular matrix (ECM) proteins, and are involved in signaling between cells and their ECM. McNamara et al (2006, ORS abstract) have reported that, in regions of curvature along the canaliculi, collagen fibrils underlying the inner wall intermittently protrude into the pericellular space and attach periodically to the cell process membrane. This intermittent attachment appears to be integrin-based. In the current paper, we shall develop a new structural model to describe these local regions of curvature with fixed integrin attachment sites and an asymmetric loading of tethering elements along the cell process membrane. We predict the magnitude of such integrin attachments and their loading rate as a function of frequency. Our model predicts that fluid induced drag forces on the tethering elements will give rise to hoop strains of >0.5% for tissue-level strains of >1000 microstrain at 1 Hz and >250 microstrain at frequencies >10 Hz. The magnitude of the tension on the integrin attachment is lower than that required for disruption, but is sufficient to trigger integrin associated signaling pathways as reported by Li et al. (2003, J Biophys. 84, 1252-62). Therefore, we propose that these integrin attachments between the osteocyte process and the canalicular wall function as the primary transmembrane machinery in the strain amplification theory for the initiation of intracellular signaling. 6110 Th, 09:30-09:45 (P38) Mechanotransduction: models how extracellular matrix proteins and integrins convert force into biochemical signals V. Vogel. Laboratory for Biologically Oriented Materials, Department of Materials, ETH Zurich, Switzerland Cells can sense and transduce a broad range of mechanical forces into distinct sets of biochemical signals that ultimately regulate cellular processes, including adhesion, proliferation, differentiation, and apoptosis. Deciphering at the nanoscale the design principles by which sensory elements are integrated into structural protein motifs whose conformations can be switched
Oral Presentations mechanically is crucial to understand the process of transduction of force into biochemical signals that are then integrated to regulate mechanoresponsive pathways. While the major focus has been on membrane proteins such as ion channels, integrins, and associated cytoplasmic complexes as mechanosensory units, a multimodular design of tandem repeats of various structural motifs is ubiquitously found among extracellular matrix proteins, as well as cell adhesion molecules, and among many intracellular players that physically link transmembrane proteins to the contractile cytoskeleton. Considering the lack of experimental techniques to determine high resolution structures of partially unraveled proteins, powerful new insights are provided by steered molecular dynamics (SMD)in which force is applied to known crystal structures of proteins after equilibrating them in a box of explicit water molecules. SMD derived high resolution structural hypotheses will be discussed how force might alter protein function. In case of fibronectin, we furthermore applied fluorescence resonance energy transfer combined with other biochemical assays, to investigate how cells can locally stretch and partially unfold fibronectin and how matrix unfolding relates to a strain-dependent exposure of cryptic sites. Using SMD, we then asked what role mechanical forces might play in the process of integrin "activation". Fibronectin's 10 th type III module (fnllll0)was thereby docked to the ~J,v153 integrin head piece. 4349 Th, 11:00-11:15 (P41) The glycocalyx as a mechanosensor for fluid shear stress '~ Yao, A. Rabodzey, C.F. Dewey Jr.. Hatsopoulos Microfluids Lab, Massachusetts Institute of Technology, Cambridge, USA Flow-induced mechanotransduction in the endothelial cells has been studied over the years with a major focus on focal adhesions. Recent studies [1] have brought in a new perspective that the glycocalyx, a structure decorating the luminal surface of vascular endothelium, may play an important role in the mechanotransduction. Our studies have shown that by modifying the amount of the glycocalyx, both short term and long term shear responses are affected significantly. It is well established that after 24 hr of laminar flow, endothelial cells align in the direction of flow [2] and their proliferation is suppressed [3]. We report here that by removing the glycocalyx using a specific enzyme heparinase III, endothelial cells no long align under flow after 24 hrs, and they proliferate as if there were no flow present. In addition, recent results obtained in our group have shown that wild-type endothelial cells rapidly respond to flow by decreasing migration speed by 50% [4] and increasing the amount of VECadherin in junction. These responses are not observed in the cells treated with heparinase II1. Interestingly, we found that heparan sulfate proteoglycans (a major component of glycocalyx) redistribute after 24 hr of flow application from a uniform surface profile to a distinct pattern with more molecules detected close to regions above the cell-cell junction and less detected above the nucleus. In summary, we conclude that the presence of the glycocalyx is necessary for the endothelial cells to respond to fluid shear, and the glycocalyx itself is affected by the flow. We suggest that the redistribution of the glycocalyx can serve as a cell adaptive mechanism by reducing the shear gradient the cell experiences. References [1] Thi M.M., et al. Proc Natl Acad Sci USA 2004; 101(47): 16483-8. [2] Dewey Jr. C.F., et al. J Biomech Eng. 1981; 103: 177-85. [3] Levesque M.J., et al. Biomaterials 1990; 11(9): 702-7. [4] Osborn E.A., et al. Am J Physiol Cell Physiol. 2006; 290(2): C444-52.
6948 Th, 11:15-11:30 (P41) The dynamics o f extracellular mechanotransduction in bronchial epithelium N. Kojic 1, P. So 1, D.J. Tschumperlin 2. 1Harvard-MIT Division of Health Sciences and Technology, Cambridge, USA, 2physiology Program, Harvard School of Public Health, Boston, USA Mechanical stress activates the epidermal growth factor receptor (EGFR) in airway epithelial cells both in culture and in constricted mouse airways. The in vitro response requires an active autocrine ligand-receptor loop localized to the basolateral surfaces of the epithelium. We have shown previously that transcellular compressive stress shrinks the lateral intercellular space (LIS) separating epithelial cells, and hypothesized that constitutively-present EGFR autocrine signaling in a collapsing LIS is sufficient to account for mechanotransduction. To evaluate the dynamics of the proposed mechanism we compared high temporal resolution biochemical measurements of EGFR activation with high speed imaging of LIS geometry during compression of cultured human bronchial epithelial cells. We used ELISAs to quantify total and phosphorylated EGFR during the first 20 minutes of compressive stress (30cmH20). Our results demonstrated a rapid change in EGFR phosphorylation peaking at a 3.4±0.2-fold increase 3 minutes after the onset of compressive stress, returning to near baseline (1.5±0.3-fold) by 20 minutes. Using a two-photon fastscan imaging technique we found that compressive stress rapidly reduced LIS