Role of intercellular adherens junctions in endothelial cell cooperative motion
1
$234 Journal of Biomechanics 2006, Vol. 39 (Suppl 1) 6410 Tu, 11:45-12:00 (P19) Actin dynamics and optical guidance of neuronal growth cones D. Koch, T. Betz, A. Ehrlicher, B. Stuhrmann, M. GSgler, J. K~s. Institute for Soft Matter Physics, University of Leipzig, Germany The understanding of the motility and the dynamics of the growth cone, the highly motile structure at the tip of a growing neurite, is a fundamental basis of unraveling the development and function of the neuronal system. In a noisy environment full of different chemical and mechanical cues the growth cone is able to successfully wire up a highly complex neuronal network. The dynamics and constant reorganisation of the growth cone structure play a crucial role in its ability to steer the advancing tip of the neurite. However, the detailed molecular mechanisms underlying growth cone motility still need to be resolved. We have shown that optical forces induced by a highly focused infrared laser beam influence the motility of a growth cone by biasing the polymerization- driven intracellular machinery. In actively extending growth cones, a laser spot placed at specific areas of the neurite's leading edge affects the direction taken by a growth cone. In an optical tweezers setup, simultaneous phase contrast and fluorescence imaging, real-time shape detection, and automated laser irradiation are combined to control growth cone motility. We have focused on the actin dynamics in the growth cone to elucidate the mechanisms underlying optical neuron guidance. At the front edge of the growth cone actin is polymerized into filaments building complex bundles and network structures that are transported back towards the center in a process termed 'retrograde flow'. We've developed a new method based on high resolution edge detection in combination with a cross-correlation algorithm to extract and quantity these actin dynamics from fluorescence observations with high spatial and temporal resolution. These novel techniques allow the investigation and manipulation of a natural biological process, the cytoskeleton driven morphological changes in a growth cone, with potential applications in the formation of neuronal networks and in understanding growth cone motility. 6500 Tu, 12:00-12:15 (P19) Role of intercellular adherens junctions in endothelial cell cooperative motion A.A. Rabodzey 1, C.E. Semino 2, R.D. Kamm 2, C.F. Dewey Jr. 1. 1Hatsopoulos Microfluids Laboratory MIT, Cambridge, USA, 2Biological Engineering Division, MIT, Cambridge, USA Intercellular junctions are important structural elements that communicate signals between cells. Adherens junction are the type of junctions responsible for the physical force of interactions; they are the most abundant intercellular structures. The major junctions-forming protein is VE-cadherin. VE-cadherin is involved in flow-induced mechanotransduction and may plays a role in the intercellular organization during crawling and vessel formation. In our previous studies we looked at the role of the adherens junctions in mechanotransduction and cell migration [1] and observed that VE-cadherin is in part responsible for early mechanotransduction in the endothelial cells [2]. In this study we used siRNA to knock down the VE-cadherin and study behavior of the adherens junctions-deficient cells. Full knockdown confirmed by western blot and immunofluorescence is achieved following 24 hr of transfection. The major advantage of using knockdowns compared to full knockouts is that we are able to use human cells whose response is well established and we achieve full suppression of VE-cadherin unlike experiments with blocking antibodies. Our current studies are directed to our previous hypothesis that VE-cadherin is an important player in cell motility, early mechanotransduction and angiogenesis. We find that average speed of HUVEC motion increases by -25% in the VE- cadherin null cells compared to normal cells. VE-cad deficient cells show faster wound closure. VE-cadherin deficient confluent cells show behavior much closer to that of subconfluent cells than confluent cells. An in vitro assay to analyze vessel formation [3] and understand the effects of adherens junction disruption on angiogenesis will also be reported. [Unlike the assay described in Dejana et al. Blood 2002, cells are migrating from the surface and are not sandwiched in between two gels]. References [1] Osborn EA, Rabodzey A, Dewey Jr CF, Hartwig JH. Am J Physiol Cell Physiol 2006 Feb; 290(2): C444-52. [2] Rabodzey AA, Yao Y, Hartwig JH, Dewey Jr CF. Endothelial cells early response to flow may be mediated by VE-cadherins Submitted. [3] Semino CE, Kamm RD, Lauffenburger DA. Exp Cell Res. 2006 Feb 1; 312(3): 289-98. Oral Presentations 5080 Tu, 12:15-12:30 (P19) Application of genetic algorithms to determine cell traction field Z. Yang, J.-S. Lin, J.H.-C. Wang. MechanoBiology Laboratory, Departments of Orthopedic Surgery, and Civil and Environmental Engineering, University of Pittsburgh, PA, USA Cell traction force plays an important role in many biological processes. Several traction force microscopy methods (TFM) have been developed to determine traction forces of individual cells on a thin, elastic polyacrylamide gel embedded with fluorescent beads. The gel in these methods is approximated as an infinite half-space in order to obtain cell traction forces. Here, we present an alternative TFM procedure to determine cell traction forces based on the genetic algo- rithms (GA). The objective function for the GA consists of three components: 1) minimization of the predicted versus measured substrate displacements; 2) the self-equilibrium of the traction forces that exert on the substrate by cells; and 3) the smoothness of the cell traction forces. The effectiveness of the GA approach in determining cell traction forces was demonstrated with numerical tests and through experimental validation. We show that the GA is computationally efficient and may serve as an effective alternative to existing TFM methods for a speedy quantification of cell traction forces. 6838 Tu, 14:00-14:15 (P22) Long range correlation of force fluctuation in an endothelial cell layer D.P. Zitterbart, C.T. Mierke, B. Fabry. Center for Medical Physics and Technology, Biophysics Group, University Erlangen, Germany Efficient wound healing requires that mechanical processes such as cell spreading, contraction and crawling are coordinated within a cell population across large distances. Here we monitored the coordination of cell movement and force fluctuation in an intact monolayer of human vascular endothelial cells. Cells from passage one or two were plated onto collagen coated elastic polyacrylamide hydrogels (E =3000Pa) and grown up to confluency. Images (300 500~m) of ongoing cell shape changes and associated changes in gel deformation were recorded every minute for a total of 30 min. For each image pair taken 1 min apart, gel embedded markers, and associated changes in cell traction forces were computed using the method of Butler et al. Am J Physiol, 2002. Thus we obtained a spatial map of the evolution of traction forces. We then computed correlation in time of the force fluctuation between any two spatial coordinates. As expected we observed correlated force fluctuations inside single cells and between neighbouring cells. Unexpectedly, however, we also found highly and significantly (p < 0.0001 ) correlated force fluctuations over long distances ranging up to 300 ~m. These findings demonstrate that changes in shape and tractions of cells within a confluent monolayer do not occur at random but are highly coordinated. We speculate that mechanochemical signal transduction processes are responsible for the behaviour and are currently testing for correlated force fluctuations after stimulation with histamine. 6873 Tu, 14:15-14:30 (P22) Force generated by the isolated smooth muscle cell in a three- dimensional collagen gel T.M. Koch 1, T.T.B. Nguyen 2, J. Pauli 1, C. Mierke 1, J.P. Butler 2, J.J. Fredberg 2, B. Fabry 1. 1Biophysics group, University of Erlangen-Nuremberg, Germany, 2physiology Program, Harvard School of Public Health, Boston, MA, USA The airway smooth muscle cell can migrate through an extracellular matrix. During migration the cell exerts forces upon the gel. We measured these forces in a three-dimensional collagen gel by extending methods for two-dimensional traction microscopy to three dimensions. Collagen gels with a Young's modulus of 75 Pa provided an elastic matrix for cells to invade. Human airway smooth muscle cells were plated on the gel surface and then allowed to invade into the gel for two days. Invaded cells assumed an elongated morphology and contracted the gels predominantly along their primary axis. To quantity gel deformation, fluorescent beads of 1 ~m diameter were embedded in the gels. The x,y,z-position of each bead was determined with a center-of-mass algorithm applied to microscopic images taken at equidistant focal depths in the gel. To obtain the undeformed state of the gels, we disrupted the actin cytoskeleton by treating the cells with Cytochalasin-D (4 ~M). Changes in bead positions between these states characterized the deformation field induced by the cells. We then fitted to the measured deformation field of the gel the deformation field that would be created by an isolated force dipole, i.e., two equal and opposite co-linear forces F applied at infinitesimal separation ~, corresponding to a dipole moment ~.E The dipole moment generated during cell invasion was on the order of 10 -12 Nm, which is comparable to that generated by maximally contracted smooth muscle cells in two-dimensional culture. Extension of traction microscopy into three dimensions allows study of physical forces generated by a cell in a natural geometry and with defined matrix properties.
Transcript of Role of intercellular adherens junctions in endothelial cell cooperative motion
$234 Journal of Biomechanics 2006, Vol. 39 (Suppl 1)
6410 Tu, 11:45-12:00 (P19) Act in dynamics and optical gu idance of neuronal growth cones D. Koch, T. Betz, A. Ehrlicher, B. Stuhrmann, M. GSgler, J. K~s. Institute for Soft Matter Physics, University of Leipzig, Germany
The understanding of the motility and the dynamics of the growth cone, the highly motile structure at the tip of a growing neurite, is a fundamental basis of unraveling the development and function of the neuronal system. In a noisy environment full of different chemical and mechanical cues the growth cone is able to successfully wire up a highly complex neuronal network. The dynamics and constant reorganisation of the growth cone structure play a crucial role in its ability to steer the advancing tip of the neurite. However, the detailed molecular mechanisms underlying growth cone motility still need to be resolved. We have shown that optical forces induced by a highly focused infrared laser beam influence the motility of a growth cone by biasing the polymerization- driven intracellular machinery. In actively extending growth cones, a laser spot placed at specific areas of the neurite's leading edge affects the direction taken by a growth cone. In an optical tweezers setup, simultaneous phase contrast and fluorescence imaging, real-time shape detection, and automated laser irradiation are combined to control growth cone motility. We have focused on the actin dynamics in the growth cone to elucidate the mechanisms underlying optical neuron guidance. At the front edge of the growth cone actin is polymerized into filaments building complex bundles and network structures that are transported back towards the center in a process termed 'retrograde flow'. We've developed a new method based on high resolution edge detection in combination with a cross-correlation algorithm to extract and quantity these actin dynamics from fluorescence observations with high spatial and temporal resolution. These novel techniques allow the investigation and manipulation of a natural biological process, the cytoskeleton driven morphological changes in a growth cone, with potential applications in the formation of neuronal networks and in understanding growth cone motility.
6500 Tu, 12:00-12:15 (P19) Role of intercellular adherens junctions in endothelial cell cooperative motion A.A. Rabodzey 1 , C.E. Semino 2, R.D. Kamm 2, C.F. Dewey Jr. 1 . 1Hatsopoulos Microfluids Laboratory MIT, Cambridge, USA, 2Biological Engineering Division, MIT, Cambridge, USA
Intercellular junctions are important structural elements that communicate signals between cells. Adherens junction are the type of junctions responsible for the physical force of interactions; they are the most abundant intercellular structures. The major junctions-forming protein is VE-cadherin. VE-cadherin is involved in flow-induced mechanotransduction and may plays a role in the intercellular organization during crawling and vessel formation. In our previous studies we looked at the role of the adherens junctions in mechanotransduction and cell migration [1] and observed that VE-cadherin is in part responsible for early mechanotransduction in the endothelial cells [2]. In this study we used siRNA to knock down the VE-cadherin and study behavior of the adherens junctions-deficient cells. Full knockdown confirmed by western blot and immunofluorescence is achieved following 24 hr of transfection. The major advantage of using knockdowns compared to full knockouts is that we are able to use human cells whose response is well established and we achieve full suppression of VE-cadherin unlike experiments with blocking antibodies. Our current studies are directed to our previous hypothesis that VE-cadherin is an important player in cell motility, early mechanotransduction and angiogenesis. We find that average speed of HUVEC motion increases by -25% in the VE- cadherin null cells compared to normal cells. VE-cad deficient cells show faster wound closure. VE-cadherin deficient confluent cells show behavior much closer to that of subconfluent cells than confluent cells. An in vitro assay to analyze vessel formation [3] and understand the effects of adherens junction disruption on angiogenesis will also be reported. [Unlike the assay described in Dejana et al. Blood 2002, cells are migrating from the surface and are not sandwiched in between two gels].
References [1] Osborn EA, Rabodzey A, Dewey Jr CF, Hartwig JH. Am J Physiol Cell Physiol
2006 Feb; 290(2): C444-52. [2] Rabodzey AA, Yao Y, Hartwig JH, Dewey Jr CF. Endothelial cells early response
to flow may be mediated by VE-cadherins Submitted. [3] Semino CE, Kamm RD, Lauffenburger DA. Exp Cell Res. 2006 Feb 1; 312(3):
289-98.
Oral Presentations
5080 Tu, 12:15-12:30 (P19) Appl icat ion of genetic algorithms to determine cell traction f ield
Z. Yang, J.-S. Lin, J.H.-C. Wang. MechanoBiology Laboratory, Departments of Orthopedic Surgery, and Civil and Environmental Engineering, University of Pittsburgh, PA, USA
Cell traction force plays an important role in many biological processes. Several traction force microscopy methods (TFM) have been developed to determine traction forces of individual cells on a thin, elastic polyacrylamide gel embedded with fluorescent beads. The gel in these methods is approximated as an infinite half-space in order to obtain cell traction forces. Here, we present an alternative TFM procedure to determine cell traction forces based on the genetic algo- rithms (GA). The objective function for the GA consists of three components: 1) minimization of the predicted versus measured substrate displacements; 2) the self-equilibrium of the traction forces that exert on the substrate by cells; and 3) the smoothness of the cell traction forces. The effectiveness of the GA approach in determining cell traction forces was demonstrated with numerical tests and through experimental validation. We show that the GA is computationally efficient and may serve as an effective alternative to existing TFM methods for a speedy quantification of cell traction forces.
6838 Tu, 14:00-14:15 (P22) Long range correlation of force fluctuation in an endothelial cell layer D.P. Zitterbart, C.T. Mierke, B. Fabry. Center for Medical Physics and Technology, Biophysics Group, University Erlangen, Germany
Efficient wound healing requires that mechanical processes such as cell spreading, contraction and crawling are coordinated within a cell population across large distances. Here we monitored the coordination of cell movement and force fluctuation in an intact monolayer of human vascular endothelial cells. Cells from passage one or two were plated onto collagen coated elastic polyacrylamide hydrogels (E =3000Pa) and grown up to confluency. Images (300 500~m) of ongoing cell shape changes and associated changes in gel deformation were recorded every minute for a total of 30 min. For each image pair taken 1 min apart, gel embedded markers, and associated changes in cell traction forces were computed using the method of Butler et al. Am J Physiol, 2002. Thus we obtained a spatial map of the evolution of traction forces. We then computed correlation in time of the force fluctuation between any two spatial coordinates. As expected we observed correlated force fluctuations inside single cells and between neighbouring cells. Unexpectedly, however, we also found highly and significantly (p < 0.0001 ) correlated force fluctuations over long distances ranging up to 300 ~m. These findings demonstrate that changes in shape and tractions of cells within a confluent monolayer do not occur at random but are highly coordinated. We speculate that mechanochemical signal transduction processes are responsible for the behaviour and are currently testing for correlated force fluctuations after stimulation with histamine.
6873 Tu, 14:15-14:30 (P22) Force generated by the isolated smooth muscle cell in a three- dimensional col lagen gel
T.M. Koch 1 , T.T.B. Nguyen 2, J. Pauli 1 , C. Mierke 1 , J.P. Butler 2, J.J. Fredberg 2, B. Fabry 1 . 1Biophysics group, University of Erlangen-Nuremberg, Germany, 2physiology Program, Harvard School of Public Health, Boston, MA, USA
The airway smooth muscle cell can migrate through an extracellular matrix. During migration the cell exerts forces upon the gel. We measured these forces in a three-dimensional collagen gel by extending methods for two-dimensional traction microscopy to three dimensions. Collagen gels with a Young's modulus of 75 Pa provided an elastic matrix for cells to invade. Human airway smooth muscle cells were plated on the gel surface and then allowed to invade into the gel for two days. Invaded cells assumed an elongated morphology and contracted the gels predominantly along their primary axis. To quantity gel deformation, fluorescent beads of 1 ~m diameter were embedded in the gels. The x,y,z-position of each bead was determined with a center-of-mass algorithm applied to microscopic images taken at equidistant focal depths in the gel. To obtain the undeformed state of the gels, we disrupted the actin cytoskeleton by treating the cells with Cytochalasin-D (4 ~M). Changes in bead positions between these states characterized the deformation field induced by the cells. We then fitted to the measured deformation field of the gel the deformation field that would be created by an isolated force dipole, i.e., two equal and opposite co-linear forces F applied at infinitesimal separation ~, corresponding to a dipole moment ~.E The dipole moment generated during cell invasion was on the order of 10 -12 Nm, which is comparable to that generated by maximally contracted smooth muscle cells in two-dimensional culture. Extension of traction microscopy into three dimensions allows study of physical forces generated by a cell in a natural geometry and with defined matrix properties.
