Our laboratory is interested in the problem of how cells move; this research is relevant to the aberrant cell motility exhibited in metastasis and to transendothelial cell migration involved in aspects of the inflammatory response. The intellectual challenge is to relate global descriptions of cell movement and force production to molecular mechanisms. We have completed a kinematic description accounting for how locomoting fish scale keratocytes maintain constant shape and speed. This model accounts for not only dynamic morphological behavior but also the behavior of the cytoskeletal meshwork and cell surface receptors. We have also developed the first quantitative assay for the strength and pattern of the traction forces exerted by moving cells and shown how the traction pattern exhibited by keratocytes can be explained. We have recently studied the adhesive contacts the motile cell makes to the substratum and how intracellular calcium is involved in traction force production by regulating cell contractility and/or adhesion. Ongoing work involves locally perturbing cell locomotion using single cell photomanipulative techniques that are operative on the micron distance scale and 100s of ms time scale as a complement to genetic manipulation. Such techniques include chromophore assisted laser inactivation [CALI] to selectively inhibit the function of molecules responsible for cell migration and adhesion and photoactivation to quickly increase the concentration of proteins or peptides that regulate the actin cytoskeleton or signal transduction pathways important for migration. Where possible our data will be used to check the predictive power of quantitative in silico models of cell locomotion developed by our theoretical collaborators.
Our efforts in cell migration have resulted in the laboratory participating in the NIH Cell Migration Consortium within the Imaging and Photomanipulation Initiative. Papers describing our studies have been published in Nature, Nature Cell Biology, J. Cell Biol., J. Biol. Chem., Cell Motility and the Cytoskeleton and Biophys. J.
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Gliding Fish Keratocyte on Glass Substratum |
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The
Chromophore-Assisted Laser
Inactivation (CALI) technique has been used to inactivate key proteins
in in vitro systems and in
living cells. The main advantage of CALI over other protein inactivation
techniques is the ability to influence the activity of specific proteins
at precisely defined times and in targeted regions of the cell. Here, we
show that laser irradiation of EGFP-a-actinin
resulted in stress fiber detachment from focal adhesions. This stress
fibers detachment from FA was observed only after focused laser
illumination of the fused EGFP-a-actinin
molecule, and laser light alone did not cause detachment of stress fibers
in normal cells.
MOVIE 1.
Typical example of "slow" detachment of stress fiber from focal
adhesion after CALI irradiation (100 msec irradiation time, 500 mW laser
beam power, 2.2mm
diameter of laser beam). The stress fibers are visualized as bright green
spots of expressed EGFP-a-actinin.
The laser irradiation of focal adhesion produced a heavy bleaching
of irradiated EGFP and as a result of this irradiation the stress fiber
detached from focal adhesion and retracted. The movement of the detached
stress fiber was initially slow, but accelerated with time. Neighboring
focal adhesions also showed some level of photobleaching but they did not
detach. MOVIE 2. An example of "fast" detachment of irradiated stress fiber from focal adhesion. . The speed of stress fiber retraction was diminishing as time of the measurement progressed to the standstill at the end of the experiment. Z. Rajfur, P. Roy, C. Otey, L. Romer, K. Jacobson. "Dissecting the link between stress fibres and focal adhesions by CALl with EGFP fusion proteins”. Nature Cell Biology, 4:286-293, 2002. P. Roy, Z. Rajfur, P. Pomorski, K. Jacobson. "Microscope-based techniques to study cell adhesion and migration". Nature Cell Biology, 4:E91-E96, 2002.
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Time-lapse video microscopy of a locomoting keratocyte following UV
laser-mediated release of caged Tb4 at the wing showing dramatic turning of the cell towards the direction of
photoactivation. Note the pivoting of the irradiated zone to the substrate as a result of photorelease of
Tb4. About 8 min after photoactivation of Tb4, normal locomotion resumed.The pivoting of the cell to the substrate results from
Tb4-induced down-regulation of cell contractility at the irradiated wing which renders the cell unable to exert retractile force. Based on
various experiments, we propose that the asymmetric down-regulation in contractility is caused by local depolymerization of actin filaments due
to sequestration of G-actin by photoreleased Tb4. P. Roy, Z. Rajfur, D. Jones, G. Marriott, L. Loew, K. Jacobson. "Local photorelease of caged thymosin b4 in locomoting keratocytes causes cell turning". Journal of Cell Biology, 153:1035-1047, 2001. |
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Before uncaging NBT-II cells move on collagen matrix (18 hours after reseeding, 1 hour recovery after microinjection). After two 200 msec consecutive laser irradiations (green dots indicate irradiation spots) lamellar protrusion was temporarily inhibited in the cells loaded with caged peptide (red lines indicate the initial position of the cell lamellipodium). |
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After uncaging |
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Phase contrast, video image of a fish epidermal keratocyte (cell outlined in black) locomoting towards the right on a bead-decorated, flexible silicone rubber substratum. Superimposed over the cell is the traction map (black arrows) generated for this experiment, which is based on the analysis of bead displacements in the substratum. Tim Oliver, Micah Dembo, and Ken Jacobson; Traction Forces in Locomoting Cells; Cell Motility and the Cytoskeleton 31:225-240, 1995. |
 Ishihara A, et al. Photoactivation of caged compounds in single, living cells: An application to the study of cell locomotion; Biotechniques, 23:268-274, 1997. |
Photoactivation of caged calcium ionosphere in fish
keratocytes. A calcium indicator, Calcium Green Dextran, was introduced into fish
keratocytes by bead loading. Cell shape indicated by the white outline positioned from
examination of original video images. The fluorescence level is presented in pseudocolor
as shown on the bar in Panel D. (A) Fluorescence reports resting level of [Ca2+]i
before photoactivation. (B) Caged calcium ionosphere was added to the cell medium
and the keratocyte exposed to the UV beam (ca. 10mm in
diameter, shown as a white circle) from the He-Cd laser for 0.1s. Immediately, a large
increase in fluorescence level corresponding to increased [Ca2+]i was
observed within the cell. (C and D) Gradually the fluorescence decreased, signifying a
return of [Ca2+]i to resting levels. C and D were at
22 and 60s, respectively, after the UV pulse. During this period, the
trailing part of the cell retracted (direction of retraction shown with an
arrow), resulting in a more rounded cell shape. Scale bar=10mm.
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