CRAC-GFP translocates to the leading edge of newly elicited pseudopods in chemotactically moving cells. Cells treated with LY429033. The micropipette is located in the center of the converging cells.
Dynamics of the CRAC-GFP redistribution. A large increment in chemoattractant was applied by increasing the microinjector pressure and the cells immediately respond. As the gradient of cAMP is restored, the polarization of CRAC-GFP is re-established.
Kinetic analysis of translocation of CRAC-GFP in response to a uniform increase in chemoattractant concentration. Frames are taken every two seconds and chemoattractant was added just before cell goes out of focus.
Latrunculin A immobolized cells were exposed to a pipette in upper portion of field. The pipette was then moved around the cells and the binding sites for PH-domains move around the cell on the inner face of the membrane.
Latrunculin immobilized cell was exposed to two opposing cyclic AMP gradients (see two micropipettes). The strength of the two gradients was alternated during the video.
Polarized translocation of PH-GFP in highly polarized cells. Micropipette was located in the lower region beyond the field. Cells were rapidly exposed to a saturating dose of attractant.
Response of CRAC-GFP to an approaching wave of chemoattractant. Pipette located on the left side of the same cell as in #6 was briefly turned on for six seconds at three times during the sequence.
Response of CRAC-GFP to an approaching wave of chemoattractant. Pipette located on the right side of the cell was briefly "turned on" for six seconds at three times during the sequence.
Translocation of CRAC-GFP in cells lacking actin filament formation. Cells chemotaxing towards pipette in lower portion of field were poisoned with Latrunculin A and followed for the next several minutes.
Single Molecule Imaging by Masahiro Ueda and Toshio Yanagida
PTEN cells expressing PH-GFP. Cells were exposed to a sudden increase in cyclic AMP (white flash). Total time of video represents ~3.5 minutes of stimulation.
An Excitable Signal Integrator Couples to an Idling Cytoskeletal Oscillator to Drive Cell Migration
Computer simulation of the STEN-CON coupling model
Level set simulation of cell movement along with the activities of the slow (Ys, in green) and fast (Yf, in red) systems as well as the merged activities. Cell membrane is driven by the signal of Yf (see Extended Experimental Procedures for simulation details). The video is shown at 20 frames/s.
RBD-GFP and LimE-RFP in undulations and protrusions
Time-lapse TIRF video of a Dictyostelium cell co-expressing RBD-GFP and LimE-RFP with a rate of 2 spf. Cytoplasmic fluorescence of RBD-GFP and LimE-RFP was subtracted to highlight areas of increased intensity. Cell boundary (in gray) was derived from the cytoplasmic fluorescence of RBD-GFP. The video is shown at 7 frames/s.
Level set simulations of the three different migratory modes.
The video also shows the trajectory of the equilibrium state on the phase plane, for the three different thresholds, where the particular state shown reflects the behavior of one of the points on the cell membrane, indicated by the moving white bar on the kymograph. Scale bars represent 10 μm.
Level set simulations of the transitions between the amoeboid to fan-shaped and the fan-shaped to oscillatory states.
The transitions are simulated with a constant threshold up to the black line, after which the threshold is gradually lowered, with the red line indicating the progress in time. The transition from amoeboid to fan-shaped is demarcated by the shift in activity from a fluctuating to a constant maxima, while the transition from fan-shaped to oscillatory occurs when the maxima shifts back to a periodic cycle. Scale bars represent 10 μm.
Time-lapse confocal videos of LimE-YFP in an amoeboid (top left), fan-shaped (right), and oscillatory (bottom left) cell. Scale bars represent 5 μm. Images were acquired every 2 sec and the videos are shown at 15 frame/sec.
Time-lapse phase video of cells before and after recruiting Inp54p
Cells expressed myr-FKBP-FKBP and mCherry-FRB-Inp54p. Rapamycin was added at time 6 min. Examples of amoeboid-to-fan-shaped and amoeboid-to-oscillatory transitions were outlined, with blue, green, and red representing amoeboid, fan-shaped, and oscillatory, respectively. Scale bar represents 20 μm. Images were acquired every 12 sec and the video is shown at 20 frame/sec.
Two-dimensional simulations on a periodic grid showing waves of activity propagating outward and collapsing, with the green and red denoting the activator and inhibitor, respectively.
The bottom panel shows how the slope of the R-nullcline is gradually lowered thereby decreasing the threshold for activation.
CynA localization during micropipette assay. Time-lapse confocal movies of 4-hour stage wild-type Ax3 cell expressing tPHCynA-KikGR before and after exposure to a micropipette containing 1 μM cAMP
CynA trans-localization upon cAMP stimulation Time-lapse confocal movies of 4-hour stage wild-type Ax3 cell expressing tPHCynA-KikGR. CynA fall off the membrane upon cAMP stimulation
ttPH-CynA and LimE localizationTime-lapse confocal movies of growth stage wild-type Ax3 cell co-expressing ttPHCynA-GFP and LimE-RFP showing that CynA is at the back of the cup of the macropinosomes.
