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Benya Lab Research Videos
All videos are downloaded as QuickTime files, each of which ranged between 1 and 10 MB in size. QuickTime can be downloaded for free from Apple.
293 Cell Videos
This cell line is a human fetal kidney cell line that we show natively expresses functional GRP receptors that are activated in an autocrine manner by natively secreted GRP. This results in activation of Focal Adhesion Kinase (FAK). These cell lines, furthermore, have been modified to express the dominant negative regulator of FAK, FRNK, under control of a doxycycline-sensitive inducible promoter. Cell images were photographed using OpenLab software (Improvision, Coventry, UK) every 5 minutes, and the images converted into a QuickTime file at native size (640x480) and using the "Video" codec. Editing was performed using Final Cut Pro 3.0 (Apple) and the final movie exported as QuickTime using the JPEG A codec at reduced size (320x240) and shown at 10 frames/sec.
Cell Motility
Confluent cells were selectively removed (i.e., wounding) and the ability to fill in the resulting gap assessed. This is a model for tissue remodeling.
Motility- Video 1: GRP activation of FAK promotes motion
Motility of 293 cells modified to contain the doxycycline-sensitive inducible promoter for FRNK under control conditions (i.e., with natively secreted GRP binding the GRP-R in an autocrine manner thereby causing FAK to be phosphorylated at Y397). Near-confluent cells were placed in serum-free media for 24 hrs, wounded using a sterile razor blade, and monitored for another 24 hr. Appreciate the rapid movement of cells to fill-in the wound guided by lamellipodia.
Motility - Video 2: Inhibiting FAK retards motion
Motility of the same cells as described above post wounding, but now in the presence of exogenous doxycycline. This activates the inducible promoter so that FRNK is present and serves as a dominant negative regulator of FAK. As compared to video 1, there is little to no motility of these cells so treated, without lamellipodia formation being affected. Culturing with a GRP-R antagonist gives the same result as incubating with doxycycline, namely, to hinder cell motility.
Cell Distensibility
Cell distensibility and blebbing are markers of greater malignancy insofar as they are biophysical markers of a cell's ability to transmigrate through a tight space, such as exists in the endothelial gaps. Movies were recorded on VHS, converted to QuickTime using the Sorensen Codec, edited in FCP, and exported using the Video codec at native size (240x180) and shown at 10 fps.
Distention- Video 3: GRP activation of FAK decreases destensibility
Under control conditions, 293 cells modified to contain the doxycycline-sensitive inducible promoter for FRNK are in serum- and doxycycline-free media so that the GRP-R is activated in an autocrine manner causing FAK to be phosphorylated at Y397. This results in marked inhibition of cell distensibility. To do this, cells were placed in serum-free media for 24 hr, disaggregated, and placed in solution for 30 min prior to attempted aspiration.
Distention- Video 4: Inhibiting FAK promotes distensibility and blebbing
Distention of the same cells as described under video 3 but now in the presence of exogenous doxycycline. This activates the inducible promoter so that FRNK is present and serves as a dominant negative regulator of FAK. As compared to video 3, cells are markedly more distensible, as well as undergo blebbing.
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