Figure 5
The cellular distribution of F-actin in control transfectants (neo) differs from that of transfectants expressing ICAM-2 WT. A) Control neo transfectants harbored transverse actin fibers. ICAM-2 WT induced juxtamembrane actin fiber distribution. ICAM-2 gsv expression did not induce a juxtamembrane localization of actin fibers. Each transfectant was plated at ~60% confluence and maintained under standard conditions at 37oC to allow the cells to adhere to chamber slides. Approximately 18 hours after plating, cells were fixed with 3.7% paraformaldehyde and incubated with FITC-conjugated phalloidin. Actin fibers were then visualized using confocal fluorescence microscopy by standard methods. Inset photomicrographs were acquired at 20x magnification and using a Zeiss Axio Observer Z.1 microscope platform in conjunction with Zen 2011 Blue imaging software (Carl Zeiss) (Photomicrographs of control cell lines (neo and WT) were published previously [13]). B) The distribution of actin fibers in cells at the leading edge of migration in scratch (wound healing) assays was similar to actin fiber distribution in stationary sub-confluent cultures of each cell line. Scratch assays were performed as described for experiments depicted in Figure 4. At 72 hours “post scratch”, cells were fixed and actin fibers visualized using FITC-phalloidin staining as described above, and fluorescence photomicrographs acquired using Nikon TE2000-U microscope with X-cite 120 mercury vapor short arc in conjunction with Q-capture ProV 5.1.1.14 software (Nikon Instruments, Inc.). Care was taken to acquire images of the control neo transfectants in areas where residual gaps from the original scratch were still visible, to confirm acquisition of the leading edge of migration.