Figure 9. Pak1TE expression rescues FA distribution and dynamics at a high adhesion strength. (A) Paxillin immunofluorescence and F-actin phalloidin staining in migrating PtK1 cells plated on high FN concentration (30 µg/ml) and expressing GFP (noted FN30 control) or GFP–Pak1TE (noted FN30 Pak1). Insets show GFP expression of the constructs. White boxes in the paxillin column indicate the positions of insets for higher magnifications shown in the rightmost column. Pak1TE expression induced the formation of thick paxillin clusters (arrows). (B) An example of GFP-paxillin fluorescence time-lapse images taken in PtK1 cells plated on FN30, control, or expressing Pak1TE. The white boxes in the whole area images (left) indicate the localization of the magnified regions shown in the right panels. Elapsed time in minutes is shown. Arrowheads and arrows indicate assembling and disassembling FAs, respectively. Note that FAs in control cells were sparsely stained and assembled/disassembled slower than those in cells expressing Pak1TE. Bars (left), 5 µm. (C) Average rate constants of FA assembly and disassembly measured from 8–15 FAs per cell ± SEM, and n ≥ 6 cells per condition. *, P = 0.018 compared with control; Student’s t test. (D) Average FA lifetime measured from the initiation of a new GFP-paxillin cluster to complete disappearance (±SEM). Although FAs in control (Ct) cells never disassembled in the 30 min of imaging, some clusters of paxillin in Pak1TE-expressing cells did completely turn over during this period.
Figure 9.

Pak1TE expression rescues FA distribution and dynamics at a high adhesion strength. (A) Paxillin immunofluorescence and F-actin phalloidin staining in migrating PtK1 cells plated on high FN concentration (30 µg/ml) and expressing GFP (noted FN30 control) or GFP–Pak1TE (noted FN30 Pak1). Insets show GFP expression of the constructs. White boxes in the paxillin column indicate the positions of insets for higher magnifications shown in the rightmost column. Pak1TE expression induced the formation of thick paxillin clusters (arrows). (B) An example of GFP-paxillin fluorescence time-lapse images taken in PtK1 cells plated on FN30, control, or expressing Pak1TE. The white boxes in the whole area images (left) indicate the localization of the magnified regions shown in the right panels. Elapsed time in minutes is shown. Arrowheads and arrows indicate assembling and disassembling FAs, respectively. Note that FAs in control cells were sparsely stained and assembled/disassembled slower than those in cells expressing Pak1TE. Bars (left), 5 µm. (C) Average rate constants of FA assembly and disassembly measured from 8–15 FAs per cell ± SEM, and n ≥ 6 cells per condition. *, P = 0.018 compared with control; Student’s t test. (D) Average FA lifetime measured from the initiation of a new GFP-paxillin cluster to complete disappearance (±SEM). Although FAs in control (Ct) cells never disassembled in the 30 min of imaging, some clusters of paxillin in Pak1TE-expressing cells did completely turn over during this period.

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