Figure 1.
Effect of Y-27632 treatment on FA reorganization in HeLa JW cells.
(A) Localization of GFP-tagged VASP, zyxin, paxillin, vinculin, talin, kindlin-2, ILK and FAK in untreated cells (control) or in the cells treated with 10 µM Y-27632 for 10, 30 or 90 min (shown are snapshots from time-lapse movies). White and black arrowheads point to peripheral and central adhesions, respectively.
(B) FA lifespan in three different cells tagged by each of the tested proteins prior to and during Y-27632 treatment, as tracked by time-lapse video microscopy. Each line marks the lifespan of an individual adhesion site, from its formation until its complete disassembly. Individual cells are marked by asterisks on the Y axes. The vertical dashed line marks the beginning of the Y-27632 treatment. Peripheral and central FAs were analyzed separately. Note the decay of existing FAs and formation of novel FAs during Y-27632 treatment.
(C) Western blots showing MLC and pMLC in untreated cells, or in cells treated with 10 µM Y-27632 for 10, 30 and 90 min Tubulin was used here as a loading control.
Figure 2.
Reversibility of the effect of Y-27632 on cell morphology.
Images of Y-27632-treated HeLa JW cells were taken prior to addition of the drug, after 30 min incubation with 10 µM Y-27632, and 2 h after the drug was washed off. The effect of Y-27632 on cell morphology was reversible, as cells regained normal morphology within 2 hours of reagent withdrawal.
Figure 3.
Differential dissociation rates of adhesome components in cells treated with Y-27632.
(A) FA lifespan (expressed as the relative number of surviving FAs) and average normalized fluorescence intensity for each protein, in peripheral and central FAs separately, as tracked by time-lapse video microsopy for a hundred individual FAs. τ values were calculated by fitting the individual decay profiles to a single-exponential function. Note that the order of exit of the different molecules is the same in all graphs, with zyxin and VASP as the first to leave and kindlin 2 as the last. (B) Based on the same dataset shown in A, the differential effect of Y-27632 on the lifespan of the FA proteins tested, in peripheral FAs, is shown for each protein, separately. FA lifespan for each protein is expressed as the percentage of total FA number over time. Untreated cells are shown as white columns; cells treated with Y27632 as black columns.
Figure 4.
Cryo-electron tomography of cells incubated with Y-27632.
REF52 cells expressing YFP-paxillin growing on EM grids, untreated (A) or treated for 3 (B), 10 (C) or 30 (D) minutes with Y-27632, were analyzed by correlative (fluorescence-cryo-electron tomography) microscopy. Individual FAs, identified by light microscopy, were subjected to cryo-ET and 3D image reconstruction. The major axis of each FA is indicated by a white arrow; some of the more prominent filaments are highlighted by a thin red line. Ten nm-thick slices through the cryo-tomograms of representative FAs indicated a major reduction in actin alignment, already observed following 3 min treatment with the drug (compare B to A: The black arrow, in B, points to an example of misaligned filaments, commonly seen after Y-27632 treatment). Upon longer treatment with Y-27632, progressive misalignment of actin filaments is apparent, accompanied by a marked reduction in filament density. The scale bar in B represents 500 nm.
Figure 5.
Peripheral FAs in Y-27632 treated cells differ from control FAs in molecular composition and lifespan.
(A) Comparison of zyxin and vinculin fluorescence intensity for FAs in untreated cells, and peripheral FAs in Y-27632 treated cells. Cells prior to and following 60 min of Y-27632 treatment were double-labeled for zyxin (left) and vinculin (center). Right: The fluorescence ratio image (zyxin/vinculin) in a logarithmic, blue-to-red spectrum scale, representing the ratio value. (B) Comparison of ILK and actin localization in FAs of untreated cells, and in peripheral FAs of Y-27632 treated cells. Two-color TIRF microscopy of the cells prior to and following 60 min of Y-27632 treatment was used to assess for ILK (left) and actin staining (center). Right: A merged image (ILK in red and actin in green), demonstrating enrichment of actin in the FA sites. (C) Normalized vinculin, zyxin, paxillin, kindlin-2 and ILK fluorescence intensities in peripheral FAs following 60 min of Y-27632 treatment, expressed as a percentage of intensity of the same protein in FAs of untreated cells. Fluorescence intensity measurements were averaged over 150 individual FAs. (D) Lifespan of peripheral FAs for each protein, expressed as the percentage of total FA number over time, averaged over 150 individual FAs for each protein.
Figure 6.
Molecular composition of the novel central adhesions formed following Rho-kinase inhibition.
(A) HeLa JW cells were treated with 10 µM Y-27632 for 60 min, and double-labeled for ILK, the most prominent component of these adhesions, as well as for additional FA components. The same field is shown for two labeled proteins; circles indicate the region of interest. (B) Relative intensity of the novel central adhesions marked by the studied proteins, following 60 min of Y-27632 treatment. The relative intensity was calculated as the percentage of fluorescence intensity measured for the corresponding protein in the untreated FAs. (C) Comparison of ILK and actin localization in FAs of untreated cells, and in the central adhesions of Y-27632 treated cells. Two-color TIRF microscopy of the cells prior to (left panel) and following 60 min of Y-27632 treatment (right panel) was used to assess the localization of ILK and actin. No correlation was found between the densities of these two proteins in the central adhesions (see inserts).
Figure 7.
Dynamics of FA plaque proteins following Y-27632 treatment.
(A) Normalized averaged FRAP curves (n=20-30) of FA plaque proteins in untreated and Y-27632 treated cells. For some proteins, no major changes were observed in the FRAP curves in untreated vs. treated cells (talin, FAK, VASP, ILK), whereas other proteins displayed higher (vinculin, kindlin-2) or lower (paxillin, zyxin) FRAP rates following Y-27632 treatment. (B) Kinetic parameters of FA proteins as derived from FRAP measurements within the first 9 min of Y-27632 treatment. The half-time to full recovery (T1/2) and mobile fraction (Rf) values of the FRAP curves prior to and following Y-27632 treatment were not indicative of the overall FA disassembly state. However, the differences between the kon and koff values derived from fitting the FRAP data to the full diffusion-exchange model represent the disassembly rate of FAs for each protein, following Y-27632 treatment. (C) Theoretical FA disassembly curves calculated from the differences in the expected disassembly rates of tested proteins from the FRAP data. (D) Actual disassembly curves measured directly from time-lapse movies, following treatment of HeLa JW cells with Y-27632. Note the remarkable similarity between the calculated and measured values.