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Fig. 2. Regulation of paxillin and talinA adhesion sites is impaired in frmA– cells. (A) TIRF images showing GFP fused paxillin (top panel) and talinA (bottom panel) localisation in wild-type (left panels) and frmA– cells (right panels). (B) The number of paxillin and talinA spots observed per cell. TIRF images of wild-type and frmA– cells expressing paxillin or talinA fused to GFP were captured 100 seconds apart over 600 seconds and the average number of paxillin- and talinA-rich spots determined. 10 or more cells were analysed for each strain in total over three separate occasions and the average ± s.e.m. is shown. (C) Using TIRF microscopy, the duration of paxillin-rich spots was followed by measuring the fluorescence intensity (Image J software) of an area where a spot would form. The fluorescence intensity values were plotted against time for spots in frmA– cells (various coloured lines) and a typical wild-type cell (black line). More than 10 cells from each strain were analysed in total over three separate occasions. (D) TIRF images of a wild-type cell expressing paxillin fused to GFP (green) and talinA fused to RFP (red), with the merged image on the right. (E) Graphical representation of the appearance and disappearance of a paxillin (green) and talinA (red) spot over time, observed using TIRF and quantified using Image J software. (F) Sequential and merged TIRF images of the appearance of talinA (red) followed by paxillin (green) at an adhesion site. White arrow highlights the spot in question.
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