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First published online 18 March 2008
doi: 10.1242/jcs.021725

Journal of Cell Science 121, 1159-1164 (2008)
Published by The Company of Biologists 2008
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The multi-FERM-domain-containing protein FrmA is required for turnover of paxillin-adhesion sites during cell migration of Dictyostelium

Hitesh Patel1, Ireen König1, Masatsune Tsujioka2, Margaret C. Frame1, Kurt I. Anderson1 and Valerie G. Brunton1,*

1 The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD, UK
2 RIKEN, Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan


Figure 1
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  		Fig. 1. Loss of FrmA impairs both cell shape and cell-substrate adhesion. (A) Single-cell-derived frmA– clones were identified by PCR of genomic DNA. PCR of wild-type genomic DNA yielded a 2 kbp product (*), whereas PCR of frmA– genomic DNA yielded a 2.4 kbp product (**). A primer set that amplified a 1 kbp fragment of eF1{alpha} was also included in the PCR reaction for FrmA as a control to show the specificity of FrmA disruption (+). At least three single-cell-derived frmA– clones were isolated and the analysis of a representative frmA– clone is shown. (B) Real-time RT-PCR was used to determine the expression of FrmA, talinB (example of a protein that is upregulated upon starvation) and eF1{alpha} (example of a protein that is not regulated by starvation) at 0 and 6 hours of starvation. Total RNA was isolated on two separate occasions and real-time RT-PCR reactions were carried out in triplicate. The average ± s.d. is shown. (C) Phase images of non-starved cells (40x objective used for all). Broad membrane protrusions (white arrows) and filopodia (yellow arrows) are indicated. (D) Cell adhesion to substrate was determined under conditions of increased shear stress. Experiments were carried out on at least four separate occasions in triplicate and the average ± s.e.m. is shown. (E) Adhesion of wild-type and frmA– cells expressing GFP, GFP:FERM(1) or GFP:FERM(2).

 

Figure 2
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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.

 

Figure 3
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  		Fig. 3. F-actin regulation is impaired by the loss of FrmA, and F-actin and FrmA colocalise. (A) Confocal images of wild-type (left) and frmA– (middle) cells fixed and stained with TRITC-phalloidin (red) and DAPI (blue) to highlight the actin cytoskeleton and the nucleus, respectively. Cross-sectional images were captured and the maximum projections shown. F-actin-rich patches were located at the cortex of frmA– cells and in particular at the cell-substrate boundary. The frmA– image closest to the cell-substrate boundary, is shown on the right with white arrows highlighting patches and yellow lines indicating the cross section being shown above and beside the layer. (B) TIRF images showing LimE{Delta}coil:GFP localisation in wild-type (left panels) and frmA– cells (right panels). (C) Confocal images of frmA–/FrmAHA cells, fixed and stained with TRITC-phalloidin and an anti-HA antibody conjugated to FITC to highlight the actin cytoskeleton and localisation of FrmAHA, respectively. Images closest to the cell-substrate boundary are shown. Specific areas (rectangles 1, 2 and 3) were further magnified and shown immediately below with arrows highlighting F-actin patches and FrmAHA colocalisation.

 

Figure 4
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  		Fig. 4. Starvation-induced development and directed cell migration is impaired in frmA– cells. (A) Transmitted light images of cells taken at 0 and 10 hours of starvation. (B) The speed of non-starved, randomly migrating cells and starved cells undergoing cell migration towards a micropipette containing 2 µM cAMP was determined and expressed as a percentage of the wild type. 15 cells were tracked (using Image J software) from each strain on three separate occasions and the average speed ± s.e.m. is shown. (C) RT-PCR of total RNA from non-starved and 6-hour-starved cells was used to determine the expression of cAR1, g{alpha}2 and FrmA. Expression of eF1{alpha} was used as a template loading control. (D) Consecutive and merged TIRF (green) and transmitted light (red) images of randomly moving non-starved cells. (E) Consecutive and merged TIRF (green) and transmitted light (red) images of 6-hour-starved cells migrating towards a micropipette containing 2 µM cAMP (white asterisks).

 

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