JCS021725 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. (A) Schematic representation of the six known FERM domain containing proteins in the Dictyostelium sequencing database. (B) The FERM domains of DdFrmA are most similar to talin FERM domains. Selected human FERM domains were aligned with the FERM domains from Dictyostelium (ClustalW) and a tree diagram drawn to show the similarity between the different FERM domains to each other.

• Supplemental Figure S2 -

  Fig. S2. (A) The circularity index of wild-type and DdfrmA^- cells was determined, where 1.0 represents a perfect circle (Image J software). The circularity of >100 cells for each clone was determined over three separate days and the average ± s.e.m is shown. (B) Using TIRF microscopy the duration of DdTalinA:GFP rich spots was followed by measuring the fluorescence intensity (Image J software) of an area where a spot would form until the spot had disappeared. The fluorescence intensity values were plotted against time for spots in DdfrmA^- (red, error bar not shown to reduce complexity) and wild-type cells (black, ± s.e.m shown). More than 10 cells from each strain were analysed in total over three separate occasions. (C) Quantification of the number of DdLimE^Δcoil:GFP rich patches. TIRF images of wild-type and DdfrmA^- cells expressing DdLimE^Δcoil:GFP were captured 100 seconds apart over 600 seconds and the average number of DdLimE^Δcoil:GFP rich patches determined. 15 or more cells were analysed for each strain in total over three separate occasions and the averages ± s.e.m is shown. (D) Using TIRF microscopy the appearance and disappearance of DdLimE^Δcoil:GFP rich patches were followed by measuring the fluorescence intensity (Image J software) of an area where a patch would form. The values of the fluorescence intensities were plotted against time. 25 patches from 10 or more wild-type cells (closed diamonds) and DdfrmA^- cells (open diamonds) were analysed in total over three separate occasions and the average intensities ± s.e.m is shown.

• Movie 1 -

  Movie 1. TIRF microscopy was used to record the formation of DdPaxillinB:GFP rich spots at the cell-substrate boundary. TIRF images of non-starving wild-type cells expressing DdPaxillinB:GFP, recorded every 2 seconds for 200 seconds.

• Movie 2 -

  Movie 2. TIRF microscopy was used to record the formation of DdPaxillinB:GFP rich spots at the cell-substrate boundary. TIRF images of DdfrmA^- cells expressing DdPaxillinB:GFP, recorded every 2 seconds for 200 seconds.

• Movie 3 -

  Movie 3. TIRF microscopy was used to record the formation of DdTalinA:GFP rich spots at the cell-substrate boundary. TIRF images of non-starving wild-type cells expressing DdTalinA:GFP, recorded every 2 seconds for 200 seconds.

• Movie 4 -

  Movie 4. TIRF microscopy was used to record the formation of DdTalinA:GFP rich spots at the cell-substrate boundary. TIRF images of DdfrmA^- cells expressing DdTalinA:GFP, recorded every 2 seconds for 200 seconds.

• Movie 5 -

  Movie 5. TIRF microscopy was used to record the formation of DdPaxillinB:GFP and DdTalinA:RFP rich spots at the cell-substrate boundary. Movie from TIRF images of non-starving wild-type cells co-expressing DdPaxillinB:GFP (green) and DdTalinA:RFP (red), recorded every 2 seconds for 200 seconds, are shown.

• Movie 6 -

  Movie 6. TIRF microscopy was used to record the formation of DdLimE^Δcoil:GFP rich patches at the cell-substrate boundary. TIRF images of non-starving wild-type cells expressing DdLimE^Δcoil:GFP, recorded every 2 seconds for 200 seconds.

• Movie 7 -

  Movie 7. TIRF microscopy was used to record the formation of DdLimE^Δcoil:GFP rich patches at the cell-substrate boundary. TIRF images of DdfrmA^- cells expressing DdLimE^Δcoil:GFP, recorded every 2 seconds for 200 seconds.