Figure 7

Filamin and mutated filamin localise to the same actin rich areas. Vegetative amoebae were viewed under a fluorescence microscope. For all but the last row, the first column contains phase contrast images, the second column contains the corresponding fields detecting green immunofluorescence using Alexa Fluor 488-conjugated anti-rabbit IgG and rabbit polyclonal antifilamin antibodies while the third column contains the corresponding field detecting actin stained with Texas Red-X phalloidin. The fourth column is an overlay of all three images and the yellow colour depicts regions where filamin and actin colocalise. Fluorescence images were obtained using an Olympus BX61TRF microscope under epifluorescence using blue light for excitation and either green or red emission filters. Images were obtained in grey scale and the colour balance adjusted post hoc to leave only the red or green channel as appropriate. As seen in column 2 filamin is found throughout the cells but is concentrated at the cell cortex. No filamin is detected in the filamin mutant HG1264. Strains expressing filaminΔ1 (which showed partial complementation of the phototaxis phenotype) and filaminΔ5 (which did not) were used as examples, as all strains expressing mutant filamins behaved in the same fashion. Column 3 illustrates that actin was found throughout the cell and was concentrated at the cell cortex. Comparison of columns 2 and 3 and examination of column 4 shows that in all strains tested filamin colocalises predominantly with actin at the cell cortex. The last row contains images of cells from the other strains tested expressing the various mutant filamins and depicts the localisation of filamin by immunofluorescence. As in all the other strains mutant filamin is concentrated at the cell cortex.