Volume 7 issue 9 September 2006
Dictyostelium RacH regulates endocytic vesicular trafficking and is required for localization of vacuolin
Baggavalli P. Somesh, Carola Neffgen, Miho Iijima, Peter Devreotes and Francisco Rivero

Supplementary Figure 1. Disruption of the racH gene. (A) Diagram of the Dictyostelium racH gene (blue) and its disruption by the blasticidin resistance cassette (bsr). A targeting vector was used with the blasticidin cassette flanked on the left by a 850 bp long arm homologous to the 5´-region and on the right by a 960 bp long arm homologous to the 3´-region of racH . Red arrows represent genes upstream and downstream of racH. (B) Screening for homologous recombinants was performed by Southern blotting. Genomic DNA was digested with EcoRI and the blot probed with a ³²P-labeled probe in the 5´ region of the racH gene. A shift of the the 2.4 kb wildtype band to a 3 kb band in the knockout is apparent.

Figure 1 (.jpg)

Supplementary Figure 2. Growth of RacH mutants in suspension. Cultures were inoculated at 2 x 10⁵ cells/ml and grown at 21ºC with shaking at 160 rpm. Cells were counted at the indicated time points. The RacH-WT overexpressor shows impaired growth. Data are the average of three independent experiments.

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Supplementary Figure 3. Distribution of membrane compartments in cells lacking RacH. AX2 and RacH-KO cells were fixed in cold methanol and incubated with monoclonal antibodies against the following membrane compartment markers: the Golgi marker comitin (mAb 110-340-2), the ER marker protein disulfide isomerase (PDI, mAb 221-135-1), a marker for the contractile vacuole/endolysosomal system (vatA, mAb 221-35-2) and a marker for the lysosomal compartment (a carbohydrate antigen, mAb 221-342-5). Images are confocal sections. Bar, 10 µm.

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Supplementary Figure 4. Cytokinesis of RacH mutants. (A) Fluorescence and phase contrast images of cells grown in shaking culture. Cells were allowed to sit for 20 min on coverslips, were fixed with picric acid/paraformaldehyde and nuclei were stained with the DNA-binding dye DAPI. Arrows indicate multinucleate cells. RacH-V13 and RacH-N18 cells (not shown) resembled RacH-WT. Scale bar 25 µm. (B) Quantitation of nuclei of cells grown in shaking suspension. Samples were processed as in A. For every strain nuclei of 250-300 cells were counted.

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Supplementary Figure 5. Development of cells lacking RacH. Cells were starved at a cell density of 5 x 10 ⁷ on phosphate agar plates and images were taken at the indicated time points using a stereomicroscope. RacH-KO cells show the same developmental behavior as AX2 cells. Bar, 1mm.

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Supplementary Figure 6. Mitochondrial membrane targeted RacH. GFP-RacH was targeted to the outer mitochondrial membrane by a fusion to the C-terminal transmembrane region of Miro. Cells were fixed with paraformaldehyde. In order to visualize the mitochondria, cells were incubated with MitoTracker Red prior to fixation. Actin was visualized with TRITC-phalloidin. Pictures are confocal sections. Bar, 10 µm.

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