Volume 7 issue 9 September 2006
Homologues of Oxysterol-Binding Proteins Affect Cdc42p- and Rho1p-Mediated Cell Polarization in S. cerevisiae
Keith G. Kozminski, Gabriel Alfaro, Shubha Dighe, and Christopher T. Beh

Supplemental Figures

Supplemental Figure S1: Quantitative analysis of OSH-dependent polarization of proteins involved in actin organization and/or assembly (complementary to Fig. 3). (Top) Polarized localization of Bni1p-GFP, GFP-Spa2p, and GFP-Arc15p to either the presumptive bud site in unbudded cells, the bud or bud tip in small budded cells, or the bud neck in large budded cells was determined in wild-type (SEY6210), oshΔ OSH4 (CBY924), and oshΔ osh4-1 (CBY926) cells at 37°C. The number of cells counted for each strain ranged from 85 to 126 for the Bni1p-GFP localization; from 62 to 132 for the GFP-Spa2p localization; and from 220 to 429 for the GFP-Arc15p localization. (Bottom) Polarized localization of GFP-Bud6p in unbudded, small, and large budded cells at 37°C (n > 72 for each morphological class counted).

Figure S1 (.tif)

Supplemental Figure S2: Quantitative analysis of septin polarization and assembly in OSH mutants (complementary to Fig. 4). (Left) Septin (GFP-Cdc3p) polarization exclusively to the presumptive bud site was scored in unbudded cells and to the bud neck in small and large budded cells in wild-type (SEY6210), oshΔ OSH4 (CBY924), and oshΔ osh4-1 (CBY926) cells at 37°C. The appearance of cortical septin patches at locations other than observed in wild-type cells was defined as unpolarized. (Right) Septin ring assembly in wild-type, oshΔ OSH4, and oshΔ osh4-1 budded cells at 37°C. Defective septin rings included those with fragmented morphology, asymmetric localization around the bud neck, and/or irregular accumulations of septins within the rings. Cells counted for both analyses ranged from 260 to 300.

Figure S2 (.tif)

Supplemental Figure S3: Quantitative analysis of Rho1p, GFP-Sec3p, and GFP-Sec4p polarized localization in OSH mutants in wild-type (SEY6210), oshΔ OSH4 (CBY925), and oshΔ osh4-1 (CBY926) cells (complementary to Fig. 7A). Rho1p polarized localization to the presumptive bud site in unbudded cells, to sites of polarized growth in small and medium budded cells, and to the bud neck in large budded cells after a shift from 23 to 37°C for 4 h (top right) or growth at 23°C (top left). GFP-Sec3p (bottom left) and GFP-Sec4p (bottom right) polarized localization to the presumptive bud site in unbudded cells, to the bud tip in small budded cells, and to the bud neck in large budded cells, after a shift from 23 to 37°C for 4 h. The number of cells counted for each morphological class ranged from 200 to 308 for the Rho1p localization; from 86 to 137 for the GFP-Sec3p localization; and from 178 to 635 for the GFP-Sec4p. For GFP-Sec4p, a cell in which localization was observed in the bud but not restricted to bud tip was not counted as polarized.

Figure S3 (.tif)

Supplemental Figure S4: Cellular defects resulting from perturbations in OSH and MSB gene dosage. (A) Unbudded cells accumulated in oshΔ osh4-1 transformants expressing P^GAL-MSB3.Differential-interference contrast (DIC) microscopy images (top panels) and corresponding DAPI-stained nuclei (bottom panels) of wild-type (SEY6210), oshΔ osh4-1 (CBY924), and oshΔ OSH4 (CBY926) cells transformed with the P^GAL-MSB3 (pCB368) plasmid after log-phase cultures were transferred from 2% raffinose to 2% galactose-containing medium at 30°C for 4 h. The corresponding percentages of unbudded, small-budded, and large-budded cells are presented in the left graph. Cells counted for each strain in this analysis ranged from 348 to 755. (B) Unbudded cell accumulation in msb3Δ msb4Δ cells transformed with multicopy OSH4 . DIC (top panels) and corresponding DAPI staining (bottom panels) for msb3Δ msb4Δ (CBY1981) log-phase cells transformed with either multicopy OSH2 (pCB239) , OSH4 (pCB241), or the vector control (pRS426) after growth in synthetic medium at 30°C. Scale bar for all panels is 10 µm. The corresponding percentages of budded and unbudded cells are presented in the right graph. Cells counted for each strain ranged from 189 to 243. In wild-type (BY4742) cells transformed with the multicopy OSH2 , OSH4, or the vector alone, no significant differences in the proportion of unbudded and budded were observed (data not shown).

Figure S4 (.tif)