Figure 4.—

Independence of septin and Rho-GEF localization to the division site. (A) Relationships among Rho-GEFs from S. pombe (http://www.genedb.org/genedb/pombe/) and S. cerevisiae (http://www.yeastgenome.org/), as well as a Rho-GEF from Drosophila (Pebble) and one from humans (ECT2) that have also been implicated in cytokinesis (Miki et al. 1993; Prokopenko et al. 1999; Tatsumoto et al. 1999; O'Keefe et al. 2001; Giansanti et al. 2004; Shandala et al. 2004). Clustal W was used to align the Rho-GEF domains and then derive a phylogenetic tree based on this alignment. (B and C) Localization and dynamic behavior of Rgf1 and Rgf3 at the division site in relation to the septins and other cell-division markers in wild-type cells. Strains JW1113 (spn1-mEGFP sad1-mRFP1), JW1124 (rgf1-GFP sad1-mRFP1), and JW1131 (rgf3-mEGFP sad1-mRFP1) were filmed in EMM5S at 25°. (B) Kymographs constructed using Image J and the movements of the spindle-pole bodies (SPBs), as marked by Sad1-mRFP1, to allow alignment at the time of SPB separation (vertical line). For the SPB, a 13.7-μm slit parallel to the long axis of the cell was used; for the other proteins, a 4.1-μm slit across the midplane of the cell was used. (C) Timeline showing the initial appearance of Spn1 (n = 8 cells) and Rgf1 (n = 8) at the division site. SPB separation is defined as time zero, and the mean time of appearance of each protein (±1 standard deviation) is plotted. The beginning of septation was scored by DIC. The data for Cdc4 are from Wu et al. (2003). (D and E) Tests of possible septin dependence of Rho-GEF localization. (D) Strains JW1124 (rgf1-GFP), JW1139 (rgf1-GFP spn1Δ), JW1131 (rgf3-mEGFP), and JW1128 (rgf3-GFP spn1Δ) were grown in EMM5S (JW1124 and JW1131) or YE5S (JW1139 and JW1128) medium at 25° and examined by DIC and/or fluorescence microscopy. (E) Strain JW1128 was examined by time-lapse microscopy. Selected DIC and GFP images (elapsed times given in minutes) are shown from a series recorded at 1-min intervals. The entire series can be viewed in File S2. (F) Cell morphology and septin localization in cells overexpressing Rgf1 or Rgf3. Strains JW1123 (spn1-mEGFP 3nmt1-rgf1) and JW1122 (spn1-mEGFP 3nmt1-rgf3) were grown under inducing conditions (EMM5S medium) for 24 hr at 25°; paired DIC and fluorescence images are shown. Arrowheads indicate a cell with a region of thickened cell wall. Bars (B and D–F), 10 μm.