Biochemical characterization of the septin complex. (A) PVP-WT and PVP-GST3, Western blot analyses of HSS made from wild-type cells (PVP-WT) or cells in which the endogenous CDC3 had been replaced with a GST-CDC3 fusion (PVP-GST3), probed with the PVP antibody. HSS, Coomassie stain of wild-type yeast HSS. SEP, Coomassie stain of polypeptides eluted from the antibody/protein A beads with PVP peptide in the presence of 1 M KCl (septin preparation). (B) Complexes purified by PVP antibody affinity from wild-type (WT) cells or cells in which the endogenous septin had been replaced with a GST-CDC10, GST-CDC11, or GST-CDC12 fusion. Cells were grown in SC-Leucine medium with galactose as a carbon source. Although the majority of Cdc12p changed mobility in the GST-CDC12 strain, some protein was still apparent at the normal Cdc12p mobility. This is most likely a breakdown product of the GST-Cdc12p fusion and not a distinct protein, as mass spectrometry (see Results) did not reveal another protein in the preparation at this molecular weight. (C) Hydrodynamic analyses of the septin preparation in the presence of 1 M KCl elution buffer. The upper gel shows analysis by sedimentation in an 8–40% sucrose gradient. The four identified septin polypeptides cosedimented at 9 S. The lower gel shows analysis by gel filtration chromatography. Fractions from both experiments were analyzed by SDS-PAGE and Coomassie blue staining.

Negative-stain EM of septin filaments. (A) Typical wild-type septin preparation adsorbed to the grid after peptide elution in the presence of 1 M KCl. (B) A preparation diluted 50-fold in 1 M KCl elution buffer before adsorption to the grid. Samples were diluted to facilitate filament measurement. (C) Histogram of the length measurements of septin filaments observed in preparations like that shown in A and B. (D) Long pairs of filaments typically observed after dialysis of a preparation like that seen in A, into 75 mM KCl elution buffer. (E) An example of the hairpin structures (arrow) observed on the ends of some filament pairs in low-salt preparations. (F) Paracrystalline arrays of laterally aligned filaments formed by GST-Cdc11p septin complexes after dialysis in 75 mM KCl elution buffer. Bar, 100 nm.

Characterization of septin deletion strains. (A) Differential-interference contrast (DIC) micrographs of wild-type, cdc10Δ, and cdc11Δ diploid cells grown at 22°C or 30°C in YPD to early log phase. (B and C) Samples were taken at 0, 45, 90, 135, 180, and 225 min from early log-phase cultures grown in YPD at 22°C (B) or 30°C (C) to examine the growth rates of wild-type, cdc10Δ, and cdc11Δ cells. Samples from each time point were sonicated and counted on a hemocytometer. The resultant cell concentration at each time point was divided by that at time 0 to give relative cell number, and plotted versus time; open circles, wild- type; closed circles, cdc10Δ; closed squares, cdc11Δ. Asterisks, relative cell number in the 225-min sample after treatment with zymolyase, as described in . Over 1,000 cells were counted from each sample in three separate experiments. Bar, 10 μm.

Characterization of septin complexes purified from cdc10Δ or cdc11Δ cells by PVP affinity chromatography in the presence of 1 M KCl. (A) The complex from cdc10Δ cells as analyzed by SDS-PAGE and Coomassie blue staining. (B) The same preparation visualized by negative-stain EM. (C) Histogram of length measurements from the sample shown in B. (D) SDS-PAGE analysis of the complex from cdc11Δ cells. (E) The cdc11Δ preparation as visualized by negative-stain EM. (F) Histogram of length measurements from the sample shown in E. Measurements of wild-type septin filaments (see Fig. ) are plotted on histograms (striped columns) for comparison of septin deletion complexes (solid columns). Bar, 100 nm.

Negative-stain EM and sedimentation assays on wild-type, cdc10Δ, and cdc11Δ septin complexes in the presence of high- or low-salt buffer. Low-salt samples were prepared by dialyzing septin complexes eluted in the presence of 1 M KCl elution buffer and PVP peptide, at a concentration of 0.07 mg/ ml, against 75 mM KCl elution buffer for 30 min at 4°C. After determining the conductivity and protein concentration of the low salt sample, a corresponding high salt sample was prepared by diluting an aliquot of PVP eluate with 1 M KCl elution buffer such that the protein concentration was equal to that of the low-salt sample. An aliquot of each sample was then examined by negative-stain EM, whereas the rest was subjected to centrifugation (90,000 rpm for 20 min) and the resulting supernatant (S) and pellet (P) samples were analyzed by SDS-PAGE, followed by staining with Coomassie brilliant blue to quantitate the formation of longer sedimentable polymers (n = 8). Bar, 100 nm.

Thin-section EM of neck regions in wild-type, cdc10Δ, and cdc11Δ cells. (A and B) Grazing sections of bud necks in cdc11Δ cells as observed in two different rounds of embedding and sectioning. (C) A grazing section of a typical cdc10Δ neck. (D) The cdc10Δ cell observed to have dots along the inside of the plasma membrane that might be neck filaments in cross section (arrowhead). Grazing (A′–C′) and cross (D′) sections of wild-type cells showing neck filaments as surface striations or as dots (arrowheads). Arrows, cellular features: NM, nuclear membrane; NP, nuclear pore; PM, plasma membrane; SPB, spindle pole body; MT, microtubule. Bar, 200 nm.

Septin localization by immunofluorescence and the corresponding phase–contrast micrographs of wild-type, cdc10Δ, and cdc11Δ cells. Note that the two cells shown in the cdc11Δ panel are from different fields of the same slide. The multinucleate cdc11Δ cells in this figure are representative of strain morphology after treatment with zymolyase. Cells were grown in YPD medium to early log-phase at 22°C. Bar, 10 μm.

Bud4p localization in wild-type, cdc10Δ, and cdc11Δ cells. Cells were grown in YPD medium to early log phase at 22°C, fixed, and processed for indirect immunofluorescence using affinity-purified Bud4p antibodies (provided by S. Sanders Massachusetts Institute of Technology, Boston, MA). Images of Bud4p staining in septin deletion strains were taken at higher exposures than the corresponding wild-type images. Bar, 10 μm.