(A) Abnormalities in cell shape and cell division in pom1-1 mutant cells. Strain JB100 cells growing exponentially at 30°C were stained with Calcofluor and bisBenzimide to visualize septa and DNA. (Asterisks) Cells with branched or angled growth; (arrows) cells with septa in abnormal positions and/or orientations; (arrowheads) cells with longitudinal or irregular septa. (B–D) Abnormal septum placement in pom1-Δ1 mutant cells. (B) Cells of wild-type strain 972 (left panels) and pom1-Δ1 strain JB110 (right panels) growing exponentially at 36°C were stained with Calcofluor and bisBenzimide (upper panels) and with rhodamine–phalloidin to visualize F-actin (lower panels). (a, b, and c) Cells with different degrees of septum misplacement, as used for the quantitative analysis in C. (C) Septation patterns were scored in >300 cells from each culture shown in B. (Left) Septum position was scored relative to the long axis of the cell as divided into five equal compartments (see examples in B). (Right) Septum angle was scored relative to the long axis of the cell. Septa with angles of 80° to 90° were designated as orthogonal and septa with smaller angles as tilted. Septa that looked very aberrant (e.g., consisting of several sheets) or were placed longitudinally in the cell (see examples in A) were scored as irregular. (D) JB110 cells growing exponentially at 30°C were treated with FITC-conjugated lectin to stain their cell walls, then washed and grown for 45 min before staining with Calcofluor (left). The cell ends that had grown during the 45 min after labeling with lectin can be identified by their unlabeled surface (right) and are marked with asterisks (left).

 Predicted sequence of Pom1p (accession no. Z50142). The kinase domain (amino acids 699–995) is shown in boldface type.

 Time-lapse analysis of the growth of a pom1-1 strain. JB100 cells growing at room temperature in YE medium were observed by DIC microscopy (see Materials and Methods). The times in minutes since the beginning of observation are indicated. Individual cells are numbered for reference in the text. (Arrows) Growing cell ends.

 Abnormal polarization of growth in pom1-Δ1 strains. (A) Strains 972 (wild type) and JB110 (pom1-Δ1) were grown at 36°C to 5 × 10⁶ cells/ml, then stained with rhodamine–phalloidin to visualize F-actin organization. Strains JB20 (cdc25-22) and JB120 (pom1-Δ1 cdc25-22) were grown to the same titer at 25°C, then shifted to 36°C for 3 hr before staining. (B) Cells from the populations shown in A were scored for unipolar or bipolar actin localization, as were cells of strains SP622 (cdc10–V50) and JB121 (pom1-Δ1 cdc10–V50) (grown as described for the cdc25-22 strains in A). For strains 972 and JB110, the percentages of cells with an actin ring were also scored. In the other strains, these percentages were <5%. At least 250 cells of each strain were evaluated. (C) Strains SP622, JB121, JB20, and JB120 were grown at 25°C to 5 × 10⁶ cells/ml, shifted to 36°C for 4 hr, and stained with Calcofluor. (D) Cells (⩾250) from each population shown in C were scored for the presence or absence of new-end growth.

 Mislocalization of growth poles and of microtubule-organizing centers in pom1-Δ1 strains. (A) Cells of strains JB110 (pom1-Δ1), JB22 (cdc11-136), and JB122 (pom1-Δ1 cdc11-136) were grown at 25°C to 5 × 10⁶ cells/ml, shifted to 36°C for 4.5 hr, and stained with Calcofluor. (B) 600 cells from each population shown in A were scored for the extent of cell branching as a function of time after the shift to 36°C. The “branched” cell population in the pom1-Δ1 single mutant consisted mainly (∼90%) of angled cells (see A, cell 1); the rest were T-shaped cells, the majority of which (∼90%) had a division scar at each end (see A, cell 2). The cdc11-136 single mutant showed <1% cell branching at all times. (C) Cells of strains 972 and JB110 were grown at 30°C to 1 × 10⁷ cells/ml, then fixed and stained with anti-tubulin antibodies and with bisBenzimide to visualize DNA. Some cells are numbered for reference in the text. (Arrowhead and arrows) Post-anaphase microtubule-organizing centers.

 Localization of Pom1p. (A–E) Immunolocalization. All strains were grown at 30°C. (A,B) Wild-type strain 972 (A) and pom1-Δ1 strain JB110 (B) were stained with antibodies to Pom1p. (C–E) The pom1–3HA strain JB111 was stained with HA-specific antibodies (C) or double-stained with HA-specific antibodies (D) and bisBenzimide to visualize the DNA (E). Arrows and arrowheads indicate structures discussed in the text. (F) Time-lapse study of Pom1p–GFP localization through the cell-cycle. Strain JB115 was grown at room temperature in EMM medium and observed by fluorescence and DIC microscopy (see Materials and Methods). The times in minutes since the beginning of observation are indicated. Individual cells are numbered for reference in the text. (White arrows) Filamentous structures; (arrowheads) central Pom1p; (black arrows) growing ends. (G–I) Comparison of Pom1p and actin localization in wild-type and mutant strains. Cells expressing Pom1p–3HA were double-stained with antibodies to HA (top) and actin (bottom). (G) Strain JB111 (wild-type except for pom1–3HA) was grown at 30°C to exponential phase before staining. (H,I) cdc10-V50 strain JB113 (H) and cdc25-22 strain JB114 (I) were grown at 25°C to 5 × 10⁶ cells/ml, then shifted to 36°C for 3 hr before staining.

 Effects of cytoskeletal inhibitors and mutations on Pom1p localization. All strains expressed Pom1p-3HA and were stained with antibodies to HA. Arrows and arrowheads indicate structures discussed in the text. (A) Strain JB111 (wild type) was grown at 30°C to 2 × 10⁶ cells/ml, and LAT-A was added to 100 μm. After 3 hr, cells were stained separately for Pom1p and for F-actin (using rhodamine–phalloidin). (B) Strain JB111 was grown at 30°C to 5 × 10⁶ cells/ml and TBZ was added to 100 μg/ml (right panels). A control culture (left panels) received no TBZ. After 1 hr, aliquots of cells from each culture were double-stained for Pom1p (top panels) and DNA (middle panels) or stained separately for microtubules (bottom panels). (C) Strain JB130 (nda3-KM311; left panel) was grown at 32°C to 5 × 10⁶ cells/ml and shifted to 20°C for 1.5 hr before staining for Pom1p. Strain JB132 (ban5-3; right panel) was grown at 25°C to 5 × 10⁶ cells/ml and shifted to 36°C for 3 hr before staining for Pom1p. (D) Strain JB131 (tea1-1) was grown at 25°C to 5 × 10⁶ cells/ml, shifted to 36°C for 3.5 hr, and stained for Pom1p. (E) Strains 972 (wild type) and JB110 (pom1-Δ1) growing exponentially at 30°C were fixed and stained with antibodies to Tea1p.