Geometric definition of terms used in this study. We define a “bud tip” as the farthest point in the bud from the midpoint of the mother-bud neck. The “original mother-bud axis” is the line perpendicular to the tangent of the mother cell at the site of bud emergence. The “angle of the bud” is the angle formed by (i) the line that intersects the bud tip and the midpoint of the mother-bud neck and (ii) the original mother-bud axis.

GFP-Bni1p localization. (a) Time-lapse analysis of Bni1p localization throughout the cell cycle. Diploid strain KY4 (bni1-Δ) harboring the GFP-BNI1 plasmid, pAGX2-BNI1, was examined by time-lapse video microscopy as described in Materials and Methods. Images were taken every ∼3 min for 2 h. Numbers indicate the time (in minutes) since the beginning of observation. Visible bud emergence occurred at around 4 min (left cell) and 19 min (right cell). (b) Bni1p localization at cytokinesis; (c) GFP-Bni1p ring at the mother-bud neck. Arrowhead, GFP-Bni1p signal at pre-bud sites; arrows, a double ring formed at the mother-bud neck. Bar, 4 μm.

Movement of GFP-Bni1p in small buds. (a) Movement of a Bni1p spot from the bud tip to the bud neck and then to the bud tip again. Strain KY4 (bni1-Δ) harboring the GFP-BNI1 plasmid pAGX2-BNI1 was subjected to time-lapse imaging at 2-min intervals for 90 min. Numbers indicate the time (in minutes) since the beginning of observation. Note that the bud growth oriented first to the left side (22 to 42 min) and then reoriented to the top (56 to 74 min). Arrowhead, a protrusion of the bud cortex as a result of the bud growth at 22 to 42 min. Bar, 4 μm. (b) Movement of fuzzy dots of GFP-Bni1p in a small bud. The same strain as that in panel a was subjected to time-lapse imaging at 15-s intervals for 15 min. Numbers indicate the time (in seconds) since the beginning of observation. At 375 s, the GFP-Bni1p signal coalesced into a cap at the bud tip. Bar, 2 μm.

Rate of Bni1p movement. Total velocities reported are derived from a compilation of 145 displacements each over a 15-s intervals observed for six representative spots as assayed by time-lapse microscopy.

Movement of a GFP-Bni1p spot and its colocalization with the apparent site of bud growth. (a) Time-lapse analysis of GFP-Bni1p movement. The visible bud emergence occurred at 27 min, and until 36 min the GFP-Bni1p spot was at the very tip of the bud (data not shown). The spot started to move to the bud neck at 39 min and stayed at the bud neck until 51 min. Then, the spot moved back to the bud tip along the right side of the bud cortex (54 to 72 min). During this period, the apparent bud growth occurred mainly on its right side, resulting in the angled bud. (b) Trace of the movement of Bni1p and outline of the bud in panel a. Each circle corresponds to the brightest point of the GFP-Bni1p signal at 3-min intervals from a 45-min time point. Each closed circle corresponds to the brightest point of GFP-Bni1p at 9-min intervals from a 48-min time point. Each curve corresponds to the outline of the bud at the same time points as the closed circles. The Bni1p localization was first strongly deviated to the right side of the bud, and after a 66-min time point, it was reoriented toward the center. Bar, 3 μm.

A defect of bni1-Δ cells in cell morphology and apical growth. (a) OHNY3 (BNI1); (b) KY4 (bni1-Δ). Cells were cultured exponentially in YPGalactose medium, which generated a thinner cell shape in wild-type cells and helped to assess apical growth. Then, cells were subjected to living stain with FITC-ConA and returned to growth for 60 min. Fixed cells were visualized by differential interference contrast (DIC) and fluorescence microscopy (FITC-ConA). Arrowheads, staining that faded out towards one end; asterisks, staining without the unlabeled region (the gradient of brightness observed in the buds of Δbni1 cells is mostly a photographic artifact because of the halo of their brighter mother cells); arrows, unlabeled regions in unbudded cells, marking pre-bud sites. Bar, 5 μm.

Polarized actin patch-independent but F-actin-dependent Bni1p localization. (a and c) Depolarization of cortical actin patches but not of Bni1p by mild heat shock. Cells of strain OHNY3 expressing GFP-Abp1p or GFP-Bni1p were cultured exponentially at 24°C and then shifted to 36°C for 30 min. Then, the cells were directly examined by fluorescent microscopy. (c) Each cell was classified either as a polarized GFP signal (solid bar), a depolarized GFP signal (hatched bar), or no concentration of GFP signal (open bar). (b and d) Alteration of the GFP-Bni1p localization by LAT-A-induced complete disruption of the actin cytoskeleton. Cells of strain OHNY3 expressing GFP-Abp1p or GFP-Bni1p were cultured exponentially at 24°C, and LAT-A was added from a 10 mM DMSO stock to a final concentration of 100 μM. An equal volume of DMSO alone was added to the control population of cells. After a 30-min incubation, the cells were directly observed by conventional fluorescent microscopy as was done in panels a and c. Bar, 5 μm.

Bni1p localization in cdc42 or rho1 mutant cells. (a) cdc42-1 temperature-sensitive mutant cells. Cells of strain KKC42-1 (cdc42-1) harboring pTAGX2-BNI1 were cultured exponentially in synthetic dextrose supplemented with 5% Casamino Acids liquid media at 24°C, shifted to 36°C for 1 h, and examined by fluorescent microscopy. Arrowheads, abnormal GFP dots in mother cells or at the bud neck. (b) rho1-104 temperature-sensitive mutant cells. Cells of strain HNY21 (rho1-104) harboring pTAGX2-BNI1 were cultured exponentially in SDA-AU liquid media at 20°C and shifted to 36°C for 1 h. Then, the cells were directly examined by fluorescent microscopy. Shown is a cell examined by light microscopy (right) and fluorescent microscopy (middle). Arrow, an abnormally thin basal part of the bud. Bar, 4 μm.

Defective Bni1p localization in spa2 or bud6 mutant cells. The strains used were TYSH1 (spa2) (a) and TFB6H3 (bud6) (b). Mutant cells harboring GFP-BNI1 plasmid pAGX2-BNI1 were examined by time-lapse video microscopy. Numbers indicate the time (in minutes) since the beginning of observation. With spa2 diploid strains, we obtained results similar to those in panel a. (a) ctl, cell of wild-type strain OHNY1 harboring pAGX2-BNI1 as a control; arrows, GFP-Bni1p signal at incipient bud sites; arrowhead, the diffuse GFP-Bni1p signal in the small bud. (b) Arrows, GFP-Bni1p signal separated into two distinct patches. Bar, 5 μm.

Mapping of the regions of Bni1p required for its proper localization and function. (a) Structures of the truncated constructs of Bni1p. Each number under the domain structure of Bni1p indicates either the first or the last amino acid residue of the region. Each horizontal bar represents a segment of Bni1p: full (aa 1 to 1954), FΔA (aa Δ133 to 245), FΔB (aa Δ826 to 987), FΔC (aa Δ1239 to 1328), FΔD (aa 1 to 1750), Fc (aa 1 to 1240), Fb (aa1 to 826), Fh (aa 1 to 642), Fi (aa 1 to 642, Δ133 to 245), and F18 (aa 490 to 1954). (b) Relative expression levels of truncated Bni1p. Various DNA fragments encoding truncated Bni1p's were cloned into pAGX2, and the resultant plasmids were used to transform cells of wild-type strain OHNY3. Transformants were subjected to Western blotting using the anti-GFP polyclonal antibody. Expression levels of mutant Bni1p relative to those of full-length Bni1p were determined by densitometry. (c) Localization of GFP-fused truncated Bni1p at the bud tip. The cells expressing truncated Bni1p were examined for GFP localization by conventional fluorescent microscopy. (d) Functional complementation of the bni1-Δ mutant phenotypes by the truncated proteins. Various DNA fragments encoding truncated Bni1p's were cloned into pRS314-P_BNI1-myc (18), and the resultant plasmids were used to transform KY4 (bni1-Δ). Transformants were subjected to morphological examinations of apical growth by the same FITC-ConA living stain used in Fig. 6, of bud neck formation by phase-contrast microscopy, and of budding pattern by chitin staining. (e) Rescue of the synthetic lethality of the bni1-Δ bnr1-Δ cells by truncated Bni1p. The same plasmids used in panel d were used to transform DKBY80B (bni1-Δ/BNI1, bnr1-Δ/BNR1). Transformants were subjected to tetrad analysis. +++, indistinguishable from the wild type; ++, mild defect; +, severe defect; −, no signal or function-like negative control. N.D., not determined.