Figure 6.

Cytoplasmic Hsl7 dot corresponds to the SPB. (A and B) Perinuclear localization of the cytoplasmic dot in G1 cells. Fluorescence microscopy was performed on wild-type cells (MJY112) expressing GFP-Hsl7 (green) from the HSL7 promoter on a CEN plasmid (YCpT-GFP-HSL7) that were grown to mid-exponential phase in SCGlc-Trp at 30°C and stained with a DNA-specific dye (DAPI) to discern the nucleus (blue). (C and D) Wild-type cells (MJY112) expressing GFP-Hsl7 from the GAL1 promoter on a CEN plasmid (YCpLG-GFP-HSL7) were grown in SCRaf-Leu at 30°C to mid-exponential phase and then induced by addition of galactose (2% final concentration). After 6 h, the cells were lightly fixed, permeabilized, stained with rat anti-α-tubulin mAb YOL134 (and an appropriate Cy3-labeled secondary antibody), counterstained with DAPI, and viewed in a fluorescence microscope, as described in MATERIALS AND METHODS. Diffuse greencytoplasmic and nuclear signal is nonspecific autofluorescence, whereas in a G1 cell the prominent green cytoplasmic dot (GFP-Hsl7) is always coincident with a bundle of astral microtubules (C), and in a preanaphase cell, GFP-Hsl7 is always at the bud neck, but can be found occasionally decorating one end of the spindle (D). (E) Wild-type cells (MJY112) expressing C-terminally c-Myc-tagged Hsl7 (Hsl7-Myc) from the GAL1 promoter on a CEN plasmid (YCpUG-HSL7-Myc) were grown in SCRaf-Ura medium at 30°C to mid-exponential phase and then induced with galactose (2% final concentration). After 6 h, cells were fixed, permeabilized, stained with mouse anti-c-Myc mAb 9E10 (visualized with an appropriate Cy3-conjugated secondary antibody) and with rabbit anti-Tub4 polyclonal antibodies (visualized with an appropriate fluorescein isothiocyanate-conjugated secondary antibody), counterstained with DAPI, and visualized in a fluorescence microscope. The cytoplasmic dot of Hsl7-myc is congruent with the γ-tubulin signal in the early G1 cell (lower left) and, in a cell at late stage of mitosis, Hsl7-myc is deposited at one of the developing SPBs (upper right). (F–H) Same cells as in E, except that Hsl7 (red) was stained with affinity-purified mouse polyclonal anti-Hsl7 antibodies (rather than with mouse anti-c-Myc mAb) before costaining with anti-Tub4 antibodies (green) and counterstaining with DAPI. Unbudded (G1) cells always show a single dot of Hsl7 congruent with Tub4 (F), whereas, characteristically, early after SPB duplication and separation Hsl7 associates assymetrically with only one SPB (G). By the time a cell has initiated mitosis, Hsl7 is found exclusively at the bud neck and is never observed at either SPB (H). Bars, 5 μm.