Microscopic analysis

Analysis of DNA, cell wall and mitochondria

Analysis of the DNA and septa were performed by fixing 1 ml of cell culture in 1/10 volume of formaldehyde for 10 minutes. After washing in PBSA, cells were visualised with the addition of DAPI or calcofluor and viewed under a fluorescence microscope. Mitochondria were visualised with 2-(4-dimethylaminostyryl)-1 -methyIpyridinium iodide (DASPMI, Sigma) (Yaffe et a l, 1996). 0.2 ml of sample was mixed with 0.5mg/ml DASPMI in water and incubated for 5 minutes at 36°C. Cells were pelletted and re­ suspended in YE.

Indirect immunofluorescence microscopy

Immunofluorescence procedures were used as described in Hagan and Hyams (1988). 5X10^ cells were filtered and fixed in methanol at -70°C for 8 minutes. After 3 washes in PEM the cell wall was digested with 0.6 mg/ml zymolyase 20T in PEMS for 70 minutes. After digestion, cells were pelletted and resuspended in l%TritonX in PEMS for 1 minute. After 3 further washes in PEM, cells were incubated in PEMBAL for 30 minutes and primary antibody was added in 50 fil of PEMBAL and left overnight. The primary antibody was washed out with PEMBAL, and the secondary antibody added in 200^1 PEMBAL and incubated in the dark for 2 hours. Finally, after washing the secondary antibody out with PEMBAL, cells were resuspended in PBSA + 0.2 /xg/ml NaNj. Immunofluorescence images were viewed with a chilled video-rated CCD camera (model C5985, Hamamatsu) connected to a computer (Apple Power Macintosh G3/400) and processed by use of Adobe(R) Photoshop (version 4). A confocal microscope LSM510 (Zeiss Co.) was also used.

Primary antibodies Gtbl Teal Sadi a-tubulin (TAT-1) HA GFP Secondary antibodies Cy3-conjugated goat Cy3-conjugated sheep

Dr. Hirohisa Masuda polyclonal 1:50 Dr. Manuel Arellano polyclonal 1:100 Dr. Iain Hagan polyclonal 1:25 Dr.Keith Gull monoclonal 1:40 BabCo (MMS-lOlR) monoclonal 1:500 Dr. Ken Sawin polyclonal 1:200

anti-rabbit IgG (Sigma C2306) 1:500 anti-mouse IgG (Sigma C2128) 1:500

Chapter 8: Materials and Methods Fluorescein-linked sheep anti-rabbit IgG (Amersham F0208) 1:40

Fluorescein-linked sheep anti-mouse IgG (Amersham F0261) 1:40 Cy5- conjugated anti rabbit IgG (Jackson 111-175-003) 1:50

Rhodamine phalloidin staining

Cells were fixed in formaldehyde (16% EM-grade MeOFl-free, Polyscience) in order to visualise rhodamine phalloidin staining of actin. 1/10 volume of PM buffer and 1/5 volume of formaldehyde were added to 10 ml of a mid log phase culture and incubated under growing conditions for 60 minutes. Cells were pelletted, washed 3 times in PM buffer and resuspended in 1% Triton X-100 for 3 minutes. After further washing in PM buffer, 5 /xl of rhodamine phalloidin (Molecular probes) was added to the pellet of cells and incubated in the dark for 1 hour at room temperature. Cells were visualised as for methanol fixed cells.

Live cell imaging

Live imaging was performed as described in Brunner and Nurse (2000), alp4-1891 cells expressing the a-tubulin atb2'^ GFP from a multicopy plasmid under the control of the n m tl promoter were used. Precultures were grown in 15 [iM thiamine before being washed three times in minimal media. After inoculation into minimal media containing 0.2 jjM thiamine cells were grown for 14 hours in 26°C before being shifted to 36°C for three hours. Live images were taken with a Zeiss LSM 510 laser scanning confocal microscope. Cells were mounted on a layer of rich media beneath a coverslip. Z-stacks (200-400 nm steps) were taken every 30 seconds for 10 minutes.

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