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Dynamic, Rho1p-dependent localization of Pkc1p to sites of polarized growth

Paul D. Andrews and Michael J. R. Stark

* Department of Biochemistry, MSI/Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, UK

Figure 1A:

Fixed yeast cells were prepared for simultaneous visualization of actin, Pkc1p-GFP and cell wall chitin and images were coloured green for Pkc1p-GFP, red for actin and blue for chitin. Overlapping actin and Pkc1p-GFP appears yellow. The movie shows a rotation of the three-dimensional images of the small-budded cells depicted in Fig. 1A. Scale bar is in micrometres.

Figure 1C:

^M The movie shows a rotation of the three-dimensional images of a large-budded cell depicted in Fig. 1C showing neck localization of Pkc1p-GFP (see legend to movie Fig. 1A for details). Scale bar is in micrometres.

Figure 4A:

Time-lapse analysis of Pkc1p-GFP localisation during the cell cycle generated with images acquired at one-minute intervals. This movie shows a large-budded cell in which Pkc1p-GFP is present at the neck until after cell separation, when it redistributes to the new bud site of the mother cell and then becomes localized to the tips of small buds. Scale bar is in micrometres.

Figure 4B:

Time-lapse analysis of Pkc1p-GFP localisation during the cell cycle generated with images acquired at one-minute intervals. This movie shows a small-budded (left) and a large-budded (right) cell. Pkc1p-GFP is present at the bud tips, fluctuating in position and intensity with time. As cells progress through the cell cycle Pkc1p-GFP is no longer observed in a localized pattern, but later still reappears at the bud neck in the cell on the right. Scale bar is in micrometres.

Figure 5A:

Time-lapse analysis of Pkc1p-GFP localisation in a group of cells in response to cell wall damage with images acquired at one-minute intervals after a brief treatment with Zymolyase. Numerous regions of non-polarized Pkc1p-GFP localization were observed within minutes of initiating cell wall damage. This unpolarized localization is in direct contrast to untreated cells, which show distinct bud-tip and neck localization (see Fig. 4 and Fig. 4 movies). Scale bar is in micrometres.

Figure 5B:

Time-lapse analysis of Pkc1p-GFP localisation in the bud of a single cell in response to cell wall damage with images acquired at one-minute intervals after a brief treatment with Zymolyase. Numerous regions of non-polarized Pkc1p-GFP localization were observed within minutes of initiating cell wall damage. Scale bar is in micrometres.

Figure 5C:

Time-lapse analysis of Pkc1p-GFP localisation in a single mother cell in response to cell wall damage with images acquired at one-minute intervals after a brief treatment with Zymolyase. Numerous regions of non-polarized Pkc1p-GFP localization were observed within minutes of initiating cell wall damage. Scale bar is in micrometres.

Figure 7A:

Time-lapse analysis of Pkc1p-GFP localisation in the neck of a single cell after treatment with 200 _M Latrunculin-A to disrupt actin polymerization. Three-dimensional image series were acquired at one-minute intervals and used to generate two-dimensional projections viewed along the mother-daughter axis. Localisation of Pkc1p remains dynamic despite disruption of actin polymerization. Scale bar is in micrometres.

Figure 7B:

Time-lapse analysis of Pkc1p-GFP localisation in the neck of a single cell after treatment with DMSO as a control for Fig. 7A. Three-dimensional image series were acquired at one-minute intervals and used to generate two-dimensional projections viewed along the mother-daughter axis. Scale bar is in micrometres.

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