Figure 5. Modulation of SIP affects the localization of SIN and its regulators.

A, B. Byr4p localization to the mitotic SPB depends on SIP. A. Wild-type and csc1Δ cells expressing Cdc7p-3xHA were stained using antibodies against the HA-epitope and against Byr4p. After staining, cells were mounted in DAPI and images acquired (n=50×3). B. csc3Δ, ppa3Δ cells grown at 24°C and paa1-L566P cells grown at 36°C were stained with antibodies against tubulin and Byr4p, mounted in DAPI medium and images acquired. C. Overexpression of Csc1p leads to a failure in cytokinesis. Csc1p was overexpressed by growing nmt1-csc1-GFP cells in minimal medium lacking thiamine for 16 hours at 24°C, fixed and stained with aniline blue (cell wall) and DAPI (DNA). The percentage of multinucleated cells was then determined (n=500×3). D. Overproduction of Csc1p leads to recruitment of Byr4p to both SPBs in mitotic cells. nmt1-csc1-GFP cells were grown and fixed as in (C) above. The cells were then stained using antibodies against Byr4p and tubulin TAT-1 antibody. Arrows indicate an increased accumulation of GFP-Csc1p (n=50×3). E. Quantitation of the percentage of anaphase SPB pairs with Byr4p on one or both the SPBs (n=50×3). F. Cdc7p and Sid1p are displaced from mitotic SPBs upon Csc1p overexpression. nmt1-csc1-GFP expressing Cdc7p-mCherry were grown in EMM medium lacking thiamine for 16 hours at 24°C, fixed with methanol and imaged. To visualize Sid1p, nmt1-csc1-GFP cells expressing Sid1p-Myc₁₃ were stained with antibodies against the Myc-epitope.