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. 2007 Dec 31;179(7):1375–1384. doi: 10.1083/jcb.200705160

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Copyright © 2007, The Rockefeller University Press

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Figure 1. — Deletion of RGA1 causes polarization and budding within the previous division site. (A) Quantitation of bud scar distribution in an asynchronous population of cells from haploid strains YEF473A (wild type), YEF2324 (rga1Δ), YEF2392 (rga2Δ bem3Δ), and YEF2380 (rga1Δ rga2Δ bem3Δ). 200 cells were counted for each strain and unbudded daughter cells were excluded. The cells with only chitin rings at the base of the growing buds were counted as having “0 bud scar.” (B) Chitin staining of wild-type and rga1Δ cells indicated in A. Double chitin rings at the neck of an rga1Δ cell (2) were visualized occasionally when the distance between the rings was large enough to be resolved by light microscopy. (C) SEM observation of bud scars. The same strains described in A were used for SEM. (D) Using the positions of the septin rings as a read-out of the budding patterns in live cells. Cells of haploid strains YZT82 (CDC3-GFP, wild type), YZT55 (rga1Δ CDC3-GFP), and YZT111 (rga2Δ bem3Δ CDC3-GFP) were grown to exponential phase in YM-P medium and observed by 3D time-lapse microscopy at 30°C. Times are given in minutes and seconds after an arbitrary starting point. Arrowheads indicate an old septin ring at the mother side of the bud neck; arrows indicate the nascent septin ring at the new bud site. Views of the 3D images from particular angles are shown: an angled side view of the bud neck of the wild-type cell, an en-face view of the mother side of the bud neck of the rga1Δ cell, and a side view of the bud neck of the rga2Δ bem3Δ cell. Please note that a clear rotation of the mother cell versus the daughter cell occurred after cytokinesis and cell separation at a time between 9 min 17 s and 11 min 45 s for the wild-type cell and between 5 min 29 s and 7 min 31 s for the rga2Δ bem3Δcell. (E) The first bud of rga1Δ daughter cells forms within the birth scar. Birth scars of representative wild-type (YEF473A) and rga1Δ (YEF2324) cells. Cells 1 and 2 represent off-center and central budding within the birth scar, respectively. Bars, 1 μm.