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. 2004 Aug;3(4):847-54.
doi: 10.1128/EC.3.4.847-854.2004.

Genetic interactions among regulators of septin organization

Amy S Gladfelter  1 , Trevin R Zyla, Daniel J Lew
Affiliations

Genetic interactions among regulators of septin organization

Amy S Gladfelter et al. Eukaryot Cell. 2004 Aug.

Abstract

Septins form a cortical scaffold at the yeast mother-bud neck that restricts the diffusion of cortical proteins between the mother and bud and serves as a signaling center that is important for governing various cell functions. After cell cycle commitment in late G(1), septins are assembled into a narrow ring at the future bud site, which spreads to form a mature septin hourglass immediately after bud emergence. Although several septin regulators have been identified, it is unclear how they cooperate to assemble the septin scaffold. We have examined septin localization in isogenic strains containing single or multiple mutations in eight septin organization genes (CDC42, RGA1, RGA2, BEM3, CLA4, GIN4, NAP1, and ELM1). Our results suggest that these regulators act largely in parallel to promote either the initial assembly of the septin ring (CDC42, RGA1, RGA2, BEM3, and CLA4) or the conversion of the ring to a stable hourglass structure at the neck (GIN4, NAP1, and ELM1). Aberrant septin localization patterns in mutant strains could be divided into apparently discrete categories, but individual strains displayed heterogeneous defects, and there was no clear-cut correspondence between the specific mutations and specific categories of defect. These findings suggest that when they are deprived of their normal regulators, septin scaffolds collapse into a limited repertoire of aberrant states in which the nature of the mutant regulators influences the probability of a given aberrant state.

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Figures

FIG. 1.
FIG. 1.
Septin phenotypes in budded cells. Cells from the strains indicated below were grown to exponential phase in yeast extract-peptone-dextrose (YEPD) at 30°C, fixed, and processed for the visualization of septin localization by use of an anti-Cdc11p antibody. The illustrative cells in the figure display a normal septin hourglass (a and b; strain DLY1), irregular neck staining (c and d; strain DLY4790), wide necks (e and f; strain MOSY148), an ectopic septin hourglass in the elongated bud in addition to a faint neck hourglass (g; strain DLY4223), faint or absent septin staining and a bent or broad neck (h and j; strain DLY4859), and septin bars at aberrantly shaped necks (i and k; strain DLY4224) or displaced into the bud (l; strain DLY4224).
FIG. 3.
FIG. 3.
Epistatic interactions among elm1, gin4, and nap1 mutants. Cells from the strains indicated below were grown to exponential phase in YEPD at 30°C, fixed, and processed for the visualization of septin localization by use of an anti-Cdc11p antibody. WT, strain DLY1; elm1Δ, strain DLY4687; gin4Δ, strain DLY4410; nap1Δ, strain DLY4790; elm1Δ gin4Δ, strain DLY4714; elm1Δ nap1Δ, strain DLY4858; gin4Δ nap1Δ, strain DLY4848; elm1Δ gin4Δ nap1Δ, strain DLY4857. Bar, 10 μm.
FIG. 4.
FIG. 4.
Synthetic interactions among cdc42, cla4, and gin4 mutants. Cells from the strains indicated below were grown at 30°C to exponential phase in minimal medium lacking uracil (to select for plasmid retention), fixed, and visualized by differential interference contrast microscopy. WT, strain DLY3067 carrying plasmid pMOSB55; cla4Δ, strain MOSY23 carrying plasmid pMOSB55; gin4Δ, strain DLY4596 carrying plasmid pMOSB55; cdc42V36T,K94E, strain DLY3067 carrying plasmid pMOSB57; cdc42Y32H, strain DLY3067 carrying plasmid pMOSB56; cla4Δ cdc42V36T,K94E, strain MOSY23 carrying plasmid pMOSB57; cla4Δ cdc42Y32H, strain MOSY23 carrying plasmid pMOSB56; gin4Δ cdc42V36T,K94E, strain DLY4596 carrying plasmid pMOSB57; gin4Δ cdc42Y32H, strain DLY4596 carrying plasmid pMOSB56.
FIG. 2.
FIG. 2.
Septin rings in unbudded cells. Cells from the strains indicated below were grown to stationary phase in YEPD at 30°C and then inoculated into fresh medium, grown for 4 h (at which point cells were entering the cell cycle), fixed, and processed for the visualization of septin localization by use of an anti-Cdc11p antibody. WT, strain DLY1; cla4Δ, strain MOSY148; elm1Δ, strain DLY4687; gin4Δ, strain DLY4410; nap1Δ, strain DLY4790; rga1Δ rga2Δ bem3Δ, strain DLY2723. Bar, 10 μm.
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