Septins exhibit mild bud neck displacement while Elm1 is absent from the bud neck in gin4∆ cells. (A) Representative images of YEF9641 (gin4∆ CDC3-GFP mRuby2-TUB1) cells with brightfield (left) and maximum-intensity projection of merged Cdc3-GFP in green and mRuby2-Tub1 in magenta (right). Scale bar = 5 μm. (B) Maximum-intensity projection images of representative WT (YEF9180), elm1∆ (YEF9935), and gin4∆ (YEF9641) cells from a time-lapse series taken with a 2-min interval with Cdc3-GFP in green and mRuby2-Tub1 in magenta at indicated times. T = 0 is bud emergence, gray dashed line is the cell periphery, yellow boxed region is the area used for measurements in C, scale bars = 2 μm. (C) Quantification of cells in B. Shown is the background subtracted intensity of Cdc3-GFP in WT (green), elm1∆ (gray), and gin4∆ (yellow) relative to the maximum value measured from the sum projection of the given number of cells for each strain. The mean is plotted with error bars being the SD. (D) Representative images of Elm1-GFP and Cdc3-mCherry in WT (YEF10440) and gin4∆ (YEF10460) cells with brightfield (left) and maximum-intensity projection of Elm1-GFP in green (middle) and Cdc3-mCherry in magenta (right). Scale bars = 5 μm. (E) Top: Immunoblot (IB) analysis of Elm1-GFP and Cdc28 from three independent replicates of total protein isolates from WT (YEF9327, sample 1), Elm1-GFP (YEF10749, sample 2), and gin4∆ Elm1-GFP (YEF10750, sample 3) cells. MW = protein marker with indicated sizes in kDa at indicated positions. Asterisk (*) indicates a non-specific band from the anti-GFP antibody. Bottom: Quantification of the ratio of Elm-GFP/Cdc28 band intensity in gin4∆ samples relative to that of WT Elm1-GFP samples for each replicate. The average is presented from the three replicates ± SD. (F) Montages of representative WT (YEF9180), elm1∆ (YEF9935), gin4∆ (YEF9641), and shs1∆ (YEF8438) cells showing maximum-intensity projections of Cdc3-GFP in green from 32 min before to 20 min after mitotic spindle break with selected frames from time-lapse series taken with a 2-min interval. T = 0 is mitotic spindle break. Scale bars = 1 μm. (G) Quantification of cells in F. Shown is the background subtracted intensity of Cdc3-GFP in WT (green), elm1∆ (gray), gin4∆ (yellow), and shs1∆ (blue) relative to the maximum value measured from the sum projection of the given number cells for each strain. The mean is plotted with error bars being the SD. (H) Quantification of cells in F. Shown is the background subtracted intensity of Cdc3-GFP in WT (green), elm1∆ (gray), gin4∆ (yellow), and shs1∆ (blue) relative to the region of interest’s area measured from the sum projection of given number cells for each strain. The mean is plotted with error bars being the SD. a.u. = arbitrary units. Source data are available for this figure: SourceData F3.
Figure 3.

Septins exhibit mild bud neck displacement while Elm1 is absent from the bud neck in gin4∆ cells. (A) Representative images of YEF9641 (gin4∆ CDC3-GFP mRuby2-TUB1) cells with brightfield (left) and maximum-intensity projection of merged Cdc3-GFP in green and mRuby2-Tub1 in magenta (right). Scale bar = 5 μm. (B) Maximum-intensity projection images of representative WT (YEF9180), elm1∆ (YEF9935), and gin4∆ (YEF9641) cells from a time-lapse series taken with a 2-min interval with Cdc3-GFP in green and mRuby2-Tub1 in magenta at indicated times. T = 0 is bud emergence, gray dashed line is the cell periphery, yellow boxed region is the area used for measurements in C, scale bars = 2 μm. (C) Quantification of cells in B. Shown is the background subtracted intensity of Cdc3-GFP in WT (green), elm1∆ (gray), and gin4∆ (yellow) relative to the maximum value measured from the sum projection of the given number of cells for each strain. The mean is plotted with error bars being the SD. (D) Representative images of Elm1-GFP and Cdc3-mCherry in WT (YEF10440) and gin4∆ (YEF10460) cells with brightfield (left) and maximum-intensity projection of Elm1-GFP in green (middle) and Cdc3-mCherry in magenta (right). Scale bars = 5 μm. (E) Top: Immunoblot (IB) analysis of Elm1-GFP and Cdc28 from three independent replicates of total protein isolates from WT (YEF9327, sample 1), Elm1-GFP (YEF10749, sample 2), and gin4∆ Elm1-GFP (YEF10750, sample 3) cells. MW = protein marker with indicated sizes in kDa at indicated positions. Asterisk (*) indicates a non-specific band from the anti-GFP antibody. Bottom: Quantification of the ratio of Elm-GFP/Cdc28 band intensity in gin4∆ samples relative to that of WT Elm1-GFP samples for each replicate. The average is presented from the three replicates ± SD. (F) Montages of representative WT (YEF9180), elm1∆ (YEF9935), gin4∆ (YEF9641), and shs1∆ (YEF8438) cells showing maximum-intensity projections of Cdc3-GFP in green from 32 min before to 20 min after mitotic spindle break with selected frames from time-lapse series taken with a 2-min interval. T = 0 is mitotic spindle break. Scale bars = 1 μm. (G) Quantification of cells in F. Shown is the background subtracted intensity of Cdc3-GFP in WT (green), elm1∆ (gray), gin4∆ (yellow), and shs1∆ (blue) relative to the maximum value measured from the sum projection of the given number cells for each strain. The mean is plotted with error bars being the SD. (H) Quantification of cells in F. Shown is the background subtracted intensity of Cdc3-GFP in WT (green), elm1∆ (gray), gin4∆ (yellow), and shs1∆ (blue) relative to the region of interest’s area measured from the sum projection of given number cells for each strain. The mean is plotted with error bars being the SD. a.u. = arbitrary units. Source data are available for this figure: SourceData F3.

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