Elm1 binding and kinase activity are required for the retention of Gin4 at the bud neck prior to cytokinesis. (A) Montages of bud neck region of representative WT (top, Strain YEF11493), elongated elm1^KD (middle, Strain YEF11496), and round elm1^KD (bottom, Strain YEF11496) cells showing maximum-intensity projections of Shs1-GFP in green and Gin4-mScarlet mRuby2-Tub1 in magenta from 35 min before to 30 min after mitotic spindle break from time-lapse series taken with a 2.5-min interval. T = 0 is mitotic spindle break. Scale bars = 1 μm. (B) Quantification of Gin4-mScarlet signal from cells in A. Shown is the integrated measured background subtracted intensity of Gin4-mScarlet in WT (blue), round elm1^KD (green), and elongated elm1^KD (yellow) cells 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. (C) Quantification of Gin4-mScarlet signal from cells in A. Shown is background subtracted intensity relative to the maximum value measured of Gin4-mScarlet in WT (blue), round elm1^KD (green), and elongated elm1^KD (yellow) cells from the sum projection of given number cells for each strain. The mean is plotted with error bars being the SD. (D) Montages of bud neck region of representative Elm1^WT-GBP (top, Strain YEF11495) and Elm1^KD-GBP (bottom, Strain YEF11494) cells showing maximum-intensity projections of Shs1-GFP in green and Gin4-mScarlet mRuby2-Tub1 in magenta from 35 min before to 30 min after mitotic spindle break from time-lapse series taken with a 2.5-min interval. T = 0 is mitotic spindle break. Scale bars = 1 μm. (E) Quantification of Gin4-mScarlet signal from cells in A and D. Shown is integrated measured background subtracted intensity of Gin4-mScarlet in WT (blue), round elm1^KD (green), elongated elm1^KD (yellow), elm1^WT-GBP (gray), and elm1^KD-GBP (orange) cells from the sum projection of given number cells for each strain. Curves for WT, round elm1^KD, and elongated elm1^KD are the same used in C and are at 30% opacity for ease of comparing the kinetic signature of elm1^WT/KD-GBP cells to previously analyzed cells. The mean is plotted with error bars being the SD. Asterisk indicates significant difference (P < 0.05) by unpaired Student’s t test when comparing gray and orange series. a.u. = arbitrary units. (F) Montages of bud neck region of representative elm1_1-420^WT-GBP (top, Strain YEF11607) and elm1_1-420^KD-GBP (bottom, Strain YEF11606) cells showing maximum-intensity projections of Shs1-GFP in green and Gin4-mScarlet mRuby2-Tub1 in magenta from 35 min before to 30 min after mitotic spindle break from time-lapse series taken with a 2.5-min interval. T = 0 is mitotic spindle break. Scale bars = 1 μm. (G) Quantification of Gin4-mScarlet signal from cells in A and F. Shown is integrated measured background subtracted intensity of Gin4-mScarlet in WT (blue), round elm1^KD (green), elongated elm1^KD (yellow), elm1_1-420^WT-GBP (navy blue), and elm1_1-420^KD-GBP (red) cells from the sum projection of given number cells for each strain. Curves for WT, round elm1^KD, and elongated elm1^KD are same used in C and are at 30% opacity for ease of comparing kinetic signature of elm1_1-420^WT/KD-GBP cells to previously analyzed cells. The mean is plotted with error bars being the SDn. a.u. = arbitrary units. (H) Model depiction of the reciprocal regulation by Gin4 and Elm1 at bud emergence (top) and prior to cytokinesis (bottom). Solid lines indicate direct interacting partners, arrows indicate direction of regulation by binding and phosphorylation (denoted by a P).