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. 2016 Nov 7;26(21):2921–2928. doi: 10.1016/j.cub.2016.08.048

Figure 2.

Figure 2

CRIB Dispersal by Latrunculin A Requires the Sty1 MAPK Pathway but Not Sty1-Dependent Gene Expression

(A) Still images from movies of Lifeact-mCherry (LA-mCh) and CRIB-3xmCitrine (CRIB-3mCit) in sty1Δ and wis1Δ cells after addition of 50 μM latrunculin A (LatA). Although LatA depolymerizes the actin cytoskeleton, CRIB does not disperse, and cells continue to elongate.

(B) Kymographs from movies of CRIB-3mCit showing rates of cell elongation in the indicated strains after addition of DMSO or LatA. The cartoon summarizes kymograph construction (see also Supplemental Experimental Procedures). The orange curves and circle represent CRIB-3mCit fluorescence at cell tips and in nucleus, respectively. The dashed box represents the region used for kymograph scans along the x-axis. Images used for kymograph analysis are z-projections, and kymograph scans measured average intensity values along a line that is five pixels wide on the y-axis (line width corresponds to height of dashed box); therefore, information from y- and z-dimensions is implicit in kymographs. Arrows indicate orientation of x and time (t) axes in kymographs. Left-hand panels in each pair of kymographs represent cells shown in Figures 1A and 2A and Movie S1. Right-hand panels show additional cells. Arrowheads indicate time of DMSO or LatA addition. Asterisks indicate disappearance of CRIB from cell tips in DMSO-treated cells due to cell division.

(C) Still images from movies of LA-mCh and CRIB-3mCit in sty1-T97A cells pre-treated with 5 μM 3-BrB-PP1 (3BrB) for 10 min prior to addition of 50 μM LatA in the continued presence of 3BrB.

(D) Still images from movies of CRIB-3mCit in atf1Δ cells after addition of 50 μM LatA. Arrowheads indicate examples of ectopic CRIB patches.

(E) Still images from movies of CRIB-3mCit in wild-type cells pre-treated with 100 μg/mL cycloheximide (CHX) for 10 min prior to addition of 50 μM LatA in the continued presence of CHX. Arrowheads indicate examples of ectopic CRIB patches. CHX treatment causes contortions of nuclei to varying extents, and therefore multiple example cells are shown.

All times shown are relative to addition of DMSO or LatA. Scale bars, 5 μm. See also Figures S1 and S2 and Movie S1.