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. 2018 Jan 24;7:e29939. doi: 10.7554/eLife.29939

Figure 5. Mira crescent size is affected by a Y-27632-sensitive mechanism that operates before NEB.

(A) Stills from Video 15. Colcemid arrested NBs were treated with 200 µM Y-27632. After >50 min Mira becomes faintly detectable apically, but retains a basal bias. LatA addition (5 µM) abolishes that asymmetric bias and Mira is uniformly distributed on the membrane. (B) Culturing colcemid-arrested NBs in 50 µM Y-27632 did not alter Mira crescent size (yellow arrowheads, quantified in E). (C) NBs polarizing in the presence of 25 µM Y-27632 show enlarged Mira crescents. Control division (−62’ to −35’) with normally sized Mira crescent and daughter cell size (−60’; yellow arrowheads, bracket, respectively). Dividing in the presence of Y-27632 (−3, NEB +1) leads to an enlarged Mira crescent (NEB +1, white arrowheads) and enlarged daughter cell size (+24’, brackets, 2). (D) NBs were allowed to polarize in the absence (upper row) or presence of 25 µM Y-27632 (middle and lower row) followed by colcemid arrest. upper row: Control NB with normal Mira crescent (yellow arrowheads) was depolarized by 1 µM LatA. Mira was displaced into the cytoplasm. middle row: adding 1 µM LatA leads to displacement of the enlarged Mira crescent (yellow arrowheads) in the cytoplasm. Lower row: adding 20 µM ML-7 drives Mira into the cytoplasm (+8’). Upon ML-7 washout, Mira recovered to an enlarged crescent (+14’, white arrowheads). (E) Quantification of Mira crescent size in the aforementioned experiments (unpaired t test). (F) Plot of the ratio of daughter cell to NB nuclei as a measure of the effect of Y-27632 on daughter cell size. NBs expressing NLSGFP were imaged by DIC to follow daughter cell birth order during three consecutive divisions [(1) pre-treatment; (2) division in the presence of 25 µM Y-27632; (3) division after drug washout]. A high-resolution z-stack of nlsGFP was recorded, and the nuclear volumes rendered and calculated using IMARIS to plot their ratio. p values: Dunn’s test. Time stamp: min. Labels as indicated. BAC{mira::mcherry-MS2} was the source of Mira::mCherry. Scale bar: 10 µm.

Figure 5.

Figure 5—figure supplement 1. Standard used to quantify Mira crescent size.

Figure 5—figure supplement 1.

(A) Schematic of workflow. NBs that have a polarity axis parallel to the imaging plane are selected. Optical planes (3–5) are collected covering 2–3 µm of the equator of the NB. (B) Fluorescence is normalized against the cytoplasmic background and straightened line plots are derived from each section. The average background and the average standard deviation is determined. Signal:>avg. background plus two times the average standard deviation. (C) 3D projections of z-sections covering the entire NBs, ctrl vs. a NB that polarized in the presence of 25 µM Y-27632. (D) Quantification of aPKC::GFP crescent size under the indicated conditions (unpaired t test). Asterisks: aPKC; yellow arrowheads: normal sized crescents; white arrowheads: enlarged Mira crescents. BAC{mira::mcherry-MS2} was the source of Mira::mCherry. Scale bar: 15 µm.