1. Volume 42, Issue 2, Pages 143-155.e5 (July 2017)
Cell Polarity Regulates Biased Myosin Activity and Dynamics during Asymmetric Cell Division via Drosophila Rho Kinase and Protein Kinase N 
Anna Tsankova, Tri Thanh Pham, David Salvador Garcia, Fabian Otte, Clemens Cabernard 
Developmental Cell 
Volume 42, Issue 2, Pages e5 (July 2017)
DOI: /j.devcel
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2. Developmental Cell 2017 42, 143-155. e5DOI: (10. 1016/j. devcel. 2017
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3. Figure 1 Biased Myosin Localization Is Dependent on Pins
(A–I) Representative image sequence of (A) a wild-type third instar neuroblast expressing Sqh:GFP (Myosin; green in overlay) and Cherry:Jupiter (microtubules [MTs]; white overlay) in (top row), (D) pinsP89/pinsP62 (middle row), and (G) UAS-Gαi (bottom row), respectively. Representative kymographs from (B) wild-type, (E) pins mutant, and (H) UAS-Gαi-expressing neuroblasts, derived from a line along the apical to basal cortex. Arrowheads highlight the apical (blue in wild-type, gray in the symmetrically dividing cells) and the basal (red in wild-type, gray in symmetrically dividing cells) cortex, respectively. The vertical dashed lines refer to the nuclear envelope break (NEB) and anaphase onset (AO), respectively. Intensity ranges are indicated with the LUT bar next to the kymographs. (C), (F), and (I) show intensity plots measured along the apical and basal cortex from the respective kymographs. The values were normalized over the global maximal intensity.
(J) Normalized cortical Sqh intensities are plotted for the indicated genotypes (blue, apical; red, basal; gray, apical or basal, since polarity is lost).
(K) The difference between the apical and basal normalized intensity, at the averaged time of the peak, for each cell is plotted as an asymmetry coefficient.
(L) Representative wild-type metaphase and pinsP89/pinsP62 neuroblasts stained with monophosphorylated Myosin (Sqh1P) and the DNA marker DAPI.
(M) Ratio of apical/basal Sqh1P intensity for wild-type and pinsP89/pinsP62.
For this and all subsequent figures, asterisks denote statistical significance, derived from two-sample equal or unequal variance t test: ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005; ns, not significant. For multiple statistical comparisons (J and K), we used Šidák correction: ∗p < (2 groups), p < (3 groups), p < (4 groups), p < (5 groups). ∗∗p < (2 groups), p < (3 groups), p < (4 groups), p < (5 groups). ∗∗∗p < (2 groups), p <  (3 groups), p < (4 groups), p < (5 groups). Time is in seconds (s); timescale bar in kymographs corresponds to 60 s. NEB, nuclear envelope breakdown (= 0 s), MTs; microtubules. Scale bars, 5 μm.
Developmental Cell  , e5DOI: ( /j.devcel )
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4. Figure 2 Asymmetric Pkn Localization Depends on Pins
(A) Representative image sequence of a third instar wild-type neuroblast, expressing Pkn:GFP (top row) and Sqh:Cherry (bottom row).
(B) Kymographs along a line from the apical to the basal cortex. Red and blue arrowheads indicate the apical cortex of Pkn and Sqh, respectively. Intensity ranges are indicated with the LUT bar next to the kymographs.
(C and D) Representative intensity plot along the apical cortex, showing Pkn (red) and Sqh (blue) (C). The time between NEB and the apical Pkn and Sqh peak was measured and plotted (D).
(E) Representative neuroblast metaphase and anaphase images, showing Pkn:GFP localization in wild-type (blue), pinsP89/pinsP62 (magenta), and UAS-Gαi (orange).
(F) Scatterplot showing apical/basal cortical Pkn:GFP intensity ratios at metaphase for the indicated genotypes.
(G) Scatterplot showing the cortical enrichment of Pkn:GFP on the non-apical cortex, in relation to cytoplasmic Pkn:GFP (ratio).
Time is in seconds (s); timescale bar in kymographs corresponds to 60 s. NEB, nuclear envelope breakdown (= 0 s); MTs, microtubules. Scale bars, 5 μm.
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5. Figure 3
Pkn Is Required for the Correct Spatiotemporal Localization and Phosphorylation of Myosin
(A–C) (A) Representative images of prometaphase and metaphase wild-type and pkn06736/Def neuroblasts expressing Sqh:GFP and stained with monophosphorylated Myosin (Sqh1P) and DAPI. Sqh:GFP (B) and Sqh1P (C) intensities were measured at the apical (green), basal (blue), and whole cortex (red) for wild-type and pkn06736/Def mutant neuroblasts.
(D) Representative image sequence of a pkn06736/Def mutant neuroblast expressing Sqh:GFP (single channel, white; merged channel. green) and Cherry:Jupiter (MTs; merged channel, white). Orange arrowheads highlight the shift in furrow position.
(E) Kymograph along a line from the apical toward the basal cortex of a pkn06736/Def mutant neuroblast. The apical and basal cortices are highlighted with a blue and red arrowhead, respectively. The intensity range is indicated by the LUT bar next to the kymograph.
(F) Normalized intensity plot, showing the intensities along the apical (blue) and basal (red) cortex.
(G and H) Scatterplots showing the duration of apical Sqh enrichment in wild-type (blue) and pkn06736/Def (gray) (G), and the temporal delay between apical peak of Sqh in relation to NEB for wild-type and pkn06736/Def (H).
Time is in seconds (s); timescale bar in kymographs corresponds to 60 s. NEB, nuclear envelope breakdown (= 0 s); MTs, microtubules. Scale bars, 5 μm.
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6. Figure 4
Apical Rok Localization Depends on Polarity Cues and Precedes Pkn Localization
(A) Representative image sequence of a wild-type neuroblast expressing Rok:GFP (white on top; green in merged channel) and Cherry:Jupiter (white in merged channel).
(B) Kymograph along a line from the apical toward the basal cortex. Apical and basal cortices are highlighted with a blue and red arrowhead, respectively.
(C) Scatterplot showing the duration of apical Rok (green), Myo (blue), Pkn (red), and Pins (dark blue) enrichment in wild-type neuroblasts.
(D) Scatterplot showing the temporal delay between NEB and the apical peak of Rok (green), Myo (blue), Pkn (red), and Pins (dark blue) in wild-type neuroblasts. The values in (C) and (D) are normalized to the total time between NEB and AO for each cell.
(E) Representative images of UAS-Gαi and pins mutant expressing Rok:GFP (white).
(F) Scatterplot showing apical (blue) and basal (red) or cortical (gray) Rok:GFP intensity ratios in relation to cytoplasmic Rok:GFP for wild-type, UAS-Gαi-expressing, and pins mutant neuroblasts.
(G) Image sequence showing a pkn06736/Def neuroblast expressing Rok:GFP (white on top, green in the merged channel) and Cherry:Jupiter (white in overlay). Orange arrowheads highlight the shift in furrow position.
(H) Apical (blue) and basal (red) intensity ratios for wild-type and pkn06736/Def mutant neuroblasts, in relation to cytoplasmic Rok levels.
Time is in seconds (s); timescale bar in kymographs corresponds to 60 s. NEB, nuclear envelope breakdown (= 0 s); MTs, microtubules. Scale bars, 5 μm.
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7. Figure 5
Pkn Mutant Neuroblasts Show Ectopic Contractions and Cell Shape Changes
(A) Curvature measurements were performed during prometaphase and metaphase on the apical neuroblast cortex.
(B and C) Representative curvature measurements for (B) a wild-type and (C) pkn06736/Def mutant neuroblast.
(D) Scatterplot showing the maximal curvature values (h) for wild-type (blue) and pkn06736/Def (beige) mutant neuroblasts.
(E–G) Curvature was measured along the apical – basal neuroblast tip from anaphase until telophase (E). Representative wild-type (F) and pkn06736/Def mutant (G) neuroblasts. Colored lines in the curvature plot correspond to the representative time points.
(H) Scatterplot showing the maximal distance between the first and final furrow. The cortical position for all curvature measurements was normalized on the respective measured distance.
Time is in seconds (s). Scale bars, 5 μm.
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8. Figure 6 Apical Cortical Constrictions Cause Ectopic Furrowing
(A and B) Representative images of a (A) wild-type and (B) pkn06736/Def mutant neuroblast expressing the Myosin activity sensor (Vt-TS-Vt). FRET histograms, showing normalized FRET ratios below the image sequences. FRET ratios were plotted for apical cortex (orange box), furrow or ectopic furrow regions (red box), and basal cortex (magenta).
(C and D) Averaged FRET ratios for the apical cortex, furrow or ectopic furrow region, and basal cortex were plotted for (C) metaphase and (D) early anaphase wild-type (blue dots) and pkn06736/Def mutant (brown dots) neuroblasts. Each spot represents a measured neuroblast.
Time is in seconds (s). Scale bars, 5 μm.
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9. Figure 7 Biased Myosin Activity Regulates Cleavage Furrow Positioning
(A–D) Constitutively activated Sqh (SqhEE) was localized to the (A) apical or (C) basal neuroblast cortex. Representative image sequence and curvature measurements along the apical-basal cortex for a neuroblast, expressing (B) ALD-SqhEE:mCherry (not shown) and Sqh:GFP (white) or (D) BLD-SqhEE:EGFP (white) and Sqh:mCherry (not shown).
(E and F) (E) Curvature measurements were also performed in mbs3/Def mutant neuroblasts, expressing Sqh:GFP (white); a representative image sequence and measurement is shown in (F).
(G and H) Scatterplots of (G) final furrow positioning and (H) furrow shift for the indicated genotypes.
(I) Representative images of wild-type and pkn06736/Def mutant neuroblasts at anaphase and telophase, stained for the polarity protein Pins (red in merged image), Miranda (green in merged image; white in single channel), and DAPI.
(J) Quantification of neuroblasts showing normal asymmetric, symmetric, or inverted asymmetric anaphase figures.
(K) Model (see text for details).
Time is in seconds (s). ALD, apical localization domain; BLD, basal localization domain; Asym, asymmetric; Sym, symmetric. Scale bars, 5 μm.
Developmental Cell  , e5DOI: ( /j.devcel )
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