Figure 5.
Figure 5. Apical myosin activation and apical constriction initiation do not require microtubules. (A) Disrupting microtubules prevents folding, despite apical myosin activation and apical constriction initiation. Time-lapse images are maximum-intensity Z-projections from representative live embryos expressing Myo::GFP (apical surface) and Gap43::mCH (membrane, subapical section) injected with DMSO (left), colchicine (5 mg/ml; middle), and Taxol (5 mg/ml; right). Apical–basal cross sections (yz) are to the right of each image. Dashed line indicates the ventral furrow. (B) Apical area initiates reduction after disrupting microtubules. Quantification of apical cell areas preconstriction (t = 0 s) and 320 s after from three representative live embryos injected with DMSO (n = 226 cells, T = 0 s; 252 cells, T = 320 s; **, P < 0.0001, unpaired t test), colchicine (n = 284 cells, T = 0 s; 353 cells, T = 320 s; **, P < 0.0001, unpaired t test), and Taxol (n = 275 cells, T = 0 s; 226 cells, T = 320 s; **, P < 0.0001, unpaired t test). The notch is the median, and bottom and top edges of the box are the 25th and 75th percentiles; whiskers extend to the most extreme data points. (C) Myosin assembly and pulses still occur after inhibiting microtubule dynamics. Graphs show apical area and myosin intensity over time in representative single cells in DMSO- (top) and Taxol-injected (bottom) embryos. (D) Disrupting microtubule dynamics does not initially interfere with the correlation between myosin increase and area reduction. Plotted is the mean time-resolved correlation function between constriction rate (positive constriction = area decrease) and myosin rate. Data are from three representative embryos injected with DMSO (n = 548 cells) or Taxol (n = 543 cells). Error bars represent the standard error of the mean. (E) Microtubule disruption does not affect apical ROCK polarity. Images show apical–basal cross sections from representative live embryos expressing ROCK::GFP and Gap43mCH injected with DMSO, colchicine, or Taxol. (F) Apical spot adherens junctions are unaffected by microtubules disruption. Images are apical surface projections of representative live embryos expressing E-cadherin::GFP and Myo::mCH injected with DMSO (left), colchicine (middle), and Taxol (right). (G) Quantification of junctional E-cadherin enrichment. Individual cells at the onset of constriction were segmented and the ratio of average raw junctional to medioapical E-cadherin::GFP intensity was calculated for two or three representative DMSO- (n = 122 cells), colchicine- (n = 67 cells), and Taxol-injected (n = 105) embryos (**, P < 0.0001, unpaired t test). Bottom and top edges of the box are the 25th and 75th percentiles, and whiskers extend to the most extreme data points; outliers are included (gray squares). (H) Apical spot junctions are pulled in during initial cell constrictions after microtubule disruption. Time-lapse images showing a single apical (cyan) and subapical (red) slice of representative embryos expressing E-cadherin::GFP injected with colchicine (left) and Taxol (right). Arrowheads point to an apical spot junction that has been pulled inwards in the second frame. Scale bars represent 15 µm (A), 10 µm (E), and 5 µm (F–H).

Apical myosin activation and apical constriction initiation do not require microtubules. (A) Disrupting microtubules prevents folding, despite apical myosin activation and apical constriction initiation. Time-lapse images are maximum-intensity Z-projections from representative live embryos expressing Myo::GFP (apical surface) and Gap43::mCH (membrane, subapical section) injected with DMSO (left), colchicine (5 mg/ml; middle), and Taxol (5 mg/ml; right). Apical–basal cross sections (yz) are to the right of each image. Dashed line indicates the ventral furrow. (B) Apical area initiates reduction after disrupting microtubules. Quantification of apical cell areas preconstriction (t = 0 s) and 320 s after from three representative live embryos injected with DMSO (n = 226 cells, T = 0 s; 252 cells, T = 320 s; **, P < 0.0001, unpaired t test), colchicine (n = 284 cells, T = 0 s; 353 cells, T = 320 s; **, P < 0.0001, unpaired t test), and Taxol (n = 275 cells, T = 0 s; 226 cells, T = 320 s; **, P < 0.0001, unpaired t test). The notch is the median, and bottom and top edges of the box are the 25th and 75th percentiles; whiskers extend to the most extreme data points. (C) Myosin assembly and pulses still occur after inhibiting microtubule dynamics. Graphs show apical area and myosin intensity over time in representative single cells in DMSO- (top) and Taxol-injected (bottom) embryos. (D) Disrupting microtubule dynamics does not initially interfere with the correlation between myosin increase and area reduction. Plotted is the mean time-resolved correlation function between constriction rate (positive constriction = area decrease) and myosin rate. Data are from three representative embryos injected with DMSO (n = 548 cells) or Taxol (n = 543 cells). Error bars represent the standard error of the mean. (E) Microtubule disruption does not affect apical ROCK polarity. Images show apical–basal cross sections from representative live embryos expressing ROCK::GFP and Gap43mCH injected with DMSO, colchicine, or Taxol. (F) Apical spot adherens junctions are unaffected by microtubules disruption. Images are apical surface projections of representative live embryos expressing E-cadherin::GFP and Myo::mCH injected with DMSO (left), colchicine (middle), and Taxol (right). (G) Quantification of junctional E-cadherin enrichment. Individual cells at the onset of constriction were segmented and the ratio of average raw junctional to medioapical E-cadherin::GFP intensity was calculated for two or three representative DMSO- (n = 122 cells), colchicine- (n = 67 cells), and Taxol-injected (n = 105) embryos (**, P < 0.0001, unpaired t test). Bottom and top edges of the box are the 25th and 75th percentiles, and whiskers extend to the most extreme data points; outliers are included (gray squares). (H) Apical spot junctions are pulled in during initial cell constrictions after microtubule disruption. Time-lapse images showing a single apical (cyan) and subapical (red) slice of representative embryos expressing E-cadherin::GFP injected with colchicine (left) and Taxol (right). Arrowheads point to an apical spot junction that has been pulled inwards in the second frame. Scale bars represent 15 µm (A), 10 µm (E), and 5 µm (F–H).

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