Figure 4.
Figure 4. Local pulses of RhoA activation underlie pulsed accumulation and disappearance of F-actin and Myosin II. (A) Micrograph of a two-cell–stage embryo expressing GFP::AHPH as a reporter for RhoA activity and NMY-2::RFP. (B) Temporal dynamics of GFP::AHPH and NMY-2::RFP accumulation during a single pulse. The time between frames is 2 s for the first five frames and 4 s thereafter. (C) Top: Normalized fluorescence intensities of GFP::AHPH and NMY-2::RFP. Bottom: A kymograph showing that local contraction (concerted movements of myosin puncta) begins after the accumulation of GFP::AHPH. The yellow box on the bottom left indicates the region used to generate the kymograph. (D) Comparison of averaged normalized fluorescence intensities versus time for active RhoA (GFP::AHPH), Myosin (NMY-2::GFP), and F-actin (GFP::UTR) from two-color data, coaligned with respect to a common reference signal (NMY-2:RFP). Data were coaligned with respect to the time at which NMY-2::RFP reaches 25% threshold. Shaded areas represent 95% confidence intervals. (E) Decomposition of the GFP::AHPH signal into a diffuse pool (green dashed line) and myosin-colocalized pool (green solid line; see Fig. S3 and Materials and methods for details). Data for NMY-2::RFP and total GFP::AHPH are the same as in D. (F) Distribution of the delays between the onset of accumulation of NMY-2::RFP, and the onset of accumulation of GFP::AHPH, NMY-2::GFP, and GFP::UTR. Onset of accumulation was measured as the time at which normalized probe intensity rose above 25% of its maximal level. (G) Distribution of the delays between the onset of disappearance of NMY-2:RFP and the onset of disappearance of GFP::AHPH, NMY-2::GFP, and GFP::UTR. The onset of disappearance was measured as the time at which normalized probe intensity fell below 75% of its maximal level. Data in D–G were averaged over the following—GFP::AHPH: n = 41 pulses in 7 embryos; GFP::UTR: n = 33 pulses in 13 embryos; NMY-2::GFP: n = 22 pulses in 4 embryos. In F and G box plots, the central mark represents the median, the box indicates the 25th and 75th percentile, the whiskers mark the minimum and maximum values, + represents outliers, and * indicates statistically significant differences with P < 0.01. Bars: (A) 5 µm; (B and C) 2 µm.

Local pulses of RhoA activation underlie pulsed accumulation and disappearance of F-actin and Myosin II. (A) Micrograph of a two-cell–stage embryo expressing GFP::AHPH as a reporter for RhoA activity and NMY-2::RFP. (B) Temporal dynamics of GFP::AHPH and NMY-2::RFP accumulation during a single pulse. The time between frames is 2 s for the first five frames and 4 s thereafter. (C) Top: Normalized fluorescence intensities of GFP::AHPH and NMY-2::RFP. Bottom: A kymograph showing that local contraction (concerted movements of myosin puncta) begins after the accumulation of GFP::AHPH. The yellow box on the bottom left indicates the region used to generate the kymograph. (D) Comparison of averaged normalized fluorescence intensities versus time for active RhoA (GFP::AHPH), Myosin (NMY-2::GFP), and F-actin (GFP::UTR) from two-color data, coaligned with respect to a common reference signal (NMY-2:RFP). Data were coaligned with respect to the time at which NMY-2::RFP reaches 25% threshold. Shaded areas represent 95% confidence intervals. (E) Decomposition of the GFP::AHPH signal into a diffuse pool (green dashed line) and myosin-colocalized pool (green solid line; see Fig. S3 and Materials and methods for details). Data for NMY-2::RFP and total GFP::AHPH are the same as in D. (F) Distribution of the delays between the onset of accumulation of NMY-2::RFP, and the onset of accumulation of GFP::AHPH, NMY-2::GFP, and GFP::UTR. Onset of accumulation was measured as the time at which normalized probe intensity rose above 25% of its maximal level. (G) Distribution of the delays between the onset of disappearance of NMY-2:RFP and the onset of disappearance of GFP::AHPH, NMY-2::GFP, and GFP::UTR. The onset of disappearance was measured as the time at which normalized probe intensity fell below 75% of its maximal level. Data in D–G were averaged over the following—GFP::AHPH: n = 41 pulses in 7 embryos; GFP::UTR: n = 33 pulses in 13 embryos; NMY-2::GFP: n = 22 pulses in 4 embryos. In F and G box plots, the central mark represents the median, the box indicates the 25th and 75th percentile, the whiskers mark the minimum and maximum values, + represents outliers, and * indicates statistically significant differences with P < 0.01. Bars: (A) 5 µm; (B and C) 2 µm.

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