Figure 4.
Analysis of dynamic distribution of spindle microtubules in live bovine zygotes. (A–F) Microtubule signal (EGFP-MAP4 or EB3-mEGFP2) from live imaging of bovine zygotes by light-sheet microscopy every 2.5 min with a spindle assembly type as described in Fig. 3 A was analyzed for 10 time points starting 2.5 min before NEBD of the leading PN (PN1) or both PNi. (A) Pseudocolor representation of EGFP-MAP4 signal within single planes through the centers of intensities at centrosomes. Corresponding lookup table is depicted in last frame of the time series. 6 of 10 analyzed time frames were selected to visualize critical time points for microtubule redistribution in early spindle assembly. Time in minutes respective to NEBD. Scale bar, 10 µm. (B–D) Measuring intensity distribution of microtubule signal along the centrosomal axis over time. (B) Scheme illustrating the measurements. After background subtraction, total microtubule intensities were calculated for 15 equidistantly distributed 2D slices within the black cuboids along the centrosomal axis for the different time points. NEBD is marked by an asterisk. Black cuboid with solid line encompasses 351 × 351 pixel–sized slices to measure entire microtubule intensity or mass along the spindle axis. Small black cuboid with dashed line encompasses 15 × 15 pixel–sized slices to indicate relative microtubule concentrations. Maximum normalized total intensities along the normalized centrosome distance were annotated. (C and D) Average distribution of maximum normalized microtubule intensities along centrosomal axis indicating relative microtubule mass within spindles over time (C; black cuboid with solid line, as described in B) and relative microtubule concentrations (D; black cuboid with dashed line, as described in B); n = 6 zygotes. Dashed lines mark the position of the 2D slice through the centrosomes (lines in dark gray) and the centrosome axis midpoint (line in light gray). Color gradient from red to blue indicates time in minutes respective to NEBD. Time of NEBD is indicated by an asterisk. (E and F) Average change of normalized total microtubule intensity for total microtubule mass (E) and relative microtubule concentrations (F) over time from NEBD until 20 min after NEBD, at centrosomes and the centrosome axis midpoint, indicated by dashed lines in dark and light gray in C and D, respectively; n = 6 zygotes. For intensity change at centrosome, mean intensity of both centrosomes was calculated.

Analysis of dynamic distribution of spindle microtubules in live bovine zygotes. (A–F) Microtubule signal (EGFP-MAP4 or EB3-mEGFP2) from live imaging of bovine zygotes by light-sheet microscopy every 2.5 min with a spindle assembly type as described in Fig. 3 A was analyzed for 10 time points starting 2.5 min before NEBD of the leading PN (PN1) or both PNi. (A) Pseudocolor representation of EGFP-MAP4 signal within single planes through the centers of intensities at centrosomes. Corresponding lookup table is depicted in last frame of the time series. 6 of 10 analyzed time frames were selected to visualize critical time points for microtubule redistribution in early spindle assembly. Time in minutes respective to NEBD. Scale bar, 10 µm. (B–D) Measuring intensity distribution of microtubule signal along the centrosomal axis over time. (B) Scheme illustrating the measurements. After background subtraction, total microtubule intensities were calculated for 15 equidistantly distributed 2D slices within the black cuboids along the centrosomal axis for the different time points. NEBD is marked by an asterisk. Black cuboid with solid line encompasses 351 × 351 pixel–sized slices to measure entire microtubule intensity or mass along the spindle axis. Small black cuboid with dashed line encompasses 15 × 15 pixel–sized slices to indicate relative microtubule concentrations. Maximum normalized total intensities along the normalized centrosome distance were annotated. (C and D) Average distribution of maximum normalized microtubule intensities along centrosomal axis indicating relative microtubule mass within spindles over time (C; black cuboid with solid line, as described in B) and relative microtubule concentrations (D; black cuboid with dashed line, as described in B); n = 6 zygotes. Dashed lines mark the position of the 2D slice through the centrosomes (lines in dark gray) and the centrosome axis midpoint (line in light gray). Color gradient from red to blue indicates time in minutes respective to NEBD. Time of NEBD is indicated by an asterisk. (E and F) Average change of normalized total microtubule intensity for total microtubule mass (E) and relative microtubule concentrations (F) over time from NEBD until 20 min after NEBD, at centrosomes and the centrosome axis midpoint, indicated by dashed lines in dark and light gray in C and D, respectively; n = 6 zygotes. For intensity change at centrosome, mean intensity of both centrosomes was calculated.

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