Figure S5.
LIN-5 controls the timely enrichment of Anillin at the equatorial cortex. (A and B) Representative images of the cortical plane from the time-lapse confocal microscopy of one-cell embryo coexpressing endogenously tagged Anillin with mNG (mNG-ANI-1; in cyan) and NMY-2 with mKate. Note that only the mNG-ANI-1 signal was imaged using confocal microscope as it was the brightest for live recording. Images show control (A) and LIN-5–depleted embryos (B) starting from 100 s after NEBD, as indicated. Also, see corresponding Videos 5 and 6. Note the significantly better clearance of the mNG-ANI-1 signal at the anterior cortex in LIN-5–depleted embryos at the time of furrow initiation w.r.t. control embryos. Also note a weak accumulation of mNG-ANI-1 signal at the equatorial membrane in the lin-5(RNAi) embryo compared with control. Time is w.r.t NEBD, deduced by studying the entry of the mNG-ANI-1 signal in the nucleus (not shown). The scale bar is 10 µm. (C) Schematic illustrating the method used to analyze cortical mNG-ANI-1 distribution, as performed by Lewellyn et al. (2010). In brief, a 50-pixel-wide line (∼1/2 the embryo width) was drawn, and embryos were divided into 20 equal-length segments from A (0%) to P (100%) to calculate the mean cortical accumulation of mNG-ANI-1 along the A-P axis (see Materials and methods for details). A: anterior; P: posterior. (D) Quantification indicates the mean cortical accumulation of mNG-ANI-1 intensity along the A-P axis (% embryo length A-P) at various time points (in seconds) after NEBD. Values were normalized by dividing by the average maximum values for controls. Control embryos are shown in a black line, and LIN-5–depleted embryos are indicated by a pink line. mNG-ANI-1 intensity is observed to be significantly decreased at 200 s on the anterior polar cortex in LIN-5–depleted embryos relative to control embryos. Also note that there is a significantly weak localization of mNG-ANI-1 at the equatorial cortical region at 200 s and at 250 s after NEBD in lin-5(RNAi) embryos in comparison with the control embryos. n is the number of embryos analyzed. Error bars are SEM; ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001, as determined by two-tailed Student’s t test. (E and F) Representative images of the central plane from the time-lapse confocal microscopy of the one-cell embryo coexpressing endogenous tagged mNG-ANI-1 (in gray) and NMY-2 with mKate. Note that only the mNG-ANI-1 signal was imaged using confocal microscope as it was the brightest for live recording. Images show control (E) and LIN-5–depleted embryos (F) starting from 100 s after NEBD, as indicated. Also, please see the corresponding Videos 7 and 8. Note the significantly better clearance of the mNG-ANI-1 signal at the anterior cortex in LIN-5–depleted embryos at the time of furrow initiation w.r.t. control embryos. Also, note a significant accumulation of mNG-ANI-1 signal at the equatorial membrane during furrow initiation in control embryo w.r.t. LIN-5–depleted embryos. Time is w.r.t NEBD, deduced by the entry of the mNG-ANI-1 signal in the nucleus. The scale bar is 10 µm. (G) Schematic illustrating the method used to analyze cortical mNG-ANI-1 distribution. In brief, a one-pixel-wide line scan along the cell cortex was drawn from A to P. Embryos were divided into 20 equal-length segments from A (0%) to P (100%), which were used to calculate the mean cortical intensity (see Materials and methods for details). A: anterior; P: posterior. (H) Quantification indicates the mean cortical intensity of mNG-ANI-1 along the line scan (% embryo length A-P) at various time points (in seconds) after NEBD. Values were normalized by dividing by the average maximum values for controls. Control embryos are shown by the black line, and LIN-5–depleted embryos are shown by the pink line. mNG-ANI-1 intensity is significantly decreased at 200 s on the anterior polar cortex relative to control embryos. Also note that there is a significantly weak localization of mNG-ANI-1 at the equatorial cortical region at 200 s and at 250 s after NEBD in lin-5(RNAi) embryos in comparison with the control embryos. n is the number of embryos analyzed. Error bars are the SEM; ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001, as determined by two-tailed Student’s t test.

LIN-5 controls the timely enrichment of Anillin at the equatorial cortex. (A and B) Representative images of the cortical plane from the time-lapse confocal microscopy of one-cell embryo coexpressing endogenously tagged Anillin with mNG (mNG-ANI-1; in cyan) and NMY-2 with mKate. Note that only the mNG-ANI-1 signal was imaged using confocal microscope as it was the brightest for live recording. Images show control (A) and LIN-5–depleted embryos (B) starting from 100 s after NEBD, as indicated. Also, see corresponding Videos 5 and 6. Note the significantly better clearance of the mNG-ANI-1 signal at the anterior cortex in LIN-5–depleted embryos at the time of furrow initiation w.r.t. control embryos. Also note a weak accumulation of mNG-ANI-1 signal at the equatorial membrane in the lin-5(RNAi) embryo compared with control. Time is w.r.t NEBD, deduced by studying the entry of the mNG-ANI-1 signal in the nucleus (not shown). The scale bar is 10 µm. (C) Schematic illustrating the method used to analyze cortical mNG-ANI-1 distribution, as performed by Lewellyn et al. (2010). In brief, a 50-pixel-wide line (∼1/2 the embryo width) was drawn, and embryos were divided into 20 equal-length segments from A (0%) to P (100%) to calculate the mean cortical accumulation of mNG-ANI-1 along the A-P axis (see Materials and methods for details). A: anterior; P: posterior. (D) Quantification indicates the mean cortical accumulation of mNG-ANI-1 intensity along the A-P axis (% embryo length A-P) at various time points (in seconds) after NEBD. Values were normalized by dividing by the average maximum values for controls. Control embryos are shown in a black line, and LIN-5–depleted embryos are indicated by a pink line. mNG-ANI-1 intensity is observed to be significantly decreased at 200 s on the anterior polar cortex in LIN-5–depleted embryos relative to control embryos. Also note that there is a significantly weak localization of mNG-ANI-1 at the equatorial cortical region at 200 s and at 250 s after NEBD in lin-5(RNAi) embryos in comparison with the control embryos. n is the number of embryos analyzed. Error bars are SEM; ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001, as determined by two-tailed Student’s t test. (E and F) Representative images of the central plane from the time-lapse confocal microscopy of the one-cell embryo coexpressing endogenous tagged mNG-ANI-1 (in gray) and NMY-2 with mKate. Note that only the mNG-ANI-1 signal was imaged using confocal microscope as it was the brightest for live recording. Images show control (E) and LIN-5–depleted embryos (F) starting from 100 s after NEBD, as indicated. Also, please see the corresponding Videos 7 and 8. Note the significantly better clearance of the mNG-ANI-1 signal at the anterior cortex in LIN-5–depleted embryos at the time of furrow initiation w.r.t. control embryos. Also, note a significant accumulation of mNG-ANI-1 signal at the equatorial membrane during furrow initiation in control embryo w.r.t. LIN-5–depleted embryos. Time is w.r.t NEBD, deduced by the entry of the mNG-ANI-1 signal in the nucleus. The scale bar is 10 µm. (G) Schematic illustrating the method used to analyze cortical mNG-ANI-1 distribution. In brief, a one-pixel-wide line scan along the cell cortex was drawn from A to P. Embryos were divided into 20 equal-length segments from A (0%) to P (100%), which were used to calculate the mean cortical intensity (see Materials and methods for details). A: anterior; P: posterior. (H) Quantification indicates the mean cortical intensity of mNG-ANI-1 along the line scan (% embryo length A-P) at various time points (in seconds) after NEBD. Values were normalized by dividing by the average maximum values for controls. Control embryos are shown by the black line, and LIN-5–depleted embryos are shown by the pink line. mNG-ANI-1 intensity is significantly decreased at 200 s on the anterior polar cortex relative to control embryos. Also note that there is a significantly weak localization of mNG-ANI-1 at the equatorial cortical region at 200 s and at 250 s after NEBD in lin-5(RNAi) embryos in comparison with the control embryos. n is the number of embryos analyzed. Error bars are the SEM; ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001, as determined by two-tailed Student’s t test.

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