Figure S3.
LIN-5 controls furrow initiation timing independent of its function in removing NMY-2 from the polar cortical region. (A and B) Representative images of the central plane from the time-lapse confocal microscopy of the one-cell embryo expressing endogenously tagged NMY-2 with GFP (GFP-NMY-2; in gray) in control (A) and LIN-5–inhibited [lin-5(RNAi); lin-5(ts)] (B) embryos starting from 100 s after NEBD, as indicated. Also, please see the corresponding Videos 3 and 4. Note that ∼100% of LIN-5–inhibited embryos (n = 11) reveal much stronger NMY-2 clearance from the anterior cortical surface during anaphase at ∼200 s in comparison with control embryos. Also, note a significantly weak accumulation of GFP-NMY-2 at the equatorial membrane at the time of furrow initiation in LIN-5–inhibited embryos. Time is w.r.t NEBD, deduced by the entry of the GFP-NMY-2 signal in the nucleus. The scale bar is 10 µm. (C) Schematic illustrating the method used to analyze cortical GFP-NMY-2 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. (D) Quantification indicates the mean cortical intensity of GFP-NMY-2 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. GFP-NMY-2 intensity is observed to be significantly decreased in LIN-5–inhibited embryos at 200 s on the anterior polar cortex relative to control embryos. Also, note that there is a significantly weak localization of GFP-NMY-2 at the equatorial membrane at 200 s and at 250 s after NEBD in LIN-5–inhibited embryos compared with the control embryos; also see Fig. 3 for cortical GFP-NMY-2 intensity measurements. 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) Examples of cortical GFP-NMY-2 accumulation in control (E) and LIN-5–inhibited (F) embryos at 200 s after NEBD at the anterior cell cortex. The scale bar is 10 μm. Boxed regions (i and ii) are the magnified view of the corresponding insets. Here, the scale bar is 5 μm. (G and H) 3D projected confocal images of one-cell embryo coexpressing GFP-NMY-2 and GFPTub. (in green) in control (A) and lin-5(RNAi) (B) embryos. The scale bar is 10 µm. (I–P) Schematic of the method (I, K, M, and O) and the quantification (J, L, N, and P) for the A/P ratio of GFP-NMY-2 fluorescence intensity at the polar cortices (I and J); A pole-to-cortex distance (K and L); P pole-to-cortex distance (M and N), and the A-to-P pole-to-cortex distance ratio (O and P), as indicated. In this and other Fig. panels, Mem. represents membrane intensity, and Bkgd. represents background intensity. A: anterior; P: posterior. n is the number of embryos analyzed. Error bars are the SEM. (Q and R) Representative images of the posterior cortical region (shown on the top left) used for the pseudokymograph analysis from the time-lapse confocal microscopy of a one-cell embryo expressing endogenously tagged NMY-2 with GFP (GFP-NMY-2; in green) in lin-5(ts) mutant embryos and depleted for LIN-5 by RNAi (Q), or codepleted for LIN-5 and HCP-4. Time is w.r.t. NEBD. The scale bar is 10 and 5 μm for the embryo and pseudokymograph, respectively. (S and T) Schematic of the method used to quantify GFP-NMY-2 fluorescence intensity at the posterior cortex over time (S). Quantification of GFP-NMY-2 enrichment at the posterior cortex in embryos depleted for LIN-5 in lin-5(ts) background (pink line), as well as depleted for LIN-5 and HCP-4 in lin-5(ts) background (blue line). Time is w.r.t. NEBD. n is the number of embryos analyzed. Error bars, SEM. ns, P > 0.05; *P < 0.05, as determined by two-tailed Student’s t test.

LIN-5 controls furrow initiation timing independent of its function in removing NMY-2 from the polar cortical region. (A and B) Representative images of the central plane from the time-lapse confocal microscopy of the one-cell embryo expressing endogenously tagged NMY-2 with GFP (GFP-NMY-2; in gray) in control (A) and LIN-5–inhibited [lin-5(RNAi); lin-5(ts)] (B) embryos starting from 100 s after NEBD, as indicated. Also, please see the corresponding Videos 3 and 4. Note that ∼100% of LIN-5–inhibited embryos (n = 11) reveal much stronger NMY-2 clearance from the anterior cortical surface during anaphase at ∼200 s in comparison with control embryos. Also, note a significantly weak accumulation of GFP-NMY-2 at the equatorial membrane at the time of furrow initiation in LIN-5–inhibited embryos. Time is w.r.t NEBD, deduced by the entry of the GFP-NMY-2 signal in the nucleus. The scale bar is 10 µm. (C) Schematic illustrating the method used to analyze cortical GFP-NMY-2 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. (D) Quantification indicates the mean cortical intensity of GFP-NMY-2 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. GFP-NMY-2 intensity is observed to be significantly decreased in LIN-5–inhibited embryos at 200 s on the anterior polar cortex relative to control embryos. Also, note that there is a significantly weak localization of GFP-NMY-2 at the equatorial membrane at 200 s and at 250 s after NEBD in LIN-5–inhibited embryos compared with the control embryos; also see Fig. 3 for cortical GFP-NMY-2 intensity measurements. 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) Examples of cortical GFP-NMY-2 accumulation in control (E) and LIN-5–inhibited (F) embryos at 200 s after NEBD at the anterior cell cortex. The scale bar is 10 μm. Boxed regions (i and ii) are the magnified view of the corresponding insets. Here, the scale bar is 5 μm. (G and H) 3D projected confocal images of one-cell embryo coexpressing GFP-NMY-2 and GFPTub. (in green) in control (A) and lin-5(RNAi) (B) embryos. The scale bar is 10 µm. (I–P) Schematic of the method (I, K, M, and O) and the quantification (J, L, N, and P) for the A/P ratio of GFP-NMY-2 fluorescence intensity at the polar cortices (I and J); A pole-to-cortex distance (K and L); P pole-to-cortex distance (M and N), and the A-to-P pole-to-cortex distance ratio (O and P), as indicated. In this and other Fig. panels, Mem. represents membrane intensity, and Bkgd. represents background intensity. A: anterior; P: posterior. n is the number of embryos analyzed. Error bars are the SEM. (Q and R) Representative images of the posterior cortical region (shown on the top left) used for the pseudokymograph analysis from the time-lapse confocal microscopy of a one-cell embryo expressing endogenously tagged NMY-2 with GFP (GFP-NMY-2; in green) in lin-5(ts) mutant embryos and depleted for LIN-5 by RNAi (Q), or codepleted for LIN-5 and HCP-4. Time is w.r.t. NEBD. The scale bar is 10 and 5 μm for the embryo and pseudokymograph, respectively. (S and T) Schematic of the method used to quantify GFP-NMY-2 fluorescence intensity at the posterior cortex over time (S). Quantification of GFP-NMY-2 enrichment at the posterior cortex in embryos depleted for LIN-5 in lin-5(ts) background (pink line), as well as depleted for LIN-5 and HCP-4 in lin-5(ts) background (blue line). Time is w.r.t. NEBD. n is the number of embryos analyzed. Error bars, SEM. ns, P > 0.05; *P < 0.05, as determined by two-tailed Student’s t test.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal