Figure 3.
NMY-2 cortical stripping by LIN-5 is not a key determinant for the cleavage furrow induction. (A and B) Representative images of the cortical plane from the time-lapse confocal microscopy of the one-cell embryo expressing endogenously tagged NMY-2 with GFP (GFP-NMY-2; in green) in control (A; n = 13) and LIN-5–inhibited [lin-5(RNAi); lin-5(ts)] (B; n = 9) embryos starting from 100 s after NEBD, as indicated. See the corresponding Videos 1 and 2. Please note that LIN-5–inhibited embryos show significant clearance of NMY-2 from the anterior cortical surface at 200 s after NEBD, which is the time when control embryos show furrow induction. Also, note a delay and a significant weak accumulation of GFP-NMY-2 at 200 and 250 s after NEBD at the equatorial cortex in the LIN-5–inhibited embryo. Time is w.r.t NEBD, deduced by the entry of the GFP-NMY-2 signal in the nucleus (not shown). n is the number of embryos analyzed. The scale bar is 10 µm. (C) Schematic illustrating the method used to analyze cortical GFP-NMY-2 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 GFP-NMY-2 along the A-P axis (see Materials and methods for details). A: anterior; P: posterior. (D) Graphs show the mean cortical intensity of GFP-NMY-2 along the A-P axis (% embryo length A-P) at various time points (in seconds) of control and LIN-5–inhibited embryos after NEBD. Values were normalized by dividing by the average maximum values for controls. GFP-NMY-2 intensity is significantly decreased in LIN-5–inhibited embryos at 200 s after NEBD on the anterior cortical region, compared with control embryos. Also, note a delay and a significant weak accumulation of GFP-NMY-2 at 200 and 250 s after NEBD at the equatorial cortex in LIN-5–inhibited embryos compared with the control embryos. See Fig. S3, A–D for GFP-NMY-2 intensity at the central plane. 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. (E and F) Kymograph analysis (E, F; see Materials and methods) of the cortical GFP-NMY-2 signal from the control embryo (E) or the LIN-5–inhibited embryo (F). The scale bar is 10 µm. (G–J) Schematics of cell cycle stages (G and I) and the corresponding representative images from the time-lapse confocal microscopy (H and J) of embryos expressing GFP-NMY-2 (shown in green in schematic, and gray in confocal images) in zyg-9(RNAi) (n = 12) (G and H) and zyg-9 (RNAi); lin-5 (RNAi) in lin-5 (ts) background (n = 17) (I and J), as indicated. The blue asterisks on the images indicate the NMY-2 clearance zone before and at the time of furrow involution. Black arrowheads indicate NMY-2 accumulation at furrow involution. n is the number of embryos analyzed. The scale bar is 10 µm. (K–M) Digitally straightened posterior membrane (K) of an embryo coexpressing GFPMem. (in gray) and mCherryTub. (in pink) to analyze the dynamics of furrow formation in the form of pseudokymographs in zyg-9 (RNAi) (L), and zyg-9 (RNAi); lin-5 (RNAi) in lin-5 (ts) background (M), as indicated. For easy comparative analysis, only GFPMem. fluorescence signal (in gray) is shown for L and M. White arrowheads indicate furrow appearance, whereas white asterisks indicate furrow dissolution. Note that 100% of the ZYG-9–depleted embryos establish a stable posterior furrow, whereas 65% (n = 17) of zyg-9(RNAi); lin-5(RNAi) in lin-5(ts) background fail to form stable posterior furrow and show oscillations in furrow ingression followed by regression. Note the re-use of the zyg-9(RNAi) embryo image shown in Fig. 3 K for Fig. S4 C. Also, see Fig. S4, C and D. n is the number of embryos analyzed. The scale bar is 10 µm. (N and O) Digitally straightened posterior membrane of an embryo expressing GFP-NMY-2 (in gray) to analyze the dynamics of GFP-NMY-2 localization in the form of pseudokymographs in zyg-9 (RNAi) (N) and zyg-9(RNAi); lin-5(RNAi) in lin-5 (ts) background (O), as indicated. White arrowheads indicate GFP-NMY-2 localization at the furrow, whereas white asterisks indicate its dissolution. The scale bar is 10 µm. (P–R) Schematic of the method used to quantify GFP-NMY-2 fluorescence intensity at involuted posterior furrow over time w.r.t. NEBD of the male pronucleus (P), in zyg-9(RNAi) (Q) and zyg-9(RNAi); lin-5(RNAi) in lin-5(ts) background (R). NEBD timing was deduced by visualizing the entry of GFP-NMY-2 in the male pronucleus. Each line represents the NMY-2 accumulation pattern of a single embryo. The dark black line in Q and the pink line in R represent the quantification of GFP-NMY-2 of the images shown in N and O, respectively. n is the number of embryos analyzed.

NMY-2 cortical stripping by LIN-5 is not a key determinant for the cleavage furrow induction. (A and B) Representative images of the cortical plane from the time-lapse confocal microscopy of the one-cell embryo expressing endogenously tagged NMY-2 with GFP (GFP-NMY-2; in green) in control (A; n = 13) and LIN-5–inhibited [lin-5(RNAi); lin-5(ts)] (B; n = 9) embryos starting from 100 s after NEBD, as indicated. See the corresponding Videos 1 and 2. Please note that LIN-5–inhibited embryos show significant clearance of NMY-2 from the anterior cortical surface at 200 s after NEBD, which is the time when control embryos show furrow induction. Also, note a delay and a significant weak accumulation of GFP-NMY-2 at 200 and 250 s after NEBD at the equatorial cortex in the LIN-5–inhibited embryo. Time is w.r.t NEBD, deduced by the entry of the GFP-NMY-2 signal in the nucleus (not shown). n is the number of embryos analyzed. The scale bar is 10 µm. (C) Schematic illustrating the method used to analyze cortical GFP-NMY-2 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 GFP-NMY-2 along the A-P axis (see Materials and methods for details). A: anterior; P: posterior. (D) Graphs show the mean cortical intensity of GFP-NMY-2 along the A-P axis (% embryo length A-P) at various time points (in seconds) of control and LIN-5–inhibited embryos after NEBD. Values were normalized by dividing by the average maximum values for controls. GFP-NMY-2 intensity is significantly decreased in LIN-5–inhibited embryos at 200 s after NEBD on the anterior cortical region, compared with control embryos. Also, note a delay and a significant weak accumulation of GFP-NMY-2 at 200 and 250 s after NEBD at the equatorial cortex in LIN-5–inhibited embryos compared with the control embryos. See Fig. S3, A–D for GFP-NMY-2 intensity at the central plane. 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. (E and F) Kymograph analysis (E, F; see Materials and methods) of the cortical GFP-NMY-2 signal from the control embryo (E) or the LIN-5–inhibited embryo (F). The scale bar is 10 µm. (G–J) Schematics of cell cycle stages (G and I) and the corresponding representative images from the time-lapse confocal microscopy (H and J) of embryos expressing GFP-NMY-2 (shown in green in schematic, and gray in confocal images) in zyg-9(RNAi) (n = 12) (G and H) and zyg-9 (RNAi); lin-5 (RNAi) in lin-5 (ts) background (n = 17) (I and J), as indicated. The blue asterisks on the images indicate the NMY-2 clearance zone before and at the time of furrow involution. Black arrowheads indicate NMY-2 accumulation at furrow involution. n is the number of embryos analyzed. The scale bar is 10 µm. (K–M) Digitally straightened posterior membrane (K) of an embryo coexpressing GFPMem. (in gray) and mCherryTub. (in pink) to analyze the dynamics of furrow formation in the form of pseudokymographs in zyg-9 (RNAi) (L), and zyg-9 (RNAi); lin-5 (RNAi) in lin-5 (ts) background (M), as indicated. For easy comparative analysis, only GFPMem. fluorescence signal (in gray) is shown for L and M. White arrowheads indicate furrow appearance, whereas white asterisks indicate furrow dissolution. Note that 100% of the ZYG-9–depleted embryos establish a stable posterior furrow, whereas 65% (n = 17) of zyg-9(RNAi); lin-5(RNAi) in lin-5(ts) background fail to form stable posterior furrow and show oscillations in furrow ingression followed by regression. Note the re-use of the zyg-9(RNAi) embryo image shown in Fig. 3 K for Fig. S4 C. Also, see Fig. S4, C and D. n is the number of embryos analyzed. The scale bar is 10 µm. (N and O) Digitally straightened posterior membrane of an embryo expressing GFP-NMY-2 (in gray) to analyze the dynamics of GFP-NMY-2 localization in the form of pseudokymographs in zyg-9 (RNAi) (N) and zyg-9(RNAi); lin-5(RNAi) in lin-5 (ts) background (O), as indicated. White arrowheads indicate GFP-NMY-2 localization at the furrow, whereas white asterisks indicate its dissolution. The scale bar is 10 µm. (P–R) Schematic of the method used to quantify GFP-NMY-2 fluorescence intensity at involuted posterior furrow over time w.r.t. NEBD of the male pronucleus (P), in zyg-9(RNAi) (Q) and zyg-9(RNAi); lin-5(RNAi) in lin-5(ts) background (R). NEBD timing was deduced by visualizing the entry of GFP-NMY-2 in the male pronucleus. Each line represents the NMY-2 accumulation pattern of a single embryo. The dark black line in Q and the pink line in R represent the quantification of GFP-NMY-2 of the images shown in N and O, respectively. n is the number of embryos analyzed.

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