Figure 3. Ring myosin compresses cortical surface along the axis perpendicular to the ring, pulling in new cortical surface at a rate proportional to the amount of ring myosin.

(A) The equatorial cortex is compressed during contractile ring assembly. Following the onset of spindle-based RhoA signaling, the initial recruitment of contractile ring proteins leads to uniform compression of cortical surface along the axis perpendicular to the forming ring across a 10 µm wide region spanning the cell equator. (left) Average flow map at (t/t_CK = −0.1) immediately after the onset of spindle-based signaling (n = 93 embryos). (middle) The surface velocity profile reveals a linear velocity gradient that spans the cell equator (−5 to +5 µm), indicating a uniform zone of cortical compression. (B) Cortical compression within the ring continues during constriction. (left graph) Plot comparing the area of the forming division plane (red) with the total cortical surface area that entered the division plane from the start of cytokinesis (purple; calculated as indicated in the schematic). (right graph) Plot comparing the rate of delivery of cortical surface into the division plane (purple) with the rate of growth of the division plane (red). The difference between the two is the rate of cortical surface compression (rate of reduction of cortical surface area; cyan). (C) The per-unit-length amount of ring myosin and the rate of cortical compression increase with the same exponential kinetics, suggesting that the rate of cortical compression may be controlled by the amount of myosin in the contractile ring/Rho zone. (top left) Representative images of the division plane in embryos expressing myosin::GFP reconstructed from 40-plane z-stacks. Gold circles mark the embryo boundary and dashed circles mark the boundaries used for ring intensity measurements. Scale bar is 10 µm. (top right) Graph plots per-unit-length myosin::GFP fluorescence for the indicated angular ranges (n = 36 embryos). (bottom left) Graph plots the rate of cortical surface compression per unit ring length (n = 93 embryos). (bottom middle) Graphs plot mean per-unit-length myosin::GFP (n = 36 embryos) and GFP::anillin (n = 26 embryos) fluorescence (n = 36 embryos) in the ring. (bottom right) Graph plots the per-unit-length rate of ring closure. Black lines are fitted single exponentials. Error bars are the SEM.

Figure 3—figure supplement 1. Arp2/3 inhibition abolishes the asymmetry in the amount of cortex entering the division plane from the anterior and posterior sides.

Graphs plot the rate of cortical flux across the anterior (light grey) and posterior (dark grey) boundaries (see schematic in Figure 3B) versus the mean for the two sides (purple) for control and arx-2(RNAi) embryos. Calculated from the average flow maps for the control (n = 93 embryos) and arx-2(RNAi) (n = 68 embryos) conditions.

Figure 3—figure supplement 2. GFP::anillin fluorescence in the ring increases exponentially during constriction.

(A) (left) Schematic of the single-copy gfp::ani-1 trangene. The transgene was re-encoded while maintaining amino acid sequence in the indicated region to render it resistant to RNAi targeting of the endogenous ani-1 gene to allow testing of the functionality of the GFP::ANI-1 fusion. (right) Graph plotting embryonic lethality demonstrates that the gfp::ani-1 transgene is functional. (B) (top) Images of the division plane in an embryo expressing GFP::anillin. Scale bar is 10 µm. (bottom) Graph plots GFP::anillin fluorescence per unit length of the ring for the indicated angular ranges. Error bars are the SEM.

Figure 3—figure supplement 3. Correcting for signal attenuation with sample depth.

Fluorescence attenuation with embryo depth was estimated from fluorescence intensity measurements made at the cell-cell boundary of two-cell embryos expressing a GFP-tagged plasma membrane marker. Cell-cell boundaries were reconstructed from 40 plane z-stacks. The intensity profile at each slice was calculated by subtracting the average background intensity estimated from dashed rectangles (left) from the cell-cell boundary region (black rectangle) at each slice and calculating the maximum intensity projection along AP axis. The effect of depth on signal was calculated from the reconstructed division planes by plotting the mean signal as a function of depth in 10 rectangular regions (white boxes) where the signal was expected to be uniform; three examples are shown here. All intensity profiles were simultaneously fitted using a single exponential. Error bars are the SD. On the right, the same cell-cell boundaries are shown after correction for depth attenuation. The scale bar is 10 µm.

Figure 3—figure supplement 4. Ring component dynamics at the four-cell stage are consistent with exponential accumulation.

(A) (left) Schematic illustrating the relative geometries of cytokinesis in one- and four-cell stage C. elegans embryos. (right) The range of ring sizes between furrow formation and contact with the midzone, which occurs at a ring radius of about 3.5 µm (perimeter ~22 µm) in all divisions and alters constriction rate and component accumulation (Carvalho et al., 2009), is much smaller at the four-cell stage than at the one-cell stage. (B) Myosin levels in the ring can only be monitored over a limited range of ring size at the four-cell stage. Images of the division plane in a representative dividing cell at the four-cell stage reconstructed from 16 × 1 µm z-stacks of an embryo expressing myosin::GFP (n = 16 embryos imaged). The range of ring sizes between the point when the folding in of the furrow first enables monitoring of ring component levels in the end-on view, and the point when the ring contacts the spindle midzone, is indicated (green; Measurement zone). (C) Graphs plotting measured mean per-unit-length myosin::GFP fluorescence in the ring at the four-cell stage. The graph on the left is reproduced from Figure 4D of Carvalho et al. (2009) where a strain with an integrated myosin::GFP transgene under an exogenous promoter was filmed. The graph on the right is new data collected in the in situ-tagged myosin::GFP strain. The measurement zone highlighted in B (from 50 to 20 µm ring perimeter is highlighted in green for both graphs). In the graph on the right, the data for rings in the measurement zone were fit to an exponential equation with the same baseline contribution as the one-cell stage data in Figure 3C (black line). Error bars are the SEM.

© 2009 Elsevier

Figure 3—figure supplement 4C (left graph) reproduced with permission from Figure 5 of Carvalho et al. (2009).