Quantitative analysis of cytokinesis in situ during C. elegans postembryonic development

Abstract

The physical separation of a cell into two daughter cells during cytokinesis requires cell-intrinsic shape changes driven by a contractile ring. However, in vivo, cells interact with their environment, which includes other cells. How cytokinesis occurs in tissues is not well understood. Here, we studied cytokinesis in an intact animal during tissue biogenesis. We used high-resolution microscopy and quantitative analysis to study the three rounds of division of the C. elegans vulval precursor cells (VPCs). The VPCs are cut in half longitudinally with each division. Contractile ring breadth, but not the speed of ring closure, scales with cell length. Furrowing speed instead scales with division plane dimensions, and scaling is consistent between the VPCs and C. elegans blastomeres. We compared our VPC cytokinesis kinetics data with measurements from the C. elegans zygote and HeLa and Drosophila S2 cells. Both the speed dynamics and asymmetry of ring closure are qualitatively conserved among cell types. Unlike in the C. elegans zygote but similar to other epithelial cells, Anillin is required for proper ring closure speed but not asymmetry in the VPCs. We present evidence that tissue organization impacts the dynamics of cytokinesis by comparing our results on the VPCs with the cells of the somatic gonad. In sum, this work establishes somatic lineages in post-embryonic C. elegans development as cell biological models for the study of cytokinesis in situ.

Figure 1. The C. elegans vulval precursor cells (VPCs) inside the living and developing animal.

(A) Schematic representation of the VPCs (P5.p, P6.p and P7.p cells) in a third larval stage (L3) worm. 3 VPC stage: undivided precursors (purple); daughter cells: 6 VPC stage (green); 12 granddaughter cells: 12 VPC stage (orange); final 22 descendants: 22 VPC stage. (B) DIC images of the VPCs at the corresponding stages shown in A. For all images, anterior is to the left and dorsal is to the top. Scale bar = 10 µm. (C) Maximum intensity projection images of worms expressing GFP-tagged non-muscle myosin II (NMY-2) in the VPCs (dotted lines) at the 3, 6, 12 and 22 VPC stages. Scale bar = 10 µm (D–E) Scatter plots of individual VPC measurements before (pre) and after (post) each round of VPC division. Cell length: ***: p<0.0001, unpaired t-test. Cell height: n.s.: p = 0.06, ***: p<0.0001, *: p = 0.02, unpaired t-test. Bars = mean with SEM. n(cells) >20 and n(worms) ≥8 for each VPC stage. (F) VPC volume = length (D)×height (E) x thickness (number of 0.6 µm steps occupied by the cells). ***: p<0.0001, unpaired t-test. Bars = mean with SEM. n(cells) >20 and n(worms) ≥8 and for each VPC stage. (G) Length of region occupied by the VPCs and their descendants. n.s.: p>0.1, one-way analysis of variance. Bars = mean with SEM. n(worms) ≥10 for each stage.

Figure 2. Contractile ring dimensions scale with the length of VPCs.

(A) GFP-tagged myosin worms at the 3 (left), 6 (middle) and 12 (right) VPC stages. Images are maximum intensity projections taken at <150 seconds following cytokinesis onset. Myosin is enriched in the contractile ring at both the basal (upper) and apical (lower) domain of the cells. Dotted lines: dividing cells. Scale bar = 5 µm. Right: 3D schematic of a dividing VPC showing contractile ring breadth in brackets. (B) Apical (left) and basal (right) contractile ring breadth plotted against VPC length for all three rounds of division. The x-axis was inverted to show the decrease in cell length through divisions. Best-fit linear regressions and their equations are shown. (C) Scatter plot of apical versus basal contractile ring breadth (data from B) for cell lengths at the 3 (purple), 6 (green) and 12 (orange) VPC stages. Colored dots: average for each stage. Scale bars = mean with SEM for both axes. n.s.: p value = 0.05, ***: p<0.0001, unpaired t-test.

Figure 3. Quantitative analysis of the kinetics of contractile ring closure in the VPCs.

(A) Maximum intensity projection images of a worm expressing myosin::GFP at the 3 VPC stage. Left: dotted box: dividing cell; arrow: contractile ring. Scale bar = 10 µm. Middle: enlargement of the dividing cell (dotted outline); dotted box and arrow: contractile ring. Scale bar = 5 µm. The cropped contractile ring is rotated 90° to generate a z, x maximum projection. Bottom: contractile ring closure over time. Scale bar = 1 µm. Right: representation of ring annotation and the parameters quantified: red = cell outline; green = contractile ring; R = cell radius; r = ring radius; Ring closure = r/R*100. (B) Average percentage of contractile ring closure over time aligned at the midpoint of closure for the first (purple), second (green) and third (orange) rounds of VPC cytokinesis. Purple/first: n(cells) = 20, n(worms) = 11. Green/second: n(cells) = 17, n(worms) = 6. Orange/third: n(cells) = 19, n(worms) = 8. Error bars = SEM. Dotted box: data for B’. (B’) Linear regression lines and their equations for 20%–80% ring closure (data from B). (C) Furrowing speed versus division plane perimeter (3 VPC: purple, 6 VPC: green and 12 VPC: orange, grey: data from ; pink: our zygote measurement). Linear regression fitted to all 8 data points. (D) Average speed of contractile ring closure over time for the three rounds of VPC cytokinesis. First round/purple, n(cells) = 20, n(worms) = 11, second/green, n(cells) = 17, n(worms) = 6, third/orange, n(cells) = 19, n(worms) = 8. **: p = 0.006 for 6 versus 12 VPC stage at time 0, unpaired t-test. Error bars = SEM. (E–F) Graphs of average percentage of ring closure and speed over time for HeLa cells (black), Drosophila S2 cells (red) and the C. elegans zygote (light green). C. elegans zygote: n(cells) = 9, Drosophila S2: n(cells) = 5, HeLa: n(cells) = 8. Error bars = SEM.

Figure 4. Strong intercellular adhesion leads to robust asymmetric contractile ring closure in the VPCs.

(A) First (left), second (middle) and third (right) rounds of VPC cytokinesis in myosin::GFP worms. First round dividing cell reproduced from Figure 3A for ease of comparison. Scale bar = 10 µm. Dotted boxes and outlines: dividing cells; arrows: contractile rings. Scale bar = 5 µm. Representation of ring annotation and the parameters quantified: red = cell outline; green = contractile ring; R = cell radius; D = distance between cell and ring centers; Asymmetry = D/R*100. 3D schematic of contractile ring closure in the VPCs. (B) First column: x, y view of dividing HeLa cell (top), Drosophila S2 cell (middle), and C. elegans zygote (bottom). Second column: corresponding x, z views. Third column: example contractile ring location over time. Scale bar = 5 µm. Fourth column: the path taken by the ring for all examples of each cell type. Last column: asymmetry versus time. (C) Average asymmetry of furrowing over the percentage of VPC ring closure (first round; purple, second; green and third; orange). First: n(cells) = 20, n(worms) = 11. Second: n(cells) = 17, n(worms) = 6. Third: n(cells) = 19, n(worms) = 8. Error bars = SEM. ***: p<0.0001, *: p = 0.03, unpaired t-test calculated at 80% closed. (D) Confocal images of control and ANI-1 depleted worms expressing myosin::GFP (3 VPC stage). Dotted lines: dividing cells; arrows: ingressing furrows. Scale bar = 5 µm. (D’) Kymographs of contractile ring closure for control (black) and ANI-1 depleted (fuchsia) worms expressing myosin::GFP. Vertical scale bar = 1 µm. Horizontal scale bar = 5 min. (E-E’) Average furrow asymmetry and ring closure speed (all three rounds of VPC cytokinesis). Control (grey), n(cells) = 56, n(worms) = 25. ani-1(RNAi) (fuchsia), n(cells) = 8, n(worms) = 4. Error bars = SEM. n.s.: p>0.1, unpaired t-test. ***: p<0.0001, unpaired t-test.

Figure 5. Tissue geometry influences the kinetics of cytokinesis in the cells of the somatic gonad.

(A) Schematic representing an L3 worm (∼31 hours post-hatching), showing the somatic gonad, VPCs and germline. Worm expressing mCherry-tagged phospholipase C (PLCδ) PH domain in the germline (top), GFP-tagged myosin in the VPCs and somatic gonad (middle), and merge image (bottom). Scale bar = 10 µm. (B) Schematic of the cells of the somatic gonad (middle) and the germline (extremities) surrounded by a continuous basement membrane (red). Worm at the 12 VPC stage expressing myosin::GFP and mCherry::LAM-1 (laminin-1) to mark basement membrane. Scale bar = 10 µm. (C) Maximum intensity projection images of the somatic gonad expressing GFP myosin. Dotted boxes: dividing cells, enlarged to the right; arrows: contractile ring. Scale bars = 10 µm (left); 5 µm (right). (D-E) Average ring closure speed and asymmetry (somatic gonad: teal blue, first VPC division: purple, second: green, and third: orange). Each VPC stage n(cells) ≥17, n(worms) ≥6. Gonad cells; n(cells) = 29, n(worms) = 12. Error bars = SEM. **: p = 0.0023, unpaired t-test, gonad cells versus 6 VPC stage. *: p = 0.06, ***: p<0.0001, n.s.: p>0.1, unpaired t-test.