Assembly of the cytokinetic contractile ring from a broad band of nodes in fission yeast - PubMed (original) (raw)
Assembly of the cytokinetic contractile ring from a broad band of nodes in fission yeast
Jian-Qiu Wu et al. J Cell Biol. 2006.
Abstract
We observed live fission yeast expressing pairs of functional fluorescent fusion proteins to test the popular model that the cytokinetic contractile ring assembles from a single myosin II progenitor or a Cdc12p-Cdc15p spot. Under our conditions, the anillin-like protein Mid1p establishes a broad band of small dots or nodes in the cortex near the nucleus. These nodes mature by the addition of conventional myosin II (Myo2p, Cdc4p, and Rlc1p), IQGAP (Rng2p), pombe Cdc15 homology protein (Cdc15p), and formin (Cdc12p). The nodes coalesce laterally into a compact ring when Cdc12p and profilin Cdc3p stimulate actin polymerization. We did not observe assembly of contractile rings by extension of a leading cable from a single spot or progenitor. Arp2/3 complex and its activators accumulate in patches near the contractile ring early in anaphase B, but are not concentrated in the contractile ring and are not required for assembly of the contractile ring. Their absence delays late steps in cytokinesis, including septum formation and cell separation.
Figures
Figure 1.
The Rlc1p spot is not essential for the assembly of the contractile ring. Elapsed time is given in minutes. (A) A time-lapse series of one cell showing that the contractile ring forms from a broad band of nodes. Cells of strain GFP-myo2 sad1-GFP (JW800) were imaged in EMM5S at 22°C. A stack of 18 fluorescence Z sections spaced at 0.4 μm was taken every 3 min and projected into a 2D image using a maximum intensity projection. The arrows indicate the SPBs. The SPB separation is defined as time zero. The whole series can be viewed in Video 1. (B–E) The assembly of contractile rings independent of the Rlc1p spot. Cells grown exponentially at 25°C were shifted to 36°C for 2–4 h, and then imaged at 25°C. (B) Myp2p and Rlc1p localize to a spot after growth at 36°C and the Rlc1p spot formation depends on Myp2p. Images are maximum intensity projections of eight 0.64-μm Z sections. Arrow indicates a cell with a spot and a contracting ring. (C) A cell of strain rlc1-EGFP myp2Δ (MLP240) forms a contractile ring normally without the Rlc1p spot. The whole series can be viewed in Video 2. (D) The Rlc1p-mYFP spot (strain JW991) incorporates into the contractile ring after the broadband formation. Arrows indicate Rlc1p spots. The micrographs are maximum intensity projections of five 0.9-μm Z sections. (E) Kymograph of the cell in D over 154 min showing the movement of the Rlc1p spot. The kymograph was constructed using the maximum intensity projections, with a 5-μm slit cross the midplane of the cell. Videos 1 and 2 are available at
http://www.jcb.org/cgi/content/full/jcb.200602032/DC1
. Bars, 5 μm.
Figure 2.
Localization of the formin Cdc12p and PCH protein Cdc15p to a broad band of nodes around the equator of cells observed by fluorescence microscopy. (A) Cdc12p localizes to a broad band of nodes (arrowhead) at the onset of mitosis in asynchronous cells expressing Cdc12p-3YFP Sad1p-CFP (strain JW1114). Only YFP images are shown. Three YFP fluorescence Z sections spaced at 0.8 μm of two cells were projected into a 2D image using a maximum intensity projection. Elapsed times are given in minutes. The whole series can be viewed in Video 3. (B and C) Cdc12p localizes to a broad band of nodes in synchronized cdc12-3GFP cdc25-22 cells (JW1118). Cells were grown at 36°C for 4 h and shifted to 24°C for 1 h. (B) The central part of one cell, as indicated, is shown with the long axis of the cell positioned horizontally. Maximum intensity projections of 20 fluorescence Z sections spaced at 0.3 μm. Time points (at 45-s intervals) 7–50 are shown. The 3D projection of the series can be viewed in Video 4. (C) Kymograph of the division site of the cell in B over 45 min showing condensation of the broad band. The kymograph was constructed using maximum intensity projections with a 5.5-μm-long slit parallel to the long axis of cell. (D) Cdc12p accumulates in a broad band of nodes in the absence of actin filaments. Strain cdc12-3GFP cdc25-22 (JW1118) was arrested at G2/M after being grown at 36°C for 4 h, treated with 100 μM Lat-A at 36°C for 30 min, and then released to 23°C for 90 min in Lat-A. 81 Z sections spaced at 0.1 μm were collected, deconvolved, and projected into 3D. The three cells are viewed at 0 and 90° to the y-axis. The whole series can be viewed in Video 5. (E) GFP-Cdc15p (strain JW1038) forms a broad band of nodes around the equator before coalescing laterally into a compact ring. A stack of 36 0.2-μm Z sections was taken every minute. The time series of the same cell is displayed in two ways; (top) sections across the midplane of the cell; (bottom) maximum intensity projections in the X–Y plane of deconvolved images. The cross-section series can be viewed in Video 7. Videos 3–5 and 7 are available at
http://www.jcb.org/cgi/content/full/jcb.200602032/DC1
. Bars, 5 μm.
Figure 3.
Localization of pairs of fluorescent fusion proteins in the broad bands of nodes, and the dependence of the node formation on Mid1p. Wild-type cells (A–E) or cdc25-22 cells (F–O) expressing pairs of CFP- and YFP-fusion proteins under the control of their native promoters were observed directly (A–C) or treated with 100 μM Lat-A for at least 30 min before observation to prevent condensation of nodes into a contractile ring (D–O). (D and E) Cells were treated with Lat-A at 23°C. (F–O) Cells with the cdc25-22 temperature-sensitive mutation were arrested at the G2/M transition by incubation at 36°C for 4 h, incubated in Lat-A at 36°C for another 30 min, and then released to 23°C in Lat-A at time zero and observed. The images, except for those on the right in A–C, are maximum intensity projections of 13 0.6- or 36 0.2-μm Z sections (with exposures of 198 ms). (A–C, left) Maximum intensity projections of the whole cells; (middle) consecutive individual sections of the division site of the cells in the leftmost images; and (right) percentage of nodes with the proteins listed before the left images. D–K show the division sites of two cells. (L and M) show two whole cells in each strain background. To reduce background autofluorescence and increase the contrast for CFP, the cells in D and G–I were photobleached briefly at 442 nm before images were taken. (M) Arrowheads indicate the SPBs. (N) Red symbols and lines use the scale on the left. All others use the scale on the right. (N and O) Graphs of the time courses of the fraction of cells with fluorescent proteins in broad bands and the separation of spindle pole bodies. Samples were taken at the indicated time, imaged, and quantified. The following strains were used: (A) mid1-YFP CFP-myo2 (JW851); (B) mYFP-rng2 CFP-myo2 (JW928); (C) mYFP-cdc15 CFP-myo2 (JW1173); (D) JW968; (E) JW929; (F) JW1024; (G) JW1023; (H) JW1181; (I) JW1183; (J) JW1216; (K) JW1026; (L) JW1118 and JW1192; (M and N) JW1220 (n = 423 cells) and JW1218 (n = 450 cells); and (O) cdc12-3YFP mCFP-myo2 (JW1183; n = 641 cells) and mid1Δ cdc12-3YFP mCFP-myo2 (JW1226; n = 343 cells). Bars, 5 μm (same bar for F–K).
Figure 4.
Localization and dynamics of Arp2/3 complex and its activators myosin I Myo1p and WASp Wsp1p in actin patches at the cell-division site. (A–C) Cells expressing mYFP-Myo1p (red) and CFP-Myo2p (green) show that endocytic actin patches containing Myo1p are spatially and temporally distinct from contractile rings labeled with Myo2p. (A) Stereo projections of 3D reconstructions made from Z series of confocal images at 0.6-μm intervals and projected at 15 and 24° tilts. The whole series can be viewed in Video 9. (B) Individual confocal sections through the division site of cell 3 from A at 0.6-μm intervals. (C) Time series at 3-s intervals in a single confocal plane showing red mYFP-Myo1p patches surrounding the green CFP-Myo2p ring. Arrowheads mark the appearance of new mYFP-Myo1p patches. (D) Stereo projections of cells expressing mCFP-Wsp1p (red) and Cdc15p-mYFP (green) show that Cdc15p appears in cortical patches at cell tips during interphase and in contractile rings during mitosis, whereas Wsp1p concentrates in actin patches throughout the cell cycle. (E and F) Localization and dynamics of mYFP-Wsp1p (red) and mCFP-Myo1p (green) in patches at the division site. (E) 3D reconstructions made from Z series of confocal images at 0.6-μm intervals. Arrowheads point out examples of patches labeled only with Wsp1p (red) or Myo1p (green). (F) Time course of the activators' assembly into patches at the division site in a single confocal plane at 3-s intervals. Arrowheads point out examples of nascent patches where appearance of Myo1p (green) lags behind Wsp1p (red) by 3–6 s. (G and H) Dynamics of mYFP-Wsp1p and ARPC5-mCFP in patches at the division site. (G) Time course of Wsp1p (red) and ARPC5 (green) patch assembly at the division site in a single confocal plane at 3-s intervals. Arrowheads mark the appearance of Wsp1p at 3 s, appearance of ARPC5 at 9 s, and initiation of patch motility at 21 s in a single patch. The whole series can be viewed in Video 10. (H) Time course of fluorescence intensity of (□) mYFP-Wsp1p and (◯) ARPC5-mCFP and total distance moved by (▪) mYFP-Wsp1p and (•) ARPC5-mCFP for the patch marked in G. (I and J) The images are maximum-intensity projections of 12 0.6-μm Z sections. Arrowheads indicate contractile rings. (I) ARPC5 does not colocalize with Myo2p in the contractile ring in cells expressing mCFP-Myo2p and ARPC5 (Arc5p)-mYFP (JW1221). (J) The concentration of ARPC5 in the contractile ring location is less than that of the formin Cdc12p. Cells expressing Cdc12p-mYFP (KV345), ARPC5-mYFP (JW1223), and Cdc12p-mYFP ARPC5-mYFP (JW1227) were imaged under identical conditions. Videos 9 and 10 are available at
http://www.jcb.org/cgi/content/full/jcb.200602032/DC1
. Bars: (A–G) 2 μm; (I and J) 5 μm.
Figure 5.
The effect of the conditional mutation arp3-c1 and activator deletion mutants on the assembly of the contractile ring during cell division. (A–C) Strains expressing mYFP-Myo2p from its native promoter in wild type (JW1110), myo1Δ (JW1248), and wsp1Δ (JW1247) were grown in YE5S medium at 25°C, washed in EMM5S, and imaged in EMM5S plus 25% gelatin at 25°C (23°C for cells in A). (A) The Myo2p ring forms normally in activator deletion mutants. Images are maximum-intensity projections of 13 0.6-μm Z sections. (B–D) Plots of the observed mean times (±1 SD) of cytokinesis events (X-axis) versus the established time of these events in wild-type cells (Wu et al., 2003). Differential interference contrast (DIC) and fluorescence images of mYFP-Myo2p in a single optical section were taken every 2 min. (B) Comparison of wild-type cells (n = 26 cells) and cells lacking myo1 (n = 29). (C) Comparison of the same wild-type sample as in B with cells lacking wsp1 (n = 20). (D) Comparison of wild-type mYFP-myo2 (JW1233; n = 18) and mYFP-myo2 arp3-c1 (JW1235; n = 12) at the restrictive temperature 19°C. Cells were grown exponentially in YE5S at 32°C, shifted to 19°C for 6 h, and then imaged at 19°C. DIC and fluorescence images in a single optical section were taken every 4 min. Bar, 5 μm.
Figure 6.
Lateral contraction model for contractile ring assembly. (A) Diagram of the assembly of a contractile ring (red) from a broad band of nodes (red) followed by relocation of actin patches (green) containing Arp2/3 complex from the ends of the cell to the cleavage site. (B and C) Scale drawings of the proteins known to be present in the nodes that assemble into the contractile ring. The numbers of protein molecules shown in each node are based on the total numbers of each protein in the broad band divided by 75 nodes, resulting in ∼20 molecules of anillin-like Mid1p, myosin II dimer, IQGAP Rng2p, and PCH protein Cdc15p, and 2 formin Cdc12p dimers (Wu and Pollard, 2005). The four nodes are separated by ∼0.7 μm as in live cells. The barbed ends of actin filaments are anchored to formin Cdc12p dimers in one node and project radially to interact with putative myosin II minifilaments in adjacent nodes.
References
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