Three myosins contribute uniquely to the assembly and constriction of the fission yeast cytokinetic contractile ring.

Article

Three Myosins Contribute Uniquely to the Assembly and Constriction of the Fission Yeast Cytokinetic Contractile Ring Graphical Abstract

Authors Caroline Laplante, Julien Berro, Erdem Karatekin, Ariel Hernandez-Leyva, Rachel Lee, Thomas D. Pollard

Correspondence [email protected]

In Brief Conventional myosin-II has long been identified as the sole source of force in cytokinesis. Laplante et al. show that three myosins function in fission yeast cytokinesis. Conventional myosin-II Myo2 is crucial to ring assembly, unconventional myosin-II Myp2 is most important for ring constriction, and type V myosin Myo51 aids the other two myosins.

Highlights d

Three myosins—Myo2, Myp2, and Myo51—participate in cytokinesis in fission yeast

d

Myo2 functions mainly in ring assembly, and Myp2 functions in ring constriction

d

Myo51 assists Myo2 and Myp2 in ring assembly and constriction

d

Myo2 and Myp2 occupy different concentric sections of the constricting ring

Laplante et al., 2015, Current Biology 25, 1–11 August 3, 2015 ª2015 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2015.06.018

Please cite this article in press as: Laplante et al., Three Myosins Contribute Uniquely to the Assembly and Constriction of the Fission Yeast Cytokinetic Contractile Ring, Current Biology (2015), http://dx.doi.org/10.1016/j.cub.2015.06.018

Current Biology

Article Three Myosins Contribute Uniquely to the Assembly and Constriction of the Fission Yeast Cytokinetic Contractile Ring Caroline Laplante,1 Julien Berro,1,2 Erdem Karatekin,3,4,5 Ariel Hernandez-Leyva,1 Rachel Lee,1 and Thomas D. Pollard1,2,6,* 1Department

of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA 3Nanobiology Institute, Yale University, West Haven, CT 06516, USA 4Department of Cellular and Molecular Physiology, Yale University, New Haven, CT 06520, USA 5Institut des Sciences Biologiques, Centre National de la Recherche Scientifique (CNRS), De ´ le´gation Paris Michel-Ange, 3 rue Michel-Ange, 75794 Paris Cedex 16, France 6Department of Cell Biology, Yale University, New Haven, CT 06520, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cub.2015.06.018 2Department

SUMMARY

Cytokinesis in fission yeast cells depends on conventional myosin-II (Myo2) to assemble and constrict a contractile ring of actin filaments. Less is known about the functions of an unconventional myosin-II (Myp2) and a myosin-V (Myo51) that are also present in the contractile ring. Myo2 appears in cytokinetic nodes around the equator 10 min before spindle pole body separation (cell-cycle time, 10 min) independent of actin filaments, followed by Myo51 at time zero and Myp2 at time +20 min, both located between nodes and dependent on actin filaments. We investigated the contributions of these three myosins to cytokinesis using a severely disabled mutation of the essential myosin-II heavy-chain gene (myo2-E1) and deletion mutations of the other myosin heavychain genes. Cells with only Myo2 assemble contractile rings normally. Cells with either Myp2 or Myo51 alone can assemble nodes and actin filaments into contractile rings but complete assembly later than normal. Both Myp2 and Myo2 contribute to constriction of fully assembled rings at rates 55% that of normal in cells relying on Myp2 alone and 25% that of normal in cells with Myo2 alone. Myo51 alone cannot constrict rings but increases the constriction rate by Myo2 in Dmyp2 cells or Myp2 in myo2-E1 cells. Three myosins function in a hierarchal, complementary manner to accomplish cytokinesis, with Myo2 and Myo51 taking the lead during contractile ring assembly and Myp2 making the greatest contribution to constriction. INTRODUCTION Myosin-II has been considered to be the main motor for cytokinesis since its discovery in the contractile ring [1] and demonstra-

tion that cleavage furrows do not form in either echinoderm embryos microinjected with antibodies to myosin-II [2] or Dictyostelium amoebae with a deletion mutation of the myosin-II gene [3]. However, cytokinesis can proceed in a variety of cells with compromised myosin-II. For example, Dictyostelium can survive without the single myosin-II gene by pulling themselves apart as they move in opposite directions on a surface [3]. Budding yeast can divide without myosin-II [4–6] or with myosin-II lacking the motor domain [7]. The ability to divide without myosin-II motor activity was attributed to the growth of the cell wall or actin filament severing and crosslinking. Mammalian COS-7 cells can complete cytokinesis with myosin-II lacking motor activity [8]. The fission yeast gene for conventional myosin-II, myo2+, is essential for viability [9, 10], but cytokinesis is remarkably normal in cells with the temperature-sensitive myo2-E1 mutation [11], which has minimal biochemical activity even at 25 C, the permissive temperature [12]. This suggested either that the remaining function of Myo2p-E1 is sufficient to generate the forces for cytokinesis or that other myosins contribute to cytokinesis. Fission yeast cells are favorable for investigating cytokinesis motors, since a large body of quantitative information is available on its division (for a review, see [13]), and the genome encodes just five myosin heavy chains: type I myosin myo1+; type II myosins myo2+ and myp2+; and type V myosins myo51+ and myo52+ [14]. Myosin molecules consist of heavy chains and light chains, and we refer to each molecule in this work by the name of its heavy chain (e.g., Myo2p is the heavy chain of the Myo2 molecule). On the other hand, we refer to the polypeptide when naming a tagged protein (e.g., mEGFP-Myo2p). Each of the five myosins has distinctive functions. Myo1 has well-characterized roles in endocytosis and mating but does not participate in cytokinesis other than its role in endocytosis during septum formation [15–17]. Myo2p and Myp2p bind light chains Cdc4p and Rlc1p, and both participate in cytokinesis [12, 18–20]. Essential Myo2 is a conventional myosin II, since the heavy chain forms a homodimeric, coiled-coil tail [21]. Myp2p is not essential for viability or cytokinesis under normal growth conditions [19, 21, 22] and is unconventional, since its exceptionally long tail folds upon itself to form an anti-parallel coiled coil [21]. Myo51 and Myo52 carry cargo along actin

Current Biology 25, 1–11, August 3, 2015 ª2015 Elsevier Ltd All rights reserved 1


Figure 1. Recruitment of Three Myosins and Actin Filaments to the Division Site in Wild-Type Cells (A) Time series of fluorescence micrographs (maximum intensity projections of 18 confocal sections, inverted grayscale lookup table [LUT]) of single cells expressing two fluorescent fusion proteins: Sad1p-RFP to label SPBs; and a second protein fused to mEGFP or 3YFP. One marker is shown in each time series. Row 1: Sad1p-RFP. Row 2: mEGFP-Myo2p. Row 3: mEGFP-Myp2p. Row 4: Myo51p-3YFP. Row 5: GFP-CHD to mark actin filaments in actin patches and the contractile ring. Scale bar, 5 mm. (B) Outcomes plot showing the fraction of cells with each of three myosins at the cleavage site over time: (C) mEGFP-Myo2p; (-) Myo51p-3YFP; and (:) mEGFP-Myp2p. (C) Color code of strains with corresponding genotype and functional myosin present used in the following figures. All times are in minutes relative to SPB separation at time zero. See also Figure S1.

filament cables, a crucial contribution to cell polarity [14]. During cell division, Myo51 redistributes from actin cables to the equator [23], and Myo52 transports vesicles containing beta-glucan synthetase Bgs1 along actin cables to the forming septum (but does not concentrate in the contractile ring) [24]. Both type V myosins require actin for their localization [23]. Our quantitative analysis of the behavior of live fission yeast cells with combinations of myosin mutations revealed that Myo2, Myp2, and Myo51 each contribute uniquely to contractile ring assembly and constriction. Myo2 is the primary myosin for ring assembly, and Myp2 and Myo51 compensate when Myo2 function is compromised. Myp2 is most important for ring constriction. Myo2 and Myp2 localize to separate concentric sub-sections of constricting rings. The presence of Myo51 improves the performance of both Myo2 and Myp2 during ring formation and constriction. Participation of multiple myosins may explain why cytokinesis is successful in other organisms with myosin-II mutations. RESULTS Myo2, Myp2, and Myo51 Accumulate at the Equator at Discrete Cell-Cycle Times Myo2p, Myp2p, and Myo51p each concentrated in the cytokinesis apparatus at different times (Figure 1A, asterisk; Figure 1B).

We used spindle pole body (SPB) separation to define cell-cycle time zero. Myo2 concentrated in cytokinetic nodes around the equator between time 10 min and time zero (Figures 1A and 1B) [25]. Formin Cdc12p accumulated in cytokinetic nodes at time zero [25] and initiated actin polymerization from nodes at time +1 min (Figure 1A), coinciding with the onset of node motions [26]. We used GFP-calponin homology domain (CHD) to mark actin filaments [27], because GFP-actin does not incorporate into the contractile ring [28]. Cytokinetic nodes condensed along with the actin filaments (Figures 1A and 2D) to form a contractile ring between time +1 and +25 min (Figure 2E) [25, 26]. Some actin filaments and small bundles of filaments persisted on the lateral borders of rings throughout constriction (Figure 1A, arrow) [26, 29]. Myo51 localized along For3p-dependent actin cables during interphase [23], giving a filamentous appearance in the cytoplasm (Figure 1A, arrow). Beginning at time zero, Myo51p concentrated in a narrow equatorial band and in filamentous structures between nodes [30] around the middle of the cell (Figures 1A, asterisk, and 1B), both dependent on actin filaments [23]. Myp2 began to accumulate in fully formed contractile rings at time +20 min (Figures 1A, asterisk, and 1B) [25]. Myo52 transports cargo including vesicles with Bgs1 b-1,3-glucan synthase along actin filament cables during interphase and mitosis, but it does not concentrate in the contractile ring [14, 23, 24, 31–33]. We confirmed previous observations [32, 33] that the Dmyo52

2 Current Biology 25, 1–11, August 3, 2015 ª2015 Elsevier Ltd All rights reserved


strain grows slowly at both 25 C and 36 C and that the Dmyo52 mutation did not enhance the myo2-E1 growth defect at either temperature, but that Dmyo52 is synthetically lethal with Dmyp2 at 36 C (Figure S1A). Node coalescence was similar in Dmyo52 Dmyp2 and Dmyo52 myo2-E1 cells to node coalescence in Dmyp2 and myo2-E1 cells, respectively (Figure S1C), evidence that Myo52 does not participate in cytokinesis beyond its role in delivering vesicles with Bgs1p to the division site. Myo2, Myp2, and Myo51 left the contractile ring at characteristic times. The Myo51p fluorescence in the ring declined shortly after the onset of ring constriction at time +35 min and disappeared over 15 min (Figures 1A, 1B, and S1F). Both Myo2 and Myp2 were retained in rings during constriction (Figure 1B), and their local concentrations increased until the ring disappeared [28, 34]. Cells with Mutations in a Single Myosin or a Pair of Myosins Are Viable Loss-of-function alleles of myo2+ and genetic deletions of myp2+ and myo51+ allowed us to evaluate how these three myosins contribute to cytokinesis (Figures 2, S1, and S2; Movies S1, S2, S3, and S4). Strains with deletion mutations of myp2+ or myo51+ were viable at both 25 C and 36 C (Figure S1A). Motegi et al. reported that a different Dmyp2 strain was temperature sensitive at 36 C [19]. Since myo2+ is essential for viability [9, 10], we used the severely disabled myo2-E1 allele [11] that grows slowly at 36 C (Figure S1A). A point mutation in the catalytic domain gives the Myo2p-E1 protein low ATPase activity and very weak affinity for actin filaments in the presence of ATP, even at 25 C [12]. Given the minimal motor activity of the Myo2p-E1 protein, its tail may provide functions that account for the viability of the strain. The Supplemental Information reports experiments on the phenotypes of three additional myo2+ mutations (Figure S2). The two strains with point mutations in the catalytic domain (myo2-S1 and myo2-S2) were viable but grew slowly with obvious cytokinetic defects at both 25 C and 36 C, whereas the neck deletion strain, myo2-DIQ1, encoding a myosin that cannot translocate actin filaments in vitro [12], had no noticeable growth or phenotypic defects at any temperature tested. Like wild-type cells [25] myo2-E1 cells assembled and constricted their contractile ring faster at 36 C than at room temperature, and since their defects were not enhanced at 36 C, we studied all of the strains at 23 C–25 C. All three double-mutant strains were viable at 25 C, but combining the myo2-E1 mutation with either the Dmyp2 or Dmyo51 mutation was synthetically lethal at 36 C (Figure S1A), as shown previously for myo2-E1 Dmyp2 [19, 35]. A large fraction of double-mutant cells accumulated in cytokinesis at 25 C, indicating that cells depending on single cytokinetic myosins have strong defects (Table 1). None of the strains with single- or double-myosin mutations differed significantly from wild-type cells in the numbers of the remaining myosin(s) in whole cells or contractile rings (Figures S1D–S1F). Time-lapse confocal microscopy of live cells with a fluorescent tag on Myo2p provided data for four measurements to evaluate contractile ring assembly and constriction: (1) the cell-cycle time when nodes started to move; (2) the distribution of instantaneous velocities of moving nodes; (3) the cell-cycle time when nodes coalesced into a contractile ring or other compact structure

where individual nodes were no longer resolved by confocal microscopy; and (4) the time course of ring constriction and disassembly. Table 1 lists all of the statistics. Outcome plots (Figures 2E, 2F, and S2A) and the SDs of the mean duration for nodes to coalesce (Table 1) illustrate the variability in the process in each cell population. The distribution of node velocities in wild-type cells had a major peak at 40 nm/s and minor peaks at 60 and 80 nm/s (Figure 2C). Wild-type cells completed the assembly of a ring between times +15 min and +25 min (Figure 2E), with a mean completion time of +18.5 ± 4.2 min (mean ± SD) on the cell-cycle clock. In an unsynchronized population of wild-type cells, 19% of the cells were in cytokinesis, meaning that they had some mEGFP-Myo2plabeled features at their equator, ranging from a broad band of nodes to a fully constricted and disassembling ring (Table 1). All six strains with mutations in one or two myosin genes had cytokinesis defects, but the double-mutant strains were more informative than strains with a single-myosin mutation, as they revealed the contributions of the single myosin present. Strains with mutations in one myosin gene revealed how the remaining pair of myosins functions together. To simplify the presentation, we refer to strains by the functional myosin(s) they expressed in bold font in addition to their genotype in italic font throughout the text, and we use a color code to identify each strain in the figures (Figure 1C). Single Cytokinesis Myosins Can Support Contractile Ring Assembly All three double mutant strains coalesced their nodes into compact structures, but the process took longer and was more variable than in wild-type cells (Figures 2A, 2B, 2D, and 2E; Table 1; Movies S2, S3, and S4). Nodes began to move at time +1 min in all three double-mutant strains as in wild-type cells, but these movements were often ineffective, and most rings formed were imperfect. Therefore, Myo2, Myp2, and Myo51 can each contribute to node coalescence in the absence of the other two myosins. Cells dependent on the motor activity of Myo2 alone (Dmyp2 Dmyo51 strain) completed node coalescence 17 min later than wild-type cells (Figure 2E; Table 1). The main peak of node velocities in these cells was 40 nm/s, like that of wild-type cells (Figure 2C), but fewer nodes moved at the higher rates observed in wild-type cells. About 80% of rings in these double mutants were bent, because they were larger than the circumference of the cell (Figure 2D, inset). Constriction subsequently reduced the circumferences of these large rings, suggesting that they were not taut when newly assembled. The Dmyp2 Dmyo51 cells had cytokinetic defects, including multiple septa and branched morphology (Figure S1B) [30]. Contractile rings assembled slowly in cells dependent on Myo51 alone (myo2-E1 Dmyp2 strain) (Figure 2E; Table 1), with a slightly longer mean time than cells with only Myo2. However, unlike cells with only Myo2, the distribution of node velocities had a single peak at 20 nm/s, so Myo51 alone cannot generate fast node movements (Figure 2C; Table 1). The newly assembled rings were often irregular in shape (Figure 2G). Of the three double-mutant strains, node coalescence was compromised most severely in cells dependent on Myp2 alone (myo2-E1 Dmyo51 strain). Nodes marked with disabled mEGFP-Myo2p-E1 began moving at cell-cycle time +1 min,



Figure 2. Contractile Ring Formation and Constriction in Wild-Type Cells and Strains Lacking One or Two Myosins (A) Confocal fluorescence micrographs (inverted grayscale LUT) of fields of asynchronous cells. Each is a single time point from Movies S1, S2, S3, and S4. The functional myosin(s) in each strain are at the top of each panel. (B) Confocal micrographs of newly formed rings in myo2-E1 Dmyo51 cells with the percentage of each type of ring in the population. (C) Graph of the distribution of instantaneous node velocities for seven genotypes and wild-type cells with 100 mM LatA. The active myosins in each strain are indicated. Nodes marked with mEGFP-Myo2p or mEGFP-Myo2p-E1 were tracked at 2-s intervals (n R 20 nodes per mutant genotype except for the LatA-treated (legend continued on next page)



Table 1. Contractile Ring Parameters

Genotypes

Mean Time Functional Peak Node Mean Time Mean Time Constriction Mean Constriction % Cytokinetic Myosins Present Velocitiesa (nm/s) Coalescenceb (min) Maturationc (min) Onsetd (min) Ratee (mm/min) Cell 25 Cf

Wild-type

Myo2, Myp2, Myo51

38, 62, 78

18.5 ± 4.2

13 ± 4

31.9 ± 2.9

0.27 ± 0.04

19

myo2-E1

Myp2, Myo51

28

24.2 ± 3.9

9±4

33.4 ± 3.1

0.28 ± 0.05

29

Dmyp2 Dmyo51

Myo2 alone

38, 68

35.5 ± 6.8

8±4

47.1 ± 10.4

0.07 ± 0.03

42

Dmyo51

Myo2, Myp2

38, 62, 72

24.8 ± 4.0

8±4

31.7 ± 4.5

0.30 ± 0.05

22

myo2-E1 Dmyp2

Myo51 alone

22

41.5 ± 11.4

indefinite

none

none

52

Dmyp2

Myo2, Myo51

38, 58

15.1 ± 3.7

30 ± 10

45.7 ± 8.3

0.20 ± 0.06

22

25

63.6 ± 10.7

11 ± 14

65.2 ± 13.5

0.07 ± 0.03

59

myo2-E1 Dmyo51 Myp2 alone

Means are ± SDs. a See Figure 2C. b All cell-cycle times are relative to SPB separation at time zero. See Figure 2E. c Calculated from the data in Figures 2E and 2F. d See Figure 2F. e See Figure 2H. f Percentage of cells with mEGFP-Myo2p in nodes or a contractile ring in populations of asynchronous cells at 25 C (n = 115 to 205 per genotype).

with a single peak at 25 nm/s in the distribution of node velocities (Figure 2C). We do not understand the mechanism of the slow node motions before Myp2 arrived at the equator at time +20 min in these cells. These cells took much longer to coalesce their nodes than wild-type cells. The first cells with complete rings appeared at time +45 min, and the last appeared at time +90 min (Figure 2E). Although 35% of these cells assembled a single, normal ring, the remaining cells had two or three separate rings, lasso-shaped rings, two laterally joined rings, or a curved bundle of mEGFP-Myo2p-E1 (n = 63 cells) (Figures 2A and 2B). These observations show that Myo2 is the main myosin for node coalescence, followed by Myo51. Myp2 normally contributes little to node coalescence, in accord with its late arrival (+20 min) in the fully formed ring in wild-type cells (Figure 1A). Pairs of Myosins Assemble Contractile Rings Better Than Single Myosins Strains with any combination of two myosins assembled contractile rings better than single myosins, showing that they normally cooperate in the process. Pairing Myo2 with either

Myo51 or Myp2 was more effective than the combination of Myp2 and Myo51. The pair of Myo2 and Myo51 in Dmyp2 cells was the most successful at ring assembly. Both myosins arrived at their normal cell-cycle times, and nodes moved with nearly normal velocities (Figure 2C). Nodes coalesced into rings slightly earlier than in wild-type cells (Figure 2E; Table 1), and the only defect was a slight bowing of the rings in about one fifth of the cells (Figure 2D, inset). Our Dmyp2 cells were morphologically normal when grown in rich medium at room temperature, and 22% of these cells were in cytokinesis, similarly to a wild-type cell population (Table 1). Therefore, Myp2 does not normally contribute to contractile ring assembly. Cells dependent on Myo2 and Myp2 (Dmyo51 strain) had a very subtle phenotype. The arrival of Myo2 in equatorial nodes at cell-cycle time 10 min and motions of these nodes after cell-cycle time +1 min were indistinguishable in wild-type cells (Figure 2C). Nevertheless, Dmyo51 cells completed the coalescence of their nodes 6 min later than wild-type cells (Figure 2E; Table 1). The Dmyo51 cells had no obvious morphological

sample, n = 12 nodes). The two-sample Kolmogorov-Smirnov test showed that, with the exception of the Myo2 Myp2 (Dmyo51) strain, the node velocity distributions of all the genotypes and LatA-treated samples differed significantly (p > 0.05) from that of the wild-type cells. (D) Time series of micrographs (inverted grayscale LUT) of strains expressing mEGFP-Myo2p or mEGFP-Myo2p-E1 and Sad1p-RFP. Insets show confocal micrographs of rings in Dmyp2 and Dmyp2 Dmyo51 cells, with the mean percent difference between the ring circumference and the cell circumference. (E and F) Outcome plots showing the accumulation over time of cells with (E) their nodes condensed into a contractile ring or other compact structure at the equator or (F) the onset of ring constriction. Time zero is at SPB separation. All cells expressed mEGFP-Myo2p or mEGFP-Myo2p-E1 to mark nodes. The legends list the functional myosins in each strain. A log-rank statistical test for outcome plots showed that each strain differed from wild-type cells (black lines); *p < 0.05, **p < 0.01, and ***p < 0.001. (G) Phenotypes of rings observed in confocal micrographs of myo2-E1 Dmyp2 relying on Myo51 and expressing mEGFP-Myo2p-E1. Left panel: Time series of a binucleate cell that failed to form a septum in the previous cell cycle, with cell-cycle time in minutes. Arrow points to a ring detaching from the cell cortex. Dashed outlines highlight the cell. Right panel: Two time points (minutes after first image) of a cell that formed a septum and divided after a long delay. Dashed outlines highlight the cell. (H) Histogram of average contractile ring constriction rates (±SD; n = 20–25 cells per genotype). The active myosins in each strain are indicated within the bars. Asterisks indicate significant statistical difference of separate strains compared to wild-type (black bar) by Student’s t test; *p < 0.05 and **p < 0.01. (I) Kymographs at 2-min intervals of contractile rings in horizontally oriented single cells generated from time series of micrographs (inverted grayscale LUT). Kymographs are aligned on the vertical line at the time nodes completed coalescence. Rings in myo2-E1Dmyp2 cells did not constrict but undulated and disassembled after node coalescence. Confocal fluorescence micrographs are maximum intensity projections of 18 confocal z-sections. Scale bars, 5 mm. See also Figure S2 and Movies S1, S2, S3, and S4.



Figure 3. Localization of Myosins in Cells with and without Myo2p Function or Actin Filaments (A) Time series of micrographs (inverted grayscale LUT) of wild-type and myo2-E1 cells expressing mEGFP-Myp2p and Sad1p-RFP (data not shown). Myp2p localizes to the equator 20 min after SPB separation in both wild-type and myo2-E1 mutant cells. (B) Time series of micrographs (inverted grayscale LUT) of myo2-E1 cells expressing Myo51p-3YFP and Sad1p-mEGFP (top) and GFP-CHD (bottom). Myo51p localizes to the equator at t = 0 min at SPB separation in myo2-E1 mutant cells as in wild-type cells (see Figure 1A). The equatorial zones of both Myo51p-3YFP and the GFP-CHD were wider in myo2-E1 cells than wild-type cells (brackets). (C) Graph of the ratio of equatorial GFP-CHD fluorescence intensity to the total cellular GFP-CHD fluorescence intensity in (black squares) wild-type cells and (green circles) myo2-E1 cells. Student’s t tests show significant differences (p < 0.05) between the ratios in wild-type and myo2-E1 cells. Error bars indicate SD. (D and E) Graphs of the width of the broad band of nodes, actin network, and Myo51 in cells expressing Rlc1p-tdTomato, GFP-CHD, or Myo51p-3YFP in (D) wildtype cells (n = 11) and (E) myo2-E1 cells (n = 11). The width of the network of actin was measured until the actin patches returned to the equator masking the network of actin filaments and bundles. Error bars indicate SD. (F) Micrographs (inverted grayscale LUT) and color merge of a cell expressing mEGFP-Myo2p-E1 (green) and mCherry-Myp2p (red). Myo2p-E1 and Myp2p do not colocalize. (G) Effect of 100 mM LatA for 10 min on the localization of mEGFP-Myo2p and mEGFP-Myp2p in cells expressing Sad1p-RFP to determine cell-cycle time: (left) mEGFP-Myo2p in a wild-type cell treated with LatA; (middle) mEGFP-Myp2p in a myo2-E1 cell treated with LatA; and (right) mEGFP-Myp2p in a myo2-E1 cell treated with DMSO. Micrographs (inverted grayscale LUT). Confocal fluorescence micrographs are maximum intensity projections of 18 confocal z-sections. Scale bars, 5 mm.

defects at room temperature, and 22% of these cells were in cytokinesis, similarly to wild-type cells (Table 1) [23, 30]. Thus, Myo51 makes a modest contribution to node coalescence. The combination of Myp2 and Myo51 (myo2-E1 strain) performed only modestly better than cells with Myo51 alone and had more problems with ring assembly than the other strains with two functional myosins. Nodes began moving at the normal time +1 min, but the node velocity distribution had only a single peak at the slow velocity of 30 nm/s (Figure 2C). The population of myo2-E1 cells began to accumulate compact rings 10 min late, and the mean time for completion was 6 min late (24.2 ± 3.9 min) (Figure 2E; Table 1). Close examination of the myo2-E1 cells revealed subtle problems with the distributions of both myosins and actin filaments. Myp2 and Myo51 accumulated at the division site at their normal cell-cycle times (asterisks in Figures 3A and 3B), but the equatorial zone of Myo51p-3YFP was broader than normal (Figures 3B, bracket, 3D, and 3E). Myp2 appeared in spots near the equator rather than in fully formed rings (Figure 3A). Most Myp2 spots were separate from nodes marked with mEGFP-Myo2p-E1 (Figure 3F). Cells treated with Latrun-

culin A (LatA) to depolymerize actin filaments did not accumulate these spots of Myp2, even though the localization of mEGFP-Myo2p to nodes was unaffected (Figure 3G). Thus, localization of both Myo51 [23, 33] and Myp2 to the equator requires actin filaments. In both wild-type and myo2-E1 mutant cells, actin accumulated steadily around the equator during the 30 min of ring assembly and maturation, peaked at the onset of ring constriction, and decreased steadily as the ring disassembled (Figure 3C). However, the GFP-CHD fluorescence at the equator was higher throughout all phases of cytokinesis in myo2-E1 mutant cells (Figure 3C), especially at the end of cytokinesis when the GFPCHD signal persisted for 15 min longer than normal (Figure 3C; Movie S5). Furthermore, the equatorial actin network was wider than normal in myo2-E1 cells (Figures 3B, bracket, 3D, and 3E), even after the nodes had coalesced. Short Maturation Periods Compensate for Slow Ring Assembly in All Myosin Mutant Strains except Dmyp2 The maturation phase, the period of time after nodes coalesce into a ring and before the ring starts to constrict, normally lasts



about 13 min (compare Figures 2E and 2F; Table 1). In many mutant strains with slow contractile ring formation [36–41], the maturation period is foreshortened by the onset of ring constriction at the normal cell-cycle time, +32 min in our experiments. In cells where node coalescence extends well beyond +32 min, constriction starts just a few minutes after a continuous ring forms [36, 39], consistent with a cell-cycle clock that delays constriction until a set time of +32 min or until the ring is complete if ring assembly exceeds this limit. Most of our mutant strains followed these patterns. The maturation periods were shorter myo2-E1 cells (9 ± 4 min), Dmyo51 cells (8 ± 4 min), and Dmyp2 Dmyo51 (8 ± 4 min), which all had delays in ring formation. The myo2-E1 Dmyo51 cells relying on Myp2 took more than an hour to form rings, well beyond the 32-min time point, and then started constricting shortly after nodes finished coalescing (Figures 2E and 2F). The rings in myo2-E1 Dmyp2 cells dependent on only Myo51 never properly constricted, so maturation was indefinitely long (Figures 2G and 2I; Table 1). The Dmyp2 cells were an exception, since they assembled rings slightly faster than wild-type cells, but they had a very prolonged maturation period of 30 ± 10 min (Figures 2E and 2F). Therefore, the absence of Myp2 delayed the onset of constriction far beyond the usual time set by the cell-cycle clock. As the rings showed no observable defect, the reason for the delay in the onset of ring constriction is unclear. Myo2 and Myo51 Support Contractile Ring Constriction by Myp2 We followed contractile ring constriction in strains with single and double mutations of the three myosins using fluorescent protein tags on either the N terminus of the Myo2p heavy chain (Figures 2F, 2H, and 2I) or the C terminus of the regulatory light chain Rlc1p (Figure S2). Rings began to constrict in wild-type cells at cell-cycle time +32 min and continued at a steady rate of 0.27 mm/min until the ring circumference decreased from 10.5 ± 0.3 mm to 4 mm (Figures 2F, 2H, and 2I; Table 1; Movie S1). In cells relying on Myp2 alone (myo2-E1 Dmyo51 strain), contractile ring constriction started very late (owing to very slow ring formation compensated by a very short maturation time) but proceeded at 55% of the rate of wild-type cells (Figures 2F, 2H, and 2I; Table 1; Movie S3). Although 38 of 63 of these cells formed abnormal rings (Figure 2B), all of these rings constricted. Cells with laterally joined rings (4% of rings observed; Figure 2B) slowly merged the two rings into one before constriction started. In the cells with lasso-shaped rings, the lateral branch disassembled as the ring constricted (Movie S3). Rings in cells expressing Myo2 alone (Dmyp2 Dmyo51 strain) constricted at 0.07 mm/min, 25% the rate of wild-type cells (Figures 2H and 2I; Table 1; Movie S2), but 14 of 37 rings regressed after constricting partially (Figure 2I). In those cells starting with rings larger than the circumference of the cell (Figure 2D, inset) constriction pulled the rings into taut, straight rings. In a few Dmyp2 Dmyo51 cells (6/37 cells), a branch marked with mEGFP-Myo2p separated from the constricting ring and reformed a new ring, similar to Dmyp2 cells. Of the strains dependent on a single myosin, only the cells of the myo2-E1 Dmyp2 strain relying on Myo51 alone failed to constrict their contractile rings (Figures 2G–2I; Movie S4). Newly assembled rings were smooth but bent. Rather than constricting,

these rings fragmented or separated into node-like structures (Figure 2G; Movie S4). A large proportion (77%, n = 77 cells) of the cells failed to separate during time-lapse movies lasting >2 hr. The strain was viable because the remaining 23% of the population eventually formed a septum that allowed the daughter cells to separate (Figure 2G). Of the cells that did not separate, 60% grew a septum juxtaposed to the irregular ring that was visible as a clear indentation. The ring material in the remaining 40% disassembled without the formation of a visible septum (Figure 2G). The presence of either Myo2 or Myo51 with Myp2 allowed for normal ring constriction, and the presence of Myo51 improved the performance of Myo2 (Figures 2H and 2I). Wang et al. also observed normal constriction rates in Dmyo51 and myo2-E1 cells [30]. Two of these strains had unique features. Rings in myo2-E1 mutant cells disassembled slowly, suggesting a role for Myo2 in contractile ring disassembly (Figures 2I, arrow, and 3C; Movie S5). Constricting rings branched in a few Dmyp2 cells (4/45 cells), and the branch formed a second ring that also constricted. This behavior suggests that Myp2 helps to maintain ring integrity during constriction. Myo2 and Myp2 Localize to Separate Concentric Ring Sub-sections during Constriction These experiments show that Myo2 is the most important myosin for ring assembly, whereas Myp2 is the most important myosin for ring constriction. Furthermore, the two myosins have different distributions around the equator [35], so we analyzed the distributions of Myo2p and Myp2p in contractile rings. We used microfabricated devices to hold yeast vertically during imaging [42, 43] to improve the resolution in the plane of the ring over digital reconstructions from series of optical sections (Figure 4). Time-lapse confocal microscopy (Movie S5) showed that Myo2 and Myp2 colocalized in the contractile ring prior to the start of constriction (Figure 4A, top row), but during constriction, the ring of Myp2 was internal to the ring of Myo2 (Figure 4A, bottom three rows). The internal Myp2 ring began constricting 4 min earlier than the peripheral Myo2 ring but constricted at a slightly slower rate than the Myo2 ring, so the two rings converged at the end of constriction. Rings of both Myo2 and Myp2 were located ahead of the leading edge of the invaginating plasma membrane marked with the SNARE protein GFP-Psy1p [44] (Figures 4E and 4F). During constriction, Myo2 remained in a perfect ring, but the distribution of Myp2p around the ring was irregular and varied over time. The ring of actin filaments marked with GFP-CHD overlapped with both mCherry-Myo2p and mCherry-Myp2p, suggesting that there are sub-regions of the contractile ring enriched in Myo2 or Myp2 (Figures 4B and 4C; Movie S5). Bundles of actin filaments containing mCherry-Myp2p peeled off from the main ring and moved centripetally (Movie S5) in wild-type cells and more so in myo2-E1 cells (Figure 4D; Movie S5). The Psy1p marker did not associate with these fragments, suggesting that plasma membrane does not follow. DISCUSSION In spite of evidence that myosin-II motor activity is not essential for cytokinesis in several model organisms [5–8, 11], previous



Figure 4. Two Layers of Myosin-II in Constricting Contractile Rings (A–E) Fluorescence micrographs (inverted grayscale LUT and color merge, maximum intensity projections of five confocal z-sections) from time lapse of cells held vertically in microfabricated chambers. Scale bars, 5 mm. (A) Time series of a wild-type cell expressing mCherry-Myo2p and mEGFP-Myp2p. The Myp2 ring began to constrict before the Myo2p ring, resulting in concentric rings with the Myp2p ring inside the Myo2p ring. Time is in minutes from time zero at the onset of constriction. (B) Wild-type cell expressing mCherry-Myo2p and GFP-CHD to mark actin filaments in a constricting ring showing overlap between the two proteins. GFP-CHD also labels endocytic patches that form on both sides of the cleavage furrow (arrow). (C) Wild-type cell expressing mCherry-Myp2p and GFPCHD to mark actin filaments showing overlap between the two proteins in a constricting ring and several punctate actin patches. (D) Micrographs at two time points of a myo2-E1 mutant cell expressing GFP-CHD and mCherry-Myp2p. (E) Cleavage furrow of a wild-type cell expressing the plasma membrane marker GFP-Psy1p and mCherry-Myp2p. (F) Cleavage furrow of a wild-type cell expressing the plasma membrane marker GFP-Psy1p and mCherry-Myo2p. Fluorescence micrographs are z-reconstructions of maximum intensity projections of 18 confocal z-sections from a cell imaged lying horizontally. See also Movie S5.

work did not provide decisive evidence that other types of myosin contribute to the assembly or constriction of the contractile ring. We show that two myosins besides conventional myosin-II participate in cytokinesis in fission yeast. Each myosin has different biochemical properties, and each makes a unique contribution to the assembly or constriction of the contractile ring. Given the participation of three myosins in a redundant system, mutations of single-myosin genes did not give a clear picture of their contributions to cytokinesis. However, their functions and limitations were obvious in strains with pairs of mutations making the cells dependent on just one of the three myosins. Multiple myosins may explain cytokinetic ring constriction in COS-7 cells with compromised myosin-II activity [8]. A Hierarchy of Myosins Contributes to Contractile Ring Assembly Normally, Myo2 and Myo51 cooperated to assemble contractile rings, and either Myo2 or Myo51 can assemble normal contractile rings without the assistance of Myp2. Myo2 is the first myosin to arrive and is a component of cytokinesis nodes, followed at time zero by Myo51, which concentrates in puncta among the broad band of nodes [23, 30, 33]. Localization of Myo51 depends on actin filaments assembled from nodes and on a complex of proteins called Rng8p and Rng9p [23, 30]. As a component of nodes, Myo2 is thought to apply force on actin filaments from other nodes [26], whereas Myo51 is more likely to act on oppositely polarized actin filaments between nodes [30]. Myo51

may promote node interactions by pulling antiparallel filaments into bundles that interact with nearby nodes, as proposed by Wang et al. [30], as well as attracting actin cables to the forming ring [45]. Node condensation by Myo51 alone has two interesting features. First, although nodes start moving near time +1 min in cells relying on either Myo2 or Myo51 alone, these strains require far more time to condense nodes into a contractile ring than cells with both Myo2 and Myo51. Therefore, even though Myo2 alone moves nodes at normal rates, condensation is slow without Myo51. Clearly, these two myosins interact productively during contractile ring formation. Second, formation of the narrow equatorial band of Myo51 depends on actin filaments [23, 33], but these initial rings of Myo51 form before nodes condense, and the distribution of Myo51 in these rings differs from actin and all other known cytokinesis components (Figure 1A). Myp2 does not participate in contractile ring assembly in wild-type cells, because it arrives after rings are complete, but in cells lacking both Myo2 and Myo51 function, Myp2 alone can move nodes at 25 nm/s, and these motions slowly form a contractile ring. However, many of these rings are defective. Purified Myo2p-E1 has little activity, even in the presence of its chaperone Rng3p [12], so residual Myo2p-E1 motor activity is unlikely to assist Myp2 in these cells. Actin filaments are necessary but not sufficient for Myp2 to concentrate around the equator, since it arrives well after actin. Therefore, other factors must limit targeting of Myp2 to the equator until time +20 min.



A Different Hierarchy of Myosins Contributes to Contractile Ring Constriction Judging from the behavior of cells with single functional cytokinetic myosins, Myp2 is the only myosin that reliably completes ring constriction on its own. This is surprising, since Myp2 is dispensable for cytokinesis under most conditions [19, 21, 22]. On the other hand, the essential Myo2 alone can only support constriction at 25% of the wild-type rate, and partially completed furrows often regressed unless Myo51 was also present. Myo51 cannot support ring constriction in the absence of Myp2 and Myo2, but it is an important accessory, as it can assist either Myo2 or Myp2 during constriction. The explanation for these observations is that each of the three myosins makes a unique contribution to constriction and furrow formation, so that pairs of the myosins are more effective than any single myosin. The tails of the myosins may be responsible, since experiments with chimeras showed that the tails of Myo2p and Myp2p each make unique contributions [35]. Computational simulations of a model of cytokinetic ring constriction with myosin clusters and anchored formins polymerizing actin filaments produce forces similar to those measured in protoplasts [29]. This model is agnostic about the type of myosin generating the forces but assumes that myosin forms clusters associated with the plasma membrane that interact with oppositely polarized actin filaments. Myo2 fulfills these criteria in that it aggregates at physiological salt concentrations [21], and multiple Myo2 molecules cluster in nodes associated with the membrane [28]. However, Myo2 is less important than the single-headed Myp2 in constricting the ring. Neither Myp2 nor Myo51 is known to associate with nodes, so they may interact with actin filaments in between their Cdc12p anchors. Myo51 forms macromolecular assemblies with Rng8p and Rng9p [30], but nothing is known about the assembly of Myp2 or its interactions with the plasma membrane. Given that Myo2 is closer to the base of the cleavage furrow than Myp2, Myo2 may have stronger physical interactions with transmembrane proteins that anchor the contractile ring than Myp2. Our evidence for three myosins participating in contractile ring assembly and constriction raises a long list of questions about mechanisms and opens up new areas of research. Addressing these questions will require more detailed information about the physical and enzymatic properties of the three myosins and their organization in the contractile ring. EXPERIMENTAL PROCEDURES Strains, Growing Conditions, and Genetic and Cellular Methods Table S1 lists the S. pombe strains used in this study. All tagged genes, except for the GFP-CHD construct, were under the control of endogenous promoters and integrated at their native chromosomal loci. N-termini of myo2+ and myp2+ were tagged by integrating their promoters into the pFA6a fission yeast integration vector [46]. Long PCR products were generated and integrated into the genome [46]. Positive clones were selected on YE5S plates with 0.1 mg/ml G418 and verified by PCR of the insert using appropriate primers, by fluorescence microscopy, and by DNA sequencing. We tagged genes in myo2-E1, myo2-S2, and myo2-DIQ12 in the same way and verified the specific point mutations or deletions by genomic DNA sequencing. Cells were grown in an exponential phase for 36–48 hr before microscopy [47]. To de-repress the expression of the Pnmt41x-GFP-CHD actin marker, cells were shifted to EMM5S 9–12 hr prior to imaging.

Microscopy and Data Analysis Cells for live-cell microscopy were collected from liquid cultures, centrifuged at 5,000 rpm, and then washed in EMM5S for imaging. Cells were spun down for 30 s at 5,000 rpm, washed in EMM5S and then in EMM5S with 0.1 mM n-propyl-gallate, and resuspended in EMM5S with 0.1 mM n-propylgallate and placed onto a thin disk of 25% porcine gelatin (Sigma-Aldrich) dissolved in EMM5S + 0.1 mM n-propyl-gallate. Cells were covered with a #1.5 coverslip, sealed with VALAP (1:1:1 Vaseline:lanolin:paraffin), and immediately imaged at room temperature (23–25 C). We imaged cells using an inverted Olympus IX-71 microscope with a 1003/ 1.40 oil objective (Olympus) with a Yokogawa CSU-X1 spinning-disk confocal unit, 488/514 and 568-nm argon ion lasers, and an electron-multiplying cooled charge-coupled device camera (EMCCD IXon 897, Andor). Maximum intensity projections of images, grayscale montages, and other image analyses were made with ImageJ (NIH). Images in the figures are maximum intensity projections of z-sections spaced at 0.34 mm. Images were analyzed with ImageJ (NIH). We defined the end of node coalescence as the time when nodes converged into a narrow ring around the equator so that individual nodes were no longer resolved by confocal microscopy. We measured the rate of ring constriction on kymographs of maximum projection images (18 z-confocal planes taken for 6.12 mm) of time-lapse datasets taken at 1- to 2-min time intervals. The diameter of a ring was measured automatically by fluorescence thresholding, and the circumference was calculated. We counted myosins in whole cells and contractile rings by measuring fluorescence intensities in stacks of 18 optical images separated by 0.34 mm of fields of cells [28]. The images were corrected for the camera noise and uneven illumination. The numbers of molecules were calculated from the fluorescence intensities and standard curves for either yellow fluorescent protein (YFP) or monomeric EGFP (mEGFP). Node Velocity Measurements A single z-plane of cells expressing a node marker was imaged by time-lapse microscopy at intervals of 110 ms, 1 s, or 2 s. Nodes were identified manually and selected using a region of interest 7 pixels (0.58 mm) in diameter with ImageJ. Nodes were tracked through time until the object disappeared (perhaps due to photobleaching of the fluorescent tag or moving out of focus) or until it fused with other another node or the assembling ring. At each time point, the node position was obtained with sub-pixel resolution using its fluorescence intensity center of mass, i.e., the brightness-weighted average of the x and y coordinates of all pixels in the selection. Velocities of nodes were calculated using MATLAB as the displacement of the particle between two consecutive time points and filtered using a moving average with a weight of 2 for the current time point and a weight of 1 for the previous and following time points. This procedure removed occasional jumps in speeds due to localization errors. Yeast Cell Holders We made a mold on a silicon wafer consisting of regular arrays of pillars, 6 or 7 mm in diameter and 14 mm high. The pillars were made of SU-8 negative resist using standard photolithography methods at the School of Engineering and Applied Science clean room (Yale University). We then crosslinked poly(dimethyl siloxane) (PDMS) on the mold, using the Sylgard 184 Silicone Elastomer Kit (Dow Corning). We mixed the base and the crosslinker at a 10:1 ratio, degassed the mixture, and poured it on top of the mold. The PDMS was cured overnight at 65 C. Yeast holders were cut from the PDMS and placed under vacuum for at least 30 min before being used with cells. A dilute solution of cells prepared as described earlier was poured on top of the PDMS mold and turned onto a circular #1.5 coverslip 40 mm in diameter. The mold was pressed against the coverslip to allow the cells to enter the chambers, and the cells were imaged immediately. Chambers 6 and 7 mm in diameter allowed space for medium around the cells.

SUPPLEMENTAL INFORMATION Supplemental Information includes two figures, one table, and five movies and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2015. 06.018.



AUTHOR CONTRIBUTIONS

14. East, D.A., and Mulvihill, D.P. (2011). Regulation and function of the fission yeast myosins. J. Cell Sci. 124, 1383–1390.

C.L. and T.D.P. designed and interpreted the experiments. C.L. conducted the experiments. J.B. wrote the node-tracking program and advised on data analysis. E.K. helped design and make the yeast holders. Undergraduate students A.H.L. and R.L. acquired movies and performed preliminary node velocity measurements. C.L. and T.D.P. wrote the paper, which was edited by J.B. and E.K.

15. Attanapola, S.L., Alexander, C.J., and Mulvihill, D.P. (2009). Ste20-kinasedependent TEDS-site phosphorylation modulates the dynamic localisation and endocytic function of the fission yeast class I myosin, Myo1. J. Cell Sci. 122, 3856–3861.

ACKNOWLEDGMENTS Research reported in this publication was supported by the National Institute of General Medical Sciences of the NIH under award number R01GM026132. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. C.L. was supported by a Long-Term Fellowship from the Human Frontier Science Program. We thank Sophie Martin, Mohan Balasubramanian, Jian-Qiu Wu, and Matthew Lord for yeast strains. We thank Rajesh Arasada and Matthew Akamatsu for helpful comments on the manuscript. Received: October 19, 2014 Revised: May 16, 2015 Accepted: June 5, 2015 Published: July 2, 2015 REFERENCES 1. Fujiwara, K., and Pollard, T.D. (1976). Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells. J. Cell Biol. 71, 848–875. 2. Mabuchi, I., and Okuno, M. (1977). The effect of myosin antibody on the division of starfish blastomeres. J. Cell Biol. 74, 251–263. 3. De Lozanne, A., and Spudich, J.A. (1987). Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science 236, 1086–1091. 4. Bi, E., Maddox, P., Lew, D.J., Salmon, E.D., McMillan, J.N., Yeh, E., and Pringle, J.R. (1998). Involvement of an actomyosin contractile ring in Saccharomyces cerevisiae cytokinesis. J. Cell Biol. 142, 1301–1312. 5. Fang, X., Luo, J., Nishihama, R., Wloka, C., Dravis, C., Travaglia, M., Iwase, M., Vallen, E.A., and Bi, E. (2010). Biphasic targeting and cleavage furrow ingression directed by the tail of a myosin II. J. Cell Biol. 191, 1333–1350. 6. Mendes Pinto, I., Rubinstein, B., Kucharavy, A., Unruh, J.R., and Li, R. (2012). Actin depolymerization drives actomyosin ring contraction during budding yeast cytokinesis. Dev. Cell 22, 1247–1260. 7. Lord, M., Laves, E., and Pollard, T.D. (2005). Cytokinesis depends on the motor domains of myosin-II in fission yeast but not in budding yeast. Mol. Biol. Cell 16, 5346–5355. 8. Ma, X., Kovaćs, M., Conti, M.A., Wang, A., Zhang, Y., Sellers, J.R., and Adelstein, R.S. (2012). Nonmuscle myosin II exerts tension but does not translocate actin in vertebrate cytokinesis. Proc. Natl. Acad. Sci. USA 109, 4509–4514. 9. Kitayama, C., Sugimoto, A., and Yamamoto, M. (1997). Type II myosin heavy chain encoded by the myo2 gene composes the contractile ring during cytokinesis in Schizosaccharomyces pombe. J. Cell Biol. 137, 1309–1319. 10. May, K.M., Watts, F.Z., Jones, N., and Hyams, J.S. (1997). Type II myosin involved in cytokinesis in the fission yeast, Schizosaccharomyces pombe. Cell Motil. Cytoskeleton 38, 385–396. 11. Balasubramanian, M.K., McCollum, D., Chang, L., Wong, K.C., Naqvi, N.I., He, X., Sazer, S., and Gould, K.L. (1998). Isolation and characterization of new fission yeast cytokinesis mutants. Genetics 149, 1265–1275. 12. Lord, M., and Pollard, T.D. (2004). UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II. J. Cell Biol. 167, 315–325. 13. Pollard, T.D., and Wu, J.Q. (2010). Understanding cytokinesis: lessons from fission yeast. Nat. Rev. Mol. Cell Biol. 11, 149–155.

16. Lee, W.L., Bezanilla, M., and Pollard, T.D. (2000). Fission yeast myosin-I, Myo1p, stimulates actin assembly by Arp2/3 complex and shares functions with WASp. J. Cell Biol. 151, 789–800. 17. Sirotkin, V., Beltzner, C.C., Marchand, J.B., and Pollard, T.D. (2005). Interactions of WASp, myosin-I, and verprolin with Arp2/3 complex during actin patch assembly in fission yeast. J. Cell Biol. 170, 637–648. 18. Le Goff, X., Motegi, F., Salimova, E., Mabuchi, I., and Simanis, V. (2000). The S. pombe rlc1 gene encodes a putative myosin regulatory light chain that binds the type II myosins myo3p and myo2p. J. Cell Sci. 113, 4157– 4163. 19. Motegi, F., Nakano, K., and Mabuchi, I. (2000). Molecular mechanism of myosin-II assembly at the division site in Schizosaccharomyces pombe. J. Cell Sci. 113, 1813–1825. 20. Naqvi, N.I., Eng, K., Gould, K.L., and Balasubramanian, M.K. (1999). Evidence for F-actin-dependent and -independent mechanisms involved in assembly and stability of the medial actomyosin ring in fission yeast. EMBO J. 18, 854–862. 21. Bezanilla, M., Forsburg, S.L., and Pollard, T.D. (1997). Identification of a second myosin-II in Schizosaccharomyces pombe: Myp2p is conditionally required for cytokinesis. Mol. Biol. Cell 8, 2693–2705. 22. Mulvihill, D.P., and Hyams, J.S. (2003). Role of the two type II myosins, Myo2 and Myp2, in cytokinetic actomyosin ring formation and function in fission yeast. Cell Motil. Cytoskeleton 54, 208–216. 23. Lo Presti, L., Chang, F., and Martin, S.G. (2012). Myosin Vs organize actin cables in fission yeast. Mol. Biol. Cell 23, 4579–4591. 24. Mulvihill, D.P., Edwards, S.R., and Hyams, J.S. (2006). A critical role for the type V myosin, Myo52, in septum deposition and cell fission during cytokinesis in Schizosaccharomyces pombe. Cell Motil. Cytoskeleton 63, 149–161. 25. Wu, J.Q., Kuhn, J.R., Kovar, D.R., and Pollard, T.D. (2003). Spatial and temporal pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis. Dev. Cell 5, 723–734. 26. Vavylonis, D., Wu, J.Q., Hao, S., O’Shaughnessy, B., and Pollard, T.D. (2008). Assembly mechanism of the contractile ring for cytokinesis by fission yeast. Science 319, 97–100. 27. Wachtler, V., Rajagopalan, S., and Balasubramanian, M.K. (2003). Sterol-rich plasma membrane domains in the fission yeast Schizosaccharomyces pombe. J. Cell Sci. 116, 867–874. 28. Wu, J.Q., and Pollard, T.D. (2005). Counting cytokinesis proteins globally and locally in fission yeast. Science 310, 310–314. 29. Stachowiak, M.R., Laplante, C., Chin, H.F., Guirao, B., Karatekin, E., Pollard, T.D., and O’Shaughnessy, B. (2014). Mechanism of cytokinetic contractile ring constriction in fission yeast. Dev. Cell 29, 547–561. 30. Wang, N., Lo Presti, L., Zhu, Y.H., Kang, M., Wu, Z., Martin, S.G., and Wu, J.Q. (2014). The novel proteins Rng8 and Rng9 regulate the myosin-V Myo51 during fission yeast cytokinesis. J. Cell Biol. 205, 357–375. 31. Grallert, A., Martıń-Garcıá, R., Bagley, S., and Mulvihill, D.P. (2007). In vivo movement of the type V myosin Myo52 requires dimerisation but is independent of the neck domain. J. Cell Sci. 120, 4093–4098. 32. Motegi, F., Arai, R., and Mabuchi, I. (2001). Identification of two type V myosins in fission yeast, one of which functions in polarized cell growth and moves rapidly in the cell. Mol. Biol. Cell 12, 1367–1380. 33. Win, T.Z., Gachet, Y., Mulvihill, D.P., May, K.M., and Hyams, J.S. (2001). Two type V myosins with non-overlapping functions in the fission yeast Schizosaccharomyces pombe: Myo52 is concerned with growth polarity and cytokinesis, Myo51 is a component of the cytokinetic actin ring. J. Cell Sci. 114, 69–79.



34. Arasada, R., and Pollard, T.D. (2014). Contractile ring stability in S. pombe depends on F-BAR protein Cdc15p and Bgs1p transport from the Golgi complex. Cell Rep. 8, 1533–1544. 35. Bezanilla, M., and Pollard, T.D. (2000). Myosin-II tails confer unique functions in Schizosaccharomyces pombe: characterization of a novel myosinII tail. Mol. Biol. Cell 11, 79–91. 36. Chen, Q., and Pollard, T.D. (2011). Actin filament severing by cofilin is more important for assembly than constriction of the cytokinetic contractile ring. J. Cell Biol. 195, 485–498. 37. Coffman, V.C., Nile, A.H., Lee, I.J., Liu, H., and Wu, J.Q. (2009). Roles of formin nodes and myosin motor activity in Mid1p-dependent contractilering assembly during fission yeast cytokinesis. Mol. Biol. Cell 20, 5195– 5210. 38. Lee, I.J., and Wu, J.Q. (2012). Characterization of Mid1 domains for targeting and scaffolding in fission yeast cytokinesis. J. Cell Sci. 125, 2973–2985. 39. Saha, S., and Pollard, T.D. (2012). Anillin-related protein Mid1p coordinates the assembly of the cytokinetic contractile ring in fission yeast. Mol. Biol. Cell 23, 3982–3992. 40. Stark, B.C., Sladewski, T.E., Pollard, L.W., and Lord, M. (2010). Tropomyosin and myosin-II cellular levels promote actomyosin ring assembly in fission yeast. Mol. Biol. Cell 21, 989–1000. 41. Tebbs, I.R., and Pollard, T.D. (2013). Separate roles of IQGAP Rng2p in forming and constricting the Schizosaccharomyces pombe cytokinetic contractile ring. Mol. Biol. Cell 24, 1904–1917.

42. Zhou, Z., Munteanu, E.L., He, J., Ursell, T., Bathe, M., Huang, K.C., and Chang, F. (2015). The contractile ring coordinates curvature-dependent septum assembly during fission yeast cytokinesis. Mol. Biol. Cell 26, 78–90. 43. Wang, L., and Tran, P.T. (2014). Visualizing single rod-shaped fission yeast vertically in micro-sized holes on agarose pad made by soft lithography. Methods Cell Biol. 120, 227–234. 44. Nakamura, T., Nakamura-Kubo, M., Hirata, A., and Shimoda, C. (2001). The Schizosaccharomyces pombe spo3+ gene is required for assembly of the forespore membrane and genetically interacts with psy1(+)-encoding syntaxin-like protein. Mol. Biol. Cell 12, 3955–3972. 45. Huang, J., Huang, Y., Yu, H., Subramanian, D., Padmanabhan, A., Thadani, R., Tao, Y., Tang, X., Wedlich-Soldner, R., and Balasubramanian, M.K. (2012). Nonmedially assembled F-actin cables incorporate into the actomyosin ring in fission yeast. J. Cell Biol. 199, 831–847. 46. Ba¨hler, J., Wu, J.Q., Longtine, M.S., Shah, N.G., McKenzie, A., 3rd, Steever, A.B., Wach, A., Philippsen, P., and Pringle, J.R. (1998). Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14, 943–951. 47. Wu, J.Q., Sirotkin, V., Kovar, D.R., Lord, M., Beltzner, C.C., Kuhn, J.R., and Pollard, T.D. (2006). Assembly of the cytokinetic contractile ring from a broad band of nodes in fission yeast. J. Cell Biol. 174, 391–402.


Mechanism of cytokinetic contractile ring constriction in fission yeast.

The contractile ring coordinates curvature-dependent septum assembly during fission yeast cytokinesis.

The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast.

Still and rotating myosin clusters determine cytokinetic ring constriction.

A New Membrane Protein Sbg1 Links the Contractile Ring Apparatus and Septum Synthesis Machinery in Fission Yeast.

Constriction model of actomyosin ring for cytokinesis by fission yeast using a two-state sliding filament mechanism.

INSIGHTS INTO THE MECHANICS OF CYTOKINETIC RING ASSEMBLY USING 3D MODELING.

Mechanoregulated inhibition of formin facilitates contractile actomyosin ring assembly.

Cytokinetic nodes in fission yeast arise from two distinct types of nodes that merge during interphase.

Form and function of the bacterial cytokinetic ring.

Detection and characterization of a ring chromosome in the fission yeast Schizosaccharomyces pombe.

Fission Yeast Exo1 and Rqh1-Dna2 Redundantly Contribute to Resection of Uncapped Telomeres.

The septation initiation network controls the assembly of nodes containing Cdr2p for cytokinesis in fission yeast.

Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast.

Aim44p regulates phosphorylation of Hof1p to promote contractile ring closure during cytokinesis in budding yeast.

The fission yeast, Schizosaccharomyces pombe.

Myo2p is the major motor involved in actomyosin ring contraction in fission yeast.

The proper splicing of RNAi factors is critical for pericentric heterochromatin assembly in fission yeast.

Genetics of the fission yeast Schizosaccharomyces pombe.

The budding yeast amphiphysin complex is required for contractile actin ring (CAR) assembly and post-contraction GEF-independent accumulation of Rho1-GTP.

The Cell Biology of Fission Yeast Septation.

The putative exchange factor Gef3p interacts with Rho3p GTPase and the septin ring during cytokinesis in fission yeast.

PomBase 2015: updates to the fission yeast database.

Rab1 interacts with GOLPH3 and controls Golgi structure and contractile ring constriction during cytokinesis in Drosophila melanogaster.