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

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1 Current Biology Supplemental Information Three Myosins Contribute Uniquely to the Assembly and Constriction of the Fission Yeast Cytokinetic Contractile Ring Caroline Laplante, Julien Berro, Erdem Karatekin, Ariel Hernandez-Leyva, Rachel Lee, and Thomas D. Pollard

2 Supplemental Data Figure S1 related to Figure 1: Genetic interactions between myosin genes and numbers of myosin polypeptides in mutant strains. (A) Growth of ten-fold dilution series of strains incubated on YE5S agar plates at 25 C or 36 C. Attempts to isolate a myo2-e1 myp2 myo51 triple mutant failed suggesting that the triple mutant is not viable. (B) DIC images of fields of myosin mutant cells grown at 25 C. Examples of cells with septation defects are marked with asterisks. Cells with branched morphologies often stack up resulting in

3 cells that are out of focus (circled cells). The myo2-e1 cells showed a mild septation defect with cells that have multiple septum when grown at room temperature on YE5S plates (data not shown). The myp2 cells were indistinguishable from wild-type cells when grown under normal growth conditions (data not shown). Scale bar: 10 µm. (C) Kymographs made from fluorescent confocal micrographs (inverted gray scale LUT) of vertically oriented cells expressing megfp-myo2p or megfp-myo2p-e1. Genotypes are in italics and the functional myosins are listed in the boxes. Node coalescence is similar to wildtype cells in myo52 myp2 and myo52 myo2-e1 cells but is prolonged in myo51 myo2-e1 and myo51 myp2 cells. Black arrow points to the time the nodes have coalesced into a ring. (D, E) Local number of molecules of megfp-myo2p (D) and megfp-myp2p (E) measured by quantitative fluorescence microscopy. Measurements represent average numbers of polypeptides in rings that have constricted by less than 20% (mean ± standard deviation). (F) Numbers of molecules of Myo51p-3YFP in constricting rings with different diameters measured by quantitative fluorescence microscopy (mean ± standard deviation).

4 Figure S2 related to Figure 2. Comparison of contractile ring formation and constriction in strains with different myosin-ii alleles. Each strain expressed Rlc1p-tdTomato, the regulatory light chain for both Myo2 and Myp2 and Sad1p-mEGFP to mark the SPB and establish time cell cycle time zero. Cells were imaged by time-lapse confocal fluorescence microscopy and analyzed for coalescence of nodes containing Rlc1p-tdTomato into contractile rings and the rates of ring constriction. The myo2- IQ1, myo2-s1 and myo2-s2 mutations each delayed the time course of node coalescence more than the myo2-e1 mutation, but all three strains eventually formed functional contractile rings. Rings constricted at the same rate as wild-type cells in the myo2- IQ1 strain but constricted slower in myo2-e1, myo2-s1 and myo2-s2 cells. (A) Outcomes plot showing the accumulation of cells with nodes condensed into a contractile ring or other compact structure around the equator as a function of time. LogRank statistical test * p > 0.05 and ** p > (B) Histogram of average contractile ring constriction rates (± standard deviations, n = 20 to 25 cells per genotype) in the myosin mutant strains. Student t-test ** p > 0.01.

5 Table S1 related to Experimental Procedures. List of strains used in this work Strain Genotype Reference JW1351 h- nmt41-gfp-chd (rng2)-leu1+ rlc1-tdtomato-natmx6 ade6-m210 leu1-32 [S1] ura4-d18 cdc25-22 TP3 h- myp2::his7+ his7-366 leu1-32 ura4-d18 ade6-m210 [S2] MBY53 h- myo2-s1 ade6-21x ura4-d18 leu1-32 [S3] MBY54 h- myo2-s2 ade6-21x ura4-d18 [S3] MLP679 h- myo2-iq 1:ura4+ leu1-32 [S4] YSM2004 h- myo51-3yfp-kanmx ade-m216 leu1-32 [S5] CL5 h- Rlc1-tdTomato-NatMX6 Sad1-mEGFP-KanMX6 ade6-21x ura4-d18 leu1-32 From Rajesh Arasada CL26 h- myo2-e1 nmt41-gfp-chd (rng2)-leu1+ rlc1-tdtomato-natmx6 ade6-m21x Crossed using JW1351 leu1-32 ura4-d18 CL55 h+ myo2-e1 Rlc1-tdTomato-NatMX6 Sad1-mEGFP-KanMX6 ade6-21x ura4-d18 leu1-32 Crossed using myo2-e1 isolated in [S6] CL97 h+ myo2-s1 Rlc1-tdTomato-NatMX6 Sad1-mEGFP-KanMX6 ade6-21x ura4-d18 CL98 h+ myo2-s2 Rlc1-tdTomato-NatMX6 Sad1-mEGFP-KanMX6 ade6-21x ura4-d18 leu1-32 CL110 h+ nmt41-gfp-chd (rng2)-leu1+ rlc1-tdtomato-natmx6 ade6-m21x leu1-32 Crossed using JW1351 ura4-d18 CL132 h- myp2::his7+ Rlc1-tdTomato-NatMX6 Sad1-mEGFP-KanMX6 ade6-21x ura4- D18 leu1-32 CL153 h+ myo2- IQ1 Rlc1-tdTomato-NatMX6 Sad1-mEGFP-KanMX6 ade6-21x ura4- D18 leu1-32 CL177 h+ KanMX6-Pmyp2-mEGFP-myp2 Sad1-RFP-KanMX6 ade6-21x ura4-d18 leu1-32 CL181 h+ KanMX6-Pmyo2-mEGFP-myo2 Sad1-RFP-KanMX6 ade6-m216 his3-d1 leu1-32 ura4-d18 CL184 h- myp2::his7+ KanMX6-Pmyo2-mEGFP-myo2-E1 Sad1-RFP-KanMX6 ade6-21x ura4-d18 leu1-32 CL188 h- KanMX6-Pmyo2-mCherry-myo2 ade6-m216 his3- CL195 myo2-e1 KanMX6-Pmyp2-mEGFP-myp2 Sad1-RFP-KanMX6 ade6-21x ura4-d18 leu1-32 CL198 nmt41-gfp-chd (rng2)-leu1+ KanMX6-Pmyo2-mCherry-myo2 ade6-m21x his3- CL200 KanMX6-Pmyp2-mCherry-myp2 KanMX6-Pmyo2-mEGFP-myo2 ade6-m216 his3-

6 CL203 myp2::his7+ KanMX6-Pmyo2-mEGFP-myo2 Sad1-mEGFP-KanMX6 ade6-m216 his3- CL208 h+ KanMX6-Pmyo2-mEGFP-myo2-E1 Sad1-RFP-KanMX6 ura4-d18 leu1-32 ade6-m216 CL210 myo2-e1 nmt41-gfp-chd (rng2)-leu1+ KanMX6-Pmyp2-mCherry-myp2 ade6- M216 his3- CL213 KanMX6-Pmyo2-mEGFP-myo2-E1 KanMX6-Pmyp2-mCherry-myp2 ade6-m216 leu1-32 ura4-d18 CL214 nmt41-gfp-chd (rng2)-leu1+ KanMX6-Pmyp2-mCherry-myp2 ade6-m21x his3- CL215 h- KanMX6:: myo51 ade6-m216 his3- CL217 h+ KanMX6:: myo52 ade6-m216 his3- CL218 h+ KanMX6-Pmyo2-mCherry-myo2-E1 ura4-d18 leu1-32 ade6-m216 CL221 KanMX6:: myo51 KanMX6-Pmyo2-mEGFP-myo2-E1 Sad1-RFP-KanMX6 ura4- D18 leu1-32 ade6-m216 his3-d18 CL222 KanMX6:: myo52 KanMX6-Pmyo2-mEGFP-myo2-E1 Sad1-RFP-KanMX6 ura4- D18 leu1-32 ade6-m216 his3-d18 CL223 KanMX6:: myo51 myp2::his7+ KanMX6-Pmyo2-mEGFP-myo2 Sad1-RFP- KanMX6 ade6-m216 his3- CL224 KanMX6:: myo52 myp2::his7+ KanMX6-Pmyo2-mEGFP-myo2 Sad1-RFP- KanMX6 ade6-m216 his3- CL228 myo51-3yfp-kanmx6 Sad1-mEGFP-KanMX6 ade6-21x CL234 myo2-e1 myo51-3yfp-kanmx6 Sad1-mEGFP-KanMX6 ade6-21x CL235 myo2-e1 myo51-3yfp-kanmx6 ade6-21x CL240 KanMX6:: myo51 KanMX6-Pmyo2-mEGFP-myo2 Sad1-RFP-KanMX6 ade6-21x CL264 KanMX6:: myo51 KanMX6-Pmyo2-mCherry-myo2-E1 KanMX6-Pmyp2-mEGFPmyp2 Sad1-RFP-KanMX6 ura4-d18 leu1-32 ade6-m216 CL266 KanMX6:: myo51 KanMX6-Pmyp2-mEGFP-myp2 Sad1-RFP-KanMX6 ura4-d18 leu1-32 ade6-m216 CL270 psy1::kanmx6 leu1+::ppsy1-gfp-psy1 KanMX6-Pmyp2-mCherry-myp2 ade6- M21x ura4-d18 CL271 psy1::kanmx6 leu1+::ppsy1-gfp-psy1 KanMX6-Pmyp2-mCherry-myp2 ade6- M21x ura4-d18 CL276 KanMX6-Pmyp2-mCherry-myo2 KanMX6-Pmyo2-mEGFP-myp2 ade6-m216 his3-

7 Supplemental References S1. 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, S2. 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. Molecular biology of the cell 8, S3. Wong, K.C., Naqvi, N.I., Iino, Y., Yamamoto, M., and Balasubramanian, M.K. (2000). Fission yeast Rng3p: an UCS- domain protein that mediates myosin II assembly during cytokinesis. Journal of cell science 113 ( Pt 13), S4. Lord, M., and Pollard, T.D. (2004). UCS protein Rng3p activates actin filament gliding by fission yeast myosin- II. The Journal of cell biology 167, S5. Lo Presti, L., Chang, F., and Martin, S.G. (2012). Myosin Vs organize actin cables in fission yeast. Molecular biology of the cell 23, S6. 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,

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