Karen L.P. McNally, Amy S. Fabritius, Marina L. Ellefson, Jonathan R. Flynn, Jennifer A. Milan, and Francis J. McNally

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1 Developmental Cell, Volume 22 Supplemental Information Kinesin-1 Prevents Capture of the Oocyte Meiotic Spindle by the Sperm Aster Karen L.P. McNally, Amy S. Fabritius, Marina L. Ellefson, Jonathan R. Flynn, Jennifer A. Milan, and Francis J. McNally Inventory of Online Supplemental Materials Figure S1. SPD-5 is not recruited to sperm centrioles until completion of meiosis in wild-type C. elegans. This figure show details of wild-type centrosome maturation relevant to figure 1. Figure S2. Specificity of antibody staining. This figure shows supplemental data related to figure 2. Figure S3. Centrosomes mature normally in tba-1(rnai) embryos. This figure shows supplemental data related to figure 3. Table S1. Frequency of centrosome maturation during meiosis. This table presents additional mutant phenotypes related to figure 1.

2 Figure S1 related to Figure 1. SPD-5 is not recruited to sperm centrioles until completion of meiosis in wild-type C. elegans. Maximum intensity projections of 3D image stacks of fixed meiotic embryos stained with DAPI (DNA) and anti- SPD-5 antibody. A. Anaphase II embryo. Inset shows that sperm DNA is highly condensed and no SPD-5 focus is apparent. B. Scission of the second polar body as indicated by the dispersal of the maternal chromosomes in the embryo. C. The spermderived DNA decondenses before the maternal DNA but no SPD-5 focus is present. D. A single SPD-5 focus has formed adjacent to the male pronucleus which is now at the cortex. Maternal chromosomes are still condensed. E. A single SPD-5 focus is next to the male pronucleus. Both male and female pronuclei have decondensed DNA but both pronuclei are still small. F. 2 large foci of SPD-5 are associated with the male pronucleus close to the cortex and DNA has started to condense in both large pronuclei. G. 2 foci of SPD-5 are associated with the male pronucleus which has moved away from the cortex and the female pronucleus has begun migrating. H. Pronuclear meeting. Bar in A = 5 μm, same scale A-H. I. Time-lapse sequence of living meiotic embryo expressing GFP::histone H2b and GFP::γ-tubulin filmed in utero. Time = extrusion of the first polar body. Sperm DNA, which also labeled with mcherry:histone H2B expressed from a sperm-specific promoter, is indicated by an arrow. A single focus of γ-tubulin (arrowhead) is first apparent at 15.5 min, after decondensation of the sperm-derived DNA and movement of the male pronucleus toward the cortex. In this sequence, the male pronucleus transiently dissociated from the centrosome (15.5 min) then reassociated with it (22 min). Embryo outlines drawn for clarity in E, H and I. Bar in I = 5 μm.

Error bars = SEM. * two-tailed p-value <.5. C. Quantification of raw pixel values of cytoplasmic anti- UNC-116 staining in wild type vs 116 (RNAi) meiotic embryos. Error bars = SEM.

3 Figure S2 related to Figure 2. Specificity of antibody staining. A. Example of fixed kca- 1(RNAi) meiotic embryos stained with anti-kca-1 and DAPI (DNA). No staining around the sperm DNA was observed in 42 kca-1(rnai) embryos. B. Quantification of raw pixel values of cytoplasmic anti-kca-1 staining in wild type vs kca-1 (RNAi) meiotic embryos. Error bars = SEM. * two-tailed p-value <.5. C. Quantification of raw pixel values of cytoplasmic anti- UNC-116 staining in wild type vs 116 (RNAi) meiotic embryos. Error bars = SEM. * twotailed p-value <.5. D. Fixed 116(RNAi) meiotic embryo showed no increased anti- UNC-116 staining intensity around the sperm DNA in meiotic embryos. Ratio indicates the fluorescence intensity around the sperm DNA divided by the fluorescence intensity of the adjacent cytoplasm. E. Fixed kca-1(rnai) meiotic embryo showed no increased anti-kca-1 staining intensity around the sperm DNA in meiotic embryos. F. Fixed meiotic embryo expressing tdimer2:yp17, DAPI (DNA) and anti-spd-5 (cytoplasm) showing an example where the sperm DNA is outside the region of packed yolk granules. Bar = 5 μm.

B. GFP:TAC-1 was recruited to sperm centrioles in 1/1 kca-1(rnai) meiotic embryos. C. Because tba-1(rnai) embryos never divide, staging mitotic embryos dissected from the worm is difficult.

4 Figure S3 related to Figure 3. Centrosomes mature normally in tba-1(rnai) and 116(f13) embryos. A. /1 tba-1(rnai) meiotic embryos had foci of maternally expressed GFP:TAC-1 associated with the sperm DNA. Lower images are enlargements of the boxed region containing the sperm DNA in the upper images. B. GFP:TAC-1 was recruited to sperm centrioles in 1/1 kca-1(rnai) meiotic embryos. C. Because tba-1(rnai) embryos never divide, staging mitotic embryos dissected from the worm is difficult. The preparation shown is of fixed embryos in the uterus of a partially dissected adult worm. The embryo labeled +1 is adjacent to the spermatheca and is in meiosis. The embryo in the +2 position is normally in the first mitotic division. The +2 embryo of this tba-1(rnai) worm has 2 foci of GFP:TAC-1 indicating that centrosome maturation occurs in the absence of cytoplasmic microtubules. Embryos have been outlined for clarity. 15/17 tba-1(rnai) embryos with mitotic chromosomes had centrosomal foci of GFP:TAC-1. 3/33 tba-1(rnai) embryos with mitotic chromosomes had centrosomal foci of GFP:γ-tubulin. D. SPD-5 was recruited to centrioles in 9/9 116(f13) pronuclear phase embryos just as in wild type. Three pronuclei were present in nearly all 116(f13) pronuclear phase embryos indicating a failure in polar body extrusion. Bar = 5 μm.

5 Table S1 related to Figure 1: Frequency of centrosome maturation during meiosis genotype wild type (n=15) kca-1(rnai) 14 (n=28) rol-1 rol-1; (RNAi) brc- 1(tm1145) brc-1; kca- 1 1(RNAi) brd- 1(ok1623) brd-1; kca- 27 1(RNAi) (n=11) szy-2(ts) 14(e57) 76(e911) 33(mn47) 16(e19) hpl- 2(tm1489) mpk-1(ts) % of embryos with SPD-5 foci at sperm DNA (n = # of embryos) % of embryos with γ-tubulin foci at sperm DNA (n = # of embryos) M I A I M II A II PN M I A I M II A II PN (n=15) 68 (n=22) (n=18) (n=17) 4 (n=16) 1 (n=32) 92 (n=11) 1 (n=9) 13 (n=15) small focus 6 (n=15) small focus 21 (n=33) small focus 7 small focus 1 1 (n=11) (n=21) Embryos were fixed and stained with anti-spd-5 antibody and scored for the presence of one or two foci of SPD-5 adjacent to the sperm DNA. For γ-tubulin, the endogenous fluorescence of GFP:γ-tubulin was used. M I = metaphase I, A I = anaphase I, M II = metaphase II, A II = anaphase II, PN = pronuclear stage. Merged cells indicate mixed stage meiotic embryos were scored. "Small focus" indicates a focus of SPD-5 staining that was smaller in diameter than those produced by kca-1(rnai) and no microtubule aster emanated from these small foci.

klp-18 (RNAi) Control. supplementary information. starting strain: AV335 [emb-27(g48); GFP::histone; GFP::tubulin] bleach

klp-18 (RNAi) Control. supplementary information. starting strain: AV335 [emb-27(g48); GFP::histone; GFP::tubulin] bleach DOI: 10.1038/ncb1891 A. starting strain: AV335 [emb-27(g48); GFP::histone; GFP::tubulin] bleach embryos let hatch overnight transfer to RNAi plates; incubate 5 days at 15 C RNAi food L1 worms adult worms