* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Supplementary Legends - Word file
Survey
Document related concepts
Therapeutic gene modulation wikipedia , lookup
Genome evolution wikipedia , lookup
Essential gene wikipedia , lookup
History of genetic engineering wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Genomic imprinting wikipedia , lookup
Ridge (biology) wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Genome (book) wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Gene expression profiling wikipedia , lookup
Biology and consumer behaviour wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Designer baby wikipedia , lookup
RNA interference wikipedia , lookup
Minimal genome wikipedia , lookup
Transcript
Supplementary Movie 1 This movie shows a C. elegans zygote at metaphase-to-anaphase transition. Note the displacement of the spindle toward the posterior (right) and the disappearance of the metaphase plate at anaphase onset. (QuickTime; 1.5 MB) Supplementary Movie 2 This movie shows asymmetric spindle severing: the anterior centrosome (left) is chopped off. The position and time of laser ablation is marked with a white circle. (QuickTime; 1.3 MB) Supplementary Movie 3 This movie shows cytokinesis in an unsevered control C. elegans zygote. (QuickTime; 2.5 MB) Supplementary Movie 4 This movie shows cytokinesis after anterior ASS: Note the formation of two distinct furrows after ASS. The severed region is highlighted with a grey bar. The first furrow does not complete, pauses, and regresses. (QuickTime; 6 MB) Supplementary Movie 5 This movie shows cytokinesis after posterior ASS: Note the furrow correction observed after ASS. The cut region is highlighted with a grey bar. The focus is rapidly changed during the correction process to reveal the position of the nuclei and the complexity of the cytokinesis furrow. (QuickTime; 3.6 MB) Supplementary Movie 6 This movie shows asymmetric spindle severing with subsequent disintegration of the chopped-off aster. The time and place of laser ablation is marked with a white circle. (QuickTime; 2.8 MB) Supplementary Movie 7 This movie shows cytokinesis after ASS with disintegration of the chopped-off aster. The posterior centrosome (highlighted with a white circle) is chopped off and disintegrated. The frames during which the aster was disintegrated were removed (see movie 6 for details of the disintegration assay). Note that the first furrow sets up further away from the remaining aster compared with wildtype or conventional ASS. (QuickTime; 3.9 MB) Supplementary Movie 8 This movie shows cytokinesis in a zen-4(RNAi) depleted one-cell embryo. Note the spindle snapping at anaphase. The furrow regresses and cytokinesis fails. (QuickTime; 2.2 MB) Supplementary Movie 9 This movie shows cytokinesis after ASS in a zen-4(RNAi) depleted embryo. The severed region is highlighted with a grey bar. The posterior aster is chopped off. Note that the ingression of the first furrow is reduced compared with wildtype (movie 4). (QuickTime; 1.8 MB) Supplementary Movie 10 This movie shows cytokinesis in a klp-7(RNAi) depleted zygote. Note the spindle snapping at anaphase. (QuickTime; 2.3 MB) Supplementary Movie 11 This movie shows cytokinesis after posterior ASS in a klp7(RNAi) depleted zygote. The severed region is highlighted with a grey bar. (QuickTime; 2.5 MB) Supplementary Movie 12 This movie shows cytokinesis in a mel-11(it26) mutant zygote. (QuickTime; 2.9 MB) Supplementary Movie 13 This movie shows cytokinesis after posterior ASS in a mel-11(it26) mutant zygote. The severed region is highlighted with a grey bar. Note that the aster-dependent furrow completes before the midzone-dependent furrow appears, leading to the formation of three cells. The furrow that separates the anterior aster from the anterior nucleus was not stable and regressed (not part of the movie). (QuickTime; 3.3 MB) Supplementary Movie 14 This movie shows cytokinesis in an NMY2::GFP strain observed by spinning disk microscopy. (QuickTime; 1.3 MB) Supplementary Movie 15 This movie shows cytokinesis after posterior ASS plus aster ablation in an NMY2::GFP strain observed by spinning disk microscopy. Note that after completion of the furrows, cortical NMY2 patches continue to flow into the midzone-positioned furrow and into the part of the aster-positioned furrow that separates the two nuclei. The part of the aster-positioned furrow that separates the posterior aster and the posterior nucleus did not show such flow, was not stable, and regressed. (QuickTime; 2.8 MB) Supplementary Table 1 Genes with known or potential involvement in the first cytokinesis of the C. elegans zygote, and their roles in redundant cleavage plane specification as analysed by asymmetric spindle severing (ASS): Shown are gene names, loci, knockout method and phenotypes after ASS. Gene knockout was performed using established protocols, either by using feeding clones obtained form the MRC Geneservice1 (labeled Ahringer) or injection of double-stranded RNA obtained from Cenix Bioscience2. For zen-4 we repeated RNAi with a different feeding clone (a gift from M. Glotzer)3. If genetic mutants were used, the strain and allele is specified. Generally, analysis after ASS was impossible if microtubule-based pulling forces were strongly reduced, if spindle assembly or positioning was severely affected, or if the cortex of the embryo displayed vigorous movements. In some cases we could obtain a result by performing ASS plus aster ablation. If both assays failed to give a result they are marked with a dash. We excluded genes from our analysis that failed in cytokinesis because of a missing furrow. If several genes acted in the same pathway, we did not test all components of this pathway but only some examples. Genes that were not tested are labeled “not tested”. The list excludes genes that are required for formation of the mitotic apparatus or that cause osmotic defects. All genes classes associated with cytokinesis and their role in cleavage plane determination as analysed by ASS are listed below: Embryonic polarity defect. The first cell division of C. elegans is asymmetric4. We hence probed polarity genes with ASS to see if polarity influences cytokinesis furrow positioning. None of the genes tested lead to a cytokinetic furrow specification defect after ASS. In order to verify that polarity is not involved in the control of cleavage plane specification, we performed ASS in a symmetrically cleaving cell (AB)4. We observed two furrows in AB (data not shown). These results establish that polarity has no effect on redundant cleavage plane specification. Reduced contractility. Several genetic defects lead to a reduced contractility of the cortex, manifested by an absence of early ruffling before the polarization of the cortex, lack of a pseudocleavage furrow, or slow cytokinesis. We wondered whether reduced contractility is a subtle manifestation of a cleavage plane determination defect. Tested genes were let-502(no pseudocleavage furrow, slow cytokinesis)5, C34C12.3 (S/TPhosphatase-like, no pseudocleavage furrow)2, C47G2.5 (phosphatase regulatory subunit, no pseudocleavage furrow)2, K06A4.3 (gelsolin-like protein, reduced pseudocleavage furrow, our unpublished data), nop-1(reduced early ruffling, no pseudocleavage furrow)6, cdc-42(reduced pseudocleavage furrow)7,8, unc-59/unc619 (septin homologues double knockout, reduced early ruffling and reduced pseudocleavage furrow, slow cytokinesis, our unpublished data), Y49E10.19 (putative Anillin homolog, reduced early ruffling)2, arx-1(slow cytokinesis, our unpublished data), ZC204.11 (kelch-like protein, reduced ruffling, reduced pseudocleavage furrow)2. All genes in this phenotypic class subjected to ASS analysis still displayed redundant cleavage plane specification and correction of the furrow were normal. We hence could not find any relation between reduced contractility and cleavage plane specification. Increased contractility – fast cytokinesis. In contrast to the first phenotypic class, the mel-11 mutation leads to increased contractility and fast ingression. In mel-11 embryos cytokinesis only takes half the time compared with wildtype embryos5. In mel11 embryos subjected to ASS the first furrow completes before the second is formed, ultimately leading to the generation of three cells. Hypercontractility – ectopic furrowing. Several mutations lead to the generation of hypercontractile and ectopic furrows during cytokinesis. Examples are csn-210, rfl-111, ubc-12, cul-111, K09H11.32, Y75B7AL.42. The effect of these genes onto the two furrows, however, is impossible to study after ASS, because of the vigorous movements of the embryo cortex during the attempts at cytokinesis. Sister-chromatid separation defect – regression of the furrow. Several gene knockouts lead to a defect in spindle midzone formation and sister chromatid separation. These mutant embryos specify and ingress a furrow but do not complete cytokinesis. Most of the genes with this phenotype belong to the air-2 (Aurora B) pathway (icp-1, bir-1, csc-1)12,13. We tested the role of air-2 in cleavage plane determination subjecting the mutant or20714 to ASS: A single furrow is specified by the asters, ingresses weakly, and finally regresses. The nonseparated chromatin has no effect on furrow positioning. The apparent lack of the second furrow can be explained by the absence of a functional spindle midzone. Spindle midzone defect – cytokinesis furrow regresses. ZEN-4 and CYK-4 constitute the centralspindlin complex which is controlled by CDC-1415-17. Depletion of these proteins leads to a spindle midzone defect, and absence of spindle microtubules, and a failure in cytokinesis. ASS in central spindle mutants reveals the same phenotype as for the sister-chromatid separation defect class. The spindle midzone does not seem to provide a signal. This defect is combined with a reduced contractility of the first furrow (maximum ingression of the first furrow is 81 3 % embryo width in wildtype and 42 4 % embryo width in zen-4(RNAi). We observed comparable results with the zen-4 mutant or15314), explaining the failure in cytokinesis. We verified the absence of midzone microtubules under our RNAi conditions by using tubulin fluorescence (data not shown). Spindle midzone defect – cytokinesis generally completes. Defects in spindle midzone formation are often accompanied by a failure in cytokinesis (reviewed in 18). Mutants in klp-7 and spd-1, however, display spindle midzone defects manifested by a snapping or breaking of the mitotic spindle, and an absence of midzone microtubules. Though, cytokinesis generally completes19,20(and this work). After ASS in spd-1 and klp-7 mutants a single furrow is specified by the asters and completes irrespective of the position of the “spindle midzone” between the two nuclei. The lack of the second furrow can be explained by the absence of a functional spindle midzone, while the completion of the first furrow suggests that the spindle midzone is dispensable for the completion of cytokinesis. However, there seem to be proteins necessary for completion of cytokinesis like ZEN-4 and CYK-4 that localize in the vicinity of the spindle midzone21,22. Cytokinesis completes but the furrow is unstable. Several mutants display apparently normal cytokinesis. After the furrow appears to have completed, the furrow rips apart and regresses. Examples for this phenotype are K04D7.123 and Y18D10A.172. Genes associated in this phenotypic class seem to be important for abscission or sealing of the membrane, a process taking place far after the cleavage plane is determined. We did not test genes required in this process. No cytokinetic furrow. Several gene knockouts (unc-60a24, mlc425, cyk-126, nmy-227, syn-428, act-22, act-52, let-212, pfn-126, rho12) lead to the complete absence of a cytokinesis furrowing. These genes are either required for furrow ingression, in general, or are involved in specification of both furrows. We did not subject these genes to our assay. 1. 2. 3. 4. 5. 6. 7. Kamath, R. S. et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231-7 (2003). Sonnichsen, B. et al. Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans. Nature 434, 462-9 (2005). Dechant, R. & Glotzer, M. Centrosome separation and central spindle assembly act in redundant pathways that regulate microtubule density and trigger cleavage furrow formation. Dev Cell 4, 333-44 (2003). Goldstein, B., Hird, S. N. & White, J. G. Cell polarity in early C. elegans development. Dev Suppl, 279-87 (1993). Piekny, A. J. & Mains, P. E. Rho-binding kinase (LET-502) and myosin phosphatase (MEL-11) regulate cytokinesis in the early Caenorhabditis elegans embryo. J Cell Sci 115, 2271-82 (2002). Rose, L. S., Lamb, M. L., Hird, S. N. & Kemphues, K. J. Pseudocleavage is dispensable for polarity and development in C. elegans embryos. Dev Biol 168, 479-89 (1995). Gotta, M., Abraham, M. C. & Ahringer, J. CDC-42 controls early cell polarity and spindle orientation in C. elegans. Curr Biol 11, 482-8 (2001). 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Kay, A. J. & Hunter, C. P. CDC-42 regulates PAR protein localization and function to control cellular and embryonic polarity in C. elegans. Curr Biol 11, 474-81 (2001). Nguyen, T. Q., Sawa, H., Okano, H. & White, J. G. The C. elegans septin genes, unc-59 and unc-61, are required for normal postembryonic cytokineses and morphogenesis but have no essential function in embryogenesis. J Cell Sci 113 Pt 21, 3825-37 (2000). Pintard, L. et al. Neddylation and deneddylation of CUL-3 is required to target MEI-1/Katanin for degradation at the meiosis-to-mitosis transition in C. elegans. Curr Biol 13, 911-21 (2003). Kurz, T. et al. Cytoskeletal regulation by the Nedd8 ubiquitin-like protein modification pathway. Science 295, 1294-8 (2002). Schumacher, J. M., Golden, A. & Donovan, P. J. AIR-2: An Aurora/Ipl1-related protein kinase associated with chromosomes and midbody microtubules is required for polar body extrusion and cytokinesis in Caenorhabditis elegans embryos. J Cell Biol 143, 1635-46 (1998). Romano, A. et al. CSC-1: a subunit of the Aurora B kinase complex that binds to the survivin-like protein BIR-1 and the incenp-like protein ICP-1. J Cell Biol 161, 229-36 (2003). Severson, A. F., Hamill, D. R., Carter, J. C., Schumacher, J. & Bowerman, B. The aurora-related kinase AIR-2 recruits ZEN-4/CeMKLP1 to the mitotic spindle at metaphase and is required for cytokinesis. Curr Biol 10, 1162-71 (2000). Mishima, M., Kaitna, S. & Glotzer, M. Central spindle assembly and cytokinesis require a kinesin-like protein/RhoGAP complex with microtubule bundling activity. Dev Cell 2, 41-54 (2002). Gruneberg, U., Glotzer, M., Gartner, A. & Nigg, E. A. The CeCDC-14 phosphatase is required for cytokinesis in the Caenorhabditis elegans embryo. J Cell Biol 158, 901-14 (2002). Mishima, M., Pavicic, V., Gruneberg, U., Nigg, E. A. & Glotzer, M. Cell cycle regulation of central spindle assembly. Nature 430, 908-13 (2004). Straight, A. F. & Field, C. M. Microtubules, membranes and cytokinesis. Curr Biol 10, R760-70 (2000). Grill, S. W., Gonczy, P., Stelzer, E. H. & Hyman, A. A. Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo. Nature 409, 630-3 (2001). Verbrugghe, K. J. & White, J. G. SPD-1 Is Required for the Formation of the Spindle Midzone but Is Not Essential for the Completion of Cytokinesis in C. elegans Embryos. Curr Biol 14, 1755-60 (2004). Raich, W. B., Moran, A. N., Rothman, J. H. & Hardin, J. Cytokinesis and midzone microtubule organization in Caenorhabditis elegans require the kinesinlike protein ZEN-4. Mol Biol Cell 9, 2037-49 (1998). Jantsch-Plunger, V. et al. CYK-4: A Rho family gtpase activating protein (GAP) required for central spindle formation and cytokinesis. J Cell Biol 149, 1391-404 (2000). 23. 24. 25. 26. 27. 28. Skop, A. R., Liu, H., Yates, J., 3rd, Meyer, B. J. & Heald, R. Dissection of the mammalian midbody proteome reveals conserved cytokinesis mechanisms. Science 305, 61-6 (2004). Ono, K., Parast, M., Alberico, C., Benian, G. M. & Ono, S. Specific requirement for two ADF/cofilin isoforms in distinct actin-dependent processes in Caenorhabditis elegans. J Cell Sci 116, 2073-85 (2003). Shelton, C. A., Carter, J. C., Ellis, G. C. & Bowerman, B. The nonmuscle myosin regulatory light chain gene mlc-4 is required for cytokinesis, anterior-posterior polarity, and body morphology during Caenorhabditis elegans embryogenesis. J Cell Biol 146, 439-51 (1999). Severson, A. F., Baillie, D. L. & Bowerman, B. A Formin Homology protein and a profilin are required for cytokinesis and Arp2/3-independent assembly of cortical microfilaments in C. elegans. Curr Biol 12, 2066-75 (2002). Guo, S. & Kemphues, K. J. A non-muscle myosin required for embryonic polarity in Caenorhabditis elegans. Nature 382, 455-8 (1996). Jantsch-Plunger, V. & Glotzer, M. Depletion of syntaxins in the early Caenorhabditis elegans embryo reveals a role for membrane fusion events in cytokinesis. Curr Biol 9, 738-45 (1999).