Chromosoma

, Volume 116, Issue 3, pp 259–274 | Cite as

Condensin I binds chromatin early in prophase and displays a highly dynamic association with Drosophila mitotic chromosomes

  • Raquel A. Oliveira
  • Stefan Heidmann
  • Claudio E. Sunkel
Research Article

Abstract

The condensed state of mitotic chromosomes is crucial for faithful genome segregation. Key factors implicated in the formation of mitotic chromosomes are the condensin I and II complexes. In Drosophila, condensin I appears to play a major role in mitotic chromosome organization. To analyze its dynamic behavior, we expressed Barren, a condensin I non-Structural Maintenance of Chromosomes subunit, as a fully functional enhanced green fluorescent protein (EGFP) fusion protein in the female and followed it during early embryonic divisions. We find that, in Drosophila, Barren-EGFP associates with chromatin early in prophase concomitantly with the initiation of chromosome condensation. Barren-EGFP loading starts at the centromeric region from where it spreads distally reaching maximum accumulation at metaphase/early anaphase. Fluorescence Recovery After Photobleaching analysis indicates that most of the bound protein exchanges rapidly with the cytoplasmic pool during prometaphase/metaphase. Taken together, our results suggest that in Drosophila, condensin I is involved in the initial stages of chromosome condensation. Furthermore, the rapid turnover of Barren-EGFP indicates that the mechanism by which condensin I promotes mitotic chromosome organization is inconsistent with a static scaffold model.

Supplementary material

412_2007_97_Fig1_ESM.gif (109 kb)
Supplementary Fig. 1

Western Blot analysis of Barren-EGFP protein levels. a A 1–2-h embryo collection of both control and Barren-EGFP expressing embryos was obtained from W1118 and UASP-Barren-EGFP III.1, α-4tub-GAL4-VP16/MKRS females, respectively. Different amounts of extract were loaded to facilitate quantification (corresponding to ten and five embryos). Western blot using a Barren specific antibody detects endogenous Barren in both extracts and ectopically expressed Barren-EGFP in Barren-EGFP embryos. A possible degradation product (asterisk) is also detected. Tubulin was used as loading control. Quantification analyses reveal that Barren-EGFP is expressed ∼1.5-fold above the endogenous levels. b Brains from wild-type larvae, from larvae that express Barren-EGFP in a wild-type background (W;UAS-Barr-GFP III.2, daGal4) and from larvae that express Barren-EGFP in a Barren-mutant background. (W;BarrL305/Df(2L)Exel7077;UAS-Barr-GFP III.2, daGal4) were dissected in PBS and resuspended in SDS sample buffer. Different amounts of extract were loaded to facilitate quantification (corresponding to ten and five brains). Western blot using a Barren-specific antibody detects ectopically expressed Barren-EGFP in similar amounts in brains from larvae that express Barren-EGFP in a mutant-and wild-type context. Endogenous Barren is strongly detected in wild-type brains, but in brains that overexpress Barren-EGFP, the endogenous protein is considerably down-regulated. A possible degradation product (asterisk) is also detected. Tubulin was used as loading control. Quantification analysis reveal that Barren-EGFP is expressed ∼2-fold above the levels detected in wild-type brains (GIF 111 kb)

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High resolution image file (TIFF 1 mb)
412_2007_97_Fig2_ESM.gif (104 kb)
Supplementary Fig. 2

ICC Timing. ICC was determined by the time strong dots of HisH2Av-mRFP1 start to be observed (at −5:40 in this example). This analysis was performed based on time-lapse microscopy analysis of embryos expressing Barren-EGFP and HisH2Av-mRFP1, while progressing throughout mitosis 12. Raw data images of the HisH2Av-mRFP1 channel (upper panel) were converted to a gradient LUT panel (lower panel) to facilitate the visualization of differences in fluorescence intensity. ICC timing was defined by the time dark orange/red pixels start to be visualized in the LUT-converted image. Analysis of different movies (n = 10) reveals that ICC occurs 6.3 ± 1.2 min (mean ± SD) before anaphase onset (GIF 106 kb)

412_2007_97_Fig2_ESM.tif (2.8 mb)
High resolution image file (TIFF 2 mb)
412_2007_97_MOESM5_ESM.avi (8.8 mb)
Movie 1In vivo analysis of syncytial nuclear divisions in Barren-EGFP and HisH2Av-mRFP1 expressing embryos. This movie shows an embryo in which Barren-EGFP (green) and HisH2Av-mRFP1 (red) were maternally deposited undergoing three consecutive syncytial embryonic divisions (mitosis 11–13). Note that barren-EGFP colocalizes with chromatin throughout mitosis (9 mb)
412_2007_97_MOESM6_ESM.avi (10.8 mb)
Movie 2In vivo analysis of postblastoderm nuclear divisions in Barren-EGFP and HisH2Av-mRFP1 expressing embryos. This movie shows mitotic domains from a postblastodermal embryo coexpressing Barren-EGFP (green) and HisH2Av-mRFP1 (red). Note that Barren-EGFP is associated with chromatin throughout mitosis (11 mb)
412_2007_97_MOESM7_ESM.avi (4.3 mb)
Movie 3In vivo analysis of the initial stages of a syncytial nuclear division in Barren-EGFP and Cid-mRFP1 expressing embryos. This movie shows an embryo in which Barren-EGFP (green) and Cid-mRFP1 (red) were maternally deposited undergoing mitosis 12. During interphase, Barren-EGFP is excluded from the nuclear space. Cid-mRFP detects dot-like structures located at the apical site of the nucleus corresponding to the centromeres. While the nuclei enter prophase, Barren-EGFP starts to be detectable inside the nuclear area specifically at the centromeric region (indicated by Cid-mRFP). Later on, Barren-EGFP signal is detectable throughout the nuclear area, suggesting Barren-EGFP localization all over chromosomal arms (4 mb)

References

  1. Bhalla N, Biggins S, Murray AW (2002) Mutation of YCS4, a budding yeast condensin subunit, affects mitotic and nonmitotic chromosome behavior. Mol Biol Cell 13:632–645PubMedCrossRefGoogle Scholar
  2. Bhat MA, Philp AV, Glover DM, Bellen HJ (1996) Chromatid segregation at anaphase requires the barren product, a novel chromosome-associated protein that interacts with topoisomerase II. Cell 87:1103–1114PubMedCrossRefGoogle Scholar
  3. Boy de la Tour E, Laemmli UK (1988) The metaphase scaffold is helically folded: sister chromatids have predominantly opposite helical handedness. Cell 55:937–944PubMedCrossRefGoogle Scholar
  4. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415PubMedGoogle Scholar
  5. Buffin E, Lefebvre C, Huang J, Gagou ME, Karess RE (2005) Recruitment of Mad2 to the kinetochore requires the Rod/Zw10 complex. Curr Biol 15:856–861PubMedCrossRefGoogle Scholar
  6. Christensen MO, Larsen MK, Barthelmes HU, Hock R, Andersen CL, Kjeldsen E, Knudsen BR, Westergaard O, Boege F, Mielke C (2002) Dynamics of human DNA topoisomerases IIalpha and IIbeta in living cells. J Cell Biol 157:31–44PubMedCrossRefGoogle Scholar
  7. Clarkson M, Saint R (1999) A His2AvDGFP fusion gene complements a lethal His2AvD mutant allele and provides an in vivo marker for Drosophila chromosome behavior. DNA Cell Biol 18:457–462PubMedCrossRefGoogle Scholar
  8. Coelho PA, Queiroz-Machado J, Sunkel CE (2003) Condensin-dependent localisation of topoisomerase II to an axial chromosomal structure is required for sister chromatid resolution during mitosis. J Cell Sci 116:4763–7476PubMedCrossRefGoogle Scholar
  9. Dej KJ, Ahn C, Orr-Weaver TL (2004) Mutations in the Drosophila condensin subunit dCAP-G: defining the role of condensin for chromosome condensation in mitosis and gene expression in interphase. Genetics 168:895–906PubMedCrossRefGoogle Scholar
  10. Earnshaw WC, Laemmli UK (1983) Architecture of metaphase chromosomes and chromosome scaffolds. J Cell Biol 96:84–93PubMedCrossRefGoogle Scholar
  11. Earnshaw WC, Halligan B, Cooke CA, Heck MM, Liu LF (1985) Topoisomerase II is a structural component of mitotic chromosome scaffolds. J Cell Biol 100:1706–1715PubMedCrossRefGoogle Scholar
  12. Foe VE (1989) Mitotic domains reveal early commitment of cells in Drosophila embryos. Development 107:1–22PubMedGoogle Scholar
  13. Freeman L, Aragon-Alcaide L, Strunnikov A (2000) The condensin complex governs chromosome condensation and mitotic transmission of rDNA. J Cell Biol 149:811–824PubMedCrossRefGoogle Scholar
  14. Fujimoto S, Yonemura M, Matsunaga S, Nakagawa T, Uchiyama S, Fukui K (2005) Characterization and dynamic analysis of Arabidopsis condensin subunits, AtCAP-H and AtCAP-H2. Planta 222:293–300PubMedCrossRefGoogle Scholar
  15. Gasser SM, Laroche T, Falquet J, Boy de la Tour E, Laemmli UK (1986) Metaphase chromosome structure. Involvement of topoisomerase II. J Mol Biol 188:613–629PubMedCrossRefGoogle Scholar
  16. Gerlich D, Hirota T, Koch B, Peters JM, Ellenberg J (2006a) Condensin I stabilizes chromosomes mechanically through a dynamic interaction in live cells. Curr Biol 16:333–344PubMedCrossRefGoogle Scholar
  17. Gerlich D, Koch B, Dupeux F, Peters JM, Ellenberg J (2006b) Live-cell imaging reveals a stable cohesin-chromatin interaction after but not before DNA replication. Curr Biol 16:1571–1578PubMedCrossRefGoogle Scholar
  18. Giet R, Glover DM (2001) Drosophila aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organize the central spindle during cytokinesis. J Cell Biol 152:669–682PubMedCrossRefGoogle Scholar
  19. Haering CH, Lowe J, Hochwagen A, Nasmyth K (2002) Molecular architecture of SMC proteins and the yeast cohesin complex. Mol Cell 9:773–788PubMedCrossRefGoogle Scholar
  20. Hagstrom KA, Holmes VF, Cozzarelli NR, Meyer BJ (2002) C. elegans condensin promotes mitotic chromosome architecture, centromere organization, and sister chromatid segregation during mitosis and meiosis. Genes Dev 16:729–742PubMedCrossRefGoogle Scholar
  21. Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, Brinkley BR, Bazett-Jones DP, Allis CD (1997) Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106:348–360PubMedCrossRefGoogle Scholar
  22. Henikoff S, Ahmad K, Platero JS, van Steensel B (2000) Heterochromatic deposition of centromeric histone H3-like proteins. Proc Natl Acad Sci USA 97:716–721PubMedCrossRefGoogle Scholar
  23. Hirano T (2005) Condensins: organizing and segregating the genome. Curr Biol 15:R265–R275PubMedCrossRefGoogle Scholar
  24. Hirano T (2006) At the heart of the chromosome: SMC proteins in action. Nat Rev Mol Cell Biol 7:311–322PubMedCrossRefGoogle Scholar
  25. Hirano T, Kobayashi R, Hirano M (1997) Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein. Cell 89:511–521PubMedCrossRefGoogle Scholar
  26. Hirota T, Gerlich D, Koch B, Ellenberg J, Peters JM (2004) Distinct functions of condensin I and II in mitotic chromosome assembly. J Cell Sci 117:6435–6445PubMedCrossRefGoogle Scholar
  27. Hudson DF, Vagnarelli P, Gassmann R, Earnshaw WC (2003) Condensin is required for nonhistone protein assembly and structural integrity of vertebrate mitotic chromosomes. Dev Cell 5:323–336PubMedCrossRefGoogle Scholar
  28. Jäger H, Rauch M, Heidmann S (2005) The Drosophila melanogaster condensin subunit Cap-G interacts with the centromere-specific histone H3 variant CID. Chromosoma 113:350–361PubMedCrossRefGoogle Scholar
  29. Kaitna S, Pasierbek P, Jantsch M, Loidl J, Glotzer M (2002) The aurora B kinase AIR-2 regulates kinetochores during mitosis and is required for separation of homologous Chromosomes during meiosis. Curr Biol 12:798–812PubMedCrossRefGoogle Scholar
  30. Kimura K, Hirano T (1997) ATP-dependent positive supercoiling of DNA by 13S condensin: a biochemical implication for chromosome condensation. Cell 90:625–634PubMedCrossRefGoogle Scholar
  31. Kimura K, Rybenkov VV, Crisona NJ, Hirano T, Cozzarelli NR (1999) 13S condensin actively reconfigures DNA by introducing global positive writhe: implications for chromosome condensation. Cell 98:239–248PubMedCrossRefGoogle Scholar
  32. Kireeva N, Lakonishok M, Kireev I, Hirano T, Belmont AS (2004) Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure. J Cell Biol 166:775–785PubMedCrossRefGoogle Scholar
  33. Lavoie BD, Tuffo KM, Oh S, Koshland D, Holm C (2000) Mitotic chromosome condensation requires Brn1p, the yeast homologue of Barren. Mol Biol Cell 11:1293–1304PubMedGoogle Scholar
  34. Lewis CD, Laemmli UK (1982) Higher order metaphase chromosome structure: evidence for metalloprotein interactions. Cell 29:171–181PubMedCrossRefGoogle Scholar
  35. Maddox PS, Portier N, Desai A, Oegema K (2006) Molecular analysis of mitotic chromosome condensation using a quantitative time-resolved fluorescence microscopy assay. Proc Natl Acad Sci USA 103:15097–15102PubMedCrossRefGoogle Scholar
  36. Maeshima K, Laemmli UK (2003) A two-step scaffolding model for mitotic chromosome assembly. Dev Cell 4:467–80PubMedCrossRefGoogle Scholar
  37. Meraldi P, Draviam VM, Sorger PK (2004) Timing and checkpoints in the regulation of mitotic progression. Dev Cell 7:45–60PubMedCrossRefGoogle Scholar
  38. Oliveira RA, Coelho PA, Sunkel CE (2005) The condensin I subunit Barren/CAP-H is essential for the structural integrity of centromeric heterochromatin during mitosis. Mol Cell Biol 25:8971–8984PubMedCrossRefGoogle Scholar
  39. Ono T, Losada A, Hirano M, Myers MP, Neuwald AF, Hirano T (2003) Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells. Cell 115:109–121PubMedCrossRefGoogle Scholar
  40. Ono T, Fang Y, Spector DL, Hirano T (2004) Spatial and temporal regulation of condensins I and II in mitotic chromosome assembly in human cells. Mol Biol Cell 15:3296–3308PubMedCrossRefGoogle Scholar
  41. Ouspenski II, Cabello OA, Brinkley BR (2000) Chromosome condensation factor Brn1p is required for chromatid separation in mitosis. Mol Biol Cell 11:1305–1313PubMedGoogle Scholar
  42. Poirier MG, Marko JF (2002) Mitotic chromosomes are chromatin networks without a mechanically contiguous protein scaffold. Proc Natl Acad Sci USA 99:15393–15397PubMedCrossRefGoogle Scholar
  43. Rorth P (1998) Gal4 in the Drosophila female germline. Mech Dev 78:113–118PubMedCrossRefGoogle Scholar
  44. Saitoh N, Goldberg IG, Wood ER, Earnshaw WC (1994) ScII: an abundant chromosome scaffold protein is a member of a family of putative ATPases with an unusual predicted tertiary structure. J Cell Biol 127:303–318PubMedCrossRefGoogle Scholar
  45. Saka Y, Sutani T, Yamashita Y, Saitoh S, Takeuchi M, Nakaseko Y, Yanagida M (1994) Fission yeast cut3 and cut14, members of a ubiquitous protein family, are required for chromosome condensation and segregation in mitosis. EMBO J 13:4938–4952PubMedGoogle Scholar
  46. Savvidou E, Cobbe N, Steffensen S, Cotterill S, Heck MM (2005) Drosophila CAP-D2 is required for condensin complex stability and resolution of sister chromatids. J Cell Sci 118:2529–2543PubMedCrossRefGoogle Scholar
  47. Schuh M, Lehner CF, Heidmann S (2007) Incorporation of Drosophila CID/CENP-A and CENP-C into centromeres during early embryonic anaphase. Curr Biol 17:237–243PubMedCrossRefGoogle Scholar
  48. Steffensen S, Coelho PA, Cobbe N, Vass S, Costa M, Hassan B, Prokopenko SN, Bellen H, Heck MM, Sunkel CE (2001) A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis. Curr Biol 11:295–307PubMedCrossRefGoogle Scholar
  49. Strunnikov AV, Hogan E, Koshland D (1995) SMC2, a Saccharomyces cerevisiae gene essential for chromosome segregation and condensation, defines a subgroup within the SMC family. Genes Dev 9:587–599PubMedGoogle Scholar
  50. Sullivan W, Ashburner A, Hawley RS (2000) Drosophila Protocols. Cold Spring Harbor, NY: CSHL, pp 141–157Google Scholar
  51. Sutani T, Yanagida M (1997) DNA renaturation activity of the SMC complex implicated in chromosome condensation. Nature 388:798–801PubMedCrossRefGoogle Scholar
  52. Tavormina PA, Come MG, Hudson JR, Mo YY, Beck WT, Gorbsky GJ (2002) Rapid exchange of mammalian topoisomerase II alpha at kinetochores and chromosome arms in mitosis. J Cell Biol 158:23–29PubMedCrossRefGoogle Scholar
  53. Vagnarelli P, Hudson DF, Ribeiro SA, Trinkle-Mulcahy L, Spence JM, Lai F, Farr CJ, Lamond AI, Earnshaw WC (2006) Condensin and Repo-Man-PP1 cooperate in the regulation of chromosome architecture during mitosis. Nat Cell Biol 8:1133–1142PubMedCrossRefGoogle Scholar
  54. Watrin E, Legagneux V (2005) Contribution of hCAP-D2, a non-SMC subunit of condensin I, to chromosome and chromosomal protein dynamics during mitosis. Mol Cell Biol 25:740–750PubMedCrossRefGoogle Scholar
  55. Wodarz A, Hinz U, Engelbert M, Knust E (1995) Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of Drosophila. Cell 82:67–76PubMedCrossRefGoogle Scholar
  56. Yeong FM, Hombauer H, Wendt KS, Hirota T, Mudrak I, Mechtler K, Loregger T, Marchler-Bauer A, Tanaka K, Peters JM, Ogris E (2003) Identification of a subunit of a novel Kleisin-beta/SMC complex as a potential substrate of protein phosphatase 2A. Curr Biol 13:2058–2064PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Raquel A. Oliveira
    • 1
    • 2
  • Stefan Heidmann
    • 3
  • Claudio E. Sunkel
    • 1
    • 4
  1. 1.Instituto de Biologia Molecular e CelularUniversidade do PortoPortoPortugal
  2. 2.Programa Doutoral em Biologia Experimental e Biomedicina (PDBEB), Center for Neuroscience and Cell BiologyUniversity of CoimbraCoimbraPortugal
  3. 3.Department of Genetics, Bayreuth Center for Molecular Biosciences (BZMB)University of BayreuthBayreuthGermany
  4. 4.Instituto de Ciências Biomédicas Abel Salazar (ICBAS)Universidade do PortoPortoPortugal

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