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

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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.

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Acknowledgements

We would like to thank Paula Coelho and Christian Lehner and all the members of the lab for comments on the manuscript. We are indebted to Monika Willert-Porada and Ingrid Otto (Chair for Materials Processing, University of Bayreuth) for access to and help with the Zeiss LSM510 confocal system. We thank Augusta Monteiro, Katharina Trunzer, and Melina Schuh for technical support and help with cloning. We also thank all the members of the labs for comments and suggestions. R.O. holds a PhD fellowship from the Fundação para a Ciência e a Tecnologia (FCT) of Portugal and is a student of the PDBEB, PhD program. The laboratory of C.E.S. is funded by grants from FCT and the TMR program of the European Union. S.H. is supported by a grant from the Deutsche Forschungsgemeinschaft (DFG He 2354/2-3).

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Authors and Affiliations

  1. Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180, Porto, Portugal

    Raquel A. Oliveira & Claudio E. Sunkel

  2. Programa Doutoral em Biologia Experimental e Biomedicina (PDBEB), Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal

    Raquel A. Oliveira

  3. Department of Genetics, Bayreuth Center for Molecular Biosciences (BZMB), University of Bayreuth, Bayreuth, Germany

    Stefan Heidmann

  4. Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Largo do Prof. Abel Salazar 4000, Porto, Portugal

    Claudio E. Sunkel

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  1. Raquel A. Oliveira
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Correspondence to Claudio E. Sunkel.

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Communicated by E.A. Nigg

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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;Barr L305 /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)

High resolution image file (TIFF 1 mb)

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)

High resolution image file (TIFF 2 mb)

Movie 1

In 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)

Movie 2

In 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)

Movie 3

In 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)

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Oliveira, R.A., Heidmann, S. & Sunkel, C.E. Condensin I binds chromatin early in prophase and displays a highly dynamic association with Drosophila mitotic chromosomes. Chromosoma 116, 259–274 (2007). https://doi.org/10.1007/s00412-007-0097-5

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