Condensin I binds chromatin early in prophase and displays a highly dynamic association with Drosophila mitotic chromosomes
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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.
KeywordsEnhance Green Fluorescent Protein Mitotic Chromosome Fluorescence Recovery After Photobleaching Chromosome Condensation Condensin Complex
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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