LETTERS Sister chromatid resolution is an intrinsic part of chromosome organization in prophase Kota Nagasaka 1,2 , M. Julius Hossain 3 , M. Julia Roberti 3 , Jan Ellenberg 3,4 and Toru Hirota 1,4 The formation of mitotic chromosomes requires both compaction of chromatin and the resolution of replicated sister chromatids. Compaction occurs during mitotic prophase and prometaphase, and in prophase relies on the activity of condensin II complexes 1,2 . Exactly when and how sister chromatid resolution occurs has been largely unknown, as has its molecular requirements. Here, we established a method to visualize sister resolution by sequential replication labelling with two distinct nucleotide derivatives. Quantitative three-dimensional imaging then allowed us to measure the resolution of sister chromatids throughout mitosis by calculating their non-overlapping volume within the whole chromosome. Unexpectedly, we found that sister chromatid resolution starts already at the beginning of prophase, proceeds concomitantly with chromatin compaction and is largely completed by the end of prophase. Sister chromatid resolution was abolished by inhibition of topoisomerase IIα and by depleting or preventing mitotic activation of condensin II, whereas blocking cohesin dissociation from chromosomes had little effect. Mitotic sister chromatid resolution is thus an intrinsic part of mitotic chromosome formation in prophase that relies largely on DNA decatenation and shares the molecular requirement for condensin II with prophase compaction. Equal segregation of the genome is the main objective of mitosis. In eukaryotes, replicated DNA molecules are held together until they are separated in mitosis. This sister chromatid cohesion is mediated primarily by cohesin complexes that embrace sister DNAs with their tripartite ring structure 3,4 as well as by catenation between sister DNAs stemming from replication 5–7 . At the onset of anaphase, proteolytic cleavage of cohesin rings by separase allows sister chromatids to be pulled apart by kinetochore microtubules 8,9 . However, in vertebrate cells, most cohesin on chromosome arms dissociates already in prophase before separase removes the remaining cohesin at centromeres 10 . This ‘prophase pathway’ proceeds through non-proteolytic removal of cohesin rings, requiring the cohesin regulator Wapl 11,12 . The progressive loss of cohesion between replicated chromosome arms goes along with chromatin compaction, requiring the condensin complexes 13,14 and ultimately results in the formation of individualized, non-overlapping masses of sister chromatids, referred to as resolution. The end product of resolution in metaphase can be recognized by light microscopy as a thin DNA-free slit between paired rod-shaped sister chromatids, which becomes especially prominent in the X-shape chromosomes attain after prolonged arrest in pro/metaphase. A seminal study with scanning electron microscopy 15 as well as a recent analysis based on fluorescence microscopy 16 found that sister chromatids became distinguishable from prometaphase onwards around the time of nuclear envelope breakdown (NEBD). When exactly in the early stages of mitosis sister resolution initiates and whether and how resolution is coordinated with chromatin compaction have however remained unknown so far. To address these gaps in our knowledge, here we developed a method to visualize the process of sister chromatid resolution by differential labelling of sister DNA molecules with two types of deoxyuridine analogue, which allowed us to quantitatively assay the process of sister resolution directly in single dividing cells. To achieve two-colour labelling of sister chromatids, diploid human RPE1 cells were first cultured in the presence of F-ara- 5-ethynyl-2 ′ -deoxyuridine (F-ara-EdU) 17 , a nucleotide suitable for click-chemistry that does not disturb cell growth 18 , for multiple rounds of replication to thoroughly label the chromatids. Next, 5-bromo-2 ′ -deoxyuridine (BrdU) 19 was incorporated into nascent DNA strands during one additional round of replication, obtaining ‘dual-labelled’ chromatids. After one more round of cell division and replication without labelled nucleotides, we obtained ‘single-labelled’ chromosomes with one sister chromatid labelled on one DNA strand with F-ara-EdU and the other chromatid with BrdU (Fig. 1a). As 1 Cancer Institute of the Japanese Foundation for Cancer Research, Division of Experimental Pathology, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan. 2 Tokyo Institute of Technology, Department of Biological Sciences, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan. 3 European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, Meyerhofstraße 1, 69117 Heidelberg, Germany. 4 Correspondence should be addressed to J.E. or T.H. (e-mail: jan.ellenberg@embl.de or thirota@jfcr.or.jp) Received 25 January 2016; accepted 1 April 2016; published online 2 May 2016; DOI: 10.1038/ncb3353 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1 © 2016 Macmillan Publishers Limited. All rights reserved