Abstract A new approach is presented which allows the in vivo visualization of individual chromosome territories in the nuclei of living human cells. The fluorescent thymi- dine analog Cy3-AP3-dUTP was microinjected into the nuclei of cultured human cells, such as human diploid fi- broblasts, HeLa cells and neuroblastoma cells. The fluo- rescent analog was incorporated during S-phase into the replicating genomic DNA. Labelled cells were further cul- tivated for several cell cycles in normal medium. This well-known scheme yielded sister chromatid labelling. Random segregation of labelled and unlabelled chroma- tids into daughter nuclei resulted in nuclei exhibiting indi- vidual in vivo detectable chromatid territories. The terri- tories were composed of subcompartments with diameters ranging between approximately 400 and 800 nm which we refer to as subchromosomal foci. Time-resolved in vivo studies demonstrated changes of positioning and shape of territories and subchromosomal foci. The hypothesis that subchromosomal foci persist as functionally distinct enti- ties was supported by double labelling of chromatin with CldU and IdU, respectively, at early and late S-phase and subsequent cultivation of corresponding cells for 5–10 cell cycles before fixation and immunocytochemical detection. This scheme yielded segregated chromatid territories with distinctly separated subchromosomal foci composed of ei- ther early- or late-replicating chromatin. The size range of subchromosomal foci was similar after shorter (2 h) and longer (16 h) labelling periods and was observed in nuclei of both living and fixed cells, suggesting their structural identity. A possible functional relevance of chromosome territory compartmentalization into subchromosomal foci is discussed in the context of present models of interphase chromosome and nuclear architecture. Introduction In recent years the nucleus has emerged as a highly com- partmentalized structure. It has been demonstrated that chromosomes form distinct territories in both animal and plant nuclei (Manuelidis 1985; Schardin et al. 1985; Cremer et al. 1988; Lichter et al. 1988; Pinkel et al. 1988; Leitch et al. 1990; for review see Cremer et al. 1993). Chromo- some territories seem to be further partitioned into dis- crete chromosomal compartments, such as chromosome arm and band-like domains, centromeric domains and telo- meric domains (Manuelidis 1990; Lawrence et al. 1993; Schedl and Grosveld 1995; Zhao et al. 1995; Kurz et al. 1996; Dietzel et al. 1998). Single active or inactive genes were visualized as apparently discrete dot-like domains (e.g. Kurz et al. 1996). The 3D-positioning of genes with- in or at the surface of chromosome territories has become a topic of active research (Cremer et al. 1993; Eils et al. 1996; Kurz et al. 1996). Nuclear proteins which are in- volved in nuclear functions, such as transcription, splic- ing, DNA replication and repair, participate in higher-or- der macromolecular domains of a size that can often be re- cognized in immunofluorescent labelling experiments as typical foci, speckles or punctate distributions of nuclear antigens (Spector 1993; Roth 1995; van Driel et al. 1995). During terminal differentiation of cells complex movements of chromatin were noted (for reviews see Manuelidis 1990; De Boni 1994). In spite of these advances we do not un- derstand the functional meaning of topological relation- ships between higher-order nuclear structures and their dyna- mics and lack a coherent and widely accepted theory of the functional chromosome territory and nuclear architecture. Studies of chromosome territory architecture using flu- orescence in situ hybridization (FISH) require fixed cells. It is not clear to what extent experimental artifacts have Daniele Zink · Thomas Cremer · Rainer Saffrich · Roger Fischer · Michael F. Trendelenburg · Wilhelm Ansorge · Ernst H. K. Stelzer Structure and dynamics of human interphase chromosome territories in vivo Hum Genet (1998) 102 : 241–251 © Springer-Verlag 1998 Received: 10 November 1997 / Accepted: 24 November 1997 RAPID COMMUNICATION D. Zink () · T. Cremer () Institut für Anthropologie und Humangenetik, LMU München, Richard-Wagner-Strasse 10/I, D-80333 Munich, Germany Tel.: +49-89-5203-381; Fax: +49-89-5203-389 R. Saffrich · W. Ansorge · E. H. K. Stelzer European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany R. Fischer · M. F. Trendelenburg Deutsches Krebsforschungszentrum, Abteilung 0195, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany