Visualization of chromosomes and nuclear envelope in living cells for molecular dynamics studies.

Cell division is a highly dynamic process in which coordinated translocation of various protein molecules is important for precise replication and distribution of intracellular structures such as centrosomes, centromere/ kinetochores, and chromosomes. For example, aurora-B and survivin, known as chromosome passenger proteins, associate with centromere/kinetochores in prometaphase and metaphase, remain in the midzone in anaphase and telophase, and finally migrate to the midbody during the cytokinesis (1). Although immunofluorescent observation of fixed cells shows certain timesections of these protein translocations, it is often difficult to follow the entire behavior of quickly migrating proteins. Green fluorescent protein (GFP) fusion technique enabled tracking of certain protein in living cells by time-lapse fluorescent microscopy and dramatically improved the protein dynamics study. However, some technical inconveniences still associate with the living cell analysis. One of the problems is that it is often difficult to precisely track the progression of mitotic stages unless chromosomes are visualized by fluorescent dye injection or differential interference contrast (DIC) microscopy. It is also difficult to assign the observed GFP signals to certain intracellular structures such as centrosomes and centromere/kinetochores, which are invisible in living unstained cells. Although DIC or phase contrast microscopy can be used to visualize chromosomes (reviewed in Reference 2) in combination with fluorescence microscopy, application of this approach to time-lapse observation usually requires a complex and expensive system that can automatically manage the two different optics. Because of these disadvantages, studies of GFP-fused protein dynamics in living cells usually require supplemental experiments on fixed cells in which some landmark structures are immunologically stained. To construct a fluorescence-only system for studying protein dynamics in mitosis, we established a human cell line expressing histone H3 and truncated importin α as fusions to cyan and red fluorescent proteins [enhanced cyan fluorescent protein (ECFP) and DsRed], respectively, to visualize the chromosomes and the nuclear envelope in living cells. These visualized structures served as spatial landmarks for specifying the relative positions of GFP-fused proteins, as well as temporal markers for tracking the progression of cell cycle stages. Mouse histone H3 cDNA, cloned in pECFP-C1 (BD Biosciences Clontech, Palo Alto, CA, USA), was introduced into a human breast tumor cell line (MDA435). Stably transformed cell lines were isolated by G418 selection, and the expression of ECFP-histone H3 was confirmed by fluorescent microscopy. A 1-kb cDNA fragment encoding the C-terminal half of human importin α1 (amino acids 251–529) was cloned into pDsRed1-C1 (BD Biosciences Clontech) and transfected into the above transformants with a puromycin-resistance plasmid (pLCpuro), followed by puromycin selection to establish a stable Visualization of chromosomes and nuclear envelope in living cells for molecular dynamics studies