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

نویسندگان

  • Takashi Fukada
  • Kimiko Inoue
  • Takeshi Urano
  • Kenji Sugimoto
چکیده

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

برای دانلود رایگان متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

High efficiency DNA mutagenesis mediated by using in vitro transcription, DNase I digestion, and RT-PCR.

porally correlated with the processes of chromosome segregation and nuclear envelope reformation. Our live image analysis with the multicolor cell line clearly indicated that survivin diffused into cytosol coincident with the chromosome segregation (435 min) and reappeared in the midzone during the nuclear envelope reformation (441–444 min). The separate images of ECFP-histone and EGFP-survivin...

متن کامل

Aurora B–mediated localized delays in nuclear envelope formation facilitate inclusion of late-segregating chromosome fragments

To determine how chromosome segregation is coordinated with nuclear envelope formation (NEF), we examined the dynamics of NEF in the presence of lagging acentric chromosomes in Drosophila neuroblasts. Acentric chromosomes often exhibit delayed but ultimately successful segregation and incorporation into daughter nuclei. However, it is unknown whether these late-segregating acentric fragments in...

متن کامل

Dynamic associations of heterochromatin protein 1 with the nuclear envelope.

To study the dynamics of mammalian HP1 proteins we have microinjected recombinant forms of mHP1alpha, M31 and M32 into the cytoplasm of living cells. As could be expected from previous studies, the three fusion proteins were efficiently transported into the nucleus and targeted specific chromatin areas. However, before incorporation into these areas the exogenous proteins accumulated in a perip...

متن کامل

Chromosomal association of Ran during meiotic and mitotic divisions.

Recent studies in Xenopus egg extracts indicate that the small G protein Ran has a central role in spindle assembly and nuclear envelope reformation. We determined Ran localization and dynamics in cells during M phase. By immunofluorescence, Ran is accumulated on the chromosomes of meiosis-II-arrested Xenopus eggs. In living cells, fluorescently labeled Ran associated with the chromosomes in Xe...

متن کامل

An Ordered Inheritance Strategy for the Golgi Apparatus: Visualization of Mitotic Disassembly Reveals a Role for the Mitotic Spindle

During mitosis, the ribbon of the Golgi apparatus is transformed into dispersed tubulo-vesicular membranes, proposed to facilitate stochastic inheritance of this low copy number organelle at cytokinesis. Here, we have analyzed the mitotic disassembly of the Golgi apparatus in living cells and provide evidence that inheritance is accomplished through an ordered partitioning mechanism. Using a Sa...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

عنوان ژورنال:
  • BioTechniques

دوره 37 4  شماره 

صفحات  -

تاریخ انتشار 2004