Time lapse images of HeLa cells over a period of approximately 87 hours using differential interference contrast imaging. Cells round up upon entry into mitosis (cell division). Images acquired by R. Ileng Kumaran and Stephen Hearn, CSHL.
Henrietta Lacks and HeLa Cells: Impact on Biological Research and Informed Consent
We have developed a cell line in which we can observe a stably integrated genetic locus as well as its mRNA and protein products using live cell microscopy and various GFP fusion proteins. This locus can be induced to initiate transcription. In the movie you will be able to see the fluorescent signal from the chromatin at the genetic locus as well as the protein product in the left panel and you will be able to see the mRNA synthesized when the genetic locus is turned on in the right panel. Both movies are showing the same cell.
When the genetic locus is not transcriptionally active the chromatin is condensed (tight blue dot in left panel) and there is no protein product seen in the cytoplasm of the cell. At this time the mRNA binding protein is diffusely distributed throughout the nucleus (right panel), as it has no substrate to bind to. When you start the movie you will see the chromatin in the left panel decondense over time and eventually you will see the protein product encoded for by the genetic locus, distributed in peroxisomes in the cytoplasm (blue dots in the cytoplasm). The protein has encoded within it a peroxisome-targeting signal. In the right panel you will notice that the mRNA binding protein concentrates at the genetic locus (transcription site) as the chromatin decondenses, since mRNA is being synthesized that has a binding site for the fluorescently tagged mRNA binding protein, and the nucleoplasmic pool now looks granular. The granular structures represent mRNPs that are moving away from the genetic locus in all directions by diffusion. They will end up in the cytoplasm where the mRNA will be translated into the protein that is targeted to peroxisomes.
From: Janicki, S.M., Tsukamoto, T., Salghetti, S.E., Tansey, W.P., Sachidanandam, R., Prasanth, K.V., Ried, T., Shav-Tal, Y., Bertrand, E., Singer, R.H. and Spector, D.L. 2004. From silencing to gene expression: Real-time analysis in single cells. Cell 116, 683-698.
HeLa cells were transiently transfected with YFP-SF2/ASF and live-cell observations were initiated 2 days post-transfection. Metaphase cells exhibit 1-2 mitotic interchromatin granule clusters (MIGs) near the metaphase plate. As the cell enters anaphase, MIGs become more abundant. SF2/ASF begins to enter daughter nuclei at telophase. Nuclear entry of YFP-SF2/ASF is nearly complete in approximately 20 min.
From: Prasanth, K.V., Sacco-Bubulya, P.A., Prasanth, S.G. and D. L. Spector. 2003. Sequential entry of components of the gene expression machinery into daughter nuclei. Molec. Biol. Cell. 14, 1043-1057.
Promyelocytic leukaemia (PML) nuclear bodies are present in most mammalian cell nuclei and are the target of PML-RAR-alpha oncoproteins in acute promyelocytic leukaemia where they are disrupted. These bodies contain numerous proteins including Sp100, SUMO-1, HAUSP(USP7), CBP, and BLM, and they have been implicated in aspects of transcriptional regulation or as nuclear storage depots. Here we show that there are three classes of PML nuclear bodies that can be distinguished based upon their dynamic properties in living cells. One class of PML bodies is particularly noteworthy in that it moves via a metabolic energy-dependent mechanism.
From: Muratani, M., D. Gerlich, S.M. Janicki, M. Gebhard, R. Eils and D.L. Spector. 2002. Metabolic energy-dependent movement of PML bodies within the mammalian cell nucleus. Nature Cell Biol. 4, 106-110.
Inhibition of ATP results in cesation of the rapidly moving PML bodies but has no effect on the other 2 classes of PML bodies.
Chromatin structure is thought to play a critical role in gene expression. Using the lac operator/repressor system and two color variants of green fluorescent protein, we developed a system to visualize a gene and its protein product directly in living cells allowing us to examine the spatial organization and timing of gene expression in vivo. Dynamic morphological changes in chromatin structure, from a condensed to an open structure, were observed upon gene activation, and targeting of the gene product/cyan fluorescent protein reporter to peroxisomes was directly visualized in living cells. Interestingly, we found that the integrated gene locus was surrounded by a promyelocytic leukemia (PML) nuclear body. The association was transcription-independent but was dependent upon the direct in vivo binding of specific proteins (EYFP/lac repressor, tetracycline receptor/VP16 transactivator) to the locus. The ability to directly visualize gene expression in living cells provides a powerful system with which to study the dynamics of nuclear events such as transcription, RNA processing, and DNA repair.
This movie shows a stably integrated genetic locus (18 Mb) in a BHK cell. The locus is colored in yellow (YFP-lac repressor). Images were taken in living cells every 10 minutes over a 7 hour period. Approximately 2 hours after the turn on of the locus, by the addition of doxycycline, the protein product is detected as a CFP fusion protein that is targeted to peroxisomes.
From: Tsukamoto, T., N. Hashiguchi, S.M. Janicki, T. Tumbar, A.S. Belmont and D.L. Spector. 2000. Visualization of gene activity in living cells. Nature Cell Biology 2, 871-878.
Overview of the dynamics of nuclear speckles enriched in pre-mRNA splicing factors. Each speckle is highly dynamic along its periphery and factors are recruited from these sites to sites of active transcription.
From: Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. The dynamics of a pre-mRNA splicing factor in living cells. Nature 387, 523-527.
From: Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. The dynamics of a pre-mRNA splicing factor in living cells. Nature 387, 523-527.
From: Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. The dynamics of a pre-mRNA splicing factor in living cells. Nature 387, 523-527.
This speckle clearly exhibits extensive dynamics along its periphery suggestive of complexes of factors leaving this nuclear region.
From: Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. The dynamics of a pre-mRNA splicing factor in living cells. Nature 387, 523-527.
From: Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. The dynamics of a pre-mRNA splicing factor in living cells. Nature 387, 523-527.
Inhibition of RNA polymerase II transcription by alpha-amanitin significantly reduces the dynamics on the periphery of nuclear speckles and the speckles round-up.
From: Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. The dynamics of a pre-mRNA splicing factor in living cells. Nature 387, 523-527.