Confocal Imaging of Cell Division

Paul S. Maddox1

1 Department of Pathology and Cell Biology, Université de Montréal, Montreal, Canada
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.11
DOI:  10.1002/0471142956.cy1211s43
Online Posting Date:  January, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Imaging of subcellular events has been key to recent advances in the basic knowledge base as well as drug discovery for disease states such as cancer. In a snowball effect, advances in imaging have spurred new investigations, which have increased the demand for new technologies. The study of cell division serves as an example of this cycle. Here, application of spinning disk confocal technology to the study of cell division is discussed. While these studies have increased understanding of fundamental mechanisms in several mitotic events, new imaging technologies in the future will unlock more secrets of cell biology. Curr. Protocol. Cytom. 43:12.11.1‐12.11.13. © 2008 by John Wiley & Sons, Inc.

Keywords: confocal imaging; live cell imaging; mitosis; cell division; microtubule; kinetochore; chromosome condensation; cytokinesis

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Spinning Disk Confocal
  • Confocal Imaging of Chromosome Condensation in C. elegans Embryos
  • Confocal Imaging of Spindle Assembly and Chromosome Dynamics
  • Confocal Imaging of Cytokinesis
  • Discussion
  • Acknowledgements
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 12.11.1 Photograph of the spinning disk confocal microscope (A). Photograph of laser, filter wheel, and fiber‐optic coupling (B).
  •   FigureFigure 12.11.2 Confocal analysis of chromosome condensation in C. elegans. The imaging and analysis regimes (AC) allow quantitative comparisons of chromosome condensation following RNAi‐based protein depletions (F). NEBD, nuclear envelope breakdown.
  •   FigureFigure 12.11.3 Confocal FSM imaging of mitosis in Drosophila embryos. Centromeres (red) are labeled with GFP‐MEIS332, while fluorescent speckled microtubules (green) were generated by microinjection of labeled tubulin (A). Box in A is the region of the spindle used for kymograph analysis (by the “box” method) in B. The speckle trajectories show that centromeres move poleward faster than speckles (B).
  •   FigureFigure 12.11.4 Confocal analysis of spindle dynamics in Xenopus egg extracts. Centromeres were labeled by addition of directly labeled anti‐CENP‐A antibody (red) and fluorescent speckled microtubules (green) were labeled by addition of labeled tubulin directly to the extract (A). The line in A was used for kymograph analysis by the “line method” revealing that, as seen in other systems, kinetochores move poleward faster than microtubule treadmilling (speckles, B).
  •   FigureFigure 12.11.5 Confocal analysis of cytokinesis in C. elegans. Single focal plane time‐lapse imaging through the middle of a dividing embryo expressing GFP‐plasma membrane marker allows gross measurement of furrow closure (A). Four‐dimensional data provides a view of cytokinesis, which revealed that furrow closure is asymmetric (B).

Videos

Literature Cited

Literature Cited
   Brenner, S. 1974. The genetics of Caenorhabditis elegans. Genetics 77:71‐94.
   Brust‐Mascher, I. and Scholey, J.M. 2002. Microtubule flux and sliding in mitotic spindles of Drosophila embryos. Mol. Biol. Cell 13:3967‐3975.
   Dykstra, B., Ramunas, J., Kent, D., McCaffrey, L., Szumsky, E., Kelly, L., Farn, K., Blaylock, A., Eaves, C., and Jervis, E. 2006. High‐resolution video monitoring of hematopoietic stem cells cultured in single‐cell arrays identifies new features of self‐renewal. Proc. Natl. Acad. Sci. U.S.A. 103:8185‐8190.
   Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. 1998. Potent and specific genetic interference by double‐stranded RNA in Caenorhabditis elegans. Nature 391:806‐811.
   Flemming, W. 1879. Archiv fur Mikroskopische Anatomie. 18:302‐436.
   Hagstrom, K.A., Holmes, V.F., Cozzarelli, N.R., and Meyer, B.J. 2002. C. elegans condensin promotes mitotic chromosome architecture, centromere organization, and sister chromatid segregation during mitosis and meiosis. Genes Dev. 16:729‐742.
   Ichihara, A., Tanaami, T., Isozki, K., Sugiyama, Y., Kosugi, Y., Mikuriya, K., Abe, M., and Uemure, I. 1996. High speed confocal fluorescence microscopy using a Nipkow scanner with microlenses for imaging of single fluorescent molecule in real time. Bioimages 4:57.
   Ichihara, T., Tanaami, H., Ishida H., and Shimizu, M. 1999. Confocal fluorescent microscopy using a Nipkow scanner. In Fluorescent and Luminescent Probes, 2nd Edition (W.T. Mason, ed.) p. 344. Academic Press, New York.
   Inoué, S. and Inoué, T. 2002. Direct‐view high‐speed confocal scanner: The CSU‐10. Methods Cell Biol. 70:87‐127.
   Inoué, S. and Spring, K. 1997. Video Microscopy. Plenum Press, New York.
   Kerrebrock, A.W., Miyazaki, W.Y., Birnby, D., and Orr‐Weaver, T.L. 1992. The Drosophila mei‐S332 gene promotes sister‐chromatid cohesion in meiosis following kinetochore differentiation. Genetics. 130:827‐841.
   Kline‐Smith, S.L., Sandall, S., and Desai, A. 2005. Kinetochore‐spindle microtubule interactions during mitosis. Curr. Opin. Cell Biol. 17:35‐46.
   Maddox, A.S. and Burridge, K. 2003. RhoA is required for cortical retraction and rigidity during mitotic cell rounding. J. Cell Biol. 160:255‐265.
   Maddox, A.S. and Oegema, K. 2003. Deconstructing cytokinesis. Nat. Cell Biol. 5:773‐776.
   Maddox, P., Desai, A., Oegema, K., Mitchison, T.J., and Salmon, E.D. 2002. Poleward microtubule flux is a major component of spindle dynamics and anaphase a in mitotic Drosophila embryos. Curr. Biol. 12:1670‐1674.
   Maddox, P., Straight, A., Coughlin, P., Mitchison, T.J., and Salmon, E.D. 2003a. Direct observation of microtubule dynamics at kinetochores in Xenopus extract spindles: Implications for spindle mechanics. J. Cell Biol. 162:377‐382.
   Maddox, P.S., Moree, B., Canman, J.C., and Salmon, E.D. 2003b. Spinning disk confocal microscope system for rapid high‐resolution, multimode, fluorescence speckle microscopy and green fluorescent protein imaging in living cells. Methods Enzymol. 360:597‐617.
   Maddox, P.S., Portier, N., Desai, A., and Oegema, K. 2006. Molecular analysis of mitotic chromosome condensation using a quantitative time‐resolved fluorescence microscopy assay. Proc. Natl. Acad. Sci. U.S.A. 103:15097‐15102.
   Maddox, A.S., Lewellyn, L., Desai, A., and Oegema, K. 2007. Anillin and the septins promote asymmetric ingression of the cytokinetic furrow. Dev Cell. 12:827‐835.
   Mitchison, T., Evans, L., Schulze, E., and Kirschner, M. 1986. Sites of microtubule assembly and disassembly in the mitotic spindle. Cell 45:515‐527.
   Mitchison, T.J. 1989. Polewards microtubule flux in the mitotic spindle: Evidence from photoactivation of fluorescence. J. Cell Biol. 109:637‐652.
   Mitchison, T.J. and Salmon, E.D. 1992. Poleward kinetochore fiber movement occurs during both metaphase and anaphase‐A in newt lung cell mitosis. J. Cell Biol. 119:569‐582.
   Murray, A.W. 1991. Cell cycle extracts. Methods Cell Biol. 36:581‐605.
   Portier, N., Audhya, A., Maddox, P.S., Green, R.A., Dammermann, A., Desai, A., and Oegema, K. 2007. A microtubule‐independent role for centrosomes and aurora a in nuclear envelope breakdown. Dev Cell. 12:515‐529.
   Ramunas, J., Montgomery, H.J., Kelly, L., Sukonnik, T., Ellis, J., and Jervis, E.J. 2007. Real‐time fluorescence tracking of dynamic transgene variegation in stem cells. Mol. Ther. 15:810‐817.
   Rieder, C.L. and Cole, R.W. 1998. Entry into mitosis in vertebrate somatic cells is guarded by a chromosome damage checkpoint that reverses the cell cycle when triggered during early but not late prophase. J. Cell Biol. 142:1013‐1022.
   Rieder, C.L. and Khodjakov, A. 2003. Mitosis through the microscope: Advances in seeing inside live dividing cells. Science 300:91‐96.
   Rieder, C.L. and Salmon, E.D. 1998. The vertebrate cell kinetochore and its roles during mitosis. Trends Cell Biol. 8:310‐318.
   Salmon, E.D., Shaw, S.L., Waters, J., Waterman‐Storer, C.M., Maddox, P.S., Yeh, E., and Bloom, K. 1998. A high‐resolution multimode digital microscope system. In Methods in Cell Biology: Video Microscopy, vol. 56. (G. Sluder and D.E. Wolf, eds.) Academic Press, London.
   Stelzer, E.H. 2000. In Imaging Neurons: A Laboratory Manual. (F.L.R. Yuste and A. Konnerth, eds.) p. 12‐1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Swedlow, J.R. 2007. Quantitative fluorescence microscopy and image deconvolution. Methods Cell Biol. 81:447‐465.
   Swedlow, J.R. and Platani, M. 2002. Live cell imaging using wide‐field microscopy and deconvolution. Cell Struct. Funct. 27:335‐341.
   Vale, R.D., Reese, T.S., and Sheetz, M.P. 1985. Identification of a novel force‐generating protein, kinesin, involved in microtubule‐based motility. Cell 42:39‐50.
   Waterman‐Storer, C.M. and Salmon, E.D. 1998. How microtubules get fluorescent speckles. Biophys. J. 75:2059‐2069.
   Waterman‐Storer, C.M. and Salmon, E.D. 1999. Fluorescent speckle microscopy of microtubules: How low can you go? FASEB J. 13:S225‐S230.
   Waterman‐Storer, C.M., Desai, A., Bulinski, J.C., and Salmon, E.D. 1998. Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells. Curr. Biol. 8:1227‐1230.
   Waterman‐Storer, C.M., Desai, A., and Salmon, E.D. 1999. Fluorescent speckle microscopy of spindle microtubule assembly and motility in living cells. Methods Cell Biol. 61:155‐173.
   Wolf, D.E., Samarasekera, C., and Swedlow, J.R. 2007. Quantitative analysis of digital microscope images. Methods Cell Biol. 81:365‐396.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library