Well-organized chromosomes: Catching nuclear proteins in action during mitosis

Research Areas:

Gene regulation, Mitosis, S. pombe

Imaging Needs:

Live-cell imaging, high spatial resolution

Imaging System:
  • Nikon Eclipse Ti inverted microscope
  • 100×, 1.4 NA objective lens
  • Yokagawa CSU10 spinning-disc confocal head
  • Hamamatsu ImageEM EM-CCD camera
  • NIH ImageJ software
  • Time-lapse images taken every 2 minutes
Measuring protein interactions in live cells

Imaging nuclear proteins in living cells requires high sensitivity and precise localization. What if you want to pan out and measure interactions within an entire living organism? Find out how Takeaki Ozawa and colleagues screen for GPCR ligands in mice in Illuminating Activity—in vitro and in vivo. Read now.

THE QUESTION

How do cells organize their chromosomes in preparation for cell division?

During mitosis, the chromosomes of the fission yeast Schizosaccharomyces pombe cluster together, attaching to the nuclear envelope at their centromeres during interphase. Similar positioning has been seen in plant and animal chromosomes prior to their segregation for cell division. In fission yeast, the chromosomes’ centromeres attach to the spindle pole body (SPB)—the yeast equivalent of the centrosome. The machinery and functional relevance of this interphase event are largely unknown.

THE BARRIERS

S. pombe provides an excellent opportunity to study centromere clustering, given sufficient resolution and sensitivity to examine the events in living cells.

THE SOLUTION

Csi1 links centromeres to the nuclear envelope for centromere clustering
Haitong Hou, Zhou Zhou, Yu Wang, Jiyong Wang, Scott P. Kallgren, Tatiana Kurchuk, Elizabeth A. Miller, Fred Chang, and Songtao Jia
J Cell Biol. 2012 Nov 199(5): 735–744. PMCID: PMC3514793.

Hou, et al,1 studied an S. pombe strain with a defect in centromere clustering to identify a novel nuclear protein essential to the process, called Csi1. Using fluorescence live-cell imaging of yeast strains expressing GFP- or mCherry-tagged Csi1, and GFP-tagged centromeric proteins, the team showed that Csi1 localizes near the nuclear envelope, centromeres, and SPB. The microscopy was performed at 25°C with a Hamamatsu ImageEM CCD camera. z stacks were collected at 0.4-μm intervals and then flattened.

Time-laspe microscopy at 2-minute intervals showed Csi1 co-located with inner nuclear envelope SUN domain protein Sad1 and centromeres through interphase and mitosis, and separated from the centromeres at their release. Further study confirmed that Csi1 and Sad1 mutations disrupt centromere clustering, and exhibit chromosome loss and other defects in chromosome segregation during mitosis.

The authors conclude that Csi1 and Sad1 work together to bring together the chromosomes at the SPB during interphase. They suggest that the clustering aids attachment of kinetochores by SPB microtubules in early mitosis, enabling healthy chromosome segregation into the daughter cells.

THE POSSIBILITIES

Hou, et al, took advantage of the high spatial resolution of Hamamatsu’s ImageEM camera to co-localize nuclear proteins essential to mitosis in the fission yeast S. pombe, and to track protein localization over time during mitosis. Find out how Takeaki Ozawa and colleagues use the ImagEM camera to detect GPCR binding using luminescence in place of fluorescence in Illuminating Activity—in vitro and in vivo.

References

  1. Hou, et al. Csi1 links centromeres to the nuclear envelope for centromere clustering. J Cell Biol. 2012 Nov 199(5): 735–744. PMCID: PMC3514793.
Inquiry about Life Science Cameras

Contact us

Scroll to Top