With a little help from a friend: Discovering a new protein partnership in cell division

Research Areas:

Cell biology, Nuclear membrane proteins, Meiosis, S. cerevisiae

Imaging Needs:

High spatial resolution, ability to detect co-localization via fluorescent protein tags (GFP family)

Imaging System:
  • Zeiss 200m inverted microscope equipped with a Yokagawa CSU-10 Confocal Scanner Unit spinning disc
  • 100x 1.4 NA oil objective lens
  • Laser excitation at 488-nm and 568-nm
  • BP 500- to 550-nm and BP 590- to 650-nm filters
  • Hamamatsu ImageEM C9000-13 Electron Multiplying CCD (EM-CCD) camera
  • ImageJ software
Imaging cellular events in real time

Find out how Fang Huang, Jeorg Bewersdorf and colleagues use the sCMOS technology in the ORCA-Flash4.0 camera to achieve video-rate imaging at nanometer scales. Read now.

THE QUESTION

How do nuclear proteins choreograph chromosome movement in mitosis and meiosis?

In almost all eukaryotic cells, inner nuclear membrane proteins containing a SUN (Sad1-UNC-84) domain bind with extra-nuclear protein partners to form bridges between the nucleoplasm and cytoplasm. These linkages join the nucleoskeleton or chromatin and the cytoplasmic cytoskeleton, and play essential roles in chromosome movement during meiosis, including centrosome attachment to the nuclear envelope.

The yeast Saccharomyces cerevisiae contains only one gene encoding a SUN domain containing protein: MPS3. Its status as the only SUN-domain gene in the organism provides a unique opportunity to study the mechanisms by which these highly conserved proteins aid nuclear movement and cell division.

THE BARRIERS

Deletion of the SUN domain from the MPS3 gene is lethal. SUN domain mutations lead to defects in duplication of the organelle equivalent to a centrosome in yeast, the spindle pole body (SPB). Investigating how MPS3 enables proper SPB formation called for high-throughput genetic techniques to identify potential partners, and highly sensitive imaging to catch candidate proteins in action at the nuclear membrane.

THE SOLUTION

Genetic Analysis of Mps3 SUN Domain Mutants in Saccharomyces cerevisiae Reveals an Interaction with the SUN-Like Protein Slp1
Jennifer M. Friederichs, Jennifer M. Gardner, Christine J. Smoyer, Christine R. Whetstine, Madelaine Gogol, Brian D. Slaughter, and Sue L. Jaspersen
G3: Genes | Genomes | Genetics. 2012; 2: 1703. PMCID: PMC3516490

Friederichs, et al,1 performed a genome-wide scan for mutations that exacerbated the effects of MPS3 mutations. Hits included many components of the spindle apparatus, as well as genes involved in lipid synthesis and membrane organization, suggesting that MPS3 may be important in nuclear membrane organization. Mutation in two genes, called SLP1 and EMP65, particularly disrupted growth in MPS3 mutant strains.

The authors used confocal fluorescence imaging to show that Slp1 and Emp65 form a protein complex associated with the ER membrane, independent of Mps3. Mutation studies showed that the Slp1/Emp65 complex is not directly required for SPB function. Using the Hamamastu ImageEM EM-CCD camera to visualize microtubules and SPBs in mCherry-TUB1 and TUB4-GFP expressing cells, the researchers showed that the SLP1 mutation disrupted Mps3 localization at the nuclear envelope. The results suggest a role for Slp1 in Mps3 localization or tethering at the nuclear membrane.

THE POSSIBILITIES

Friederichs, et al, relied on theultra-low-light capabilities of Hamamatsu’s ImageEM EM-CCD camera to localize important nuclear proteins in S. cerevisiae. Learn what’s possible with the faster speeds and wider fields-of-view of Hamamatsu’s ORCA-Flash4.0 technology, in Exciting Advances Push the Limits of Visualization.

References

  1. Friederichs, et al. Genetic Analysis of Mps3 SUN Domain Mutants in Saccharomyces cerevisiae Reveals an Interaction with the SUN-Like Protein Slp1. G3: Genes | Genomes | Genetics. 2012 Dec 2:1703-1718. PMCID: PMC3516490.
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