Proteomic and Cell Biological Analysis of Centrosomes and Telomeres

Normal cell division requires that chromosomes are accurately replicated and precisely segregated to daughter cells.  Carcinogenesis is strongly correlated with aneuploidy, or imbalances in chromosome segregation, but the mechanisms causing genomic instability have not yet been clearly defined.  To better understand these processes, I have focused on examining cell division machinery, especially the centrosome, a structure that helps form the mitotic spindle for faithful chromosome segregation, and the telomere, the ends of linear chromosomes whose length correlates with carcinogensis and aging.  Despite the biological importance of centrosomes and telomeres and their clinical importance in cancer, during which both structures become visibly abnormal (please see images below from Marx, Science, 2002), identification of proteins associating with these structures have only recently become possible with the development of bioinformatic and proteomic methods.

Normal bipolar spindle in a diploid human fibroblast (two centrosomes, yellow; DNA on metaphase plate, blue; microtubules, green)	Abnormal multipolar spindle in carcinoma cell (multiple centrosomes, red; condensed DNA, blue; microtubules, green)

My dissertation work involved identifying proteins related to the budding yeast centrosome component Spc110p.  Spc110p, a rod-shaped coiled-coil protein, was known to play a critical role in anchoring the mitotic spindle apparatus to the yeast centrosome, and evidence suggested conservation of Spc110p.  Taking a bioinformatic approach, I used fungal Spc110p-related sequences in low-stringency BLAST searches to identify Spc110p homologues in fission yeast (S. pombe), mice, and humans. We subsequently demonstrated that the human Spc110p homologue, kendrin/pericentrin, is overexpressed in cervical and colon cancer cells but is unique in its restricted localization to the ends of mitotic spindle poles, even in cells with multipolar spindles.

I have subsequently focused on analyzing the function of the fission yeast homologue, which I named Pcp1p. Fission yeast serves as an excellent model system allowing facile genetic analysis, live cell subcellular localization, and rapid sample generation for biochemistry and mass spectrometry.  Furthermore, fission yeast cell division mechanisms have been shown to be remarkably similar to those in humans.  Intriguingly, overexpression of Pcp1p causes fission yeast cells to exhibit a phenotype highly reminiscent of human carcinoma cells including supernumerary centrosomes, mitotic spindle defects, and missegregation of chromosomes (please see image at left). I used a proteomic approach to identify novel proteins that are recruited to the abnormal Pcp1p-containing centrosomal structures.  I reasoned that this approach might reveal both new centrosomal components, and perhaps also proteins that are improperly recruited to abnormal centrosomal structures.  To facilitate this approach, I used a strategy in which immunopurified eptitope-tagged protein complexes containing Pcp1p are analyzed by tandem mass spectrometry (LC-MS/MS) to reveal the sequence of isolated proteins.

I subsequently have employed genetic approaches such as gene knockouts, and cell biological techniques, including fluorescence microscopy and fluorescence resonance energy transfer, to characterize the function of the candidate proteins.  One novel enriched protein, which I named Ccq1p, localizes to both the centrosome and telomere during different stages of growth.  Reduced Ccq1p levels cause abnormal telomeric shortening and cell division defects, both hallmarks of human cancer cells.  In normal fission yeast cells, Ccq1p appears to play a pivotal role in facilitating the clustering of telomeres to the centrosome during meiosis, which occurs as a conserved event from yeast to humans.  In fission yeast cells with extra (abnormal) centrosomal structures, titration of Ccq1p away from telomeres leads to catastrophic telomeric shortening that eventually impedes proper cell division.  Failure to divide properly leads to further accumulation of centrosomes, potentiating a vicious cycle. It will be very interesting to test whether similar mechanisms act in human cancers to promote tumorigenesis.

	Fission yeast cell with supernumerary centrosomes (green), misshapen spindles (red), and abnormal chromosomal masses (blue); cell outline (white) [Flory et al., Cell Growth and Differentiation, 2002]
		Ion trap mass spectrometer reveals sequences of peptides; peptides are then matched to proteins in a sequence database

•   Many novel and interesting candidate Pcp1p-interacting proteins already identified by mass spectrometry await biological characterization.  We will employ gene tagging and knockout approaches to generate strains for analysis, and subsequently will use fluorescence microscopy, biochemical and genetic strategies to define functions for these proteins.  Many of these proteins show exciting structural features and have not been characterized previously.

• I have added various epitope tags to Ccq1p in preparation for analysis of its binding partners by mass spectrometry.  This analysis will likely reveal novel components of the fission yeast telomere, a critically important structure in the cell whose components are not yet well defined.  Candidate molecules may be examined using the techniques described above.  Ccq1p function will also be probed by combining ccq1 knockout alleles with other loss-of-function mutants to define roles for Ccq1p in telomeric protection and cell cycle regulation.

• We are also using cell biological and molecular methods to identify candidate human homologues of Ccq1p in cultured human cells.  Such a protein could potentially play important roles in normally preventing deleterious anaphase bridging events, which likely lead to the sorts of chromosome segregation and aneuploidy seen during carcinogenesis.

• We are also engaged in several collaborations with outside groups involving proteomic characterization of affinity-enriched biological complexes.  We are also applying the power of mass spectrometry and bioinformatics in collaboration with the Weir and Rice Groups at Wesleyan University in an effort to characterize alternative translational expression mechanisms in the budding yeast Saccharomyces cerevisiae.

Recent Publications

Rundle NT, Nelson J, Flory MR, Joseph J, Th’ng J, Aebersold R, Dasso M, Anderson RJ, Roberge M. (2006). An ent-kaurene that inhibits mitotic chromosome movement and binds the kinetochore protein ran-binding protein 2. ACS Chemical Biology 1:443.

Flory MR, Lee H, Bonneau R, Mallick P, Serikawa K, Morris DR, Aebersold, R. (2006) Quantitative proteomic analysis of the budding yeast cell cycle using acid-cleavable isotope-coded affinity tag reagents, Proteomics, 6:6146.

Knee KM, Roden CK, Flory MR, Mukerji, I (2007) The role of b93 Cys in the inhibition of Hb S fiber formation Biophysical Chemistry 187:181-193.

Prakash A, Piening B, Whiteaker J, Zhang H, Paulovich A, Watts J, Martin D, Flory M, Aebersold R, Goodlett D, Schwikowski B. (2007) Assessing reproducibility of mass spectrometry experiments, manuscript submitted.

King NL, Deutsch EW, Ranish JA, Nesvizhskii AI, Eddes JS, Mallick P, Eng J, Desiere F, Flory M, Martin DB, Kim K, Lee H, Raught B, Aebersold R. 2006. Analysis of the S. cerevisiae proteome with PeptideAtlas, Genome Biology 7:R106.

Eddes JS, King NL, Deutsch EW, Nesvizhskii AI, Aebersold R. 2006. A dataset of high quality unassigned tandem mass spectrometry spectra extracted from the Yeast Peptide Atlas, manuscript submitted.

Prakash A, Mallick P, Whiteaker J, Zhang H, Paulovich A, Flory M, Lee Hookeun, Aebersold R, Schwikowski B. 2005. Signal maps for mass spectrometry-based comparative proteomics.  Mol. Cell Proteomics 5:423-32.

Flory, M.R., Carson, A., Muller, E., and R. Aebersold. 2004. An SMC domain protein links telomeres to the meiotic centrosome in fission yeast. Molecular Cell, 16:619-30. 

MacKay, V.L., Li, X., Flory, M.R., Turcott, E., Law, G.L., Serikawa, K.A., Xu, X.L., Lee, H., Goodlett, D.R., Aebersold, R.A., and D.R. Morris. 2004. Gene expression in yeast responding to mating pheromone: analysis by high resolution translation state analysis and quantitative proteomics. Mol. Cell. Proteomics 3:478-489.

Flory, M.R. and T.N. Davis. 2003. The centrosomal proteins pericentrin and kendrin are encoded by alternatively spliced products of one gene. Genomics, 82(3):401-5.

Flory, M.R. and R. Aebersold. 2003. Proteomic approaches for the identification of cell cycle-related drug targets.  Chapter 15 in Progress in Cell Cycle Research: Cell Cycle Regulators as Therapeutic Targets, Ed. Meijer, L., Jezequel, A. and M. Roberge, Volume 5.

Flory, M.R., Griffin, T.J., Martin, D. and R. Aebersold. 2002. Advances in quantitative proteomics using stable isotope tags. Trends in Biotechnology 20(12S):S23-9.

Flory, M.R., Joseph, J. D., Morphew, D.M., Means, A.R. and T.N. Davis. 2002. Pcp1p, an Spc110p-related calmodulin target at the centrosome of the fission yeast Schizosaccharomyces pombe.  Cell Growth and Differentation 13(2):47-58.

Flory, M.R., Moser, M.J., Monnat, R.J. and T.N. Davis. 2000. Identification of a human centrosomal calmodulin-binding protein that shares homology with pericentrin. PNAS 97(11):5919-23.

Graduate Students:

James Arnone
Tina Motwani