Molecular and functional analysis of kinetochores

Our research focuses on kinetochores; highly conserved proteinaceous assemblies that hierarchically form from more than 100 proteins onto the centromeric regions of duplicated chromosomes (sister chromatids). Kinetochores regulate sister chromatid segregation during cell division by performing four essential activities: they I) associate each chromatid pair with the tips of the spindle microtubules, II) act as a sensor of microtubule binding, III) regulate the onset of sister chromatid segregation via the proofreading spindle assembly checkpoint, and IV) maintain microtubule attachment during cycles of microtubule shrinkage and growth, thereby generating forces that are required for chromatid movement along the spindle.
The presence of abnormal chromosome numbers (aneuploidy) is a molecular hallmark of cancer cells. Precisely how aneuploidy is generated remains unclear. Due to their essential roles in chromosome segregation, kinetochore dysfunctions are a likely cause of aneuploidy. Thus, a better understanding of kinetochore composition, formation, activity, and regulation is bound to reveal new insights into the mechanisms underlying tumorigenesis.
We study the kinetochores of budding yeast (Saccharomyces cerevisiae) and human cells by integrating a variety of methodologies:

Genetics: mutant suppressor screens, synthetic lethality/viability screens, two-hybrid interaction screens, creation of temperature-sensitive mutants, epigenetic pathway mapping.
Pharmacogenetics: compound library screening to identify small ligands that interfere with certain kinetochore activities using in vitro and in vivo assays.
Biochemistry: hydrodynamics (chromatography, sedimentation ultracentrifugation), chromatin immunoprecipitation, affinity purification of kinetochore components from cell extracts, chromatin immunoprecipitation (ChIP), ChIP on CHIP.
Cell cycle analysis: molecular dissection of kinetochore protein activity in arrested-and-released yeast and human cell cultures via live-cell imaging, immunofluorescence, ChIP, FACS, Western hybridization, etc.
Crystallography: production, crystallization and structural analysis of recombinant kinetochore proteins and complexes.
High-resolution imaging: time-lapse videomicroscopy of yeast and human cells, single-molecule TIRF imaging of recombinant kinetochore proteins and complexes using in vitro assays.

The knowledge obtained from our research will contribute to the understanding of how DNA is correctly transmitted from one generation to the next. In addition, it will stimulate the development of (i) diagnostic tools to screen for kinetochore-based cancer susceptibility markers (mutations in kinetochore components), and (ii) novel anticancer drugs (molecules that interfere with kinetochore assembly or function).

1)Screpanti E, Santaguida S, Nguyen T, Silvestri R, Gussio R, Musacchio A, Hamel E, De Wulf P. A screen for kinetochore-microtubule interaction inhibitors identifies novel antitubulin compounds. PLoS One. 2010 Jul 15;5(7):e11603.
2)Pagliuca C., Draviam V.M., Marco E., Sorger P.K., and De Wulf P.
Roles for the conserved Spc105p/Kre28p complex in kinetochore-microtubule binding and the spindle assembly checkpoint.
PLoS One, Oct 28;4(10):e7640 (2009)
3)De Wulf P., Montani F., and Visintin R.
Protein phosphatases take the mitotic stage.
Current Opinion in Cell Biology, Sep 18. [Epub ahead of print]. (2009)
4)Cohen R.L., Espelin C.W., De Wulf P., Sorger P.K., Harrison S.C., and Simons K.T.
Structural and functional dissection of Mif2p, a conserved DNA-binding kinetochore protein.
Molecualr Biology of the Cell, 19:4480-4491 (2008)
5)Fukagawa T., and De Wulf P.
Kinetochore composition, formation and organization. In: "The Kinetochore: from Molecular Discoveries to Cancer Therapy". Eds. De Wulf P., and Earnshaw W.C.
Springer Publ., New York, pp. 133-191 (2008)
6)De Wulf P., and Visintin R.
Cdc14B and APC/C tackle DNA damage.
Cell, 134: 210-212. (2008)
7)Ciferri C., Pasqualato S., Screpanti E., Varetti G., Santaguida S., Dos Reis G., Maiolica A., Polka J., De Luca J.G., De Wulf P., Salek M., Rappsilber J., Moores C.A., Salmon E.D., and Musacchio A.
Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex.
Cell, 133: 427-39. (2008).
8)Miranda JJ.M., De Wulf P., Sorger P.K., and Harrison S.C.
The yeast DASH complex decorates microtubules as a closed ring.
Nature Structural and Molecular Biology, 12: 138-143. (2005)

No projects are available to students for the current accademic year.