Our lab studies how a dividing cell segregates its replicated chromosomes to generate two daughters with a correct genetic content. Errors made during the process can lead to offspring carrying an abnormal number of chromosomes (aneuploidy), which may result in cell death, genetic syndromes (e.g., Down), infertility or cancer (most malignant cells are aneuploid). By studying the proteins that orchestrate chromosome segregation we will better understand (i) a most essential biological activity, (ii) how mistakes made during the event contribute to the above anomalies, and (iii) how drug targeting certain aspects of the process can selectively eradicate cancer cells. Since the chromosome segregation process is conserved between all higher species we study its biology both in the budding yeast Saccharomyces cerevisiae and in human cells.
Our work focuses on the kinetochore, a conserved protein complex the size of a ribosome (~60 subunits in yeast, ~100 in human cells) that assembles on the centromeric sequence of each chromosome, and on the centromeric chromatin itself. Kinetochores attach the replicated chromosomes onto the mitotic spindle structure and orchestrate chromosome segregation along the regressing spindle microtubules. We also study additional genomic regions (e.g., rDNA) that are subject to specific regulation during chromosome segregation, hence allowing for their timely transmission with the rest of the genome.
Our research implements genetics, biochemistry, molecular biology, high-resolution live-cell microscopy, bioinformatics, systems network analysis, computational modelling of drug-protein interactions, and drug development.
- Regulation of kinetochore assembly on replicated centromeres
In yeast, kinetochores disassemble in early S phase to allow the replication fork to move through the centromeric sequence. Following centromere replication, kinetochores quickly reassemble to attach the chromosomes onto the spindle. We have identified an evolutionary conserved cell cycle activity that determines the timely re-assembly of kinetochores onto the replicated centromeres. We are currently dissecting the molecular mechanisms that underlie this regulatory pathway.
- A response pathway that counteracts kinetochore toxicity
Cancer cells often pathologically express kinetochores subunits. Their excess levels disturb the assembly and faithful activity of the 3D kinetochore structure, resulting in chromosome mis-segregation. Indeed, studies with mice have shown that even a single overexpressed kinetochore protein can trigger cancerogenesis.
By performing genetic screens with yeast, we recently identified a conserved response pathway that antagonizes mis-expressed kinetochore proteins. The pathway acts in parallel to the spindle assembly checkpoint, which monitors chromosome-spindle alignment. Noteworthy, this new response pathway is chemically activatable, which implies a novel clinical strategy to selectively target cancer cells suffering from kinetochore protein overexpression. We are dissecting this pathway in cells and are reconstituting it biochemically to delineate it at the molecular level.
- Kinetochore-inhibiting anti-cancer drugs
Cancer cells driven by a variety of genetic mutations (e.g., the K-Ras oncogene) are highly susceptible to interference with kinetochore activity. Using the crystal structures of human kinetochore subunits we computationally dock chemotype libraries onto them to in silico identify ligands that inhibit the activity of these proteins. Via biochemical studies, cell cycle analyses and phenotypic evaluations of genetically well-defined primary cancer cell lines we study how these drugs can selectively eradicate mutation-addicted cancer cells.
- Peter De Wulf, PI
- Indri Erliandri, Post-Doctoral Researcher
- Ksenia Smurova, Post-Doctoral Researcher
- Giovanna Berto, MSc Thesis Student
- Marta Andolfato, BSc Thesis Student
- Beatrice Tremonti, BSc Thesis Student
- Alessandro Abram, BSc Thesis Student
- Melissa D'Agostini, BSc Thesis Student
- Dr. David Baroni (University of Padova, Italy)
- Dr. Dana Branzei (IFOM-FIRC Institute of Molecular Oncology, Italy)
- Dr. Sébastien Ferreira-Cerca (University of Regensburg, Germany)
- Dr. Saverio Minucci (European Institute of Oncology, Italy)
- Dr. Romano Silvestri (University of Rome - La Sapienza, Italy)
- Dr. Tomoyuki Tanaka (University of Dundee, The Wellcome Institute, UK)
- Dr. Esti Yeger-Lotem (Ben-Gurion University of the Negev, Israel)
Smurova K., De Wulf P. Centromere and pericentromere transcription: roles and regulation... in sickness and in health. Frontiers in Genetics. Invited Review.
Iacovella M.G., Bremang M., Basha O., Giaco L., Carotenuto W., Golfieri C., Szakal B., Dal Maschio M., Infantino V., Beznoussenko G.V., Chinnu R.J., Visintin C., Mironov A.A., Visintin R., Branzei D., Ferreia-Cerca S., Yeger-Lotem E., De Wulf P. (2018). Integrating Rio1 activities discloses its nutrient-activated network in Saccharomyces cerevisiae. Nucleic Acids Research, 46: 7586-7611.
Iacovella M.G., Golfieri C., Massari L.F., Busnelli S., Pagliuca C., Dal Maschio M., Infantino V., Visintin R., Mechtler K., Ferreira-Cerca S., De Wulf P. (2015). Rio1 promotes rDNA stability and downregulates RNA polymerase I to ensure rDNA segregation. Nature Communications, 6:6643.
Thapa K.S., Oldani A., Pagliuca C., De Wulf P., Hazbun T.R. (2015). The Mps1 kinase modulates the recruitment and activity of Cnn1CENP-T at Saccharomyces cerevisiae kinetochores. Genetics, 200:79-90.
Bock L.J., Pagliuca C., Kobayashi N., Grove R.A., Oku Y., Shrestha K., Alfieri C., Golfieri C., Oldani A., Dal Maschio M., Bermejo R., Hazbun T.R., Tanaka T.U., De Wulf P. (2012). Cnn1 inhibits the interactions between the KMN complexes of the yeast kinetochore. Nature Cell Biology, 14:614-624.
Nguyen T.L., Cera M.T., Pinto A., Lo Presti L., Hamel E., Conti P., Gussio R., De Wulf P. (2012). Evading Pgp activity in drug-resistant cancer cells: a structural and functional study of antitubulin furan metotica compounds. Molecular Cancer Therapeutics, 11:1103-1111.
Screpanti E., Santaguida S., Nguyen T.L., Silvestri R., Gussio R., Musacchio A., Hamel E., De Wulf P. (2010). A screen for kinetochore-microtubule interaction inhibitors identifies novel antitubulin compounds. PLoS One, 5:e11603.
De Wulf P., Montani F., Visintin R. (2009) Protein phosphatases take the mitotic stage. Current Opinion in Cell Biology, 21:806-815.
Pagliuca C., Draviam V.M., Marco E., Sorger P.K., De Wulf P. (2009). Roles for the conserved Spc105p/Kre28p complex in kinetochore-microtubule binding and the spindle assembly checkpoint. PLoS One, 4:e7640.
Fukagawa T., De Wulf P. (2009). Kinetochore composition, formation and organization. In: "The Kinetochore: from Molecular Discoveries to Cancer Therapy". Eds. De Wulf P., Earnshaw W.C. Springer Publ., New York City, p. 133-191.
Ciferri C., Pasqualato S., Screpanti E., Maiolica A., Polka J., DeLuca J.B., De Wulf P., Salek M., Rappsilber J., Moores C.A., Salmon E.D., Musacchio A. (2008). Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex. Cell, 133:427-439.
Cohen R.L., Espelin C.W., De Wulf P., Sorger P.K., Harrison S.C., Simons K.T. (2008). Structural and functional dissection of Mif2p, a conserved DNA-binding kinetochore protein. Molecular Biology of the Cell, 19:4480-4491.
De Wulf P., Visintin R. (2008). Cdc14B and APC/C tackle DNA damage. Cell, 134:210-212.
Miranda JJ.M., De Wulf P., Sorger P.K., Harrison S.C. (2005). The yeast DASH complex forms closed rings on microtubules. Nature Structural and Molecular Biology, 12:138-143.
De Wulf P., McAinsh A.D., Sorger P.K. (2003). Hierarchical assembly of the budding yeast kinetochore from multiple subcomplexes. Genes and Development, 17:2902-2921.