Activities

During mitosis, kinetochores orchestrate chromosome transmission from the mother into the daughter cells. Kinetochores are protein complexes containing 60 (yeast) to >100 components (humans). At mitotic entry, kinetochores assemble on the centromeres (CEN) of the replicated chromosomes (sister chromatids) to bi-orient them on the mitotic spindle, a dynamic array of centrosomes (spindle poles), microtubules and microtubule-associated proteins. Sister chromatid alignment is supervised by the spindle assembly checkpoint (SAC), which will delay cells in mitosis even if only one sister chromatid pair is not bi-oriented. Following satisfaction of the SAC and separation of all bi-oriented sisters (by dissolution of the cohesion linkages), kinetochores, motor proteins and spindle regression move the chromosomes into the daughter cells, generating offspring with a correct number of chromosomes. Errors made during chromosome segregation e.g., due to a lack or overexpression of a single kinetochore protein, can lead to daughter cells with an abnormal number of chromosomes (aneuploidy). Most often, aneuploidy results in death (e.g., the termination of a developing fetus). However, depending on which chromosome is affected, aneuploid organisms can be viable but will suffer from physical and mental disabilities, as exemplified by the Down syndrome. As most solid tumors are aneuploid it has been hypothesized that chromosome missegregation drives or supports the cancer transformation process. By identifying, functionally dissecting, and structurally analyzing kinetochore proteins and their regulators we can better understand the basis of aneuploidy disease, including cancer, and convert major players into cancer biomarkers and anticancer drug targets.

As chromosome segregation and the proteins involved are evolutionary conserved we study them in the yeast Saccharomyces cerevisiae. Using yeast allows for a quick genetic identification of proteins involved in chromosome segregation and for dissecting functional relationships between the involved proteins. In addition, progression through the yeast cell cycle can easily be manipulated with small compounds or via mutations in regulatory proteins. Importantly, insights obtained with yeast can be extrapolated to human cells.

Besides kinetochores we also study proteins regulating rDNA segregation in Saccharomyces cerevisiae. At anaphase entry, kinetochores initiate the segregation of >99% of the genome.
However, the rDNA locus separates at the end of anaphase. This is because cells must actively transcribe rDNA to generate ribosomes and sustain viability. In anaphase, rDNA becomes repressed allowing for local condensation and segregation before the next cell cycle initiates. Hence, the segregation of the genome is coordinated in time and space.

To study chromosome segregation we implement a multi-dimensional approach. To identify new proteins involved in chromosome segregation we perform protein affinity purifications and yeast two-hybrid screens. Alternatively, we identify new proteins and establish functional relationships with established kinetochore proteins using robotic genetic interaction screens (SGA). Protein localization and recruitment studies are performed by ChIP (Chromatin Immunoprecipiation), ChIP-CHIP (ChIP followed by microarray (CHIP) hybridization) and quantitative live-cell fluorescence imaging.
4D based protein localization, activity and copy number analyses during cell division are performed using time-lapse widefield DeltaVision deconvolution fluorescence microscopy or spinning disk confocal microscopy. Protein turnover in yeast is probed by FRAP (Fluorescence Recovery after Photobleaching). Phenotypic effects of mutations on chromosome segregation and mitosis are studied in synchronous cell cycles by indirect immunofluorescence microscopy, chromosome spreads, study of sister chromatid alignment using integrated pericentromeric GFP arrays, and FACS analysis (to probe the DNA status of the cells). At the molecular level, we study yeast protein activity biochemically in yeast cell extracts or in in vitro assays using recombinant proteins produced in bacteria, yeast or insect cells. Assays include co-IPs, in vitro protein-centromere binding studies using gel retardation analysis, in vitro protein reconstitution and affinity measurements using ITC or chromatography, kinase assays followed by mass spectroscopic identification of phosphorylated residues. Structural protein studies are performed by hydrodynamics (gel filtration plus glycerol gradient density velocity ultracentrifugation) and crystallographic analysis of recombinant proteins following crystallization at our in-house crystallography facility.

By integrating a variety of methodologies we wish to obtain a most detailed understanding of the proteins and regulators that drive chromosome segregation in yeast, and ultimately, in human cells.