Molecular basis of chromosome segregation
c/o IFOM-IEO Campus
Via Adamello, 16 - 20139 Milan, Italy
Tel. +39 02 57489829 - +39 02 57489871
Fax +39 0294375990
Our research interests focus on a stage of the eukaryotic cell cycle known as mitosis. It is during mitosis that the replicated chromosomes of the mother cell divide equally to create two daughter cells with a perfectly defined copy of the genetic material. Mitosis is a phenomenally complex process. Polymeric structures known as microtubules play a central role in mitosis. Microtubules are dynamic tubulin polymers that interact with a wealth of different proteins, including molecular motors that use the microtubules as tracks for the intracellular movement of different cargoes. During mitosis, the microtubules change their organization, and assemble an extraordinary structure known as the mitotic spindle. The mitotic spindle is devoted to the capture of chromosomes and to their division in subsequent phases of mitosis. The capture process starts in prometaphase, and continues until all chromosomes align in the middle of the spindle, the metaphase plate. This marks a phase known as metaphase, which is followed by the process of chromosome segregation to opposite spindle poles, known as anaphase.
fig.1: Key steps in the regulation of mitotic progression [ more info]
After DNA replication, the chromosomes consist of two identical copies of the same chromosome glued together (Figure 1). These glued chromosomes are known as sister chromatids. A protein complex known as Cohesin exerts the cohesive force holding the sister chromatids together. The sister chromatids in each pair link to microtubules originating from opposite poles of the mitotic spindle, a condition known as biorientation. In this manner, loss of cohesion ensuing at the end of metaphase as a result of the cleavage of cohesin by a protease named Separase will allow the separated sisters to migrate to opposite spindle poles under the forces exerted by several microtubule motors and by the dynamic nature of microtubules.
fig.2: Kinetochores and checkpoint signalling [ more info]
This only apparently simple trick allows cells to inherit precisely the same number and type of chromosome. The process, however, is rather complex in essence and still poorly understood. Despite steady progress, it remains largely unclear how microtubules capture the sister chromatids and form a stable link with them. Complex protein scaffolds, known as kinetochores, are essential to regulate this phenomenon (Figure 2). Kinetochores assemble on specialized heterochromatin in the centromere region of the chromosome that contains a nucleosomal core marked by the histone H3-like variant CENP-A. Kinetochores contain a large (>60) number of proteins, which assemble into a solid structure bridging the chromosomes and the microtubules. Although several microtubule-binding proteins reside at the kinetochore during mitosis, none of them seems to be strictly required for microtubule-chromosome attachment, so that a defined microtubule receptor at the kinetochore has not been identified yet.
fig.3: Checkpoints [ more info]
During mitosis, kinetochores also host a safety device known as the spindle assembly checkpoint (Figure 3). This device is designed to sense the occupancy of microtubules on kinetochores (known as attachment) and the forces opposing cohesion that result from bi-orientation. Tension between sister chromatids can be visualized as an increase in the distance between the sister kinetochores in bi-oriented chromosomes relative to unattached or mono-oriented chromosomes (Figure 2). It is believed that tension allows the spindle checkpoint to recognize bipolar orientation from incorrect configurations, such as syntelic attachment, that would result in improper segregation of the sister chromatids, a disastrous event that most often causes cell death, but that may also be important for the development of malignancies by creating unbalances in chromosome number and gene dosage. Before all sister chromatid pairs have achieved bi-orientation on the mitotic spindle, the spindle assembly checkpoint generates a 'wait anaphase' signal that inhibits anaphase, allowing more time for attachment or for the correction of attachment errors (Figure 1). It was proven by Conly Rieder and colleagues in 1995 that the last unattached kinetochore emits a signal that halts anaphase throughout the spindle to which that kinetochore belongs, and that its laser ablation drives the cell into precocious anaphase. This experiment shows that the 'wait anaphase' signal originates at kinetochore, consistently with the observation that all the spindle checkpoint proteins are recruited to kinetochores during mitosis (Musacchio and Hardwick, 2002).
Our laboratory is interested in understanding the inner workings of the spindle assembly checkpoint and its interaction with kinetochores. We use a combination of methods ranging from structural analysis using protein X-ray crystallography, to biochemistry and cell biology to gain an understanding of the molecular bases of the recruitment of the spindle checkpoint components to the kinetochores, of their role in the generation of a signal that halts anaphase. We have been interested both in the 'attachment sensing' and in the 'tension sensing' branch of the spindle assembly checkpoint. The 'attachment sensing' branch of the checkpoint requires the function of proteins known as Mad1 and Mad2. An important project in our laboratory deals with the demonstration of a model that we have named the "Mad2 template" model (Sironi et al., 2001; Sironi et al., 2002; De Antoni et al., 2005). We are also actively involved in the structural characterization of proteins involved in the control of mitosis, such as the Aurora B kinase, of which we have reported the crystal structure (Sessa et al., 2005). Furthermore, we are actively involved in the reconstitution and characterization of kinetochore proteins and complexes, including the Ndc80 complex and Lis1 (Ciferri et al., 2005; Tarricone et al., 2004).