General concepts: Cells are the universal thread of biological matter, and their division is of outmost importance for organismal development and for the propagation of life across generations. The reductional division of cells, known as meiosis, gives rise to gametes, whose encounter restores the genetic content (ploidy) of organisms. The equational division of cells, known as mitosis, provides the daughter cells with faithful copies of the genome. Both process are accurate and closely regulated. Our laboratory studies the molecular mechanisms of cell division, their regulation, and their deregulation in the most common disease of cell division, cancerous transformation.
The segregation of chromosomes during mitosis and meiosis is among the most exciting examples of the ability of biological matter in cells to create functional structures capable of harnessing energy to carry out complex functions. Crucial to this function is the ability of dynamic polymeric structures, known as microtubules, to form the mitotic spindle by interacting with a wealth of different proteins, including molecular motors that use the microtubules as tracks for the intracellular movement of different cargoes. During mitosis, nanometer-size tubulin and their binding partners self-organize in the mitotic spindle, which spans several microns. The mitotic spindle is devoted to the capture of chromosomes and to their division in subsequent phases of mitosis.
The capture of chromosomes 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. After DNA replication, the chromosomes consist of two identical copies of the same chromosome “glued” together, the sister chromatids. A protein complex known as cohesin exerts the cohesive force that holds 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. This way, loss of cohesion ensuing at the end of metaphase as a result of cohesin cleavage by a protease allows the separated sisters to migrate to opposite spindle poles. This (only apparently) simple trick allows cells to inherit precisely the same number and type of chromosome at every subsequent division.
Kinetochore organization The process, however, is rather complex in essence and still poorly understood. A protein scaffold, known as the kinetochore, plays an essential function in it (Santaguida & Musacchio, 2009). Kinetochores assemble on specialized nucleosomes marked by the histone H3-like variant CENP-A, which are only present at the centromere of each chromosome (Figure 1). Kinetochores form highly elongated structures (spanning at least ~120 nm) that reach out from the chromosome to bind microtubules. Kinetochores may contain a large number of different proteins (>100 in humans), each in multiple copies. A 10-protein assembly known as the KMN network (from the initials of its three subcomplexes, the 2-subunit Knl1 complex, the 4-subunit Mis12 complex, and the 4-subunit Ndc80 complex) provides the core microtubule attachment site. The Ndc80 complex contains a microtubule-binding site that is essential for chromosome segregation. Structural and functional analysis from our group has contributed to develop a rather advanced molecular understanding of the mechanism of microtubule binding by the Ndc80 complex and of its regulation (Ciferri et al. 2005; Ciferri et al. 2008; Petrovic et al. 2010; Alushin et al. 2010; Petrovic et al. 2014; Figures 2 and 3).
More recently, we extended our analysis to the interactions that dock the KMN network to the centromeric chromatin. A 15- or 16-subunit complex known as the constitutive centromere associated network (CCAN) creates a bridge between the centromeric nucleosomes containing CENP-A and the KMN network. Besides reporting a direct interaction between CENP-C and the Mis12 complex (Steensgaard et al. 2004; Screpanti et al. 2011), we have now started to reconstitute large parts of the CCAN by recombinant expression of its subunits. In a very recent highlight, we have been able to characterize the role of the pseudo GTPase, CENP-M, in kinetochore assembly (Basilico et al. submitted).
Feedback control of mitosis A striking aspect of kinetochores is their ability to coordinate microtubule attachment with the output of feedback mechanisms controlling cell cycle progression, and the accuracy of kinetochore-microtubule attachment (Musacchio & Salmon 2007). These feedback mechanisms are respectively known as spindle assembly checkpoint (SAC) and error correction. Their exact relationship remains one of the most controversial topics in the biology of mitosis (Nezi & Musacchio 2009). The importance of error correction and the spindle checkpoint is easily explained. Their ablation, which can be achieved with different methods in different organisms, leads to chromosome segregation errors, causing cells to inherit an incorrect number of chromosomes. This condition, known as aneuploidy, is hardly tolerated by normal cells, but is instead typical of cancer cells.
The SAC senses some aspect of the occupancy of microtubules on kinetochores (known as “attachment”) or lack thereof. Its effector, a protein complex named MCC (for Mitotic Checkpoint Complex), binds and inhibits an enzyme (known as the APC/C) whose activity is required for mitotic exit (Primorac & Musacchio, 2013). When the APC/C is inhibited, cells remain arrested in mitosis and cannot proceed into anaphase. Thus, checkpoint activity results in additional time for attachment. Indeed, the last unattached kinetochore in a cell emits a signal that is sufficient to halt anaphase. As shown by Rieder and colleagues as early as 1995, laser ablation of the last unattached kinetochore drives the cell into a 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. Furthermore, the experiment strongly suggests that the “wait anaphase” signal generated at that kinetochore is diffusible, i.e. it can have a global effect on anaphase progression.
At least 10-12 different proteins have been implicated in checkpoint signaling, including the products of the mitotic arrest deficient (MAD) and budding uninhibited by benomyl (BUB) genes (Figure 4). In our studies, we focused on the molecular interactions causing the recruitment of SAC proteins to kinetochores. For instance, we initially concentrated on the role of Mad1 as a Mad2 receptor at kinetochores, on the role of Mad2 dimerization on its activation during checkpoint signaling, and on the role of p31comet as a negative regulator of Mad2 function (Sironi et al. 2002; De Antoni et al. 2005; Vink et al. 2006; Mapelli et al. 2006; Nezi et al. 2006; Mapelli et al. 2007; Simonetta et al. 2009; Varetti et al. 2011). The interactions we have discovered brought to light the existence of a scheme for checkpoint protein activation, known as the Mad2-template model, which is discussed in Figure 4 in more detail.
More recently, we have extensively studied the mechanism of recruitment of Bub1 to kinetochores, the role of Bub3 in this process, and the function of the RZZ complex (Civril et al. 2010; Krenn et al. 2012; Primorac et al. 2013; Krenn et al. 2014). We believe that the study of the protein interactions that regulate the recruitment and residency of checkpoint proteins at kinetochores has the potential to reveal the detailed molecular requirements of this process.
In addition, we spent considerable effort in characterizing small-molecule modulators of the checkpoint response and their interaction with protein kinases. We initially identified the small-molecule Reversine as an inhibitor of spindle checkpoint signaling (Santaguida et al. 2010) that targets the Mps1 kinase. Subsequently, we concentrated on the more enigmatic role of the Aurora B kinase in this process (Santaguida et al. 2011; De Antoni et al. 2012). We characterized the structure of certain mitotic kinases and their mechanism of binding to defined inhibitors (Sessa et al. 2005; Villa et al., 2009)
Error correction is believed to monitor spindle forces at kinetochores. It is believed that incorrect kinetochore-microtubule attachments fail to generate tension, activating a pathway of correction that eventually severs or removes incorrectly-bound microtubules, allowing correction and promoting new, hopefully correct, attachments, as well as checkpoint signaling. Forces acting on mitotic chromosomes can be visualized as an increase in the distance between the sister kinetochores in bi-oriented chromosomes relative to unattached or mono-oriented chromosomes. Recent data, however, indicate that kinetochores themselves can “stretch” internally upon binding to microtubules, and that the degree of intra-kinetochore stretching correlates with error correction and checkpoint status. These observation suggest the possibility that kinetochores act as sensors of tension, regulating the spindle checkpoint as well as the error correction pathway accordingly.
Future plans Our work on kinetochore reconstitution is reaching a critical point at which we can handle recombinant versions of most kinetochore and checkpoint proteins. On the one hand, we would like to use this material to perform a detailed structural characterization of kinetochores and of their complex functions. Furthermore, we want to approach “synthetically” the problem of mitotic cell division by in vitro reconstitution of fundamental processes, such as checkpoint signaling and microtubule binding. We believe that these efforts have the potential to reveal the essence of crucial mechanisms that drive chromosome segregation in all eukaryotic cells. In the future, we envision our reconstituted kinetochores to become included as parts of a “synthetic cell” created in the laboratory and capable of self-propagation in vitro.
In our research, we are using a combination of methods that include, broadly speaking, classical molecular biology, biochemistry, biophysics, structural biology, chemical biology, and cell biology. We carry out structural analysis using protein X-ray crystallography and electron microscopy (EM). We are proficient in a number of approaches of recombinant protein production (e.g. bacteria, insect cells). We apply protein purification procedures to generate recombinant versions of our proteins of interest, and biophysical characterization techniques to assess homogeneity and stability and also to characterize the interactions in which our proteins engage (e.g. isothermal titration calorimetry, microscale thermophoresis, ultracentrifugation). Finally, we use light microscopy to visualize the dynamics of certain processes in cells or for testing the consequence of molecular perturbations, and to monitor reconstituted reactions in vitro through approaches like fluorescence resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP).
Alushin GM, Ramey VH, Pasqualato S, Ball DA, Grigorieff N, Musacchio A & Nogales E. (2010) The Ndc80 kinetochore complex forms oligomeric arrays along microtubules. Nature 467:805-10
Ciferri C, De Luca J, Monzani S, Ferrari Karin, Ristic D, Wyman C, Stark H, Kilmartin J, Salmon ED & Musacchio A (2005) Architecture of the human Hec1/Ndc80 complex, a critical constituent of the outer kinetochore, J Biol Chem 280, 29088-29095
Ciferri C, Pasqualato S, Screpanti E, Varetti G, Santaguida S, Dos Reis G, Maiolica A, Polka J, De Luca JG, De Wulf P, Salek M, Rappsilber J, Moores CA, Salmon ED & Musacchio A (2008) Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex. Cell 133, 427-439
Civril F, Wehenkel A, Giorgi FM, Santaguida S, Di Fonzo A, Grigorean G, Ciccarelli FD & Musacchio A (2010) Structural analysis of the RZZ complex reveals common ancestry with multisubunit vesicle tethering machinery. Structure 18:616-26
De Antoni A, Pearson CG, Cimini D, Canman J, Sala V, Nezi L, Mapelli M, Sironi L, Faretta M, Salmon ED & Musacchio A, (2005) The Mad1/Mad2 Complex as a Template for Mad2 Activation in the Spindle Assembly Checkpoint, Curr. Biol. 15, 214-225
De Antoni A, Maffini S, Knapp S, Musacchio A* & Santaguida S* (2012) A small-molecule inhibitor of Haspin alters the kinetochore functions of Aurora B. J Cell Biol 199:269-84 (* co-corresponding author)
Krenn V, Wehenkel A, Li X, Santaguida S & Musacchio A (2012) Structural analysis reveals features of the spindle checkpoint kinase Bub1-kinetochore subunit Knl1 interaction, J Cell Biol 196:451-67
Krenn V, Overlack K, Primorac I, van Gerwen S & Musacchio A (2014) KI motifs of human Knl1 enhance assembly of comprehensive spindle checkpoint complexes around MELT repeats. Curr Biol 24:29-39. doi: 10.1016/j.cub.2013.11.046.
Mapelli M, Filipp FV, Rancati G, Massimiliano L, Nezi L, Stier G, Hagan RS, Confalonieri S, Piatti S, Sattler M & Musacchio A (2006) Determinants of conformational dimerization of Mad2 and its inhibition by p31(comet) EMBO J 25, 1273-8
Mapelli M, Massimiliano L, Santaguida S & Musacchio A (2007) The Mad2 Conformational Dimer: Structure and Implications for the Spindle Assembly Checkpoint. Cell 131, 730-43
Musacchio A & Salmon ED (2007) The spindle assembly checkpoint in space and time. Nature Rev Mol Cell Biol 8, 379-93
Nezi L, Rancati G, De Antoni A, Pasqualato S, Piatti S & Musacchio A (2006) Accumulation of Mad2-Cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of Mad2 in Saccharomyces cerevisiae. J Cell Biol 174, 39-51
Nezi L & Musacchio A (2009) Sister chromatid tension and the spindle assembly checkpoint. Curr Opin Cell Biol 21(6):785-95
Petrovic A, Mosalaganti S, Keller J, Mattiuzzo M, Overlack K, Krenn V, De Antoni A, Wohlgemuth S, Cecatiello V, Pasqualato S, Raunser S & Musacchio A (2014) Modular Assembly of RWD Domains on the Mis12 Complex Underlies Outer Kinetochore Organization. Mol Cell 53:591-605. doi: 10.1016/j.molcel.2014.01.019.
Petrovic A, Pasqualato S, Dube P, Krenn V, Santaguida S, Cittaro D, Monzani S, Massimiliano L, Keller J, Tarricone A, Maiolica A, Stark H & Musacchio A (2010) The MIS12 complex is a protein interaction hub for outer kinetochore assembly. J Cell Biol 190:835-52
Primorac I, Weir JR, Chiroli E, Gross F, Hoffmann I, van Gerwen S, Ciliberto A & Musacchio A (2013) Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling, Elife 2:e01030. doi: 10.7554/eLife.01030.
Primorac I & Musacchio A (2013) Panta rhei: The APC/C at steady state. J Cell Biol. 201:177-89. doi: 10.1083/jcb.201301130.
Santaguida S & Musacchio A (2009) The life and miracles of kinetochores. EMBO J 28:2511-31
Santaguida S, Tighe A, D'Alise AM, Taylor SS & Musacchio A (2010) Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine. J Cell Biol 190:73-87
Santaguida S, Vernieri C, Villa F, Ciliberto A & Musacchio A (2011) Evidence that Aurora B is implicated in spindle checkpoint signalling independently of error correction. EMBO J 30:1508-19
Screpanti E, De Antoni A, Alushin GM, Petrovic A, Melis T, Nogales E & Musacchio A (2011) Direct binding of Cenp-C to the Mis12 complex joins the inner and outer kinetochore. Curr Biol 21:391-8
Sessa F, Mapelli M, Ciferri C, Tarricone C, Areces LB, Schneider TR, Stukenberg PT & Musacchio A (2005) Mechanism of Aurora-B activation by INCENP and inhibition by Hesperadin, Mol Cell 18, 379-391
Simonetta M, Manzoni R, Mosca R, Mapelli M, Massimiliano L, Vink M, Novak B, Musacchio A* & Ciliberto A* (2009) The influence of catalysis on Mad2 activation dynamics. PLoS Biology 7, e10 (*corresponding authors)
Sironi L, Mapelli M, Knapp S, De Antoni A, Jeang K-T & Musacchio A (2002) Structure of the tetrameric Mad1-Mad2 core complex: implications of a ‘safety belt’ binding mechanism for the spindle checkpoint, EMBO J, 21, 2496-2506
Steensgaard P, Garrè M, Muradore I, Transidico P, Nigg E, Kitagawa K, Earnshaw WC, Faretta M & Musacchio A, (2004) Sgt1 is required for human kinetochore assembly, EMBO Rep 5, 626-631
Varetti G, Guida G, Santaguida S, Chiroli E & Musacchio A (2011) Homeostatic control of mitotic arrest. Mol. Cell 44: 710-720
Villa F, Capasso P, Tortorici M, Forneris M, de Marco A, Mattevi A & Musacchio A (2009) Crystal structure of the catalytic domain of Haspin, an atypical kinase implicated in chromatin organization. PNAS 106:20204-9
Vink M, Simonetta M, Transidico P, Ferrari K, Mapelli M, De Antoni A, Massimiliano L, Ciliberto A, Faretta M, Salmon ED & Musacchio A (2006) In vitro FRAP identifies the minimal requirements for Mad2 kinetochore dynamics. Curr Biol 16, 755-66