Contact

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Prof. Dr. Andrea Musacchio

Phone:+49 (231) 133-2100Fax:+49 (231) 133-2199

Secretary

Antje Peukert

Phone:+49 (231) 133 - 2101Fax:+49 (231) 133 - 2199

Mechanistic Cell Biology

Research Interest

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.

<strong>Kinetochore organization.</strong> Kinetochores are assembled on dense centromeric chromatin containing the histone H3 variant CENP-A. Directly adjacent to the centromeric chromatin is the CCAN (constitutive centromere associated network), which consists of 15 or 16 subunits (pink). A direct contact of CENP-C with the Mis12 complex localizes the subunits of the KMN network (yellow) to kinetochores. The KMN binds microtubules and it also recruits and regulates the component of the spindle assembly checkpoint. Zoom Image
Kinetochore organization. Kinetochores are assembled on dense centromeric chromatin containing the histone H3 variant CENP-A. Directly adjacent to the centromeric chromatin is the CCAN (constitutive centromere associated network), which consists of 15 or 16 subunits (pink). A direct contact of CENP-C with the Mis12 complex localizes the subunits of the KMN network (yellow) to kinetochores. The KMN binds microtubules and it also recruits and regulates the component of the spindle assembly checkpoint. [less]

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.

<strong>Biochemical reconstitution.</strong> The KMN network can be reconstituted with recombinant components. In this example, the 4-subunit Mis12 complex (Petrovic et al. 2010) was combined with the so-called Ndc80bonsai complex (Ciferri et al. 2008) and with the C-terminal region of Knl1 (Petrovic et al. 2014) to obtain a 7-subunit complex that was subsequently studied by electron microscopy (in collaboration with Shyamal Mosalaganti and Stefan Raunser at MPI Dortmund). Zoom Image
Biochemical reconstitution. The KMN network can be reconstituted with recombinant components. In this example, the 4-subunit Mis12 complex (Petrovic et al. 2010) was combined with the so-called Ndc80bonsai complex (Ciferri et al. 2008) and with the C-terminal region of Knl1 (Petrovic et al. 2014) to obtain a 7-subunit complex that was subsequently studied by electron microscopy (in collaboration with Shyamal Mosalaganti and Stefan Raunser at MPI Dortmund). [less]

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).

<strong>Structural analysis.</strong> The combination of high-resolution analysis of smaller structural units by X-ray crystallography and of low resolution analysis of larger assemblies allows building more detailed models of kinetochore proteins. In this case, the structures of Ndc80bonsai complex (Ciferri et al. 2008) and of the C-terminal region of Knl1 (Petrovic et al. 2014) was fitted into a density obtained by electron microscopy on the sample shown in Figure 2 (in collaboration with Shyamal Mosalaganti and Stefan Raunser at MPI Dortmund). Zoom Image
Structural analysis. The combination of high-resolution analysis of smaller structural units by X-ray crystallography and of low resolution analysis of larger assemblies allows building more detailed models of kinetochore proteins. In this case, the structures of Ndc80bonsai complex (Ciferri et al. 2008) and of the C-terminal region of Knl1 (Petrovic et al. 2014) was fitted into a density obtained by electron microscopy on the sample shown in Figure 2 (in collaboration with Shyamal Mosalaganti and Stefan Raunser at MPI Dortmund). [less]

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.

<strong>Schematic of mitosis and feedback control by the SAC.</strong> The SAC is active in prometaphase and turns off when chromosomes are bi-oriented (metaphase). Kinetochores are shown as green or red dots, depending on whether or not they are bound to microtubules, respectively. The inset shows that SAC proteins are recruited to &ldquo;red&rdquo; kinetochores. There, they assemble the MCC, which binds and inhibits the APC/C. The APC/C is required for the metaphase to anaphase transition. SAC inhibition of APC/C keeps cells in mitosis. B) Mad1/C-Mad2 forms a 2:2 tetramer. The Mad2 template model posits that Mad1/C-Mad2 at kinetochores acts as a receptor for O-Mad2. In turn, the interaction facilitates the transformation of O-Mad2 into C-Mad2 bound to Cdc20. The &ldquo;conformational dimerization&rdquo; of O-Mad2 with C-Mad2 is required for the rapid transformation of O-Mad2 into C-Mad2 bound to Cdc20. There is no input of energy in this reaction, except for the free energy of binding of Mad2 to Cdc20. C) A more detailed depiction of the Mad1/C-Mad2 complex. The N-terminal region of Mad1 forms an extended coiled-coil [CC; see Sironi et al. (2002) EMBO J 21, 2496-2506]. Mad2 binds a Mad2-binding motif of Mad1 (in green) positioned after the N-terminal coiled-coil with the help of its &ldquo;seatbelt&rdquo; (in red). Two additional anti-parallel helices position the CTD of Mad1 near the Mad2-binding region. Note that the RLK motif neighbors the CTD. The SAC kinase Mps1 phosphorylates the kinetochore protein Knl1 to promote recruitment of Bub1-Bub3. In addition, Mps1 phosphorylates Bub1 to elicit an interaction with Mad1/C-Mad2. We propose that the complex of Mad1/C-Mad2 and Bub1/Bub3 represents the catalytic core required for rapid MCC formation. The substrates of this catalytic platform are O-Mad2, BubR1/Bub3, and Cdc20. Note that Cdc20 contains a Mad2-binding motif related to that present in Mad1. BubR1/Bub3 is evolutionarily and structurally related to Bub1/Bub3. Thus, the MCC (Cdc20/C-Mad2/BubR1/Bub3) may represent a copy of a template consisting of Mad1/C-Mad2/Bub1/Bub3. The coloring scheme emphasizes the copy to template relationship of the MCC product with the catalytic platform. Zoom Image
Schematic of mitosis and feedback control by the SAC. The SAC is active in prometaphase and turns off when chromosomes are bi-oriented (metaphase). Kinetochores are shown as green or red dots, depending on whether or not they are bound to microtubules, respectively. The inset shows that SAC proteins are recruited to “red” kinetochores. There, they assemble the MCC, which binds and inhibits the APC/C. The APC/C is required for the metaphase to anaphase transition. SAC inhibition of APC/C keeps cells in mitosis. B) Mad1/C-Mad2 forms a 2:2 tetramer. The Mad2 template model posits that Mad1/C-Mad2 at kinetochores acts as a receptor for O-Mad2. In turn, the interaction facilitates the transformation of O-Mad2 into C-Mad2 bound to Cdc20. The “conformational dimerization” of O-Mad2 with C-Mad2 is required for the rapid transformation of O-Mad2 into C-Mad2 bound to Cdc20. There is no input of energy in this reaction, except for the free energy of binding of Mad2 to Cdc20. C) A more detailed depiction of the Mad1/C-Mad2 complex. The N-terminal region of Mad1 forms an extended coiled-coil [CC; see Sironi et al. (2002) EMBO J 21, 2496-2506]. Mad2 binds a Mad2-binding motif of Mad1 (in green) positioned after the N-terminal coiled-coil with the help of its “seatbelt” (in red). Two additional anti-parallel helices position the CTD of Mad1 near the Mad2-binding region. Note that the RLK motif neighbors the CTD. The SAC kinase Mps1 phosphorylates the kinetochore protein Knl1 to promote recruitment of Bub1-Bub3. In addition, Mps1 phosphorylates Bub1 to elicit an interaction with Mad1/C-Mad2. We propose that the complex of Mad1/C-Mad2 and Bub1/Bub3 represents the catalytic core required for rapid MCC formation. The substrates of this catalytic platform are O-Mad2, BubR1/Bub3, and Cdc20. Note that Cdc20 contains a Mad2-binding motif related to that present in Mad1. BubR1/Bub3 is evolutionarily and structurally related to Bub1/Bub3. Thus, the MCC (Cdc20/C-Mad2/BubR1/Bub3) may represent a copy of a template consisting of Mad1/C-Mad2/Bub1/Bub3. The coloring scheme emphasizes the copy to template relationship of the MCC product with the catalytic platform. [less]

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.

Techniques

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).

Selected Reading

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

 
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