Andrea Musacchio

The spindle-assembly checkpoint in space and time

In eukaryotes, the spindle-assembly checkpoint (SAC) is a ubiquitous safety device that ensures the fidelity of chromosome segregation in mitosis. The SAC prevents chromosome mis-segregation and aneuploidy, and its dysfunction is implicated in tumorigenesis. Recent molecular analyses have begun to shed light on the complex interaction of the checkpoint proteins with kinetochores — structures that mediate the binding of spindle microtubules to chromosomes in mitosis. These studies are finally starting to reveal the mechanisms of checkpoint activation and silencing during mitotic progression.

Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector

Practically any oligopeptide can be exposed on the surface of the bacteriophage capsid by fusion to the major coat protein of filamentous bacteriophages. A phage expressing a particular peptide tag can be selected from a mixture of tens of millions of clones, exposing oligopeptides of random sequence, by affinity purification with a protein ligand. In this respect, pVIII can be used as an alternative and complement to the exposition vectors based on the product of gene III (pIII). We have constructed a phagemid vector that contains gene VIII under the control of the pLac promoter. This vector can be conveniently used to construct libraries of oligopeptides with a random amino acid sequence. An antipeptide monoclonal antibody was used to affinity-purify phagemids exposing oligopeptides which can interact with the monoclonal antibody. DNA sequencing of the amino terminus of gene VIII of the recovered clones predicts the synthesis of hybrid proteins whose aminoterminal amino acid sequence is related to that of the oligopeptide used to raise the antibody. In other words, only oligopeptides that bind a very small portion of the immunoglobulin G surface are affinity-purified by this method, implying that the antigen binding site possesses molecular properties that renders it much stickier than the remainder of the molecule.

The spindle checkpoint: structural insights into dynamic signalling.

Chromosome segregation is a complex and astonishingly accurate process whose inner working is beginning to be understood at the molecular level. The spindle checkpoint plays a key role in ensuring the fidelity of this process. It monitors the interactions between chromosomes and microtubules, and delays mitotic progression to allow extra time to correct defects. Here, we review and integrate findings on the dynamics of checkpoint proteins at kinetochores with structural information about signalling complexes.

Kinetochore Microtubule Dynamics and Attachment Stability Are Regulated by Hec1

Mitotic cells face the challenging tasks of linking kinetochores to growing and shortening microtubules and actively regulating these dynamic attachments to produce accurate chromosome segregation. We report here that Ndc80/Hec1 functions in regulating kinetochore microtubule plus-end dynamics and attachment stability. Microinjection of an antibody to the N terminus of Hec1 suppresses both microtubule detachment and microtubule plus-end polymerization and depolymerization at kinetochores of PtK1 cells. Centromeres become hyperstretched, kinetochore fibers shorten from spindle poles, kinetochore microtubule attachment errors increase, and chromosomes severely mis-segregate. The N terminus of Hec1 is phosphorylated by Aurora B kinase in vitro, and cells expressing N-terminal nonphosphorylatable mutants of Hec1 exhibit an increase in merotelic attachments, hyperstretching of centromeres, and errors in chromosome segregation. These findings reveal a key role for the Hec1 N terminus in controlling dynamic behavior of kinetochore microtubules.

The life and miracles of kinetochores

Kinetochores are large protein assemblies built on chromosomal loci named centromeres. The main functions of kinetochores can be grouped under four modules. The first module, in the inner kinetochore, contributes a sturdy interface with centromeric chromatin. The second module, the outer kinetochore, contributes a microtubule‐binding interface. The third module, the spindle assembly checkpoint, is a feedback control mechanism that monitors the state of kinetochore–microtubule attachment to control the progression of the cell cycle. The fourth module discerns correct from improper attachments, preventing the stabilization of the latter and allowing the selective stabilization of the former. In this review, we discuss how the molecular organization of the four modules allows a dynamic integration of kinetochore–microtubule attachment with the prevention of chromosome segregation errors and cell‐cycle progression.

Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine

Addition of reversine to dividing cells ejects Mad1 and the RZZ complex from unattached kinetochores and prevents resolution of incorrect chromosome–microtubule attachments (see also related papers by Hewitt et al. and Maciejowski et al. in this issue).

Crystal structure of the tetrameric Mad1–Mad2 core complex: implications of a ‘safety belt’ binding mechanism for the spindle checkpoint

The spindle checkpoint protein Mad1 recruits Mad2 to unattached kinetochores and is essential for Mad2–Cdc20 complex formation in vivo but not in vitro. The crystal structure of the Mad1–Mad2 complex reveals an asymmetric tetramer, with elongated Mad1 monomers parting from a coiled‐coil to form two connected sub‐complexes with Mad2. The Mad2 C‐terminal tails are hinged mobile elements wrapping around the elongated ligands like molecular ‘safety belts’. We show that Mad1 is a competitive inhibitor of the Mad2–Cdc20 complex, and propose that the Mad1–Mad2 complex acts as a regulated gate to control Mad2 release for Cdc20 binding. Mad1–Mad2 is strongly stabilized in the tetramer, but a 1:1 Mad1–Mad2 complex slowly releases Mad2 for Cdc20 binding, driven by favourable binding energies. Thus, the rate of Mad2 binding to Cdc20 during checkpoint activation may be regulated by conformational changes that destabilize the tetrameric Mad1–Mad2 assembly to promote Mad2 release. We also show that unlocking the Mad2 C‐terminal tail is required for ligand release from Mad2, and that the ‘safety belt’ mechanism may prolong the lifetime of Mad2–ligand complexes.

SH3 — an abundant protein domain in search of a function

Src‐homology 3 is a small protein domain of about 60 amino acid residues. It is probably made of β‐sheets. SH3 is present in a large number of eukaryotic proteins which are involved in signal transduction, cell polarization and membrane—cytoskeleton interactions. Here we review its occurrence and discuss possible functions of this domain.

Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1–C-Mad2 core complex

To satisfy the mitotic checkpoint and drive chromosome congression, the Mps1 kinase lets go of kinetochores by phosphorylating itself in trans (see also related papers by Maciejowski et al. and Santaguida et al. in this issue).

Atomic Structure of Clathrin: A β Propeller Terminal Domain Joins an α Zigzag Linker

Clathrin triskelions form the lattice that organizes recruitment of proteins to coated pits and helps drive vesiculation of the lipid bilayer. We report the crystal structure at 2.6 A resolution of a 55 kDa N-terminal fragment from the 190 kDa clathrin heavy chain. The structure comprises the globular “terminal domain” and the linker that joins it to the end of a triskelion leg. The terminal domain is a seven-blade β propeller, a structure well adapted to interaction with multiple partners, such as the AP-1 and AP-2 sorting adaptor complexes and the nonvisual arrestins. The linker is an α-helical zigzag emanating from the propeller domain. We propose that this simple motif may extend into the rest of the clathrin leg.