Towards building a chromosome segregation machine

All organisms, from bacteria to humans, face the daunting task of replicating, packaging and segregating up to two metres (about 6 × 109 base pairs) of DNA when each cell divides. This task is carried out up to a trillion times during the development of a human from a single fertilized cell. The strategy by which DNA is replicated is now well understood. But when it comes to packaging and segregating a genome, the mechanisms are only beginning to be understood and are often as variable as the organisms in which they are studied

A Sensitized Emission Based Calibration of FRET Efficiency for Probing the Architecture of Macromolecular Machines

Macromolecular machines participate in almost every cell biological function. These machines can take the form of well-defined protein structures such as the kinetochore, or more loosely organized protein assemblies like the endocytic coat. The protein architecture of these machines—the arrangement of multiple copies of protein subunits at the nanoscale, is necessary for understanding their cell biological function and biophysical mechanism. Defining this architecture in vivo presents a major challenge. High density of protein molecules within macromolecular machines severely limits the effectiveness of super-resolution microscopy. However, this density is ideal for Forster Resonance Energy Transfer (FRET), which can determine the proximity between neighboring molecules. Here, we present a simple FRET quantitation scheme that calibrates a standard epifluorescence microscope for measuring donor–acceptor separations. This calibration can be used to deduce FRET efficiency fluorescence intensity measurements. This method will allow accurate determination of FRET efficiency over a wide range of values and FRET pair number. It will also allow dynamic FRET measurements with high spatiotemporal resolution under cell biological conditions. Although the poor maturation efficiency of genetically encoded fluorescent proteins presents a challenge, we show that its effects can be alleviated. To demonstrate this methodology, we probe the in vivo architecture of the γ-Tubulin Ring. Our technique can be applied to study the architecture and dynamics of a wide range of macromolecular machines.Electronic supplementary materialThe online version of this article (doi:10.1007/s12195-013-0290-y) contains supplementary material, which is available to authorized users

In Vivo Protein Architecture of the Eukaryotic Kinetochore with Nanometer Scale Accuracy

The kinetochore is a macromolecular protein machine [1] that links centromeric chromatin to the plus-ends of one or more microtubules (MT), and segregates chromosomes during cell division. Its core structure consists of eight multi-component protein complexes, most of which are conserved in all eukaryotes. We use an in vivo two-color fluorescence microscopy technique to determine, for the first time, the location of these proteins along the budding yeast kinetochore axis at nanometer resolution. Together with kinetochore protein counts [2, 3], these localizations predict the 3-D protein architecture of a kinetochore-microtubule attachment, and provide new functional insights. We also find that the kinetochore becomes much shorter in anaphase as metaphase tension is lost. Shortening is mainly due to a decrease in the length of the Ndc80 complex, which may result either from intra-molecular bending of the Ndc80 complex at the kink within the stalk region of the Ndc80/Nuf2 dimer [4, 5] or from a change in its orientation relative to the microtubule axis. Conformational changes within the Ndc80 and Mtw1 complexes may serve as mechanical cues for tension-dependent regulation of MT attachment and the spindle assembly checkpoint. The geometry of the core structure of the budding yeast kinetochore reported here is remarkably similar to that found in mammalian kinetochores, indicating that kinetochore structure is conserved in eukaryotes with either point or regional centromeres

Mechanisms of force generation by end-on kinetochore-microtubule attachments

Generation of motile force is one of the main functions of the eukaryotic kinetochore during cell division. In recent years, the KMN network of proteins (Ndc80 complex, Mis12 complex and KNL-1 complex) has emerged as a highly conserved core microtubule-binding complex at the kinetochore. It plays a major role in coupling force generation to microtubule plus-end polymerization and depolymerization. In this review, we discuss current theoretical mechanisms of force generation, and then focus on emerging information about mechanistic contributions from the Ndc80 complex in eukaryotes, and the microtubule-binding Dam1/DASH complex from fungi. New information has also become available from super-resolution light microscopy on the protein architecture of the kinetochore-microtubule attachment site in both budding yeast and humans, which provides further insight into the mechanism of force generation. We briefly discuss potential contributions of motors, other microtubule-associated proteins, and microtubule depolymerases. Using the above evidence, we present speculative models of force generation at the kinetochore

Design Features of a Mitotic Spindle: Balancing Tension and Compression at a Single Microtubule Kinetochore Interface in Budding Yeast

Accurate segregation of duplicated chromosomes ensures that daughter cells get one and only one copy of each chromosome. Errors in chromosome segregation result in aneuploidy and have severe consequences on human health. Incorrect chromosome number and chromosomal instability are hallmarks of tumor cells. Hence, segregation errors are thought to be a major cause of tumorigenesis. A study of the physical mechanical basis of chromosome segregation is essential to understand the processes that can lead to errors. Tremendous progress has been made in recent years in identifying the proteins necessary for chromosome movement and segregation, but the mechanism and structure of critical force generating components and the molecular basis of centromere stiffness remain poorly understood

Molecular architecture of the kinetochore-microtubule attachment site is conserved between point and regional centromeres

Point and regional centromeres specify a unique site on each chromosome for kinetochore assembly. The point centromere in budding yeast is a unique 150-bp DNA sequence, which supports a kinetochore with only one microtubule attachment. In contrast, regional centromeres are complex in architecture, can be up to 5 Mb in length, and typically support many kinetochore-microtubule attachments. We used quantitative fluorescence microscopy to count the number of core structural kinetochore protein complexes at the regional centromeres in fission yeast and Candida albicans. We find that the number of CENP-A nucleosomes at these centromeres reflects the number of kinetochore-microtubule attachments instead of their length. The numbers of kinetochore protein complexes per microtubule attachment are nearly identical to the numbers in a budding yeast kinetochore. These findings reveal that kinetochores with multiple microtubule attachments are mainly built by repeating a conserved structural subunit that is equivalent to a single microtubule attachment site

Vertebrate kinetochore protein architecture: protein copy number

The stoichiometry of kinetochore components is determined, suggesting conservation between multiple microtubule-binding vertebrate and single microtubule-binding yeast kinetochores.To define the molecular architecture of the kinetochore in vertebrate cells, we measured the copy number of eight kinetochore proteins that link kinetochore microtubules (MTs [kMTs]) to centromeric DNA. We used a fluorescence ratio method and chicken DT40 cell lines in which endogenous loci encoding the analyzed proteins were deleted and complemented using integrated green fluorescent protein fusion transgenes. For a mean of 4.3 kMTs at metaphase, the protein copy number per kMT is between seven and nine for members of the MT-binding KNL-1/Mis12 complex/Ndc80 complex network. It was between six and nine for four members of the constitutive centromere-associated network: centromere protein C (CENP-C), CENP-H, CENP-I, and CENP-T. The similarity in copy number per kMT for all of these proteins suggests that each MT end is linked to DNA by six to nine fibrous unit attachment modules in vertebrate cells, a conclusion that indicates architectural conservation between multiple MT-binding vertebrate and single MT-binding budding yeast kinetochores

Molecular architecture of a kinetochore–microtubule attachment site

Kinetochore attachment to spindle microtubule plus-ends is necessary for accurate chromosome segregation during cell division in all eukaryotes. The centromeric DNA of each chromosome is linked to microtubule plus-ends by eight structural-protein complexes1–9. Knowing the copy number of each of these complexes at one kinetochore–microtubule attachment site is necessary to understand the molecular architecture of the complex, and to elucidate the mechanisms underlying kinetochore function. We have counted, with molecular accuracy, the number of structural protein complexes in a single kinetochore–microtubule attachment using quantitative fluorescence microscopy of GFP-tagged kinetochore proteins in the budding yeast Saccharomyces cerevisiae. We find that relative to the two Cse4p molecules in the centromeric histone1, the copy number ranges from one or two for inner kinetochore proteins such as Mif2p2, to 16 for the DAM–DASH complex8,9 at the kinetochore–microtubule interface. These counts allow us to visualize the overall arrangement of a kinetochore–microtubule attachment. As most of the budding yeast kinetochore proteins have homologues in higher eukaryotes, including humans, this molecular arrangement is likely to be replicated in more complex kinetochores that have multiple microtubule attachments

Adaptor Autoregulation Promotes Coordinated Binding within Clathrin Coats

Membrane traffic is an essential process that allows protein and lipid exchange between the endocytic, lysosomal, and secretory compartments. Clathrin-mediated traffic between the trans-Golgi network and endosomes mediates responses to the environment through the sorting of biosynthetic and endocytic protein cargo. Traffic through this pathway is initiated by the controlled assembly of a clathrin-adaptor protein coat on the cytosolic surface of the originating organelle. In this process, clathrin is recruited by different adaptor proteins that act as a bridge between clathrin and the transmembrane cargo proteins to be transported. Interactions between adaptors and clathrin and between different types of adaptors lead to the formation of a densely packed protein network within the coat. A key unresolved issue is how the highly complex adaptor-clathrin interaction and adaptor-adaptor interaction landscape lead to the correct spatiotemporal assembly of the clathrin coat. Here we report the discovery of a new autoregulatory motif within the clathrin adaptor Gga2 that drives synergistic binding of Gga2 to clathrin and the adaptor Ent5. This autoregulation influences the temporal and/or spatial location of the Gga2-Ent5 interaction. We propose that this synergistic binding provides built-in regulation to ensure the correct assembly of clathrin coats

The microtubule-based motor Kar3 and plus end–binding protein Bim1 provide structural support for the anaphase spindle

In budding yeast, the mitotic spindle is comprised of 32 kinetochore microtubules (kMTs) and ∼8 interpolar MTs (ipMTs). Upon anaphase onset, kMTs shorten to the pole, whereas ipMTs increase in length. Overlapping MTs are responsible for the maintenance of spindle integrity during anaphase. To dissect the requirements for anaphase spindle stability, we introduced a conditionally functional dicentric chromosome into yeast. When centromeres from the same sister chromatid attach to opposite poles, anaphase spindle elongation is delayed and a DNA breakage-fusion-bridge cycle ensues that is dependent on DNA repair proteins. We find that cell survival after dicentric chromosome activation requires the MT-binding proteins Kar3p, Bim1p, and Ase1p. In their absence, anaphase spindles are prone to collapse and buckle in the presence of a dicentric chromosome. Our analysis reveals the importance of Bim1p in maintaining a stable ipMT overlap zone by promoting polymerization of ipMTs during anaphase, whereas Kar3p contributes to spindle stability by cross-linking spindle MTs