Christopher W. Carroll

Protein Architecture of the Human Kinetochore Microtubule Attachment Site

Chromosome segregation requires assembly of kinetochores on centromeric chromatin to mediate interactions with spindle microtubules and control cell-cycle progression. To elucidate the protein architecture of human kinetochores, we developed a two-color fluorescence light microscopy method that measures average label separation, Delta, at <5 nm accuracy. Delta analysis of 16 proteins representing core structural complexes spanning the centromeric chromatin-microtubule interface, when correlated with mechanical states of spindle-attached kinetochores, provided a nanometer-scale map of protein position and mechanical properties of protein linkages. Treatment with taxol, which suppresses microtubule dynamics and activates the spindle checkpoint, revealed a specific switch in kinetochore architecture. Cumulatively, Delta analysis revealed that compliant linkages are restricted to the proximity of chromatin, suggested a model for how the KMN (KNL1/Mis12 complex/Ndc80 complex) network provides microtubule attachment and generates pulling forces from depolymerization, and identified an intrakinetochore molecular switch that may function in controlling checkpoint activity.

Dual recognition of CENP-A nucleosomes is required for centromere assembly

CENP-C and CENP-N recognize distinct structural elements of CENP-A nucleosomes, providing a foundation for the assembly of other centromere and kinetochore components.

Centromere assembly requires the direct recognition of CENP-A nucleosomes by CENP-N

Centromeres are specialized chromosomal domains that direct kinetochore assembly during mitosis. CENP-A (centromere protein A), a histone H3-variant present exclusively in centromeric nucleosomes, is thought to function as an epigenetic mark that specifies centromere identity. Here we identify the essential centromere protein CENP-N as the first protein to selectively bind CENP-A nucleosomes but not H3 nucleosomes. CENP-N bound CENP-A nucleosomes in a DNA sequence-independent manner, but did not bind soluble CENP-A–H4 tetramers. Mutations in CENP-N that reduced its affinity for CENP-A nucleosomes caused defects in CENP-N localization and had dominant effects on the recruitment of CENP-H, CENP-I and CENP-K to centromeres. Depletion of CENP-N using siRNA (short interfering RNA) led to similar centromere assembly defects and resulted in reduced assembly of nascent CENP-A into centromeric chromatin. These data suggest that CENP-N interprets the information encoded within CENP-A nucleosomes and recruits other proteins to centromeric chromatin that are required for centromere function and propagation.

In vitro centromere and kinetochore assembly on defined chromatin templates

During cell division, chromosomes are segregated to nascent daughter cells by attaching to the microtubules of the mitotic spindle through the kinetochore. Kinetochores are assembled on a specialized chromatin domain called the centromere, which is characterized by the replacement of nucleosomal histone H3 with the histone H3 variant centromere protein A (CENP-A). CENP-A is essential for centromere and kinetochore formation in all eukaryotes but it is unknown how CENP-A chromatin directs centromere and kinetochore assembly. Here we generate synthetic CENP-A chromatin that recapitulates essential steps of centromere and kinetochore assembly in vitro. We show that reconstituted CENP-A chromatin when added to cell-free extracts is sufficient for the assembly of centromere and kinetochore proteins, microtubule binding and stabilization, and mitotic checkpoint function. Using chromatin assembled from histone H3/CENP-A chimaeras, we demonstrate that the conserved carboxy terminus of CENP-A is necessary and sufficient for centromere and kinetochore protein recruitment and function but that the CENP-A targeting domain—required for new CENP-A histone assembly—is not. These data show that two of the primary requirements for accurate chromosome segregation, the assembly of the kinetochore and the propagation of CENP-A chromatin, are specified by different elements in the CENP-A histone. Our unique cell-free system enables complete control and manipulation of the chromatin substrate and thus presents a powerful tool to study centromere and kinetochore assembly.

The Doc1 subunit is a processivity factor for the anaphase-promoting complex

Ubiquitin-mediated proteolysis of securin and mitotic cyclins is essential for exit from mitosis. The final step in ubiquitination of these and other proteins is catalysed by the anaphase-promoting complex (APC), a multi-subunit ubiquitin-protein ligase (E3). Little is known about the molecular reaction resulting in APC-dependent substrate ubiquitination or the role of individual APC subunits in the reaction. Using a well-defined in vitro system, we show that highly purified APC from Saccharomyces cerevisiae ubiquitinates a model cyclin substrate in a processive manner. Analysis of mutant APC lacking the Doc1/Apc10 subunit (APCdoc1Δ) indicates that Doc1 is required for processivity. The specific molecular defect in APCdoc1Δ is identified by a large increase in apparent KM for the cyclin substrate relative to the wild-type enzyme. This suggests that Doc1 stimulates processivity by limiting substrate dissociation. Addition of recombinant Doc1 to APCdoc1Δ fully restores enzyme function. Doc1-related domains are found in mechanistically distinct ubiquitin-ligase enzymes and may generally stimulate ubiquitination by contributing to substrate–enzyme affinity.

The APC Subunit Doc1 Promotes Recognition of the Substrate Destruction Box

BACKGROUND Accurate chromosome segregation during mitosis requires the coordinated destruction of the mitotic regulators securin and cyclins. The anaphase-promoting complex (APC) is a multisubunit ubiquitin-protein ligase that catalyzes the polyubiquitination of these and other proteins and thereby promotes their destruction. How the APC recognizes its substrates is not well understood. In mitosis, the APC activator Cdc20 binds to the APC and is thought to recruit substrates by interacting with a conserved target protein motif called the destruction box. A related protein, called Cdh1, performs a similar function during G1. Recent evidence, however, suggests that the core APC subunit Doc1 also contributes to substrate recognition. RESULTS To better understand the mechanism by which Doc1 promotes substrate binding to the APC, we generated a series of point mutations in Doc1 and analyzed their effects on the processivity of substrate ubiquitination. Mutations that reduce Doc1 function fall into two classes that define spatially and functionally distinct regions of the protein. One region, which includes the carboxy terminus, anchors Doc1 to the APC but does not influence substrate recognition. The other region, located on the opposite face of Doc1, is required for Doc1 to enhance substrate binding to the APC. Importantly, stimulation of binding by Doc1 also requires that the substrate contain an intact destruction box. Cells carrying DOC1 mutations that eliminate substrate recognition delay in mitosis with high levels of APC substrates. CONCLUSIONS Doc1 contributes to recognition of the substrate destruction box by the APC. This function of Doc1 is necessary for efficient substrate proteolysis in vivo.

Dynamics of CENP-N kinetochore binding during the cell cycle

Accurate chromosome segregation requires the assembly of kinetochores, multiprotein complexes that assemble on the centromere of each sister chromatid. A key step in this process involves binding of the constitutive centromere-associated network (CCAN) to CENP-A, the histone H3 variant that constitutes centromeric nucleosomes. This network is proposed to operate as a persistent structural scaffold for assembly of the outer kinetochore during mitosis. Here, we show by fluorescence resonance energy transfer (FRET) that the N-terminus of CENP-N lies in close proximity to the N-terminus of CENP-A in vivo, consistent with in vitro data showing direct binding of CENP-N to CENP-A. Furthermore, we demonstrate in living cells that CENP-N is bound to kinetochores during S phase and G2, but is largely absent from kinetochores during mitosis and G1. By measuring the dynamics of kinetochore binding, we reveal that CENP-N undergoes rapid exchange in G1 until the middle of S phase when it becomes stably associated with kinetochores. The majority of CENP-N is loaded during S phase and dissociates again during G2. We propose a model in which CENP-N functions as a fidelity factor during centromeric replication and reveal that the CCAN network is considerably more dynamic than previously appreciated.

Enzymology of the anaphase-promoting complex.

The anaphase-promoting complex (APC) is an ubiquitin-protein ligase that promotes mitotic progression by catalyzing the ubiquitination of numerous proteins, including securin and cyclin. Its complex subunit composition and extensive regulation make the APC an active subject of investigation for both cell biologists and enzymologists. This chapter describes a system for the reconstitution and quantitative analysis of APC activity from budding yeast in vitro. We focus in particular on the measurement of processive ubiquitination, which complements traditional analysis of the reaction rate as a means to elucidate the molecular details of substrate recognition and ubiquitination by the APC.

Loss of optimality in cross correlators

In simple binary detection, cross correlation gives the best possible output signal-to-noise ratio only if the background noise is white. But, because of the ease of implementation, optical cross correlators are often used even in nonwhite-noise situations. General expressions and tight bounds are derived to quantify the loss in signal-to-noise ratio when a cross correlator is used instead of the truly optimal filter. Such a quantification should enable the designer of any optical detection scheme to determine whether the extra effort involved in using the truly optimal filters is worth the signal-to-noise ratio improvements.

Acetylation of histone H4 lysine 5 and 12 is required for CENP-A deposition into centromeres

Centromeres are specified epigenetically through the deposition of the centromere-specific histone H3 variant CENP-A. However, how additional epigenetic features are involved in centromere specification is unknown. Here, we find that histone H4 Lys5 and Lys12 acetylation (H4K5ac and H4K12ac) primarily occur within the pre-nucleosomal CENP-A–H4–HJURP (CENP-A chaperone) complex, before centromere deposition. We show that H4K5ac and H4K12ac are mediated by the RbAp46/48–Hat1 complex and that RbAp48-deficient DT40 cells fail to recruit HJURP to centromeres and do not incorporate new CENP-A at centromeres. However, C-terminally-truncated HJURP, that does not bind CENP-A, does localize to centromeres in RbAp48-deficient cells. Acetylation-dead H4 mutations cause mis-localization of the CENP-A–H4 complex to non-centromeric chromatin. Crucially, CENP-A with acetylation-mimetic H4 was assembled specifically into centromeres even in RbAp48-deficient DT40 cells. We conclude that H4K5ac and H4K12ac, mediated by RbAp46/48, facilitates efficient CENP-A deposition into centromeres.