Insights from the reconstitution of the divergent outer kinetochore of Drosophila melanogaster

Accurate chromosome segregation during mitosis and meiosis is crucial for cellular and organismal viability. Kinetochores connect chromosomes with spindle microtubules and are essential for chromosome segregation. These large protein scaffolds emerge from the centromere, a specialized region of the chromosome enriched with the histone H3 variant CENP-A. In most eukaryotes, the kinetochore core consists of the centromere-proximal constitutive centromere-associated network (CCAN), which binds CENP-A and contains 16 subunits, and of the centromere-distal Knl1 complex, Mis12 complex, Ndc80 complex (KMN) network, which binds microtubules and contains 10 subunits. In the fruitfly, Drosophila melanogaster, the kinetochore underwent remarkable simplifications. All CCAN subunits, with the exception of centromeric protein C (CENP-C), and two KMN subunits, Dsn1 and Zwint, cannot be identified in this organism. In addition, two paralogues of the KMN subunit Nnf1 (Nnf1a and Nnf1b) are present. Finally, the Spc105R subunit, homologous to human Knl1/CASC5, underwent considerable sequence changes in comparison with other organisms. We combined biochemical reconstitution with biophysical and structural methods to investigate how these changes reflect on the organization of the Drosophila KMN network. We demonstrate that the Nnf1a and Nnf1b paralogues are subunits of distinct complexes, both of which interact directly with Spc105R and with CENP-C, for the latter of which we identify a binding site on the Mis12 subunit. Our studies shed light on the structural and functional organization of a highly divergent kinetochore particle

Reconstitution of a 26-Subunit human kinetochore reveals cooperative microtubule binding by CENP-OPQUR and NDC80

The approximately thirty core subunits of kinetochores assemble on centromeric chromatin containing the histone H3 variant CENP-A and connect chromosomes with spindle microtubules. The chromatin proximal 16-subunit CCAN (constitutive centromere associated network) creates a mechanically stable bridge between CENP-A and the kinetochore's microtubule-binding machinery, the 10-subunit KMN assembly. Here, we reconstituted a stoichiometric 11-subunit human CCAN core that forms when the CENP-OPQUR complex binds to a joint interface on the CENP-HIKM and CENP-LN complexes. The resulting CCAN particle is globular and connects KMN and CENP-A in a 26-subunit recombinant particle. The disordered, basic N-terminal tail of CENP-Q binds microtubules and promotes accurate chromosome alignment, cooperating with KMN in microtubule binding. The N-terminal basic tail of the NDC80 complex, the microtubule-binding subunit of KMN, can functionally replace the CENP-Q tail. Our work dissects the connectivity and architecture of CCAN and reveals unexpected functional similarities between CENP-OPQUR and the NDC80 complex. [Abstract copyright: Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Mammalian oocytes store proteins for the early embryo on cytoplasmic lattices

Mammalian oocytes are filled with poorly understood structures called cytoplasmic lattices. First discovered in the 1960s and speculated to correspond to mammalian yolk, ribosomal arrays, or intermediate filaments, their function has remained enigmatic to date. Here, we show that cytoplasmic lattices are sites where oocytes store essential proteins for early embryonic development. Using super-resolution light microscopy and cryoelectron tomography, we show that cytoplasmic lattices are composed of filaments with a high surface area, which contain PADI6 and subcortical maternal complex proteins. The lattices associate with many proteins critical for embryonic development, including proteins that control epigenetic reprogramming of the preimplantation embryo. Loss of cytoplasmic lattices by knocking out PADI6 or the subcortical maternal complex prevents the accumulation of these proteins and results in early embryonic arrest. Our work suggests that cytoplasmic lattices enrich maternally provided proteins to prevent their premature degradation and cellular activity, thereby enabling early mammalian development

The MIS12 complex is a protein interaction hub for outer kinetochore assembly

The NSL1 subunit structures interactions between the MIS12, NDC80, and KNL1 kinetochore complexes (see also a related paper by Maskell et al. in this issue)

Investigation of higher-order self-assembly of the DNA binding protein H-NS

H-NS, a DNA binding protein widely distributed in Gram-negative bacteria, acts as a global transcriptional regulator for the expression of a large number of genes. The 136 residue protein has a modular organisation with two structurally independent domains responsible for DNA binding and protein-protein interactions, respectively. Self-association of the H-NS polypeptide is intimately linked to the formation of a specific nucleoprotein structure capable of inhibiting transcription. Attempts at deciphering the structure/function relationship of the H-NS polypeptide have highlighted the convoluted relationship between the various regions in the primary sequence. A combination of complementary techniques was used to investigate self-assembly of the Salmonella typhimurium H-NS polypeptide. The results thus obtained suggest a possible model for the H-NS oligomerisation and highlight the importance of secondary structure in mediating a higher-order structure formation. The assembly of H-NS oligomers occurs through the association of the dimeric intermediate via two protein interfaces. The initial dimer formation occurs through the coiled-coil motif residing in the amino terminal domain (residues 1-49). The dimers thus formed associate in a head-to-tail fashion through the region encompassing residues 64-89 and 1-20. Mutagenesis studies underline the importance of the short amino-terminal helix H2 in supporting the higher-order oligomer formation. Hydrodynamic and modelling studies of the H-NS homologue, StpA, are also presented

Molecular requirements for the inter-subunit interaction and kinetochore recruitment of SKAP and Astrin

Accurate chromosome segregation during cell division is crucial for propagating life and protects from cellular transformation. The SKAP: Astrin heterodimer localizes to spindle microtubules and to mature microtubule-kinetochore attachments during mitosis. Depletion of either subunit disrupts spindle structure and destabilizes kinetochore-microtubule attachments. Here, we identify molecular requirements for the inter-subunit interaction of SKAP and Astrin, and discuss requirements for their kinetochore recruitment. We also identify and characterize a microtubule-binding domain in SKAP, distinct from the SXIP motif that mediates end binding (EB) protein binding and plus end tracking, and show that it stimulates the growth-rate of microtubules, possibly through a direct interaction with tubulin. Mutations targeting this microtubule-binding domain impair microtubule plus-end tracking but not kinetochore targeting, and recapitulate many effects observed during depletion of SKAP. Collectively, our studies represent the first thorough mechanistic analysis of SKAP and Astrin, and significantly advance our functional understanding of these important mitotic proteins

Insights from biochemical reconstitution into the architecture of human kinetochores

Chromosomes are carriers of genetic material and their accurate transfer from a mother cell to its two daughters during cell division is of paramount importance for life. Kinetochores are crucial for this process, as they connect chromosomes with microtubules in the mitotic spindle(1). Kinetochores are multi-subunit complexes that assemble on specialized chromatin domains, the centromeres, that are able to enrich nucleosomes containing the histone H3 variant centromeric protein A (CENP-A)(2). A group of several additional CENPs, collectively known as constitutive centromere associated network (CCAN)(3-6), establish the inner kinetochore, whereas a ten-subunit assembly known as the KMN network creates a microtubule-binding site in the outer kinetochore(7,8). Interactions between CENP-A and two CCAN subunits, CENP-C and CENP-N, have been previously described(9-11), but a comprehensive understanding of CCAN organization and of how it contributes to the selective recognition of CENP-A has been missing. Here we use biochemical reconstitution to unveil fundamental principles of kinetochore organization and function. We show that cooperative interactions of a seven-subunit CCAN subcomplex, the CHIKMLN complex, determine binding selectivity for CENP-A over H3-nucleosomes. The CENP-A: CHIKMLN complex binds directly to the KMN network, resulting in a 21-subunit complex that forms a minimal high-affinity linkage between CENP-A nucleosomes and microtubules in vitro. This structural module is related to fungal point kinetochores, which bind a single microtubule. Its convolution with multiple CENP-A proteins may give rise to the regional kinetochores of higher eukaryotes, which bind multiple microtubules. Biochemical reconstitution paves the way for mechanistic and quantitative analyses of kinetochores

Data Collection and Refinement Statistics.

<p>Data for the highest-resolution shell are given in parentheses.</p><p><i>R</i>_<i>merge</i> = Σ_<i>h</i>Σ_<i>i</i>|<i>I</i>_{<i>h</i>,<i>i</i>} − ⟨<i>I</i>_<i>h</i>⟩|/Σ_<i>h</i>Σ_<i>i</i><i>I</i>_{<i>h</i>,<i>i</i>}, where the outer sum (h) is over the unique reflections and the inner sum (i) is over the set of independent observations of each unique reflection.</p><p><i>R</i>_<i>work</i> = ∑_<i>hkl</i>||<i>F</i>_<i>obs</i>| − |<i>F</i>_<i>calc</i>||/∑<i>F</i>_<i>obs</i>, where F_obs and F_calc are the observed and calculated structure factors of the respective reflections hkl.</p><p>R_free is equivalent to R_work but is calculated on a random set of reflections corresponding to 5% of all reflections and that are excluded from refinement.</p><p>*As defined by MolProbity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144673#pone.0144673.ref052" target="_blank">52</a>]</p><p>Data Collection and Refinement Statistics.</p