An optimized method for 3D fluorescence co-localization applied to human kinetochore protein architecture

Two-color fluorescence co-localization in 3D (three-dimension) has the potential to achieve accurate measurements at the nanometer length scale. Here, we optimized a 3D fluorescence co-localization method that uses mean values for chromatic aberration correction to yield the mean separation with ~10 nm accuracy between green and red fluorescently labeled protein epitopes within single human kinetochores. Accuracy depended critically on achieving small standard deviations in fluorescence centroid determination, chromatic aberration across the measurement field, and coverslip thickness. Computer simulations showed that large standard deviations in these parameters significantly increase 3D measurements from their true values. Our 3D results show that at metaphase, the protein linkage between CENP-A within the inner kinetochore and the microtubule-binding domain of the Ndc80 complex within the outer kinetochore is on average ~90 nm. The Ndc80 complex appears fully extended at metaphase and exhibits the same subunit structure in vivo as found in vitro by crystallography

A quantitative description of Ndc80 complex linkage to human kinetochores

The Ndc80 complex, which mediates end-on attachment of spindle microtubules, is linked to centromeric chromatin in human cells by two inner kinetochore proteins, CENP-T and CENP-C. Here to quantify their relative contributions to Ndc80 recruitment, we combine measurements of kinetochore protein copy number with selective protein depletion assays. This approach reveals about 244 Ndc80 complexes per human kinetochore (∼14 per kinetochore microtubule), 215 CENP-C, 72 CENP-T and only 151 Ndc80s as part of the KMN protein network (1:1:1 Knl1, Mis12 and Ndc80 complexes). Each CENP-T molecule recruits ∼2 Ndc80 complexes; one as part of a KMN network. In contrast, ∼40% of CENP-C recruits only a KMN network. Replacing the CENP-C domain that binds KMN with the CENP-T domain that recruits both an Ndc80 complex and KMN network yielded functional kinetochores. These results provide a quantitative picture of the linkages between centromeric chromatin and the microtubule-binding Ndc80 complex at the human kinetochore

How the kinetochore couples microtubule force and centromere stretch to move chromosomes

The Ndc80 complex (Ndc80, Nuf2, Spc24, Spc25) is a highly conserved kinetochore protein essential for end-on anchorage to spindle microtubule plus-ends and for force generation coupled to plus-end polymerization and depolymerization. Spc24/Spc25 at one end of the Ndc80 complex binds the kinetochore. The N-terminal tail and CH domains of Ndc80 bind microtubules, and an internal domain binds microtubule-associated proteins (MAPs) such as the Dam1 complex. To determine how the microtubule and MAP binding domains of Ndc80 contribute to force production at the kinetochore in budding yeast, we have inserted a FRET tension sensor into the Ndc80 protein about halfway between its microtubule binding and internal loop domains. The data support a mechanical model of force generation at metaphase where the position of the kinetochore relative to the microtubule plus-end reflects the relative strengths of microtubule depolymerization, centromere stretch and microtubule binding interactions with Ndc80 and Dam1 complexes

Prolonged Mitosis of Neural Progenitors Alters Cell Fate in the Developing Brain

Embryonic neocortical development depends on balanced production of progenitors and neurons. Genetic mutations disrupting progenitor mitosis frequently impair neurogenesis; however, the link between altered mitosis and cell fate remains poorly understood. Here we demonstrate that prolonged mitosis of radial glial progenitors directly alters neuronal fate specification and progeny viability. Live imaging of progenitors from a neurogenesis mutant, Magoh(+/-), reveals that mitotic delay significantly correlates with preferential production of neurons instead of progenitors, as well as apoptotic progeny. Independently, two pharmacological approaches reveal a causal relationship between mitotic delay and progeny fate. As mitotic duration increases, progenitors produce substantially more apoptotic progeny or neurons. We show that apoptosis, but not differentiation, is p53 dependent, demonstrating that these are distinct outcomes of mitotic delay. Together our findings reveal that prolonged mitosis is sufficient to alter fates of radial glia progeny and define a new paradigm to understand how mitosis perturbations underlie brain size disorders such as microcephaly

Decoding Polo-like kinase 1 signaling along the kinetochore–centromere axis

Protein kinase signaling along the kinetochore-centromere axis is crucial to assure mitotic fidelity, yet its spatial coordination is obscure. Here, we examined how pools of human Polo-like kinase 1 (Plk1) within this axis control signaling events to elicit mitotic functions. To do this, we restricted active Plk1 to discrete subcompartments within the kinetochore-centromere axis using chemical genetics and decoded functional and phosphoproteomic signatures of each. We observe distinct phosphoproteomic and functional roles, suggesting that Plk1 exists and functions in discrete pools along this axis. Deep within the centromere, Plk1 operates to assure proper chromosome alignment and segregation. Thus, Plk1 at the kinetochore is a conglomerate of an observable bulk pool coupled with additional functional pools below the threshold of microscopic detection/resolution. Although complex, this multiplicity of locales provides an opportunity to decouple functional and phosphoproteomic signatures for a comprehensive understanding of Plk1’s kinetochore functions

The Architecture of CCAN Proteins Creates a Structural Integrity to Resist Spindle Forces and Achieve Proper Intrakinetochore Stretch

Constitutive Centromere Associated Network (CCAN) proteins, particularly CENP-C, CENP-T and the CENP-H/-I complex, mechanically link CENP-A-containing centromeric chromatin within the inner kinetochore to outer kinetochore proteins, like the Ndc80 complex, that bind kinetochore microtubules. Accuracy of chromosome segregation depends critically upon Aurora B phosphorylation of Ndc80/Hec1. To determine how CCAN protein architecture mechanically constrains intrakinetochore stretch between CENP-A and Ndc80/Hec1 for proper Ndc80/Hec1 phosphorylation, we used super-resolution fluorescence microscopy and selective protein depletion. We found that at bi-oriented chromosomes in late prometaphase cells, CENP-T is stretched ~16 nm to the inner end of Ndc80/Hec1, much less than expected for full-length CENP-T. Depletion of various CCAN linker proteins induced hyper-intrakinetochore stretch (an additional 20-60 nm) with corresponding significant decreases in Aurora B phosphorylation of Ndc80/Hec1. Thus, proper intrakinetochore stretch is required for normal kinetochore function and depends critically on all the CCAN mechanical linkers to the Ndc80 complex

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

Spindle assembly checkpoint proteins are positioned close to core microtubule attachment sites at kinetochores

Depletion analyses and nanometer-scale mapping of spindle assembly checkpoint proteins reveal how these proteins are integrated within the substructure of the kinetochore.Spindle assembly checkpoint proteins have been thought to reside in the peripheral corona region of the kinetochore, distal to microtubule attachment sites at the outer plate. However, recent biochemical evidence indicates that checkpoint proteins are closely linked to the core kinetochore microtubule attachment site comprised of the Knl1–Mis12–Ndc80 (KMN) complexes/KMN network. In this paper, we show that the Knl1–Zwint1 complex is required to recruit the Rod–Zwilch–Zw10 (RZZ) and Mad1–Mad2 complexes to the outer kinetochore. Consistent with this, nanometer-scale mapping indicates that RZZ, Mad1–Mad2, and the C terminus of the dynein recruitment factor Spindly are closely juxtaposed with the KMN network in metaphase cells when their dissociation is blocked and the checkpoint is active. In contrast, the N terminus of Spindly is ∼75 nm outside the calponin homology domain of the Ndc80 complex. These results reveal how checkpoint proteins are integrated within the substructure of the kinetochore and will aid in understanding the coordination of microtubule attachment and checkpoint signaling during chromosome segregation

In silico APC/C substrate discovery reveals cell cycle-dependent degradation of UHRF1 and other chromatin regulators

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease. Copyright

Spindle microtubules generate tension-dependent changes in the distribution of inner kinetochore proteins

The N and C termini of CENP-T undergo tension-dependent separation, suggesting that CENP-T elongation is responsible for changes in the shape of the inner kinetochore