Bayesian inference of multi-point macromolecular architecture mixtures at nanometre resolution.

Gaussian spot fitting methods have significantly extended the spatial range where fluorescent microscopy can be used, with recent techniques approaching nanometre (nm) resolutions. However, small inter-fluorophore distances are systematically over-estimated for typical molecular scales. This bias can be corrected computationally, but current algorithms are limited to correcting distances between pairs of fluorophores.

Evidence for a HURP/EB free mixed-nucleotide zone in kinetochore-microtubules.

Current models infer that the microtubule-based mitotic spindle is built from GDP-tubulin with small GTP caps at microtubule plus-ends, including those that attach to kinetochores, forming the kinetochore-fibres. Here we reveal that kinetochore-fibres additionally contain a dynamic mixed-nucleotide zone that reaches several microns in length. This zone becomes visible in cells expressing fluorescently labelled end-binding proteins, a known marker for GTP-tubulin, and endogenously-labelled HURP - a protein which we show to preferentially bind the GDP microtubule lattice in vitro and in vivo.

Kinetochore tracking in 3D from lattice light-sheet imaging data with KiT.

Motivation: Lattice light-sheet microscopy (LLSM) is revolutionizing cell biology since it enables fast, high-resolution extended imaging in three dimensions combined with a drastic reduction in photo-toxicity and bleaching. However, analysis of such datasets still remains a major challenge.

Results: Automated tracking of kinetochores, the protein complex facilitating and controlling microtubule attachment of the chromosomes within the mitotic spindle, provides quantitative assessment of chromosome dynamics in mitosis.

Subcellular Euclidean distance measurements with multicolor fluorescence localization imaging in cultured cells.

This protocol measures the 3D Euclidean distance (Δ) between two/three fluorescently labeled kinetochore components in fixed samples using Kinetochore Delta software (KiDv1.0.1, MATLAB based).