ilas 2 tirf module  (Gataca Inc)

Journal: bioRxiv

Article Title: Stable kinetochore-microtubule attachment requires loop-dependent Ndc80-Ndc80 binding

doi: 10.1101/2022.08.25.505310

Figure Lengend Snippet: A ) Cartoon of a chromosome pair during mitosis with sister kinetochores that are attached (green) and unattached (red) to microtubules. The unattached kinetochores trigger a spindle assembly checkpoint (SAC) signal. One outer kinetochore contains a lawn of Ndc80 complexes, resulting in many Ndc80 complexes binding to a single microtubule. B ) Prediction of the full-length Ndc80 structure with residues that comprise the tail, th e kink, the loop, and the tetramerisation domain indicated. The box shows the loop region at a 6x magnification. See for more information. C, D ) Low-angle Pt/C shadowing of Mis12:Ndc80 (panel C) and Mis12:Ndc80 Δloop (Δ431-463) (panel D) complexes. The Mis12 complex appears as a 20 nm rod-like extension and marks Ndc80 and Ndc80 Δloop . Calculated and (theoretical) masses are indicated in the legend. These complexes were fluorescently labelled. See Suppl . the SPC24:SPC25 side of the Ndc80 complex. E ) Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) profiles of for more information. F ) Total Internal Reflection Fluorescence (TIRF) microscopy was used to investigate Ndc80 Alexa488 complexes on taxol-stabilized microtubules that were attached to a passivated glass surface. Kymographs show Ndc80 complexes at a concentration of 0.2 nM with (FL, blue) or without (ΔL, orange) the loop. Scale bars: vertical (5 μm), horizontal (5 s). G ) Quantification of Ndc80 residence times for data as in panel F. Solid line represents a single exponential fit. H ) One-dimensional diffusion of Ndc80 complexes (with n indicated) on microtubules. Traces were split into segments of 0.5 s and averaged (see Materials and Methods). Mean values (circles) and SEM (shaded area) are shown. I ) Distribution of the initial brightness of Ndc80 complexes on stabilized microtubules. J ) Typical fields of view showing decoration of taxol-stabilized microtubules (cyan) incubated with full-length or loopless Ndc80 (yellow) at the indicated concentration. Images show an average projection of 200 frames. The contrast between individual fluorescent channels (inverted grayscale) was fixed. Auto-contrast was used for the composite images to highlight the differences in the uniformity of Ndc80 decoration. K ) Distribution of the initial brightness of Ndc80 complexes added at indicated concentrations to taxol-stabilized microtubules.

Article Snippet: All other experiments were performed with a Nikon Ti-E microscope equipped with a Plan Apo 100 × 1.45 NA TIRF oil-immersion objective, iLas 2 ring TIRF module (Roper Scientific) and a Evolve 512 EMCCD camera (Roper Scientific).

Techniques: Binding Assay, Size-exclusion Chromatography, Fluorescence, Microscopy, Concentration Assay, Diffusion-based Assay, Incubation

Journal: bioRxiv

Article Title: Stable kinetochore-microtubule attachment requires loop-dependent Ndc80-Ndc80 binding

doi: 10.1101/2022.08.25.505310

Figure Lengend Snippet: A ) Schematic of a cold-shock assay following an electroporation experiment. Immunofluorescence images showing the attachment status of kinetochores to microtubules in cells electroporated with recombinant Ndc80-wt, Ndc80-ΔL, or Ndc80-M5 complexes. The number of cells with multipolar spindles and the total number of analysed cells are shown. Some signal from the tubulin channel is visible in the CENP-C channel. Scale bar: 5 μm. B ) Total Internal Reflection Fluorescence (TIRF) microscopy was used to investigate Ndc80 Alexa488 complexes (0.6 nM) added to trimeric Ndc80 TMR (10 pM) on fluorescent taxol-stabilized microtubules that were attached to a passivated glass surface. Typical kymographs showing virtually motionless Ndc80 trimers (magenta) and transiently binding Ndc80 monomers (yellow). Wild-type (wt) monomers associate with wt trimers (left), but not with M5 trimers (right). Scale bars: vertical (100 s), horizontal (5 μm). C ) Quantification of the intensity of the monomeric Ndc80 associating with microtubule-bound trimeric Ndc80 (see Materials and Methods for details). A threshold for binding was set at an intensity equivalent to one Alexa488 copy. Intensities well above 1 (yellow) could thus reflects multiple monomers binding simultaneously. D ) Fraction of time there was at least one monomer (added to solution at a concentration of 0.6 nM) present at the microtubule-bound trimer (10 pM), tested in various combinations of wild-type (wt), loopless (ΔL) and M5 monomers or trimers. All analysed traces of Ndc80 trimers are shown ( n is shown in the legend). Horizontal lines show median values and statistical significance was determined using a two-tailed Mann-Whitney test. P-values: 1 (wt-trimer + wt-monomer) vs 2 (M5-trimer + wt-monomer): 7·10 −7 (***); 2 vs 3: 0.17 (n.s.); 1 vs 3: 1·10 −6 (***); 1 vs 4: 3·10 −15 (***); 3 vs 4: 1·10 −8 (***); 1 vs 5: 1·10 −13 (***); 4 vs 5 0.23 (n.s.).

Article Snippet: All other experiments were performed with a Nikon Ti-E microscope equipped with a Plan Apo 100 × 1.45 NA TIRF oil-immersion objective, iLas 2 ring TIRF module (Roper Scientific) and a Evolve 512 EMCCD camera (Roper Scientific).

Techniques: Electroporation, Immunofluorescence, Recombinant, Fluorescence, Microscopy, Binding Assay, Concentration Assay, Two Tailed Test, MANN-WHITNEY