ilas 3 ring tirf illumination system  (Gataca Inc)

Journal: The EMBO Journal

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

doi: 10.15252/embj.2022112504

Figure Lengend Snippet: A Cartoon of a chromosome pair during mitosis with sister kinetochores that are attached (green) and unattached (red) to microtubules. 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, the hinge, the loop, and the tetramerization domain indicated. The box shows the loop region at a 6× magnification. See Fig 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 the SPC24:SPC25 side of the Ndc80 complex. E Size exclusion chromatography coupled with multi‐angle light scattering (SEC‐MALS) profiles of fluorescently labeled Ndc80 and Ndc80 Δloop . Calculated (and theoretical) masses are indicated. See Appendix Fig 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. 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 brightness of Ndc80 complexes at indicated concentrations on taxol‐stabilized microtubules. Source data are available online for this figure.

Article Snippet: Experiments reported in Figs and were performed using a Nikon Eclipse Ti2 microscope equipped with a Plan Apo 100 × 1.45 NA TIRF, iLas 3 ring TIRF illumination system (GATACA), and an Andor iXon 897 EMCCD camera.

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

Journal: The EMBO Journal

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

doi: 10.15252/embj.2022112504

Figure Lengend Snippet: 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‐∆L, or Ndc80‐M5 complexes. The number of cells with multipolar spindles and the total number of analyzed cells are shown. Some signal from the tubulin channel is visible in the CENP‐C channel. Scale bar: 5 μm. 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). Quantification of the intensity of the monomeric Ndc80 associating with microtubule‐bound trimeric Ndc80. 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. 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 analyzed traces of Ndc80 trimers are shown ( n = 42, 63, 33, 30, 34 for conditions 1–5). 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.). Source data are available online for this figure.

Article Snippet: Experiments reported in Figs and were performed using a Nikon Eclipse Ti2 microscope equipped with a Plan Apo 100 × 1.45 NA TIRF, iLas 3 ring TIRF illumination system (GATACA), and an Andor iXon 897 EMCCD camera.

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