ring tirf illumination  (Nikon)

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

Journal: bioRxiv

Article Title: Cell wall remodeling drives engulfment during Bacillus subtilis sporulation

doi: 10.1101/087858

Figure Lengend Snippet: (A) Sporulating cells stained with a green fluorescent derivative of penicillin V (BOCILLIN-FL). Bright foci are observed at the LE of the engulfing membrane. Membranes were stained with FM 4-64 (red). (B) Average BOCILLIN-FL (green) and FM 4-64 (red) fluorescence intensities along forespore contours plotted as a function of the degree of engulfment. Cells are binned according to percentage of engulfment. BOCILLIN-FL signal is enriched at the LE throughout engulfment ( n = 125). (C) Cell-specific localization of the peptidoglycan biosynthetic machinery. GFP tagged versions of different B. subtilis PBPs and actin-like proteins (ALPs) were produced from mother cell-(MC) or forespore-(FS) specific promoters. (D) Six different localization patterns were observed upon cell-specific localization of PBPs and ALPs. For each pair of images, left panel shows overlay of membrane and GFP fluorescence, while the right panel only shows GFP fluorescence. Pictures of representative cells displaying the different patterns are shown (top, GFP fusion proteins transcribed from spoIIR promoter for forespore-specific expression, and from spoIID promoter for mother cell-specific expression). The six different patterns are depicted in the bottom cartoon and proteins assigned to each one are indicated. Membranes were stained with FM 4-64. See Figure 2-figure supplement 1 for cropped fields of all PBPs we assayed. Transglycosylase (TG), transpetidase (TP), carboxipetidase (CP), endopeptidase (EP), actin-like protein (ALP). (E) TIRF microscopy of forespore-produced GFP-MreB in four different forespores (i to iv). In every case, the leftmost picture is an overlay of the forespore membranes (shown in white) and the tracks followed by individual TIRF images of GFP-MreB (color encodes time, from blue to red). Sporangia are oriented with the forespores up. For the rst sporangia (i), snapshots from TIRF timelapse experiments taken 8 s apart are shown. Arrows indicate GFP-MreB foci and are color coded to match the trace shown in the left panel. Rightmost panel for each forespore shows a kymograph representing the fluorescence intensity along the line joining the leading edges of the engulfing membrane over time (from top to bottom; total time 100 s). Average focus speed ( n = 14) is indicated at the bottom. Timelapse movies of the examples presented here and additional sporangia are shown in Video 2. (F) Localizaiton of GFP-SpoIIP in untreated sporangia, or in sporangia treated with bacitracin (50 μ g/ml) or cephalexin (50 g/ml). (G) Fraction of GFP-SpoIIP fluorescence at LE of the engulfing membrane. Bars represent the average and standard error of 85 untreated sporangia, 38 sporangia treated with bacitracin (50 μ g/ml), and 67 sporangia treated with cephalexin (50 μ g/ml). (H) Model for PG synthesis and degradation at the LE of the engulfing membrane. New PG is synthesized ahead of the LE of the engulfing membrane by forespore-associated PG biosynthetic machinery, and is subsequently degraded but the mother-cell DMP complex. We propose that DMP has specificity for the peptide cross-links that join the newly synthesized PG with the lateral cell wall (orange), which leads to the extension of the septal PG around the forespore. Scale bars 1 μ m.

Article Snippet: Laser power was set to 15 %, and exposure time was 200 ms. (ii) An Applied Precision OMX Structured Illumination microscopy equipped with a Ring-TIRF system and a UApoN 1.49NA objective, immersion oil n = 1.518.

Techniques: Staining, Fluorescence, Produced, Expressing, Microscopy, Synthesized