Quantitative mapping of dense microtubule arrays in mammalian neurons
- Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Netherlands
Figures
Figure 1 with 4 supplements
Radial distribution of microtubule subtypes in dendrites imaged using STED microscopy.
(A-B) Representative single planes in XY (left) and YZ cross-sections along the yellow dashed line (right) of a dendrite imaged with 2D (A) and 3D (B) STED. Scale bar 5 µm (XY) and 2 µm (YZ). (C) Qua…
Figure 1—figure supplement 1
Thickness of dendrites imaged using STED.
(A-B) Distribution of dendrite’s cross-section area for the data shown in Figure 1(F)–(G).
Figure 1—figure supplement 2
Resolution of STED.
(A) Measurements of STED 2D lateral resolution for different microtubule subsets in cell body images (Figure 2B) using decorrelation analysis (x-axis) or Gaussian fitting of microtubule profiles …
Figure 1—figure supplement 3
Intensity analysis along the whole length of individual dendrites.
(A) Fluorescent intensity of different microtubule subsets along the length of a dendrite, normalized by an average value in the first proximal 5 µm of dendrite’s length in each channel. Plot lines …
Figure 1—figure supplement 4
Detyrosinated and delta-2 microtubules subsets.
(A) Representative images of COS-7 cells stained for detyrosinated, acetylated, and tyrosinated tubulin. Yellow square box indicates the region displayed in the zoom in the lower row. Scale bar 10 …
Figure 2 with 2 supplements
Analysis pipeline for detection and intensity quantification of individual (post-translationally modified) microtubules.
(A) Vertical cross-section along a neuronal cell body (soma). Dashed rectangle marks the volume (sub-z-stack) under the nucleus used for microtubule filament detection (maximum intensity projection) …
Figure 2—figure supplement 1
Analysis of microtubule segments intensities.
(A) Zoom-in of cell body area in total tubulin channel from Figure 2B, showing individual microtubule segments detections in different colors. Scale bar 5 µm. (B) Histograms of background corrected …
Figure 2—figure supplement 2
Heatmaps of microtubule subsets using STED.
(A) Three-color heatmap overlay of normalized intensity distributions of single microtubule segments detected in total tubulin, (yellow, n=9050), tyrosinated (cyan, n=10281 segments), and acetylated …
Figure 3
Estimation of absolute numbers of (modified) microtubules in dendrites using single-microtubule intensities from the soma.
(A) Three-color overlay of maximum intensity projection of a 2D STED z-stack including the whole volume of dendrites (total tubulin in yellow, tyrosinated in cyan and acetylated in magenta). Dashed …
Figure 4 with 1 supplement
Expansion Microscopy improves quantification of the radial distribution of microtubule modifications in dendrites.
(A) Representative volumetric 3D rendering of total tubulin and its posttranslational modifications in a dendrite imaged using ExM. Scale bar 2 μm (physical size post-expansion 8.3 μm). (B) Represent…
Figure 4—figure supplement 1
Characterization of dendrites imaged using ExM.
(A) (top) Representative single YZ cross-section of the dendrite from Figure 4A (E)-(F) and (bottom) corresponding maximum intensity XY projections of subvolume marked by dashed yellow lines. Scale …
Figure 5 with 5 supplements
Direct estimation of microtubule numbers in dendrites using FlipExM.
(A) Top: Schematics of gel reorientation for FlipExM imaging. Bottom: Simulated z-stacks illustrating the advantages of FlipExM for imaging of dendritic microtubules. A set of simulated …
Figure 5—figure supplement 1
Estimation of single microtubule intensity per channel.
(A-B) Histograms of background subtracted integrated intensity of individual microtubules cross-sections detected and quantified in tyrosinated (A) or acetylated (B) tubulin channel for the dendrite …
Figure 5—figure supplement 2
Heatmaps of microtubule subsets using FlipExM.
(A) Heatmaps of normalized single microtubule cross-section intensities in tyrosinated and acetylated channels for the detections performed in the specified channel built separately (data is the …
Figure 5—figure supplement 3
Percentage of microtubule subsets per dendrite.
Percentage of tyrosinated, acetylated, and non-modified microtubules as a fraction of total microtubule number per dendrite as a function of dendrite’s cross-section area (6 cells, N=two independent …
Figure 5—figure supplement 4
Number of microtubules along the length of a dendrite.
The number of total, tyrosinated and acetylated microtubules along the length of the dendrite shown in Figure 5B (left y-axis). The black line represents corresponding area of dendrite’s …
Figure 5—figure supplement 5
Different crosstalk estimation methods for FlipExM data.
(A)-(C) Comparison of different methods for quantification of tyrosination levels of acetylated MTs (α) and acetylation levels of tyrosinated MTs (β). In the left panels (same data as in Figure …
Videos
Video 1
Illustration of radial intensity distribution analysis in dendrites acquired using STED microscopy.
Video 2
3D volumetric rendering of a dendrite imaged using ExM (same as in Figure 4A).
Scale bar corresponds to the physical post expansion size.
Video 3
Illustration of gel sample reorientation and microscope’s PSF for FlipExM imaging.
Video 4
3D volumetric rendering of a dendrite imaged using FlipExM (same as in Figure 5B).
Scale bar corresponds to the physical post expansion size.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Cercopithecus aethiops) | COS7 | Lukas Kapitein lab | Cell line has not been authenticated, but mycoplasm testing was done fewer than 26 weeks before experiments | |
| Biological sample (Rattus norvegicus domestica) | Primary hippocampal neuron culture | Lukas Kapitein lab | Cultured on embryonic day 18 | |
| Antibody | Mouse monoclonal anti-Acetylated tubulin | Sigma | Cat#T7451, [6-11B-1], RRID:AB_609894 | IF Expansion (1:200), IF confocal/STED (1:600) |
| Antibody | Rat monoclonal anti-Tyrosinated tubulin | Abcam | Cat#Ab6160, [YL1/2], RRID:AB_305328 | IF Expansion (1:100), IF confocal/STED (1:250) |
| Antibody | Rabbit recombinant anti-alpha tubulin | Abcam | Cat# 52866, [EP1332Y] RRID:AB_869989 | IF Expansion (1:250), IF confocal/STED (1:500) |
| Antibody | Rabbit polyclonal anti-detyrosinated tubulin | Merck | Cat# AB3210, | IF STED (1:500) |
| Antibody | Rabbit anti-Δ2 tubulin | Millipore | Cat# AB3203, RRID:AB_177351 | IF STED (1:500) |
| Antibody | Alexa 594 Goat Anti-Rat IgG (H+L) | Molecular Probes, Life Technologies | Cat#A11007, RRID:AB_10561522 | IF Expansion (1:250), IF confocal/STED (1:500) |
| Antibody | Alexa 488 Goat Anti-Rabbit IgG (H+L) | Molecular Probes, Life Technologies | Cat#A11034, RRID:AB_2576217 | IF Expansion (1:250), IF confocal/STED (1:500) |
| Antibody | Alexa 594 Goat Anti-Mouse IgG (H+L) | Molecular Probes, Life Technologies | Cat#A11032, RRID:AB_2534091 | IF Expansion (1:250), IF confocal/STED (1:500) |
| Antibody | Alexa 488 Goat Anti-Rat IgG (H+L) | Molecular Probes, Life Technologies | Cat#A11006, RRID:AB_2534074 | IF Expansion (1:250), IF confocal/STED (1:500) |
| Antibody | Abberior Star 635P goat anti-mouse IgG (H+L) | Abberior GmbH | Cat#ST635P-1001–500 UG | IF Expansion (1:250), IF confocal/STED (1:500) |
| Antibody | Abberior Star 635P goat anti-rabbit IgG (H+L) | Abberior GmbH | Cat#ST635P-1002–500 UG | IF Expansion (1:250), IF confocal/STED (1:500) |
| Chemical compound,drug | Triton X-100 | Sigma | Cat#X100 | |
| Chemical compound,drug | Gluteraldehyde (8%) | Sigma | Cat#G7526 | |
| Chemical compound,drug | Paraformaledehyde (16%) | Electron Microscopy Sciences | Cat#15710 | EM-grade |
| Chemical compound,drug | Acryloyl-X | Thermo Fisher | Cat#A20770 | |
| Chemical compound,drug | Sodium Acrylate | Sigma Aldrich | Cat#408220 | |
| Peptide, recombinant protein | Proteinase K | ThermoFisher | Cat#EO0492 | |
| Software, algorithm | Huygens Professional software version 17.04 | Scientific Volume Imaging, the Netherlands | RRID:SCR_014237 | |
| Software, algorithm, | Correlescence plugin v.0.0.4 (2021) | Eugene Katrukha | https://github.com/ekatrukha/Correlescence https://doi.org/10.5281/zenodo.4534715 | |
| Software, algorithm, | CurveTrace plugin ver.0.3.5 (2021) | Eugene Katrukha | https://github.com/ekatrukha/CurveTrace https://doi.org/10.5281/zenodo.4534721 | |
| Software, algorithm | ComDet plugin v.0.5.3 (2021) | Eugene Katrukha | https://github.com/ekatrukha/ComDet https://doi.org/10.5281/zenodo.4281064 | |
| Other | Silicone mold for gels 13 mm | Sigma-Aldrich | Cat#GBL664107 |
