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Microtubule Tip Tracking and Tip Structures at the Nanometer Scale Using Digital Fluorescence Microscopy

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Abstract

Microtubules (MTs) are central to fundamental cellular processes including mitosis, polarization, and axon extension. A key issue is to understand how MT-associated proteins and therapeutic drugs, such as the anticancer drug paclitaxel, control MT self-assembly. To facilitate this research, it would be helpful to have automated methods that track the tip of dynamically assembling MTs as observed via fluorescence microscopy. Through a combination of digital fluorescence imaging with MT modeling, model-convolution, and automated image analysis of live and fixed MTs, we developed a method for MT tip tracking that includes estimation of the measurement error. We found that the typical single-frame tip tracking accuracy of GFP-tubulin labeled MTs in living LLC-PK1α cells imaged with a standard widefield epifluorescence digital microscope system is ~36 nm, the equivalent of ~4.5 tubulin dimer layers. However, if the MT tips are blunt, the tip tracking accuracy can be as accurate as ~15 nm (~2 dimer layers). By fitting a Gaussian survival function to the MT tip intensity profiles, we also established that MTs within living cells are not all blunt, but instead exhibit highly variable tapered tip structures with a protofilament length standard deviation of ~180 nm. More generally, the tip tracking method can be extended to track the tips of any individual fluorescently labeled filament, and can estimate filament tip structures both in vivo and in vitro with single-frame accuracy on the nanoscale.

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Acknowledgments

We thank Dominique Seetapun for technical assistance. The research was conducted with support from NIH (GM071522 and GM076177), and NSF (MCB-0615568).

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Authors and Affiliations

  1. Department of Biomedical Engineering, University of Minnesota, 7-132 Hasselmo Hall, 312 Church Street SE, Minneapolis, MN, 55455, USA

    Alexei O. Demchouk, Melissa K. Gardner & David J. Odde

  2. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

    Melissa K. Gardner

Authors
  1. Alexei O. Demchouk
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  2. Melissa K. Gardner
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  3. David J. Odde
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Corresponding author

Correspondence to David J. Odde.

Additional information

Associate Editors Yingxiao Wang and Peter J. Butler oversaw the review of this article.

Electronic supplementary material

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Supplementary Fig. 1: Point spread function (PSF) and simulation of a MT tip.

(A) Simulated fine-grid PSF, using numerical aperture of 1.49 and wavelength of 513 nm, with pixel size of 2.5 nm. (B) PSF intensity profile (blue markers). A Gaussian function (red line) can be used to approximate the intensity, which analytically obeys a 2D Airy function (dashed blue line). The Gaussian fit to a noiseless PSF has a standard deviation of ~ 72 nm. (C) Start of the MT simulation: a speckle-free, blunt simulated MT on a fine-grid, pixel size = 2.5 nm. (D) Fine-grid blunt MT. Each pixel of the MT is given a 10% probability to have a value of 1, otherwise its value is set to 0. This 10% probability accounts for the incomplete labeling of tubulin in eGFP-α tubulin labeled cells. The stochastic incorporation of labeled subunits gives rise to the “speckle” noise along the MT. Inset: a magnified MT tip. (E) Speckled MT shown in Fig. 1d convolved with the PSF shown in 1A. (F) Model-convolved MT from Fig. 1e after coarse-graining and addition of Gaussian white noise. Coarse-graining is done so that one pixel in the final simulated image represents 42 nm in the specimen plane. The coarse-grained image is obtained by averaging a 17 × 17 array of 2.5 nm fine-grid pixels to form a single 42 nm coarse-grained pixel. Gaussian white noise is added to the coarse-grained pixels to match the variability in background intensity observed experimentally.

Supplementary Fig. 2: Estimation of MT speckle level.

MT noise was plotted as a function of the speckle parameter for the 15th frame of fixed cell MT movies (speckle level estimation in live cell MTs is similar). The simulation starts with a speckle parameter of 0.002 (0.2%) and increases it incrementally by 0.002, each time recording the standard deviation of the pixel intensities along the backbone of the MT. Parameters for background intensity, signal above background, and background noise remain constant. For high speckle parameter values, the noise along the MT is dominated by background Gaussian white noise, while for low speckle parameter values it is dominated by the speckle noise. The parameter is adjusted until the noise along the MT matches the level observed experimentally (black dashed line). The red line shows the best-fit power law to the simulations represented by the blue dots. The point of intersection between the red line and the dashed black line represents the estimated speckle parameter (black circle).

Supplementary Fig. 3: Fixed cell MT simulation.

(A) σPF+PSF distributions for experimental (dark blue) and simulated (light blue) fixed cell MTs. (B) Accuracy of tip position estimation. Dashed lines represent accuracy analysis with a presence of a stage drift, solid lines represent detrended accuracy analysis.

Supplementary Fig. 4: Example kymographs of live cell background.

Due to diffusion in live cells, the background appears relatively uniform with no apparent streaks throughout the movies (in comparison to kymographs from fixed cell background, Supplementary Fig. 5). Top panel: live cell region, white lines indicate regions where kymographs were obtained (max intensity). Bottom panels: five kymographs from the cell region. Horizontal bar = 1000 nm, vertical bar = 10 s.

Supplementary Fig. 5: Example kymographs of fixed cells background.

Same as Supplementary Fig. 4, except for fixed MTs. Due to lack of diffusion in fixed cells, the background appears relatively nonuniform with persistent fluorescent streaks that remain stationary throughout the movies. Top panel: fixed cell region, white lines indicate regions where kymographs were obtained (max intensity). Bottom panels: seven kymographs from the cell region. Horizontal bar = 1000 nm, vertical bar = 10 s.

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Demchouk, A.O., Gardner, M.K. & Odde, D.J. Microtubule Tip Tracking and Tip Structures at the Nanometer Scale Using Digital Fluorescence Microscopy. Cel. Mol. Bioeng. 4, 192–204 (2011). https://doi.org/10.1007/s12195-010-0155-6

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