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A simple photoactivation and image analysis module for visualizing and analyzing axonal transport with high temporal resolution

Nature Protocols volume 7, pages 62–68 (2012)Cite this article

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Abstract

We describe a strategy for analyzing axonal transport of cytosolic proteins (CPs) using photoactivatable GFP—PAGFP—with modifications of standard imaging components that can be retroactively fitted to a conventional epifluorescence microscope. The photoactivation and visualization are nearly simultaneous, allowing studies of proteins with rapidly mobile fractions. Cultured hippocampal neurons are transfected with PAGFP-tagged constructs, a discrete protein population within axons is photoactivated, and then the activated population is tracked by live imaging. We show the utility of this method in analyzing axonal transport of CPs that have inherent diffusible pools and distinguish this transport modality from passive diffusion and vesicle transport. The analytical tools used to quantify the motion are also described. Aside from the time needed for preparation of neuronal cultures/transfection, the experiment takes 2–3 h, during which time several axons can be imaged and analyzed. These methods should be easy to adopt by most laboratories and may also be useful for monitoring CP movement in other cell types.

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Figure 1: Equipment setup and experimental design.
Figure 2: Photoactivation strategy to study axonal transport.
Figure 3: A strategy for quantifying the axonal transport of soluble proteins.

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Acknowledgements

S.R. acknowledges the Larry Hillblom Foundation, the March of Dimes Foundation and the University of California, San Diego Alzheimer's Disease Research Center for their support (P50AG005131), as well as G. Patterson and J. Lippincott-Schwartz for sharing the PAGFP construct.

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Authors and Affiliations

  1. Department of Neurosciences, University of California, San Diego, La Jolla, California, USA

    Subhojit Roy, Yong Tang & David A Scott

  2. Department of Pathology, University of California, San Diego, La Jolla, California, USA

    Subhojit Roy, Yong Tang & David A Scott

  3. Department of Biomedical Engineering, Lane Center for Computational Biology, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA

    Ge Yang

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Contributions

S.R. and D.A.S. conceived the imaging experiments, performed the data analyses and wrote this paper. G.Y. originally suggested the use of the intensity-center analysis algorithm to reveal bulk motion, wrote the initial MATLAB codes (that were added to by D.A.S.) and helped in writing relevant parts of this paper. Y.T. performed imaging experiments, analyzed data and provided key insights during troubleshooting.

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Correspondence to Subhojit Roy.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Step-wise depiction of intensity center shift quantification.(A) Following the photoactivation of cytosolic protein in the axon, the average background intensity adjacent to the activated zone was subtracted from each frame of the time-lapse movie. Images show (from top-bottom) an axon in the first frame of a time-lapse movie immediately following photoactivation; the same image following subtraction of background intensity (207.3 A.F.U). (B) The photoactivated axon was thresholded to determine the equal intensity boundaries of the photoactivated region for kymograph generation. Images show (from top-bottom) a pseudocolor image of the axon from (A) depicting the intensity fall off at the boundaries of the photoactivated zone; the thresholded photoactivated zone; a 20px polyline drawn in the proximal to distal direction over the length of the photoactivated zone. Average intensity linescans were then generated by calculating the average intensity of pixels over the width of the polyline and were used to create kymographs. (C) The kymographs shown were generated from average intensity linescans of the photoactivated axonal region from successive frames from a time-lapse movie. The table inset shows the intensities of pixels across each linescan in row vectors representing each time-point in time-lapse movies, which make up the kymographs. The pseudocolor kymograph shows the anterograde biased movement of the photoactivated fluorescent mass towards the distal end of the axon, and the plot to the right shows the calculated intensity center shift of the photoactivated GFP population over a 60s time-lapse movie. (PNG 142 kb)

Supplementary Video 1

Principles of intensity-center analysis. A temporally-linked composite showing the mobility of the PAGFP:synapsin after photoactivation (greyscale, top); the resultant pseudocolor kymograph in the center (middle); and the intensity-center data below (the quantitative center is marked with a red dot to facilitate visualization of the shift). Note the shift in the curves (displacement of the red dot) corresponds to the anterogradely-biased transport of the fluorescent plume as seen in the kymograph and the greyscale image. Gaussian curves are shown for ease of visualization, but the data are similar to that from raw intensity-values. (MOV 5619 kb)

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Roy, S., Yang, G., Tang, Y. et al. A simple photoactivation and image analysis module for visualizing and analyzing axonal transport with high temporal resolution. Nat Protoc 7, 62–68 (2012). https://doi.org/10.1038/nprot.2011.428

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