Abstract
A clear consensus concerning the mechanisms of intracellular secretory vesicle trafficking in astrocytes is lacking in the physiological literature. A good characterization of vesicle trafficking that may assist researchers in achieving that goal is the trajectory angle, defined as the angle between the trajectory of a vesicle and a line radial to the cell’s nucleus. In this study, we provide a precise definition of the trajectory angle, describe and compare two methods for its calculation in terms of measureable trafficking parameters, and give recommendations for the appropriate use of each method. We investigated the trafficking of vesicles containing excitatory amino acid transporter 2 (EAAT2) fluorescently tagged with enhanced green fluorescent protein (EGFP) to quantify and validate the precision of each method. The motion of fluorescent puncta—taken to represent vesicles containing EAAT2-EGFP—was found to be typical of secretory vesicle trafficking. An exact method for calculating the trajectory angle of these puncta produced no error but required large computation time. An approximate method reduced the requisite computation time but produced an error depending on the inverse of the ratio of the punctum’s initial distance from the nucleus centroid to its maximal displacement. Fitting this dependence to a power function allowed us to establish an exclusion distance from the centroid, beyond which the approximate method is less likely to produce an error above an acceptable 5 %. We recommend that the exact method be used to calculate the trajectory angle for puncta closer to the nucleus centroid than this exclusion distance.
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Abbreviations
- CB1R:
-
Cannabinoid receptor 1
- DIC:
-
Differential interference contrast
- EAAT2:
-
Excitatory amino acid transporter 2
- EGFP:
-
Enhanced green fluorescent protein
- Emd:
-
Emerald green fluorescent protein
- FITC:
-
Fluorescein isothiocyanate
- GFAP:
-
Glial fibrillary acidic protein
- HBSS:
-
Hank’s balanced salt solution
- IQR:
-
Interquartile range
- pro-ANP:
-
pro-Atrial natriuretic peptide
- RT:
-
Room temperature
- TGN:
-
Trans-Golgi network
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Acknowledgments
We thank Dr. Jeffrey D. Rothstein for providing the EAAT2-EGFP plasmid. We also thank Erik Malarkey for his assistance with the initial vesicle trafficking protocols. This study was funded by the National Institutes of Health (The Eunice Kennedy Shriver National Institute of Child Health and Human Development award HD078678).
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The authors declare that they have no conflict of interest.
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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.
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Supplementary material 1 (MPG 1679 kb)
Online Resource 1 Mobility of fluorescent puncta representing intracellular vesicles carrying EAAT2-EGFP. The astrocyte from Fig. 1 is shown. Puncta were imaged and tracked over a period of 40 s. Movement of trafficking puncta can be observed (see tracks Fig. 1e). Tracks representative of both non-directional and directional puncta are present (see Fig. 2a). Images were taken at a rate of 1.2 frames s−1, and the resulting time-lapse image was compressed to a 10 s movie (5 frames s−1). Image size, 145.7 μm by 108.9 μm.
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Cavender, C.E., Gottipati, M.K. & Parpura, V. Trafficking of excitatory amino acid transporter 2-laden vesicles in cultured astrocytes: a comparison between approximate and exact determination of trajectory angles. Amino Acids 47, 357–367 (2015). https://doi.org/10.1007/s00726-014-1868-y
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DOI: https://doi.org/10.1007/s00726-014-1868-y