Abstract
Comparing the distribution of cytoplasmic particles and organelles between different experimental conditions can be challenging due to the heterogeneous nature of cell morphologies. The border-to-border distribution method was created to enable the quantitative analysis of fluorescently labeled cytoplasmic particles and organelles of multiple cells from images obtained by confocal microscopy. The method consists of four steps: (1) imaging of fluorescently labeled cells, (2) division of the image of the cytoplasm into radial segments, (3) selection of segments of interest, and (4) population analysis of fluorescence intensities at the pixel level either as a function of distance along the selected radial segments or as a function of angle around an annulus. The method was validated using the well-characterized effect of brefeldin A (BFA) on the distribution of the vesicular stomatitis virus G protein, in which intensely labeled Golgi membranes are redistributed within the cytoplasm. Surprisingly, in untreated cells, the distribution of fluorescence in Golgi membrane-containing radial segments was similar to the distribution of fluorescence in other G protein-containing segments, indicating that the presence of Golgi membranes did not shift the distribution of G protein towards the nucleus compared to the distribution of G protein in other regions of the cell. Treatment with BFA caused only a slight shift in the distribution of the brightest G protein-containing segments which had a distribution similar to that in untreated cells. Instead, the major effect of BFA was to alter the annular distribution of G protein in the perinuclear region.
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Abbreviations
- BFA:
-
Brefeldin A
- BTBDM:
-
Border-to-border distribution method
- ER:
-
Endoplasmic reticulum
- MTOC:
-
Microtubule organizing center
- PNH:
-
Perinuclear high
- PNL:
-
Perinuclear low
- VSV:
-
Vesicular stomatitis virus
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Acknowledgments
This study was supported by grants from the United States National Institute of Allergy and Infectious Diseases R01 AI015892 and R01 AI105012 (DSL) and National Cancer Institute RO1 CA127621 (DAO). We also acknowledge the support of the Cellular Imaging Shared Resource and the Cell and Virus Vector Core Laboratory of the Comprehensive Cancer Center of Wake Forest University supported by NCI grant P30 CA012197.404
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The authors declare that they have no competing interests.
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Yacovone, S.K., Ornelles, D.A. & Lyles, D.S. The border-to-border distribution method for analysis of cytoplasmic particles and organelles. Cell Tissue Res 363, 351–360 (2016). https://doi.org/10.1007/s00441-015-2265-x
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DOI: https://doi.org/10.1007/s00441-015-2265-x