We developed a computational imaging approach that describes the three-dimensional spatial organization of endomembranes from micromanipulation-normalized mammalian cells with probabilistic density maps. Applied to several well-known marker proteins, this approach revealed the average steady-state organization of early endosomes, multivesicular bodies or lysosomes, endoplasmic reticulum exit sites, the Golgi apparatus and Golgi-derived transport carriers in crossbow-shaped cells. The steady-state organization of each tested endomembranous population was well-defined, unique and in some cases depended on the cellular adhesion geometry. Density maps of all endomembrane populations became stable when pooling several tens of cells only and were reproducible in independent experiments, allowing construction of a standardized cell model. We detected subtle changes in steady-state organization induced by disruption of the cellular cytoskeleton, with statistical significance observed for just 20 cells. Thus, combining micropatterning with construction of endomembrane density maps allows the systematic study of intracellular trafficking determinants.
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We acknowledge L. Sengmanivong of the Nikon Imaging Centre at Institut Curie–Centre National de la Recherche Scientifique and V. Fraisier of the Plate-forme Imagerie Cellulaire et Tissulaire–Infrastructures en Biologie Santé et Agronomie Imaging Facility for their extensive help with microscopes and in particular their help using the deconvolution service of the facility. We thank J.-B. Sibarita for advice on image analysis including use of the multidimensional image analysis program and fruitful discussion during early phases of the project; I. Brito for statistical advice; W. Hong (Institute of Molecular and Cell Biology, Singapore) for providing the Sec13 antibody; M. Piel, A. Azioune and J. Fink for help with microprinting; and G. Egea, S. Miserey, A. Echard and J. Enninga for critical reading of the manuscript. K.S. received funding from the Fondation pour la Recherche Médicale en France and Association pour la Recherche sur le Cancer. This project was supported by grants from the Centre National de la Recherche Scientifique and Institut Curie.
The authors declare no competing financial interests.
Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Notes 1–2 (PDF 2412 kb)
Maximum intensity projection of the deconvolved fluorescence of GFP-Rab6-positive cells (n = 82) under control conditions. (AVI 2507 kb)
Maximum intensity projection of the deconvolved fluorescence of GFP-Rab6-positive cells (n = 47) after nocodazole treatment. (AVI 1383 kb)
Maximum intensity projection of the deconvolved fluorescence of GFP-Rab6-positive cells (n = 50) after cytochalasin D treatment. (AVI 1667 kb)
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Schauer, K., Duong, T., Bleakley, K. et al. Probabilistic density maps to study global endomembrane organization. Nat Methods 7, 560–566 (2010) doi:10.1038/nmeth.1462
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