Probabilistic density maps to study global endomembrane organization.
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.
Unité Mixte de Recherche 144, Centre National de la Recherche Scientifique, Institut Curie, Laboratory Molecular Mechanisms of Intracellular Transport, Paris, France. kristine.schauer@curie.fr <kristine.schauer@curie.fr>
Kristine
K
Schauer
Tarn
T
Duong
Kevin
K
Bleakley
Sabine
S
Bardin
Michel
M
Bornens
Bruno
B
Goud
D015415:Biomarkers; D002460:Cell Line; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology; D020868:Gene Silencing; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology*; D015739:Nocodazole / Q000494:pharmacology; D021381:Protein Transport; D015203:Reproducibility of Results; D050257:Tubulin Modulators / Q000494:pharmacology
D015415:Biomarkers; D050257:Tubulin Modulators; D015739:Nocodazole