The Endomembrane network

The intracellular membrane system of eukaryotic cells comprises a variety of endomembranes including nuclear envelope, endoplasmic reticulum, Golgi apparatus, endosomes and tubular-vesicular transport carriers, connected by multiple routes. Many studies have highlighted the complexity of transport pathways that permit constant exchange between intracellular compartments.

Although the organization of the intracellular membrane system is not known, it is intimately linked to cell cytoskeleton architecture. Membranous transport carriers shuttling between compartments move along microtubules or are tethered and displaced on actin networks via molecular motors. One difficulty in studying endomembrane organization is that the connection between transport and cytoskeleton goes both ways: transport events also strongly influence cell cytoskeleton architecture.

Moreover, unlike the compact Golgi apparatus in mammalian cells and the endocytic recycling compartment, endosomes and various transport carriers are spatially dispersed and difficult to quantify. The large number of structures belonging to endomembranous populations makes studying their organization challenging.

Kristin Schauer and co-workers addressed this challenging task using the crossbow adhesive micropatterns and developed a mathematical algorithm to generate probabilistic density maps of the different endosome compartments.

Thanks to the normalizing effect of the micropatterns, great statistical relevance was obtained by analyzing and computing n cells (n=35-82) into a single 3D representation of the compartment.

Lysosome compartment

Early endosomes

Endoplasmic reticulum exit sites

Trans Golgi, secretion and retrograde transport

 

 

Analyzing drug effects on the endomembrane network As an example, the results on the right illustrate the effect of Cytochalasin D on the Rab6-positive endosome compartment. Looking at images of individual cells (left) yields little insight on the effect of this drug. However, when generating the density maps over 50-82 normalized cells, the effect becomes evident with a significant decrease of late endosomes at the plasma membrane over the adhesive edge. (More drug effect analyses can be found in the Nature Methods paper) The study demonstrates that significant morphological alterations of the endomembrane compartments can be consistently detected with as few as 20 cells. To determine the advantage of cellular standardization by micropatterning, Schauer et al. performed the same statistical test on nonpatterned cells. For strong phenotypes, such as Golgi dispersion after nocodazole treatment, they could detect significant differences, although data for eight times more cells were needed. In contrast, subtle differences, such as those seen upon treatment with cytochalasin D, were not detectable in unconstrained cells, whatever the number of cells that were analyzed.   Graph giving the average P value as a function of the number of cells that were analyzed

Reference:

Probabilistic density maps to study global endomembrane organization
Kristine Schauer, Tarn Duong, Kevin Bleakley, Sabine Bardin, Michel Bornens & Bruno Goud

Nature Methods 2010