Single Particle Trajectories Reveal Active Endoplasmic Reticulum Luminal Flow

Abstract

The Endoplasmic Reticulum (ER) is a contiguous membrane-bound network of sheet-like reservoirs and tubules extending throughout the cell. This morphology is maintained by membrane shaping proteins and supports the distribution of ER luminal content to distant sites. Motion of ER-luminal proteins was generally considered as passive, although measurements of protein mobility using fluorescence recovery after photo-bleaching (FRAP) have previously uncovered an energy dependence that is difficult to reconcile with passive diffusion. Based on recent advances in super-resolution microscopy, allowing to image individual ER-luminal proteins, we present a finer view of the ER luminal motion at the single molecule level. Using the Langevin equation together with local estimators of motion parameters, we found that luminal motion can be divided in two spatially separated categories: a slow diffusive-like motion at tubule junctions and a fast and directed motion in the tubules. The latter is present in multiple cell types, disappears after applying ATP-depletion treatments, is not detected when imaging ER membrane proteins and is coherent with transversal tubule contractions observed in fast Structured Illumination Microscopy (fSIM) experiments. Furthermore, this specific jump-diffusion motion allowed us to develop an algorithm, based on local trajectory density, to reconstruct the ER network of a cell from individual trajectories. Studying the characteristics of these networks from multiple cells, over the 36sec duration of the recordings, revealed that they form a main strongly connected component allowing redistribution of the luminal content over the entire ER. Finally, an analysis of the transient dynamic of the network showed that luminal content redistribution supported locally by alternating periods of uni-directional flows in tubules. Overall this analysis highlights the capacity of super-resolved single particle trajectories analysis to provide new insights about the dynamics of cellular structures.