Section: New Results

3D tracking of endocytic and exocytic events using lattice light sheet microscopy

Participants : Cesar Augusto Valades Cruz, Ludovic Leconte, Jean Salamero, Charles Kervrann.

Figure 6. 3D tracking of Gal3-Atto647n (red) vs AP2-eGFP (green) adaptor protein in SUM159 cell.

The study of the whole cell dynamics of endocytic/exocytic-recycling events has proven difficult until recently because of lack of sensitivity, limited speed, photobleaching and phototoxicity associated with conventional imaging modalities. The Lattice Light Sheet Microscope (LLSM) allows to overcome these difficulties, yet reaching high spatial resolution. 3D images are captured for several minutes at a high acquisition frequency, and enables the study of signaling, transport, and stochastic self-assembly in complex environments. In addition, this imaging technique and 3D-tracking allow us to characterize the molecular machineries involved in the exocytosis and endocytosis mechanisms. We have the opportunity to observe a series of sequential events corresponding to the fusion with the plasma membrane (exocytosis) and the formation of endocytic carriers, including the trafficking of vesicles throughout the entire membrane system. We have got preliminary results of the coordination of vesicle recycling from the endosomal recycling compartment up to the plasma membrane using LLSM imaging and 3D tracking. In addition, we introduced a quantitative analysis of endocytosis dynamics of AP2 adaptor complex, Galectin-3 (see Figure 6) and Transferrin using single particle tracking analysis of 3D+time data. These case studies clearly demonstrated the advantage of lattice light sheet microscopy for imaging endocytic/exocytic events in single cells.

Software: THOTH and CPAnalysis (see Sections 6.2 and 6.6).

Collaborators: C. Wunder and L. Johannes (Institut Curie, PSL Research University, Cellular and Chemical

                          Biology, U1143 INSERM / UMR 3666 CNRS).