Oral Presentation The 42nd Lorne Conference on Protein Structure and Function 2017

Whole cell imaging and analysis of all endocytic events at single molecule resolution. (#172)

Senthil Arumugam 1 , Srigokul Upadhyayula 2 , Ludger Johannes 3 , Eric Betzig 4 , Tomas Kirchhausen 2
  1. University of New South Wales, Randwick, NSW, Australia
  2. Harvard Medical School, Harvard University, Boston, MA, USA
  3. Chemical Biology Unit, Institut Curie, Paris, Ile de France, France
  4. Janelia Research Campus, Ashburn, VA, USA

So far, attempts to follow in real time endocytic events in cultured cells have been limited to events occurring on the ventral (attached) surface or relatively small regions of the dorsal (free) surface. The advent of the powerful lattice light-sheet microscope (LLSM) based on ultrathin light sheets for real time 3D imaging with single molecule sensitivity (Chen et al., 2014)1 provided us with the unique opportunity to simultaneously explore the dynamics of cargo uptake mediated by the clathrin-dependent and clathrin-independent routes within the same cell. We first implemented a LLSM system based on ultrathin light sheets for real time 3D imaging with single molecule sensitivity suitable to study live cells. We used LLSM for direct visualization and molecular counting during endocytosis of fluorescently tagged shiga toxins2 (purported to be involved in CME and CIE) and transferrin (classical CME cargo) in genome-edited cells expressing fluorescently tagged chimeras of clathrin light-chain A, the endocytic adaptor AP-2, and endophilin (constituent of the clathrin-dependent and independent routes)3,4. Using two distinct approaches, we measure clathrin-mediated endocytosis (CME), clathrin independent/ fast endophilin mediated endocytosis (FEME) in whole cells. We investigated the CIE using two different methodologies: (a) an image analysis based algorithm that assays CIE and (b) based on recruitment of endophilin to clathrin independent carriers / FEME processes at the plasma membrane as reported previously for STx We found that Shiga toxin was captured by a very large number of clathrin-coated structures. Further, we observed at the whole cell level that shiga toxin increases the formation of clathrin coated structures and endophilin assemblies.

  1. 1. Chen BC, et al. (2014) Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346(6208):1257998.
  2. 2. Ling H, et al. (1998) Structure of the shiga-like toxin I B-pentamer complexed with an analogue of its receptor Gb3. Biochemistry 37(7):1777-1788.
  3. 3. Boucrot E, et al. (2015) Endophilin marks and controls a clathrin-independent endocytic pathway. Nature 517(7535):460-465
  4. 4. Renard HF, et al. (2015) Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis. Nature 517(7535):493-496.