Forschung

Mikroskopische Anatomie und Strukturbiologie

Research

The Department of Microscopic Anatomy and Structural Biology led by Benoît Zuber develops and applies advanced methods to explore the fine structure of organs, cells, and proteins. To this end we mainly use cryo-electron microscopy and serial block face scanning electron microscopy

Time-resolved structural study of exocytosis

Isolated neuronal synapse

In our brain, information travels along the axon of neurons in the form of electrical signals. When the signal reaches the end of the axon, chemical messagers (neurotransmitters) are released and are detected by the neigbouring neuron, which in turn processes the signal. This happens at a specialized and asymetric interneuronal contact site called the synapse. Neurotransmitters are contained in membrane-bounded synaptic vesicles. Typically a synapse contains hundreds of vesicles. When an electrical signal arrives to the synapse, it triggers the entry of calcium in the presynaptic neuron, which in turn triggers the fusion of one or a few vesicles with the plasma membrane, resulting in the release of neurotransmitters in the extracellular space between the two neurons.

A number of questions about the mechanism of fusion remain open. We approach them with an original method where we analyze by cryo-electron tomography (LINK) the 3-dimensional ultrastructure of native isolated synapses (synaptosomes) that were fixed by vitrification milliseconds after exocytosis has been triggered. We focus in particular on the structure of membranes, as well as on the modification occurring to the dense network of filaments interconnecting the vesicles.

Collaborators: Vladan Lucic (MPI Biochemistry, Martinsried), Harvey McMahon (MRC LMB, Cambridge), Henning Stahlberg (University of Basel).

Membrane patches isolated from neuroendocrine cells

Neuroendocrine cells are another type of cells exhibiting calcium-triggered exocytosis. In this case large dense core vesicles filled with hormones fuse with the plasma membrane and release their content in the blood stream. Although the proteins involved in neuroendocrine secretion are in part identical and in part homolog to the neuronal proteins, the kinetics of neuroendocine secretion are slower those of neuronal secretion. In order to study the ultrastructural changes taking place in neuroendocrine secretion we have developed a system where the plasma membrane and associated structures, such as docked vesicles and the actin cortex, are isolated directly on an electron microscopy grid. The method, termed iMEM (isolation of Membrane patches for cryo-Electron Microscopy), allows a quick preparation of thin specimens directly observable by cryo-EM or cryo-ET. Furthermore, since the cytoplasmic side of the plasma membrane is exposed, physiological events can be easily triggered chemically, and intracellular structures can be labelled with gold-coupled labels.

Reconstituted SNARE fusion

The SNARE proteins mediate exocytosis membrane fusion. A proteoliposome in vitro reconstitution system initiated by J. Rothman’s lab has allowed to biochemically dissect how a population of proteoliposome mimicking synaptic vesicles fuse with a population of proteoliposome mimicking the plasma membrane. We analyse structural changes occuring at the level of the membranes and of the proteins by cryo-electron tomography of this system in order to better understand the function of the fusion machinery.

Collaborators: Harvey McMahon (MRC LMB, Cambridge)

 

Pore-forming toxin aerolysin

Many pathogenic bacteria produce protein toxins. The largest class of toxins consists of pore-forming toxins. They are secreted by bacteria in a water-soluble form and after binding to a receptor on target cells, typically eukaryotic cells, they insert and form a pore in their membrane. The cells become leaky, which may eventually lead to cell death. Production of pore-forming proteins is not only found in bacteria but also in other kingdoms of life, where they may assume different roles, e.g. in the defense against pathogens by the immune system. Fresh water bacteria Aeromonas hydrophila produce toxin aerolysin, which is the archetype of a large family of pore-forming proteins present in all kingdoms of life. Secreted water-soluble aerolysin binds to glycosylphosphatidylinositol (GPI) anchored membrane proteins, which are found in all eukaryotes. There, aerolysin C-terminal part gets cleaved off by host cell proteases, it forms a homoheptameric complex. The complex then rapidly forms a β-barrel pore in the plasma membrane. We have used single particle cryo-electron microscopy to obtain the atomic structure of aerolysin variants (mutants) blocked at different stages of the pore formation process Iacovache et al. (2016) Nature communications 7:12062. Our structures revealed that a loop refolds to form of long β-barrel, that a major collapse of the protein leads to membrane insertion of the barrel and, finally, that the cytoplasmic tip of the β-barrel folds outwards to form a rivet within the hydrophobic part of the membrane and thereby firmly anchor the pore in the membrane. Our aim is now to further dissect the mechanism of pore formation to understand in greater details how a water-soluble protein can insert in a membrane bilayer.

Collaborators: Gisou van der Goot (EPFL), Matteo Dal Perraro (EPFL), Nuria Cirauqui (EPFL/Federal University of Rio de Janeiro)