David Tareste, INSERM researcher (Biographical sketch)
Phone: +33 1 57 27 80 38
Agnès de Lacroix, research engineer
Shruti Mittal, postdoctoral fellow
Membrane fusion is central to many fundamental physiological processes, including neuritogenesis, synaptic transmission, viral infection, and mitochondrial dynamics. During all these events, two cellular compartments, initially separated and delimited by lipid bilayers, have to connect and merge in order to mix their membrane and aqueous components. Since biological membranes are designed to be stable, such fusion events are energetically costly and require the intervention of specialized proteins which help membranes to advance through the successive stages leading to fusion (Figure 1A). These proteins are also assisted by various regulatory factors (lipids or proteins) which facilitate fusion (for example, by inducing favorable membrane curvature or by creating micro-domains displaying a high local density of active proteins), and ensure that fusion occurs at the right time and place (for example, by trapping and releasing the fusion machinery in response to particular signals).
All intracellular membrane fusion events – except those involving mitochondria – are orchestrated by proteins from the SNARE family. The core of the SNARE fusion machinery consists of the assembly between cognate SNARE proteins, initially residing in the apposing membranes, which brings the lipid bilayers into close proximity and triggers their fusion (Figure 1B).
Figure 1. (A) The various stages of membrane fusion; adapted from (Jahn et al., Curr Opin Cell Biol 2002). (B) The core principle of SNARE-mediated membrane fusion.
Question and strategy
Our goal is to elucidate the molecular mechanisms of intracellular membrane fusion machineries using the in vitro reconstitution of candidate fusion proteins into a wide variety of membrane environments with defined and tunable biophysical properties (e.g. supported lipid bilayers, liposomes of various sizes), combined with membrane imaging, adhesion and fusion assays (e.g. confocal imaging, surface force measurements, giant liposomes micromanipulation, FRET-based lipid mixing assay; Figure 2).
Figure 2. Membrane adhesion and fusion assays. (A) Surface Forces Apparatus. (B) Giant liposomes micromanipulation. (C) FRET-based lipid mixing assay.
SNAREs of neurotransmission
Surface Force Apparatus (SFA) experiments between membrane-embedded SNARE proteins allowed us to determine the structural and energetic landscape of SNAREpins as they assemble across membranes (Figure 3A). Notably, they revealed that the energy accumulated during folding of a single SNAREpin corresponds closely with the energy required to drive membrane hemifusion. This approach also allowed us to understand how the regulatory protein Complexin (CPX) involved in Calcium-triggered exocytosis clamps SNAREpin assembly and therefore membrane fusion.
We also developed a large liposome/giant liposome assay that offers the possibility of following liposome adhesion and fusion independently in the same experiment through confocal microscopy (Figure 3B). Using this assay, we have demonstrated that the synaptic SM protein Munc18-1 plays (at least) two roles in membrane fusion: (i) to promote vesicle-plasma membrane adhesion, and (ii) to help overcoming the energetic barrier for fusion.
Figure 3. (A) Properties of SNAREpin assembly revealed through SFA experiments . (B) Adhesion and fusion of fluorescent t-SNARE large liposomes with initially non- fluorescent v-SNARE giant liposomes in the absence or the presence of Munc18-1.
SNAREs of neuritogenesis and Mitofusins
(Collaboration with the Rojo lab, Institut de Biochimie et Génétique Cellulaires, Bordeaux)
We now want to extend our current biophysical understanding of SNARE fusion machineries by studying the key molecular mechanisms and the regulatory factors of SNARE proteins involved in neurite outgrowth (neuritogenesis). In addition, we want to elucidate the mode of action of the other, largely unknown, intracellular fusion machinery: that controlling mitochondrial dynamics, which does not use SNAREs but some proteins called Mitofusins that are solely dedicated to mitochondrial fusion. The results obtained – combined with concepts that have emerged from a decade of valuable research on extracellular viral fusion and intracellular synaptic vesicle exocytosis – should reveal which elements (functional protein domains, lipid environment, etc.) and molecular mechanisms are conserved amongst all membrane fusion reactions.
Figure 4. Schematic representation of a neuron (left) and a mitochondrion (right).