Geert van den Bogaart

Membrane protein sequestering by ionic protein–lipid interactions

Neuronal exocytosis is catalysed by the SNAP receptor protein syntaxin-1A, which is clustered in the plasma membrane at sites where synaptic vesicles undergo exocytosis. However, how syntaxin-1A is sequestered is unknown. Here we show that syntaxin clustering is mediated by electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Using super-resolution stimulated-emission depletion microscopy on the plasma membranes of PC12 cells, we found that PIP2 is the dominant inner-leaflet lipid in microdomains about 73 nanometres in size. This high accumulation of PIP2 was required for syntaxin-1A sequestering, as destruction of PIP2 by the phosphatase synaptojanin-1 reduced syntaxin-1A clustering. Furthermore, co-reconstitution of PIP2 and the carboxy-terminal part of syntaxin-1A in artificial giant unilamellar vesicles resulted in segregation of PIP2 and syntaxin-1A into distinct domains even when cholesterol was absent. Our results demonstrate that electrostatic protein–lipid interactions can result in the formation of microdomains independently of cholesterol or lipid phases.

Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions

Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science.

One SNARE complex is sufficient for membrane fusion

In eukaryotes, most intracellular membrane fusion reactions are mediated by the interaction of SNARE proteins that are present in both fusing membranes. However, the minimal number of SNARE complexes needed for membrane fusion is not known. Here we show unambiguously that one SNARE complex is sufficient for membrane fusion. We performed controlled in vitro Förster resonance energy transfer (FRET) experiments and found that liposomes bearing only a single SNARE molecule are still capable of fusion with other liposomes or with purified synaptic vesicles. Furthermore, we demonstrated that multiple SNARE complexes do not act cooperatively, showing that synergy between several SNARE complexes is not needed for membrane fusion. Our findings shed new light on the mechanism of SNARE-mediated membrane fusion and call for a revision of current views of fusion events such as the fast release of neurotransmitters.

Lateral diffusion of membrane proteins

We measured the lateral mobility of integral membrane proteins reconstituted in giant unilamellar vesicles (GUVs), using fluorescence correlation spectroscopy. Receptor, channel, and transporter proteins with 1-36 transmembrane segments (lateral radii ranging from 0.5 to 4 nm) and a alpha-helical peptide (radius of 0.5 nm) were fluorescently labeled and incorporated into GUVs. At low protein-to-lipid ratios (i.e., 10-100 proteins per microm(2) of membrane surface), the diffusion coefficient D displayed a weak dependence on the hydrodynamic radius (R) of the proteins [D scaled with ln(1/R)], consistent with the Saffman-Delbruck model. At higher protein-to lipid ratios (up to 3000 microm(-2)), the lateral diffusion coefficient of the molecules decreased linearly with increasing the protein concentration in the membrane. The implications of our findings for protein mobility in biological membranes (protein crowding of approximately 25,000 microm(-2)) and use of diffusion measurements for protein geometry (size, oligomerization) determinations are discussed.

Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment

Synaptic-vesicle exocytosis is mediated by the vesicular Ca2+ sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca2+ through PIP2. This interaction allows both Ca2+-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca2+ triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca2+ influx bringing the vesicle membrane close enough for membrane fusion.

Synaptotagmin-1 may be a distance regulator acting upstream of SNARE nucleation

Synaptotagmin-1 triggers Ca2+-sensitive, rapid neurotransmitter release by promoting interactions between SNARE proteins on synaptic vesicles and the plasma membrane. How synaptotagmin-1 promotes this interaction is unclear, and the massive increase in membrane fusion efficiency of Ca2+-bound synaptotagmin-1 has not been reproduced in vitro. However, previous experiments have been performed at relatively high salt concentrations, screening potentially important electrostatic interactions. Using functional reconstitution in liposomes, we show here that at low ionic strength SNARE-mediated membrane fusion becomes strictly dependent on both Ca2+ and synaptotagmin-1. Under these conditions, synaptotagmin-1 functions as a distance regulator that tethers the liposomes too far from the plasma membrane for SNARE nucleation in the absence of Ca2+, but while bringing the liposomes close enough for membrane fusion in the presence of Ca2+. These results may explain how the relatively weak electrostatic interactions between synaptotagmin-1 and membranes substantially accelerate fusion.

On the Mechanism of Pore Formation by Melittin

The mechanism of pore formation of lytic peptides, such as melittin from bee venom, is thought to involve binding to the membrane surface, followed by insertion at threshold levels of bound peptide. We show that in membranes composed of zwitterionic lipids, i.e. phosphatidylcholine, melittin not only forms pores but also inhibits pore formation. We propose that these two modes of action are the result of two competing reactions: direct insertion into the membrane and binding parallel to the membrane surface. The direct insertion of melittin leads to pore formation, whereas the parallel conformation is inactive and prevents other melittin molecules from inserting, hence preventing pore formation.

Long Unfolded Linkers Facilitate Membrane Protein Import Through the Nuclear Pore Complex

Natively unfolded linkers facilitate nuclear membrane protein import. Active nuclear import of soluble cargo involves transport factors that shuttle cargo through the nuclear pore complex (NPC) by binding to phenylalanine-glycine (FG) domains. How nuclear membrane proteins cross through the NPC to reach the inner membrane is presently unclear. We found that at least a 120-residue-long intrinsically disordered linker was required for the import of membrane proteins carrying a nuclear localization signal for the transport factor karyopherin-α. We propose an import mechanism for membrane proteins in which an unfolded linker slices through the NPC scaffold to enable binding between the transport factor and the FG domains in the center of the NPC.

Molecular sieving properties of the cytoplasm of Escherichia coli and consequences of osmotic stress

We determined the diffusion coefficients (D) of (macro)molecules of different sizes (from ∼0.5 to 600 kDa) in the cytoplasm of live Escherichia coli cells under normal osmotic conditions and osmotic upshift. D values decreased with increasing molecular weight of the molecules. Upon osmotic upshift, the decrease in D of NBD‐glucose was much smaller than that of macromolecules. Barriers for diffusion were found in osmotically challenged cells only for GFP and larger proteins. These barriers are likely formed by the nucleoid and crowding of the cytoplasm. The cytoplasm of E. coli appears as a meshwork allowing the free passage of small molecules while restricting the diffusion of bigger ones.

Protein mobility and diffusive barriers in Escherichia coli : consequences of osmotic stress

The effect of osmotic stress on the intracellular diffusion of proteins in Escherichia coli was studied, using a pulsed version of fluorescence recovery after photo‐bleaching, pulsed‐FRAP. This method employs sequences of laser pulses which only partly bleach the fluorophores in a cell. Because the cell size and geometry are taken into account, pulsed‐FRAP enables to measure diffusion in very small cells of different shapes. We found that upon an osmotic upshock from 0.15 to 0.6 Osm, imposed by NaCl or sorbitol, the apparent intracellular diffusion (D) of mobile green fluorescent protein (GFP) decreased from 3.2 to 0.4 μm2 s−1, whereas the membrane permeable glycerol had no effect. Exposing E. coli cells to higher osmolalities (> 0.6 Osm) led to compartmentalization of the GFP into discrete pools, from where the GFP could not escape. Although free diffusion through the cell was hindered, the mobility of GFP in these pools was still relatively high (D∼0.4 μm2 s−1). The presence of osmoprotectants restored the effect of osmotic stress on the protein mobility and apparent compartmentalization. Also, lowering the osmolality from 0.6 Osm back to 0.15 Osm restored the mobility of GFP. The implications of these findings in terms of heterogeneities and diffusive barriers inside the cell are discussed.