Fluorescence imaging of lamellipodin-mediated biomolecular condensates on solid supported lipid bilayer membranes.

Biomolecular condensates play a major role in numerous cellular processes, including several that occur on the surface of lipid bilayer membranes. There is increasing evidence that cellular membrane trafficking phenomena, including the internalization of the plasma membrane through endocytosis, are mediated by multivalent protein-protein interactions that can lead to phase separation. We have recently found that proteins involved in the clathrin-independent endocytic pathway named Fast Endophilin Mediated Endocytosis can undergo liquid-liquid phase separation (LLPS) in solution and on lipid bilayer membranes. Here, the protein solution concentrations required for phase separation to be observed are significantly smaller compared to those required for phase separation in solution. LLPS is challenging to systematically characterize in cellular systems in general, and on biological membranes in particular. Model membrane approaches are more suitable for this purpose as they allow for precise control over the nature and amount of the components present in a mixture. Here we describe a method that enables the imaging of LLPS domain formation on solid supported lipid bilayers. These allow for facile imaging, provide long-term stability, and avoid clustering of vesicles and vesicle-attached features (such as buds and tethers) in the presence of multi-valent membrane interacting proteins.

Purification of Recombinant Human Amphiphysin 1 and its N-BAR Domain.

Bin/Amphiphysin/Rvs (BAR) proteins are known as classical membrane curvature generators during endocytosis. Amphiphysin, a member of the N-BAR sub-family of proteins that contain a characteristic amphipathic sequence at the N-terminus of the BAR domain, is involved in clathrin-mediated endocytosis. Full-length amphiphysin contains a ~ 400 amino acid long disordered linker connecting the N-BAR domain and a C-terminal Src homology 3 (SH3) domain. We express and purify recombinant amphiphysin and its N-BAR domain along with an N-terminal glutathione-S-transferase (GST) tag. The GST tag allows extraction of the protein of interest using affinity chromatography and is removed in the subsequent protease treatment and ion-exchange chromatography steps. In the case of the N-BAR domain, cleavage of the GST tag was found to cause precipitation. This issue can be minimized by adding glycerol to the protein purification buffers. In the final step, size exclusion chromatography removes any potential oligomeric species. This protocol has also been successfully used to purify other N-BAR proteins, such as endophilin, Bin1, and their corresponding BAR domains. Graphical overview.

Multivalent interactions between molecular components involved in fast endophilin mediated endocytosis drive protein phase separation.

A specific group of transmembrane receptors, including the ß1-adrenergic receptor (ß1-AR), is internalized through a non-clathrin pathway known as Fast Endophilin Mediated Endocytosis (FEME). A key question is: how does the endocytic machinery assemble and how is it modulated by activated receptors during FEME. Here we show that endophilin, a major regulator of FEME, undergoes a phase transition into liquid-like condensates, which facilitates the formation of multi-protein assemblies by enabling the phase partitioning of endophilin binding proteins. The phase transition can be triggered by specific multivalent binding partners of endophilin in the FEME pathway such as the third intracellular loop (TIL) of the ß1-AR, and the C-terminal domain of lamellipodin (LPD). Other endocytic accessory proteins can either partition into, or target interfacial regions of, these condensate droplets, and LPD also phase separates with the actin polymerase VASP. On the membrane, TIL promotes protein clustering in the presence of endophilin and LPD C-terminal domain. Our results demonstrate how the multivalent interactions between endophilin, LPD, and TIL regulate protein assembly formation on the membrane, providing mechanistic insights into the priming and initiation steps of FEME.

Kinetics of pore formation in stearoyl-oleoyl-phosphatidylcholine vesicles by pH sensitive cell penetrating peptide GALA.

In order to engineer endosomal escape of drug carrying liposomes into the cytoplasm of target cells, the kinetics of bilayer poration by cell penetrating peptides needs to be well understood. To this end, we have studied pH-dependent pore formation in stearoyl-oleoyl-phosphatidylcholine vesicles as a function of concentration of the peptide GALA. Using laser scanning confocal microscopy, we measured the rate of fluorophore transport from the suspending medium into giant unilamellar vesicles across bilayer pores induced by GALA under acidic pH conditions. We also measured the mean pore size of GALA-induced pores in large unilamellar vesicles by electron microscopy. We fitted a mathematical model of pore formation kinetics to the measured rate of fluorophore transport across the giant vesicle bilayer to estimate the rate of pore formation as a function of GALA concentration. We observed that the number of pores per vesicle and the pore density increased with increasing GALA concentration.

Mechanical and transport properties of chitosan-zwitterionic phospholipid vesicles.

Chitosan is a polysaccharide that has shown promise in liposomal drug delivery because of certain desirable properties such as muco-adhesivity, biodegradability and low toxicity. In this study, chitosan-bearing 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles were prepared using inverse phase precursor method to measure their mechanical and transport properties. We show that while an increase in chitosan: lipid molar ratio in the vesicle bilayer at pH 7 led to a substantial increase in its bending modulus, chitosan-mediated change in bending modulus was diminished at pH 4.5. Water permeability across the vesicle bilayer, as well as phospholipid diffusivity within supported lipid bilayers, were also found to decrease with increasing chitosan: lipid molar ratio. Together, these findings demonstrate that incorporation of chitosan in phospholipid bilayers modulates the mechanical and transport properties of liposomes which may affect their in vivo circulation time and drug release rate.

PEG-grafted phospholipids in vesicles: Effect of PEG chain length and concentration on mechanical properties.

Incorporation of low molecular weight poly-ethylene glycol (PEG) - grafted phospholipids in vesicle bilayers is known to increase the circulation time of liposomal drug delivery vehicles. Mechanical properties of giant unilamellar DPPC vesicles containing varying concentrations of DSPE-PEG (PEG MW: 550, 1000 and 2000) were measured by micropipette aspiration assay or osmotic swelling. While the area compressibility modulus did not change significantly, the bending modulus and water permeability of the bilayer was found to increase with increasing mole fraction of DSPE-PEG. This increase was more pronounced for higher molecular weight PEG. The measured bending modulus agreed with that predicted by scaling theory only at low mole fractions of DSPE-PEG. The water permeability was also measured as a function of the increase in area per lipid (due to steric repulsion between PEG chains), and for the same area per lipid, the PEG chain with MW 550 provided a greater resistance to water transport across the vesicle membrane compared to PEG 1000 and 2000. Lysis tension of the membrane, determined by osmotic lysis method at different loading rates showed a decrease in membrane strength on inclusion of the polymer lipid. These results suggest that liposome lifetime in the circulation and the rate of drug delivery are affected by the molecular weight and concentration of PEG in the bilayer.