Second Membrane Physiology Symposium, April 30 to May 1, 2025, Napa, California, USA

'Shooting gallery' for membrane proteins provides new insights into complexities of their function and structural dynamics.

Monomeric alpha-synuclein (aSyn) is a well characterised protein that importantly binds to lipids. aSyn monomers assemble into amyloid fibrils which are localised to lipids and organelles in insoluble structures found in Parkinson’s disease patient’s brains. Previous work to address pathological aSyn-lipid interactions has focused on using synthetic lipid membranes, which lack the complexity of physiological lipid membranes. Here, we use physiological membranes in the form of synaptic vesicles (SV) isolated from rodent brain to demonstrate that lipid-associated aSyn fibrils are more easily taken up into iPSC-derived cortical i3Neurons. Lipid-associated aSyn fibril characterisation reveals that SV lipids are an integrated part of the fibrils and while their fibril morphology differs from aSyn fibrils alone, the core fibril structure remains the same, suggesting the lipids lead to the increase in fibril uptake. Furthermore, SV enhance the aggregation rate of aSyn, yet increasing the SV:aSyn ratio causes a reduction in aggregation propensity. We finally show that aSyn fibrils disintegrate SV, whereas aSyn monomers cause clustering of SV using small angle neutron scattering and high-resolution imaging. Disease burden on neurons may be impacted by an increased uptake of lipid-associated aSyn which could enhance stress and pathology, which in turn may have fatal consequences for neurons.

John A. Raven.

Sphingolipids and membrane biology as determined from genetic models

Large scale in vivo recordings to study neuronal biophysics

The Lipid-Modifying Enzyme SMPDL3B Negatively Regulates Innate Immunity.

This paper unifies and outlines some important points made by recent experimental work, dealing with MS ion channels and growth cone motility. The considerable work carried out in cell biology, neurochemistry and membrane biophysics in the past decade allows us to imagine the neurite growth in term of known molecules and chains of simple mechanisms. On the other hand, it is becoming increasingly clear the real complexity of the growth cone behavior. There remain important gaps in our understanding of the role of MS channels in growth cone motility. At present, a major limitation for physiological work is the lack of specific blockers. An encouraging development of molecular genetic studies of Drosophila SAK channels (60) will probably provide valuable tools for straightforward experiments.

Supplementary material from "A new approach for investigating the response of lipid membranes to electrocompression by coupling droplet mechanics and membrane biophysics"

Membrane Biophysics - Planar Lipid Bilayers and Spherical Liposomes

Cellular membrane alterations are commonly observed in many diseases, including Alzheimer's disease (AD). Membrane biophysical properties, such as membrane molecular order, membrane fluidity, organization of lipid rafts, and adhesion between membrane and cytoskeleton, play an important role in various cellular activities and functions. While membrane biophysics impacts a broad range of cellular pathways, this review addresses the role of membrane biophysics in amyloid-β peptide aggregation, Aβ-induced oxidative pathways, amyloid precursor protein processing, and cerebral endothelial functions in AD. Understanding the mechanism(s) underlying the effects of cell membrane properties on cellular processes should shed light on the development of new preventive and therapeutic strategies for this devastating disease.

Fluorescent sterols as tools in membrane biophysics and cell biology

The discovery of spin echoes by Erwin Hahn in 1950 [ 19] has provided seminal ideas for many of the important concepts of modern NMR. For example, the observation of transient ‘free induction decay’ (FID) signals, following one or more short r.f. pulses applied at a frequency close to the average nuclear Larmor frequencyw0,led ultimately to Fourier transform NMR spectroscopy. Also, today it is impossible to conceive of preparing complex spin systems in well defined, ‘interesting’, non-equilibrium states without using pulse sequences of one sort or another. Biological and model membranes are two systems for which NMR, and especially deuterium (2H) NMR, has provided special insights in recent years. The2H NMR techniques that have proved to be fruitful up to now in elucidating the properties of membranes are very close to those described in Hahn’s original work, so that this is a particularly suitable place to discuss recent work in this area. We shall discuss several studies in our laboratory of membrane dynamics using2H NMR and shall try to place this within the perspective of current developments in membrane biophysics, which is a vibrant and dynamic field at the present time. Membrane biophysics is in the process of generating an enormous literature, so that our selection of background material is necessarily personal, being severely limited by space considerations to questions that we deem to be of potential interest to a general audience and, at the same time, amenable to study in our own laboratory.

We have used a combination of histochemical, electrophysiological, and behavioral approaches to study signal transduction, membrane biophysics, and chemosensory function in the neurons of the mouse Grueneberg ganglion (GG) olfactory subsystem. The GG is a recently appreciated collection of ~1,000 clustered primary olfactory neurons located at the anterior tip of the mammalian nasal cavity. Despite their far-forward position, GG neurons are fully trapped beneath a keratinized epithelium and are wrapped by glial cells. This raises the question of how they contribute to the sense of smell. We found that GG neurons have key components of cGMP signal transduction pathway and are molecularly similar to GC-D neurons, which project to the enigmatic necklace glomeruli in the olfactory bulb. In electrophysiological analyses, individual GG neurons spontaneously discharged action potentials in one of three distinct temporal patterns that were stable for >20 min. An auxiliary fast-inactivating Na+ current accounted for the various discharge patterns in computer simulations of the neuronal ionic currents. Despite differences in baseline activity, the majority of GG neurons responded to specific mammalian pheromones. In behavioral experiments, we found that the weaning of adolescent mice induced GG activity; however, the effects did not depend on ambient temperature or the presence of other animals. Because GG neurons reside on a dense vascular bed, have specialized access to serum contents, and directly responded to pressure ejections of serum, their activity can likely be modulated by internally circulating hormones or proteins associated with specific physiological states such as stress. Taken together, our results demonstrate unusual molecular and functional aspects of a morphologically and anatomically atypical olfactory nerve.

A new method for quantifying lipid-lipid interactions within biomimetic membranes undergoing electrocompression is demonstrated by coupling droplet mechanics and membrane biophysics. The membrane properties are varied by altering the lipid packing through the introduction of cholesterol. Pendant drop tensiometry is used to measure the lipid monolayer tension at an oil-water interface. Next, two lipid-coated aqueous droplets are manipulated into contact to form a bilayer membrane at their adhered interface. The droplet geometries are captured from two angles to provide accurate measurements of both the membrane area and the contact angle between the adhered droplets. Combining the monolayer tension and contact angle measurements enables estimations of the membrane tension with respect to lipid composition. Then, the membrane is electromechanically compressed using a transmembrane voltage. Electrostatic pressure, membrane tension and the work necessary for bilayer thinning are tracked, and a model is proposed to capture the mechanics of membrane compression. The results highlight that a previously unaccounted for energetic term is produced during compression, potentially reflecting changes in the lateral membrane structure. This residual energy is eliminated in cases with cholesterol mole fractions of 0.2 and higher, suggesting that cholesterol diminishes these adjustments.

Due to the lack of measurement techniques suitable for examining compartments of intact, living cells, membrane biophysics is almost exclusively investigated in the plasma membrane despite the fact that its alterations in intracellular organelles may also contribute to disease pathogenesis. Here, we employ a novel, easy-to-use, confocal microscopy-based approach utilizing F66, an environment-sensitive fluorophore in combination with fluorescent organelle markers and quantitative image analysis to determine the magnitude of the molecular order-related dipole potential in the plasma membrane and intracellular organelles of various tumor and neural cell lines. Our comparative analysis demonstrates considerable intracellular variations of the dipole potential that may be large enough to modulate protein functions, with an inward decreasing gradient on the route of the secretory/endocytic pathway (plasma membrane >> lysosome > Golgi > endoplasmic reticulum), whereas mitochondrial membranes are characterized by a dipole potential slightly larger than that of lysosomes. Our approach is suitable and sensitive enough to quantify membrane biophysical properties selectively in intracellular compartments and their comparative analysis in intact, living cells, and, therefore, to identify the affected organelles and potential therapeutic targets in diseases associated with alterations in membrane lipid composition and thus biophysics such as tumors, metabolic, neurodegenerative, or lysosomal storage disorders.