Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large number of studies have focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, in which the preferential associations between cholesterol and saturated lipids drive the formation of relatively packed (or ordered) membrane domains that selectively recruit certain lipids and proteins. Recent studies have yielded new insights into this mechanism and its relevance in vivo, owing primarily to the development of improved biochemical and biophysical technologies.

The mystery of membrane organization: composition, regulation and roles of lipid rafts

The plasma membrane has a highly asymmetric distribution of lipids and contains dynamic nanodomains many of which are liquid entities surrounded by a second, slightly different, liquid environment. Contributing to the dynamics is a continuous repartitioning of components between the two types of liquids and transient links between lipids and proteins, both to extracellular matrix and cytoplasmic components, that temporarily pin membrane constituents. This make plasma membrane nanodomains exceptionally challenging to study and much of what is known about membrane domains has been deduced from studies on model membranes at equilibrium. However, living cells are by definition not at equilibrium and lipids are distributed asymmetrically with inositol phospholipids, phosphatidylethanolamines and phosphatidylserines confined mostly to the inner leaflet and glyco- and sphingolipids to the outer leaflet. Moreover, each phospholipid group encompasses a wealth of species with different acyl chain combinations whose lateral distribution is heterogeneous. It is becoming increasingly clear that asymmetry and pinning play important roles in plasma membrane nanodomain formation and coupling between the two lipid monolayers. How asymmetry, pinning, and interdigitation contribute to the plasma membrane organization is only beginning to be unraveled and here we discuss their roles and interdependence.

/pdf/interleaflet-coupling-pinning-and-leaflet-asymmetry-major-53s8fnunco.pdf

Interleaflet Coupling, Pinning, and Leaflet Asymmetry-Major Players in Plasma Membrane Nanodomain Formation.

Super-resolution microscopy techniques enable optical imaging in live cells with unprecedented spatial resolution. They unfortunately lack the temporal resolution required to directly investigate cellular dynamics at scales sufficient to measure molecular diffusion. These fast time scales are, on the other hand, routinely accessible by spectroscopic techniques such as fluorescence correlation spectroscopy (FCS). To enable the direct investigation of fast dynamics at the relevant spatial scales, FCS has been combined with super-resolution stimulated emission depletion (STED) microscopy. STED-FCS has been applied in point or scanning mode to reveal nanoscale diffusion behavior of molecules in live cells. In this protocol, we describe the technical details of performing point STED-FCS (pSTED-FCS) and scanning STED-FCS (sSTED-FCS) measurements, from calibration and sample preparation to data acquisition and analysis. We give particular emphasis to 2D diffusion dynamics in cellular membranes, using molecules tagged with organic fluorophores. These measurements can be accomplished within 4-6 h by those proficient in fluorescence imaging.

/pdf/measuring-nanoscale-diffusion-dynamics-in-cellular-membranes-3f29vji42v.pdf

Measuring nanoscale diffusion dynamics in cellular membranes with super-resolution STED-FCS.

HIV-1 Gag protein assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly PI(4,5)P2, at the inner leaflet of this membrane. Here, we address the question whether Gag is able to trap specifically PI(4,5)P2 or other lipids during HIV-1 assembly in the host CD4+ T lymphocytes. Lipid dynamics within and away from HIV-1 assembly sites were determined using super-resolution microscopy coupled with scanning fluorescence correlation spectroscopy in living cells. Analysis of HIV-1–infected cells revealed that, upon assembly, HIV-1 is able to specifically trap PI(4,5)P2 and cholesterol, but not phosphatidylethanolamine or sphingomyelin. Furthermore, our data showed that Gag is the main driving force to restrict the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. This is the first direct evidence highlighting that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol as a membrane nanoplatform for virus assembly.

https://advances.sciencemag.org/content/advances/5/10/eaaw8651.full.pdf

Fatma Betul Can

Papers

A comparative study on fluorescent cholesterol analogs as versatile cellular reporters