Interleaflet Coupling of Lipid Nanodomains - Insights From in vitro Systems

Abstract

The plasma membrane is a complex system, consisting of two layers of lipids and proteins compartmentalized into small structures called nanodomains. Despite the asymmetric composition of both leaflets, coupling between the layers is surprisingly strong. This can be evidenced, for example, by recent experimental studies performed on phospholipid giant unilamellar vesicles showing that nanodomains formed in the outer layer are perfectly registered with those in the inner leaflet. Similarly, microscopic phase separation in one leaflet can induce phase separation in the opposing leaflet that would otherwise be homogeneous. In this review, we summarize the current theoretical and experimental knowledge that led to the current view that domains are - irrespective of their size - commonly registered across the bilayer. Mechanisms inducing registration of nanodomains suggested by theory and calculations are discussed. Furthermore, domain coupling is evidenced by experimental studies based on the sparse number of methods that can resolve registered from independent nanodomains. Finally, implications that those findings using model membrane studies might have for cellular membranes are discussed.

Keywords: biomembranes; domain registration; interleaflet coupling; membrane asymmetry; nanodomains; phase separation; plasma membranes.

FIGURE 1

Most probable driving forces for the registration of nanodomains. (A) Membrane undulations, (B) chain interdigitations, (C) registration of nanodomains by cholesterol, and (D) line tension.

FIGURE 2

Domain anti-registration versus partial registration. (A) Domains are classified as antiregistered when the domains localized in one leaflet (gray squares) are aligned with the nondomain region in the other leaflet (yellow squares). Such an arrangement is possible only if the domain and nondomain subregions occupy an equal area of the bilayer. (B) Higher amount of one of the regions inevitably leads to transversal overlap between the domain and nondomain subregions (yellow-gray squares surrounded by solid red).

FIGURE 3

Schematic representation of the direct visualization of lipid domains by fluorescence microscopy and AFM. (Top) Domain formation within GUVs can be assessed by fluorescence microscopy if, for example, a lipid dye with no affinity for the domains is used. For registered domains, the bulk membrane fluorescence is recorded while the domains have no intensity. Unregistered domains would present half the fluorescence intensity of the bulk membrane whenever there is no corresponding domain within the opposing leaflet. (Bottom) Using AFM, L_o nanodomains, for example, can be detected by measuring the height of the bilayer, since they will be thicker than the bulk membrane. Unregistered domains would however present an intermediate membrane height resulting from the opposing bulk membrane not contributing to the thickness increase.

FIGURE 4

Basic principles of MC-FRET for detection of nanodomains. (A) At a sufficiently high acceptor concentration in the bilayer, FRET between donors (green stars) and acceptors (red stars) occurs. (B) If donors and acceptors with high affinity for nanodomains are used, the presence of nanodomains leads to accumulation of donors and acceptors in the nanodomains, and consequently to a more efficient FRET. (C) The efficiency of FRET on a homogeneous vs a heterogeneous membrane with nanodomains.

FIGURE 5

FRET and domain registration. In a lipid bilayer, FRET (indicated by orange arrows) occurs within the same leaflet (intra-FRET) but also from one leaflet to the other one (inter-FRET). The efficiency of FRET, measured by time-resolved spectroscopy, depends on mutual organization of nanodomains. The efficiency of FRET is the lowest when nanodomains are in antiregistration (A), intermediate when they are independent (B), and the highest when the nanodomains are registered (C). Time-resolved fluorescence decays of donors in the presence of acceptors shown on panel (D) report on the kinetics of deexcitation and contain information about the size and concentration of nanodomains and their interleaflet organization.

FIGURE 6

Plasma membrane asymmetry. Schematic representation of the plasma membrane lipid composition. Distribution in both leaflets is depicted as a percentage of the total lipid class. Based on (Zachowski, 1993; Lingwood, 2011; Fujimoto and Parmryd, 2017).