Size and mobility of lipid domains tuned by geometrical constraints

Abstract

In the plasma membrane of eukaryotic cells, proteins and lipids are organized in clusters, the latter ones often called lipid domains or "lipid rafts." Recent findings highlight the dynamic nature of such domains and the key role of membrane geometry and spatial boundaries. In this study, we used porous substrates with different pore radii to address precisely the extent of the geometric constraint, permitting us to modulate and investigate the size and mobility of lipid domains in phase-separated continuous pore-spanning membranes (PSMs). Fluorescence video microscopy revealed two types of liquid-ordered (l o ) domains in the freestanding parts of the PSMs: (i) immobile domains that were attached to the pore rims and (ii) mobile, round-shaped l o domains within the center of the PSMs. Analysis of the diffusion of the mobile l o domains by video microscopy and particle tracking showed that the domains' mobility is slowed down by orders of magnitude compared with the unrestricted case. We attribute the reduced mobility to the geometric confinement of the PSM, because the drag force is increased substantially due to hydrodynamic effects generated by the presence of these boundaries. Our system can serve as an experimental test bed for diffusion of 2D objects in confined geometry. The impact of hydrodynamics on the mobility of enclosed lipid domains can have great implications for the formation and lateral transport of signaling platforms. diffusion | hydrodynamics | lipid rafts | membrane dynamics | pore-spanning membranes T he plasma membrane of eukaryotic cells compartmentalizes into lipid domains that enable the selective recruitment of specific proteins (1, 2). These domains are an important feature for regulating biological functions such as apical sorting, protein trafficking, and the clustering of proteins during signaling. The most prominent concept for membrane organization, the lipid raft theory, relates lipid phase separation [driven by interactions between cholesterol (chol), sphingolipids, and saturated phospholipids] to membrane protein partitioning and regulation (2, 3). Lipid domains such as rafts are difficult to observe in biological membranes due to their small size and dynamic nature (4). To understand the phase separation phenomena and the dynamics of lipid domains, the phase behavior of giant plasma membrane-derived vesicles (5, 6) as well as ternary model membranes containing two phospholipid species and chol have been investigated extensively in the last decade. In these model membranes, lipid phase separation between liquid-ordered (l o ) and liquid-disordered (l d ) phases is readily observed at low temperature (7-9). These raft-like domains grow several micrometers in size in giant unilamellar vesicles (GUVs) (7), while their mobility is preserved, whereas lipid domains in supported lipid bilayers are smaller but are largely immobile and do not form energy-minimized round shapes (10). Explanations for the size difference of lipid domains in native plasma membranes and model membranes ran...

Phase separation in pore-spanning membranes induced by differences in surface adhesion

“…At this stage, l b -or l o -domains become discernible, even though the time resolution was not sufficient to resolve the circular appearance of such domains in the f-PSMs that we have visualized previously. 44 Circular domains are indicative of a significant line tension, which has been determined by Schwille and coworkers for a similar lipid composition (DOPC/SM C 18:0 /Chol, 2 : 2 : 1) to be 1.2 pN. 60 Tsai and Feigenson 61 later demonstrated that an increase in the Chol content slightly reduces the line tension in such membrane mixtures.…”

“…19,[33][34][35][36][37][38][39][40][41][42] As yet, PSMs composed of PC/SM/Chol have been formed on gold functionalized pore rims, and hence, owing to the quenching of the fluorescence by the gold layer, s-PSMs could not be fluorescently observed lacking the information about l o /l d -phase separation of the entire PSM. 43,44 To overcome this drawback, we here make use of SiO x=1-2 functionalized porous silicon substrates 33 in combination with two fluorescent (l d -and l o -) markers allowing the visualization of f-PSMs and s-PSMs. Our results on DOPC/ SM/Chol PSMs with different Chol contents highlight the importance of the differences in adhesion of particular membrane lipids considerably altering the phase behaviour.…”

SNARE-Mediated Fusion of Single Chromaffin Granules with Pore-Spanning Membranes

“…The PSM is a membrane with two different areas (s-PSMs and f-PSMs), in which the membrane components diffuse differently (30,31). Moreover, at the boundary of s-PSM and f-PSM, the membrane is locally bent (29). It is conceivable that the topography of the PSM also influences the diffusion behavior.…”

“…To overcome the drawback of fully immobile docked vesicles and to observe possible intermediate fusion states by fluorescence microscopy in a time-resolved manner, we used planar pore-spanning membranes (PSMs) (28,29). These membranes consist of freestanding PSM (f-PSM) and supported PSM (s-PSM) areas and thus allow a comparison of the diffusive and fusion behavior of docked vesicles on the two different membrane types under otherwise exact same conditions.…”

Geometry‐Dependent Nonequilibrium Steady‐State Diffusion and Adsorption of Lipid Vesicles in Micropillar Arrays

“…Embryonic growth and development mechanisms also depend on diffusion, as does tumor growth and metastasis. Adsorbed liposomes onto structured surfaces can form fluid, supported lipid bilayers as realistic models for biomembranes capable of mimicking cellular functions such as transport and signaling . Micro‐ and nanopatterned surfaces can be utilized to mimic the geometries and scales of these environments, and to elucidate how biological phenomena that rely on diffusive transport are impacted in these spatially restricted environments …”