Current approaches to studying membrane organization

Abstract

The local structure and composition of the outer membrane of an animal cell are important factors in the control of many membrane processes and mechanisms. These include signaling, sorting, and exo- and endocytic processes that are occurring all the time in a living cell. Paradoxically, not only are the local structure and composition of the membrane matters of much debate and discussion, the mechanisms that govern its genesis remain highly controversial. Here, we discuss a swathe of new technological advances that may be applied to understand the local structure and composition of the membrane of a living cell from the molecular scale to the scale of the whole membrane.

Keywords: electron microscopy; membrane; proteins.

Figure 1.. A chemical view of the cell membrane.

( a) Image of membrane bilayer exhibits a patchwork mosaic of the distribution of lipids in the cell membrane and captures the lateral heterogeneity of the organization of membrane components in live cells. ( b) This bilayer is anchored to the cortical actin meshwork as visualized by rapid-freeze deep-etch tomographic renderings of the cortical surface closest to the membrane. ( c) Scanning electron microscopy image of a fibroblast cell. ( d, i) The distribution of metabolically incorporated 15N-sphingolipids in the plasma membrane region indicated above represented as the detected sphingolipid-specific 15N-enrichment with NanoSIMS. Orange and yellow regions represent plasma membrane domains that are enriched with 15N-sphingolipids. ( d, ii) The distribution of 18O-enrichment showing that the metabolically incorporated 18O-cholesterol is distributed relatively uniformly in the plasma membrane. Images ( a), ( b), and ( c) and ( d) are reproduced with permission from , , and , respectively.

Figure 2.. A dynamic view of the membrane.

( a) Picket-fence model where transmembrane proteins anchored to the actin membrane skeleton meshwork effectively act as rows of pickets and temporarily confine the movement of lipids and proteins through steric hindrance and circumferential slowing (packing or frictional) effects. ( b) Two representative trajectories of 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE) lipids on a living cell membrane recorded at a time resolution of 25 μs (40,500 frames/s) for a period of 56 ms (2,250 frames) where plausible compartments are shown in different colors. ( c) Schematic Voronoi lattices (purple) representative of actin-based compartment sizes together with simulated diffusion trajectories (cyan). Scale bar: 250 nm. ( d) Dependency of the apparent diffusion coefficient of di-palmitoyl phosphoethanolamine (DPPE) lipids on the area of observation (blue line). Images reproduced with permission from ( a, b) and ( c, d).

Figure 3.. A super-resolution view of membrane associated focal adhesions.

( a) Schematic model of the molecular architecture of focal adhesions. This model is based on the protein position measurement by interferometric photoactivatable localization microscopy (iPALM). The exquisite sensitivity of iPALM to register axial distances could determine the orientation of talin within focal adhesion. ( b– e) Top and side views of iPALM images of focal adhesions (white boxes, top-view panels) and corresponding z histograms. Color encodes distance from the coverglass surface in nanometers. Placing the fluorescent probe at the two ends of the talin rod show that the N-terminus of talin rod localizes close to the cytoplasmic tails of integrin ( b, c), whereas the C-terminus can localize up to 40 nm higher ( d, e). Scale bars: 5 μm ( b, d) and 500 nm ( c, e). Images reproduced with permission from . Abbreviations: ECM, extracellular matrix.

Figure 4.. A spatial view of the cell membrane: hierarchical organization of proteins.

( a) Quantitative analysis of the spatial distribution of glycosyl phosphatidylinositol-anchored proteins (GPI-APs). On flat regions of relatively constant fluorescence intensity (grayscale), the anisotropy images (pseudocolored, where low values indicate increased numbers of clusters) display a hierarchical distribution of GPI-AP (e.g., in the form of nanoclusters and characteristic distances between nanocluster-rich regions). Homo-Förster resonance energy transfer ( h-FRET) imaging reports on the molecular proximity of like fluorophores at the 1- to 10-nm scale but imaging is still diffraction-limited and has no access to the region between 10 and 300 nm. ( b) A super-resolution technique such as near-field scanning optical microscopy (NSOM) can provide access to these spatial scales and revealed GPI-AP nanoclusters to organize in 150- to 300-nm sized regions. Three-dimensional projection of a fluorescence intensity NSOM image with nanodomains (black arrows) and monomers (white arrows) of GPI-APs is shown. Contour dashed lines on the two-dimensional image illustrate the preference of nanodomains to concentrate on specific sites as hotspots. Images reproduced with permission from ( a) and ( b).

Figure 5.. Space and time resolution of current methods.

Representation of the landscape that current methods occupy in the space and time axes. Abbreviations: FCS, fluorescence correlation spectroscopy; FRET, Förster resonance energy transfer; NSOM, near-field scanning optical microscopy; PALM, photoactivatable localization microscopy; SPT, single particle tracking; STED, stimulated emission depletion; STICS, spatio-temporal image correlation spectroscopy; STORM, stochastic optical reconstruction microscopy.