Plasma Membrane Plasticity of Xenopus laevis Oocyte Imaged with Atomic Force Microscopy

Abstract

Proteins are known to form functional clusters in plasma membranes. In order to identify individual proteins within clusters we developed a method to visualize by atomic force microscopy (AFM) the cytoplasmic surface of native plasma membrane, excised from Xenopus laevis oocyte and spread on poly-L-lysine coated glass. After removal of the vitelline membrane intact oocytes were brought in contact with coated glass and then rolled off. Inside-out oriented plasma membrane patches left at the glass surface were first identified with the lipid fluorescent marker FM1-43 and then scanned by AFM. Membrane patches exhibiting the typical phospholipid bilayer height of 5 nm showed multiple proteins, protruding from the inner surface of the membrane, with heights of 5 to 20 nm. Modelling plasma membrane proteins as spherical structures embedded in the lipid bilayer and protruding into the cytoplasm allowed an estimation of the respective molecular masses. Proteins ranged from 35 to 2,000 kDa with a peak value of 280 kDa. The most frequently found membrane protein structure (40/μm²) had a total height of 10 nm and an estimated molecular mass of 280 kDa. Membrane proteins were found firmly attached to the poly-L-lysine coated glass surface while the lipid bilayer was found highly mobile. We detected protein structures with distinguishable subunits of still unknown identity. Since X. laevis oocyte is a generally accepted expression system for foreign proteins, this method could turn out to be useful to structurally identify specific proteins in their native environment at the molecular level.

Imaging CFTR in its native environment

“…We developed a method to isolate the plasma membrane of oocytes on a glass support to study membrane proteins and lipids by AFM [67,83]. After removal of the vitelline membrane, intact oocytes are brought into contact with poly-L-lysine-coated glass and then rolled off.…”

“…Instead of cytoskeleton, we found large areas of lipid membrane without any protruding proteins indicating a flow of lipids over the surface after isolation. It was shown that a phospholipid bilayer lacking proteins could be removed by repetitive scanning, whereas the lipid bilayer that contains proteins resists multiple scanning [83]. Figure 8 shows a complex scenario indicating high plasticity of plasma membranes.…”

Steroids dilate nuclear pores imaged with atomic force microscopy

“…NPC conformational changes, induced by physiological stimuli, can be detected by atomic force microscopy (AFM) (Binnig and Quate, 1986), a nanoresolution technique needing no complex preparation procedure of the biological sample. AFM has proved to be a suitable approach for imaging of single molecules (Hinterdorfer et al, 1996;Engel and Muller, 2000;Hansma et al, 2003;Sitko et al, 2003) and native biomembranes (Franco-Obregon et al, 2000;Schillers et al, 2000;Schä fer et al, 2002;Fotiadis et al, 2003;Oberleithner et al, 2003). Latter technique has been applied more and more frequently over the past few years for identifying processes of nuclear pore regulation (Oberleithner et al, 1994StehnoBittel et al, 1995b;Perez-Terzic et al, 1996;Rakowska et al, 1998;Stoffler et al, 1999b;Wang and Clapham, 1999;Shahin et al, 2001;Moore-Nichols et al, 2002;Jaggi et al, 2003).…”

Direct Aldosterone Action on Mouse Cardiomyocytes Detected with Atomic Force Microscopy

“…However, images taken from whole cells do not allow to distinguish between nanoenfoldings of the plasma membrane lipid bilayer or membrane protein protrusions. Such a distinction is feasible only after plasma membrane isolation [36]. In this study, we put the focus on whole cells instead of membrane patches.…”