Stacking in lipid vesicle-tubulin mixtures is an artifact of negative staining.

Abstract

Multilamellar stacking seen in negatively stained lipid vesicle-tubulin mixtures has been attributed to lipid-protein interactions (Caron, J . M., and R. D . Berlin, 1979, 1. Cell Biol. 81 :665-671) . We show that this stacking is produced by the phosphotungstic acid used for staining, independent of the presence of tubulin in the sample . The morphology of negatively stained single bilayer vesicles obtained from dimyristoyl phosphatidylcholine or egg lecithin is specifically dependent upon the choice of metal stain. Uranyl oxalate maintains the appearance of unilamellar vesicles . After staining with sodium tungstate, the lipids form a network of multilayered lamellae with a periodicity of -55 Á. Phosphotungstic acid produces stacks of flattened vesicles with a period of -115 Á as well as broader multilamellar structures having a 55 Á repeat . The stain-determined morphology is not markedly altered by sample concentration, incubation time, or temperature, or by the presence of tubulin .Caron and Berlin (4) recently reported that tubulin and the high molecular weight proteins associated with microtubule assembly selectively adsorb to unilamellar dimyristoyl phosphatidylcholine liposomes. Negative staining of the proteinlipid mixtures with phosphotungstic acid (PTA) showed stacks of flattened liposomes and/or larger multilamellar structures resulting from fusion of the stacks .We have found that identical images of stacked vesicles and multilamellar structures can be produced with negatively stained liposomes that contain no tubulin . The appearance of these negatively stained lipid preparations is specifically dependent on the metal stain used, rather than on the presence of protein . The possibility of a morphology being induced by the stain must be critically evaluated in this and other negatively stained lipoprotein systems . MATERIALS AND METHODS Preparation of LipidsDimyristoyl phosphatidylcholine (DMPC) and egg lecithin were obtained from Sigma Chemical Co., St . Louis, Mo. Lipids were suspended in chloroform, dried overnight under vacuum to remove solvent, and swollen for 6-18 h in phosphate-buffered 0.1 N saline (pH 6.75; 2 mM EDTA). The multilayered liposomes (10 mg of lipid/0.6 ml of buffer) were sonicated in a bath sonicater at 26°-30°C until nearly optically clear. Preparation of TubulinOnce-and twice-cycled tubulin proteins were prepared from homogenates of gray matter from adult cattle, following the procedures of Caron and Berlin (4). THE JOURNAL OF CELL BIOLOGY " VOLUME 86 SEPTEMBER 1980 881-884 © The Rockefeller University Press " 0021-9525/80/09/0881/04 $1 .00 Lipid-Tubulin MixturesThe cold-soluble proteins resulting from one or two cycles of microtubule assembly were incubated with sonicated DMPC or egg lecithin vesicles at 30°C for at least 30 min. Lipid and protein were each suspended in 0.1 M NaCl, 0.02 M phosphate, pH 6.75, 2 mM EDTA, and mixed in ratios of 10 mg of lipid/0.5 mg of protein. Several specimens were prepared with higher protein content (10 mg of lipid/5 mg of protein...

Electron Tomography: A Three‐Dimensional Analytic Tool for Hard and Soft Materials Research

“…[169] Other studies showed that the stain could introduce distortions to some molecules, such as general aggregation, molecular dissociation, and flattening. [167,170,171] For example, a common artifact due to negative staining of lipid-related proteins is that particles stacked and packed together into a structure called a rouleaux. [163,172–176] Recently, an optimized negative-staining (OpNS) method [177–179] was proposed via a refined conventional protocol (Figure 10).…”

Optimized negative-staining electron microscopy for lipoprotein studies

“…NS-EM also delivers images that contain high SNR, and an intermediate (near a nanometer) resolution [3] for reconstruction of three-dimensional (3D) models [20]. However, certain effects of the heavy-metal stain produce undesirable outcomes due to interactions with the specimen, such as aggregation, molecular dissociation, and artifacts of stacking [4, 21, 22]. …”

“…An obvious artifact of lipoprotein structure observed for decades is how the lipoprotein particles regularly present stacking rouleau in NS images [35–40]. This is significant in that rouleau formation is absent from serum analysis, native-gel, small-angle scattering, and cryo-EM studies [12, 22, 29, 30, 36, 39, 41–44]. Considering that rouleau formation displayed in NS images could lead to inaccurate interpretations of lipoprotein structure and function, a thorough investigation or comparison of the NS protocols used to examine the lipoprotein structure is necessary.…”

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

“…Traditional NS, unfortunately, can produce artifacts induced by stain-protein interactions, such as general aggregation, molecular dissociation, flattening and stacking 8,21,22 . For lipid related proteins, such as lipoproteins 16,23–30 , a common artifact results in particles that are stacked and packed together into a rouleaux (Figure 1) 31–36 .…”

“…For lipid related proteins, such as lipoproteins 16,23–30 , a common artifact results in particles that are stacked and packed together into a rouleaux (Figure 1) 31–36 . Many lipoprotein studies, such as nondenaturing polyacrylamide gradient gel electrophoresis, cryo-EM studies 13,29,37–40 , mass spectrometry 39,41 , and small angle X-ray diffraction data 42 all show lipoprotein particles are isolated particles instead of naturally stacked together forming a rouleaux 21,29,30,35,42–45 . The observation of rouleaux formation by conventional NS is possibly caused by dynamic interactions between lipoproteins composed of apolipoproteins (apo) and phospholipids that are structurally flexible in solution 13,29,30,46–49 and sensitivity to the standard NS protocol.…”