Rapid isolation and lipid characterization of plasma membranes from normal and malignant lymphoid cells of mouse

A rapid isolation method was developed for plasma membranes from mouse lymphoid cells such as lymph node lymphocytes, thymocytes, radiation-induced thymoma cells and L1210 cells. Lysates of these lymphoid cells were prepared by Dounce homogenization under hypotonic conditions and directly layered on sucrose step density gradients containing 2 mM CaCl₂ and 5 mM MgCl₂, and centrifuged at $\text{52 000 ×}\text{g}$ for 1 h. Plasma membrane fractions appeared at the interface between 20 and 42% sucrose in the gradients. The procedure permitted purified membranes from cells to be obtained within 3 h, and the preparations appeared to be uniform by electron microscopy. Specific activities of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATpase}$, Mg²⁺-ATPase and 5′-nucleotidase of the isolated plasma membranes were enriched 23- to 61-fold, 12- to 15-fold and 18- to 34-fold, respectively, in comparison with those of the corresponding cell homogenates. Cholesterol content of the malignant cell membranes was lower than that of the normal membranes and the molar ratio of cholesterol to phospholipid of the malignant cell membranes was also lower than that of the normal membranes. A decreased plasmalogen content was observed in the malignant plasma membranes, together with a higher percentage of phosphatidylethanolamine and a lower percentage of phosphatidylserine. In the normal cell membranes, thymocytes contained a higher percentage of phosphatidylcholine and a lower percentage of sphingomyelin than those of the lymph node lymphocytes. At all temperature ranges (5 to 40°C) the plasma membranes of the malignant cells had lower microviscosity than those of the normal cells.     

Resolution of plasma membrane lipid fluidity in intact cells labelled with diphenylhexatriene

The partitioning of fluorescence probes into intracellular organelles poses a major problem when fluorescence methods are applied to evaluate the fluidity properties of cell plasma membranes with intact cells. This work describes a method for resolution of fluidity parameters of the plasma membrane in intact cells labelled with the fluorescence polarization probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The method is based on selective quenching, by nonradiative energy transfer, of the fluorescence emitted from the plasma membrane after tagging the cell with a suitable membrane impermeable electron acceptor. Such selective quenching is obtained by chemical binding of 2,4,6-trinitrobenzene sulfonate (TNBS), or by incorporation of $\text{N-}\text{bixinoyl}$ glucosamine (BGA) to DPH-labelled cells. The procedures for determination of lipid fluidity in plasma membranes of intact cells by this method are simple and straightforward.     

Lipid composition and microviscosity of subcellular fractions from rabbit thymocytes. Differences in the microviscosity of plasma membranes from subclasses of thymocytes

There are indications from freeze-fracture experiments that subclasses of rabbit thymocytes show different mobilities of plasma membrane components. Consequently, one would expect differences in the fluidity of the plasma membrane. For this reason, rabbit thymocytes were separated on a Ficoll/Metrizoate gradient yielding three subclasses representing various levels of cell differentiation. These thymocyte subclasses did not show any significant differences in the degree of fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. The fluorescence polarization of the plasma membrane may be overshadowed by the contribution of all cellular lipids due to penetration of the fluorescent probe into the cell. Therefore, plasma membranes were isolated from rabbit thymocytes using a cell-disrupting pump, differential centrifugation, and sucrose density gradient centrifugation. As shown by biochemical and electron microscopical analyses, plasma membranes with a high degree of purity were obtained. As expected the plasma membrane fractions showed a higher microviscosity than the other subcellular fractions. This was attributed to a higher cholesterol to phospholipid molar ratio and a higher degree of saturation of phospholipid fatty acid chains.Subsequently, the microviscosity was measured of plasma membrane preparations obtained from two main subclasses of thymocytes representing mature and immature lymphocytes. The immature thymocytes yielded two plasma membrane fractions with higher microviscosity than the mature cells.     

Lipid structural order parameters (reciprocal of fluidity) in biomembranes derived from steady-state fluorescence polarization measurements

This paper presents an interpretation of fluorescence polarization measurements in lipid membranes which are labelled with the apolar probe 1,6-diphenyl-1,3,5-hexatriene. The steady-state fluorescence anisotropy, $\text{r}{}_{\mathrm{<mtext>S</mtext>}}$, is resolved into a fast decaying or kinetic component, $\text{r}{}_{\mathrm{<mtext>f</mtext>}}$, and an infinitely slow decaying or static component, $\text{r}{}_{\mathrm{\infty }}$. The latter contribution, which predominates in biological membranes, is exclusively determined by the degree of molecular packing (order) in the apolar regions of the membrane; $\text{r}{}_{\mathrm{\infty }}$ is proportional to the square of the lipid order parameter. An empirical relation between $\text{r}{}_{\mathrm{<mtext>S</mtext>}}$ and $\text{r}{}_{\mathrm{\infty }}$ is presented, which is in agreement with a prediction based on a theory of rotational dynamics in liquid crystals. This relation enabled us to estimate a lipid structural order parameter directly from simple steady-state fluorescence polarization measurements in a variety of isolated biological membranes. It is shown that major factors determining the order parameter in biomembranes are the temperature, the cholesterol and sphingomyelin content and (in a few systems) the membrane intrinsic proteins.     

Analysis of membrane fractions from Mycoplasma gallisepticum

Membrane fractions have been isolated from Mycoplasma gallisepticum following a procedure derived from that described by Maniloff, J. and Quinlan, D.C. (J. Bacteriol. (1974) 120, 495–501). A light fraction F₁ was obtained which contained structures resembling the bleb-infrableb apparatus characteristic of M. gallisepticum. It was enriched in DNA and had an electrophoretic profile different from that of unfractionated membranes. Cholesterol-to-phospholipid ratios higher than two and elevated values of the ratio of saturated to unsaturated fatty acids were other characteristics of this fraction. The two other fractions isolated (F_II and F_IV) also differed from intact membranes by their cholesterol and phospholipid content as well as by their saturation ratios. The membrane fluidity of F_II and F_IV, estimated by fluorescence polarization, was similar to that of unfractionated membranes while a slight but significant difference was recorded for the light fraction. Possible relationships between the lateral heterogeneity of the M. gallisepticum membrane and the obtainment of fractions are discussed.     

Pulsed-laser creation and characterization of giant plasma membrane vesicles from cells

Femtosecond-pulsed laser irradiation was found to initiate giant plasma membrane vesicle (GPMV) formation on individual cells. Laser-induced GPMV formation resulted from intracellular cavitation and did not require the addition of chemical stressors to the cellular environment. The viscosity, structure, and contents of laser-induced GPMVs were measured with fluorescence microscopy and single-particle tracking. These GPMVs exhibit the following properties: (1) GPMVs grow fastest immediately after laser irradiation; (2) GPMVs contain barriers to free diffusion of incorporated fluorescent beads; (3) materials from both the cytoplasm and surrounding media flow into the growing GPMVs; (4) the GPMVs are surrounded by phospholipids, including phosphatidylserine; (5) F-actin is incorporated into the vesicles; and (6) caspase activity is not essential for GPMV formation. The effective viscosity of 65 nm polystyrene nanoparticles within GPMVs ranged from 32 to 434 cP. The nanoparticle diffusion was commonly affected by relatively large, macromolecular structures within the bleb.     

Fluorescence polarization study on the increase of membrane fluidity of human erythrocyte ghosts induced by synthetic water-soluble polymers

The effect of water-soluble polymers on the membrane fluidity of human erythrocyte ghosts was investigated and was compared with that of concanavalin A by means of the fluorescence polarization technique. 8-Anilino-1-naphthalene sulfonic acid sodium salt and 1,6-diphenyl-1,3,5-hexatriene were used as probe molecules. The membrane fluidity was increased by the addition of polycations with concentrations of less than $\text{2 · 10}{}^{\mathrm{?3}}$ wt% 60 min after mixing. The fluidity changes were affected by the chemical structure (hydrophobicity, charge density, etc.) of polycations. Thus, the membrane fluidity increased markedly with increasing charge density on the chain backbone of polycations. On the other hand, nonionic polymers such as poly(ethylene glycol) and $\text{poly}\text{(N-}\text{vinyl}\text{-2-}\text{pyrrolidone}$) changed the membrane fluidity in a biphasic manner. That is, the fluidity of human erythrocyte ghost was temporarily increased and then decrease. For example, 20 wt% of poly(ethylene glycol) gave a maximum fluidity 15 min after mixing with erythrocyte ghosts. A similar fluidity change was observed by adding concanavalin A. Such fluidity changes were not observed when lipid bilayer vesicles were used instead of cell membranes. These results suggested that the increase of membrane fluidity resulted from the intramembraneous aggregation of membrane-bound proteins which was induced by the added polymers. Cell agglutination was also induced by the addition of a large amount of polymers. This agglutination was considered to be due to the intermembraneous aggregation of membrane-bound proteins.