Giant Membrane Vesicles Research Articles

A Transgenic Mouse Model to Study Glucose Transporter 4myc Regulation in Skeletal Muscle

Skeletal muscle is the major site for dietary glucose disposal, taking up glucose via glucose transporter 4 (GLUT4). Although subcellular fractionation studies demonstrate that insulin increases GLUT4 density in sarcolemma and transverse tubules, fractionation cannot discern GLUT4 vesicle-membrane association from insertion and exofacial exposure. Clonal muscle cultures expressing exofacially tagged GLUT4 have allowed quantification of GLUT4 exposure at the cell surface, its exocytosis, endocytosis, and partner proteins. We hypothesized that transgenic expression of GLUT4myc in skeletal muscles would provide a useful model to investigate GLUT4 biology in vivo. A homozygous mouse colony was generated expressing GLUT4myc driven by the muscle creatine kinase (MCK) promoter. GLUT4 protein levels were about 3-fold higher in hindlimb muscles of MCK-GLUT4myc transgenic mice compared with littermates (P < 0.05). Insulin (12 nm, 30 min) induced a 2.1-fold increase in surface GLUT4myc detected by immunofluorescence of the exofacial myc epitope in nonpermeabilized muscle fiber bundles (P < 0.05). Glucose uptake and surface GLUT4myc levels were 3.5- and 3-fold higher, respectively, in giant membrane vesicles blebbed from hindlimb muscles of insulin-stimulated transgenic mice compared with unstimulated counterparts (P < 0.05). Muscle contraction also elevated both parameters, an effect partially additive to insulin's. GLUT4myc immunoprecipitation with anti-myc antibodies avoids interfering with associated intracellular binding proteins. Tether, containing a UBX domain, for GLUT4 coimmunoprecipitated with GLUT4myc and insulin stimulation significantly decreased such association (P < 0.05). MCK-GLUT4myc transgenic mice are thus useful to quantify exofacial GLUT4 exposure at the sarcolemma and GLUT4 binding partners in skeletal muscle, essential elements in the investigation of muscle GLUT4 regulation in physiological and pathological states in vivo.

Membrane flow patterns in multicomponent giant vesicles induced by alternating electric fields†Electronic supplementary information (ESI) available: Vesicle preparation procedure, numerical calculations and confocal microscopy movies of domain motion. See DOI: 10.1039/b811876kClick here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.

Alternating electric (AC) fields induce circular patterns of lipid transport in membranes of giant vesicles. The flow is visualized by fluorescently labelled lipid domains.

Adhesion induced non-planar and asynchronous flow of a giant vesicle membrane in an external shear flow.

We show the existence of a flow at the surface of strongly adhering giant lipid vesicles submitted to an external shear flow. The surface flow is divided into two symmetric quadrants and presents two stagnation points (SP) on each side of the vesicle meridian plane. The position of these stagnation points depends strongly on the adhesion strength, characterized by the ratio of the contact zone diameter to the vesicle diameter. Contrary to the case of non-adhesive vesicles, streamlines do not lie in the shear plane. By avoiding the motionless contact zone, streamlines result in three-dimensional paths, strongly asymmetric away from the SP. Additional shearing dissipation may occur on the membrane surface as we observed that the mean rotational velocity of the membrane increases towards the vesicle SP, and is mainly determined by the adhesion induced vesicle shape.

Transition from long- to short-lived transient pores in giant vesicles in an aqueous medium

We have observed large pores in the membrane of giant vesicles in an aqueous medium. The lifetime of the pores can reach 2 min and their size (a few micrometers) enables their visualization by fluorescence microscopy. These pores are obtained thanks to a destabilization of the membrane due to the synergistic action of a cone-shaped and nitrobenzodiazole (NBD) labeled phospholipid illuminated in the presence of dithionite. The opening of the pore occurs immediately after illumination starts so that it can be accurately triggered. A concomitant decrease of the vesicle radius is observed; we interpret it as a solubilization of the membrane. Depending on the rate of this solubilization, long- or short-lived pores were observed. At the transition between both regimes for a 30 microm vesicle, the solubilization rate was about 1/300 s{-1} . In order to interpret these observations, we have revisited the current model of pore opening to take into account this solubilization. This proposed model along with simulations enables us to prove that solubilization explains why the large long-lived pores are observed even in an aqueous medium. The model also predicts the solubilization rate at the transition between a single long-lived pore and a cascade of short-lived pores.

Sulfo-N-succinimidyl esters of long chain fatty acids specifically inhibit fatty acid translocase (FAT/CD36)-mediated cellular fatty acid uptake.

Sulfo-N-succinimidyl esters of LCFAs are a powerful tool to investigate the functional significance of plasmalemmal proteins in the LCFA uptake process. This notion is based on the following observations. First, sulfo-N-succinimidyl oleate (SSO) was found to inhibit the bulk of LCFA uptake into various cell types, i.e. rat adipocytes, type II pneumocytes and cardiac myocytes. Second, using cardiac giant membrane vesicles, in which LCFA uptake can be investigated in the absence of mitochondrial beta-oxidation, SSO retained the ability to largely inhibit LCFA uptake, indicating that inhibition of LCFA transsarcolemmal transport is its primary action. Third, SSO has no inhibitory effect on glucose and octanoate uptake into giant membrane vesicles derived from heart and skeletal muscle, indicating that its action is specific for LCFA uptake. Finally, SSO specifically binds to the 88 kDa plasmalemmal fatty acid transporter FAT, a rat homologue of human CD36, resulting in an arrest of the transport function of this protein. In addition to its inhibitory action at the plasma membrane level, evidence is presented for the lack of a direct inhibitory effect on subsequent LCFA metabolism. First, the relative contribution of oxidation and esterification to LCFA uptake is not altered in the presence of SSO. Second, isoproterenol-mediated channeling of LCFAs into oxidative pathways is not affected by sulfo-N-succinimidyl palmitate (SSP). As an example of its application, we used SSP to study the role of FAT/CD36 in contraction- and insulin-stimulated LCFA uptake by cardiac myocytes, showing that this transporter is a primary site of regulation of cellular LCFA utilization.

Giant membrane vesicles as a model to study cellular substrate uptake dissected from metabolism.

In order to use giant vesicles for substrate uptake studies in metabolically important tissues, we characterized giant vesicles isolated from heart, liver, skeletal muscle and adipose tissue. We investigated which cell types and which plasma membrane regions are involved in giant vesicle formation and we examined the presence of transporters for metabolic substrates. Analysis of giant vesicles with markers specific for distinct cell types and distinct domains of the plasma membrane reveals that the plasma membrane of parenchymal cells, but not endothelial cells, are the source of the vesicle membranes. In addition, plasma membrane regions enriched in caveolae and involved in docking of recycling vesicles from the endosomal compartment are retained in giant vesicles, indicating that KCl-induced alterations in recycling processes are involved in giant vesicle formation. Giant vesicles contain vesicular lumen consisting of the soluble constituents of the cytoplasm including, fatty-acid binding proteins. Furthermore, giant vesicles isolated from heart, liver, skeletal muscle and adipose tissue are similar in size (10-15 microm) and shape and do not contain subcellular organelles, providing the advantage that substrate fluxes in the different organs can be studied independently of the surface/volume ratio but most importantly in the absence of intracellular metabolism.

Dynamic strengths of molecular anchoring and material cohesion in fluid biomembranes

Building on Kramers' theory for reaction kinetics in liquids and using laboratory experiments, we show how strengths of molecular anchoring and material cohesion in fluid-lipid membranes increase with rate of force and tension loading. Expressed on a scale of log(loading rate), the dynamic spectra of pull-out forces and rupture tensions image the microscopic and mesoscopic energy barriers traversed in molecular extraction and membrane failure. To capture such images, we have pulled single molecules from membranes with force rates from 1 to 104 pN s-1 and ruptured giant membrane vesicles with tension rates from 10-2 to 102 mN m-1 s-1.

Shape Changes of Self-Assembled Actin Bilayer Composite Membranes

We report the self-assembly of thin actin shells beneath the membranes of giant vesicles. Ion-carrier mediated influx of Mg2+ induces actin polymerization in the initially spherical vesicles. Buckling of the vesicles and the formation of blisters after thermally induced bilayer expansion is demonstrated. Bilayer flickering is dominated by tension generated by its coupling to the actin cortex. Quantitative flicker analysis suggests the bilayer and the actin cortex are separated by 0.4 \mum to 0.5 \mum due to undulation forces.

Glucose transport and transporters in muscle giant vesicles: differential effects of insulin and contractions

Collagenase treatment of skeletal muscle results in the formation of large spheres of membranes (3-30 microns diam). A procedure is described for purification and concentration of these giant membrane vesicles prepared from rat muscle. Morphological observations, marker enzyme analysis, and immunoblotting demonstrate that the vesicles are of plasma membrane origin and that sarcoplasmic reticulum, T-tubules, and mitochondrial inner membranes are absent from the preparation. Western blots demonstrate that the vesicles contain GLUT-4 glucose transporters, whereas GLUT-1 could not be detected. Vesicles prepared from control muscle display specific transport of D-glucose with a maximum velocity (Vmax) for glucose influx of approximately 2,500 pmol.mg plasma membrane protein-1.s-1 and an apparent Michaelis constant (Km) of 16 mM measured at zero-trans conditions at room temperature. Muscle contractions in vivo doubled the Vmax of vesicle glucose transport and membrane GLUT-4 content but did not change Km. In contrast, in vivo administration of insulin did not affect vesicle glucose transport or membrane GLUT-4 content. The combination of insulin and contractions caused similar changes as did contractions alone. It is concluded that the present vesicle population contains membrane components almost exclusively derived from the plasma membrane and contains very little if any GLUT-1 but substantial amounts of GLUT-4. Thus the preparation allows the study of transport kinetics of pure GLUT-4 transporters. The procedure for preparing vesicles probably results in activation of the glucose transport system similar to the activation by insulin but not by contractions.(ABSTRACT TRUNCATED AT 250 WORDS)