Role of lipid rafts in agrin-elicited acetylcholine receptor clustering.

Emerging concepts of membrane organization point to the compartmentalization of the plasma membrane into distinct lipid microdomains. This lateral segregation within cellular membranes is based on cholesterol-sphingolipid-enriched microdomains or lipid rafts which can move laterally and assemble into large-scale domains to create plasma membrane specialized cellular structures at specific cell locations. Such domains are likely involved in the genesis of the postsynaptic specialization at the neuromuscular junction, which requires the accumulation of acetylcholine receptors (AChRs), through activation of the muscle specific kinase MuSK by the neurotropic factor agrin and the reorganization of the actin cytoskeleton. We used C2C12 myotubes as a model system to investigate whether agrin-elicited AChR clustering correlated with lipid rafts. In a previous study, using two-photon Laurdan confocal imaging, we showed that agrin-induced AChR clusters corresponded to condensed membrane domains: the biophysical hallmark of lipid rafts [F. Stetzkowski-Marden, K. Gaus, M. Recouvreur, A. Cartaud, J. Cartaud, Agrin elicits membrane condensation at sites of acetylcholine receptor clusters in C2C12 myotubes, J. Lipid Res. 47 (2006) 2121-2133]. We further demonstrated that formation and stability of AChR clusters depend on cholesterol. We also reported that three different extraction procedures (Triton X-100, pH 11 or isotonic Ca++, Mg++ buffer) generated detergent resistant membranes (DRMs) with similar cholesterol/GM1 ganglioside content, which are enriched in several signalling postsynaptic components, notably AChR, the agrin receptor MuSK, rapsyn and syntrophin. Upon agrin engagement, actin and actin-nucleation factors such as Arp2/3 and N-WASP were transiently recovered within raft fractions suggesting that the activation by agrin can trigger actin polymerization. Taken together, the present data suggest that AChR clustering at the neuromuscular junction relies upon a mechanism of raft coalescence driven by agrin-elicited actin polymerization.

Formation of unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscopy and 31P-nuclear magnetic resonance.

It has been reported that repetitive freeze-thaw cycles of aqueous suspensions of dioleoylphosphatidylcholine form vesicles with a diameter smaller than 200 nm. We have applied the same treatment to a series of phospholipid suspensions with particular emphasis on dioleoylphosphatidylcholine/dioleoylphosphatidic acid (DOPC/DOPA) mixtures. Freeze-fracture electron microscopy revealed that these unsaturated lipids form unilamellar vesicles after 10 cycles of freeze-thawing. Both electron microscopy and broad-band 31P NMR spectra indicated a disparity of the vesicle sizes with a highest frequency for small unilamellar vesicles (diameters < or =30 nm) and a population of larger vesicles with a frequency decreasing exponentially as the diameter increases. From 31P NMR investigations we inferred that the average diameter of DOPC/DOPA vesicles calculated on the basis of an exponential size distribution was of the order of 100 nm after 10 freeze-thaw cycles and only 60 nm after 50 cycles. Fragmentation by repeated freeze-thawing does not have the same efficiency for all lipid mixtures. As found already by others, fragmentation into small vesicles requires the presence of salt and does not take place in pure water. Repetitive freeze-thawing is also efficient to fragment large unilamellar vesicles obtained by filtration. If applied to sonicated DOPC vesicles, freeze-thawing treatment causes fusion of sonicated unilamellar vesicles into larger vesicles only in pure water. These experiments show the usefulness of NMR as a complementary technique to electron microscopy for size determination of lipid vesicles. The applicability of the freeze-thaw technique to different lipid mixtures confirms that this procedure is a simple way to obtain unilamellar vesicles.

Assembly of connexins and MP26 in lens fiber plasma membranes studied by SDS-fracture immunolabeling.

The SDS-fracture immunolabeling technique, unlike conventional freeze-fracture, provides direct evidence for the biochemical nature of membrane constituents. SDS-fracture immunolabeling shows that during differentiation of lens fiber cells the onset of junctional assembly is characterized by the presence of small clusters and linear arrays comprising connexins alpha3 and alpha8. At this initial stage MP26, a major fiber membrane constituent, appears to be colocalized with these two connexins. The application of double-immunogold labeling reveals that when large junctional plaques are assembled MP26 becomes mainly associated with the periphery of the junctional domains. This type of distribution suggests that MP26 may play a role in the clustering and gathering of connexons. In aged nuclear fiber membranes connexins, MP26 and their proteolytic derivatives form an orthogonal lattice of repeating subunits.

Further identification of the exoplasmic face particles on the freeze-fractured thylakoid membranes: a study using double and triple mutants from Chlamydomonas reinhardtii lacking various photosystem II subunits and the cytochrome b6/f complex.

About 20% of the exoplasmic face (EF) particles present in the freeze-fractured thylakoid membranes of the wild type strain of Chlamydomonas reinhardtii remain in mutants lacking photosystem II (PSII) because of the absence of either one of the two PSII subcomplexes CP43 or D1/D2/CP47. We show that about half of these residual EF particles can be accounted for by PSII subcomplexes still present in such mutants, and by cytochrome (cyt) b6/f complexes. Analysis of double mutants lacking both types of protein complexes points to an association of cyt b6/f complexes with PSII subcomplexes in some of these EF particles and to a requirement in cyt b6/f complexes for the translocation of each of the two PSII subcomplexes (the CP43 subunit and the D1/D2/CP47 subcomplex) from the unstacked to the stacked regions of the thylakoid membranes.

Posttranslational events leading to the assembly of photosystem II protein complex: a study using photosynthesis mutants from Chlamydomonas reinhardtii.

We studied the assembly of photosystem II (PSII) in several mutants from Chlamydomonas reinhardtii which were unable to synthesize either one PSII core subunit (P6 [43 kD], D1, or D2) or one oxygen-evolving enhancer (OEE1 or OEE2) subunit. Synthesis of the PSII subunits was analyzed on electrophoretograms of cells pulse labeled with [14C]acetate. Their accumulation in thylakoid membranes was studied on immunoblots, their chlorophyll-binding ability on nondenaturating gels, their assembly by detergent fractionation, their stability by pulse-chase experiments and determination of in vitro protease sensitivity, and their localization by immunocytochemistry. In Chlamydomonas, the PSII core subunits P5 (47 kD), D1, and D2 are synthesized in a concerted manner while P6 synthesis is independent. P5 and P6 accumulate independently of each other in the stacked membranes. They bind chlorophyll soon after, or concomitantly with, their synthesis and independently of the presence of the other PSII subunits. Resistance to degradation increases step by step: beginning with assembly of P5, D1, and D2, then with binding of P6, and, finally, with binding of the OEE subunits on two independent high affinity sites (one for OEE1 and another for OEE2 to which OEE3 binds). In the absence of PSII cores, the OEE subunits accumulate independently in the thylakoid lumen and bind loosely to the membranes; OEE1 was found on stacked membranes, but OEE2 was found on either stacked or unstacked membranes depending on whether or not P6 was synthesized.

Organization of the photosystem II centers and their associated antennae in the thylakoid membranes: a comparative ultrastructural, biochemical, and biophysical study of Chlamydomonas wild type and mutants lacking in photosystem II reaction centers.

We investigated the ultrastructure of thylakoid membranes that lacked either some or all of their Photosystem II centers in the F34SU3 and F34 mutants of Chlamydomonas reinhardtii. We obtained the following results: (a) There are no particles of the 160-A size class on the EF faces of the thylakoids in the absence of Photosystem II centers (as in F34); the F34SU3 contains 50% of the wild-type number of PSII centers and EF particles. (b) The density of the particles on the PF faces of the thylakoids is higher in the mutants than in the wild type. (c) The fluorescence analysis shows that the organization of the pigments is the same regardless of whether 50% of the PSII centers are temporarily inactivated (by preilluminating the wild type) or are actually missing from the thylakoid membrane (F34SU3). Our results, therefore, support a model in which: (a) each 160-A EF particle has only one PSII center surrounded by light-harvesting complexes and (b) part of the PSH antenna is associated with 80-A PF particles in both of the mutants and the wild type.

Ultrastructure - function relationship in Chlamydomonas reinhartii thylakoids, by means of a comparison between the wild type and the F34 mutant which lacks the photosystem II reaction center.

The F34 mutant strain of Chlamydomonas reinhartii is deficient in photosystem II reaction centers. The E fracture faces of the thylakoid membranes of this mutant show a considerable reduction in the number of particles present ant in their size compared with the wild type. We conclude that the polypeptides associated with photosystem II reaction centers, which are missing in SDS polyacrylamide gel electrophoresis patterns of proteins from this mutant strain, are part of the EF particles and are required for assembly of these particles.

Development and regulation of the phosphorylase-glycogen complex in myogenic cells of the L6 line.

The development of the phosphorylase-glycogen complex was studied in cells of the myogenic line L6 at different stages of differentiation. Our results indicate that the complex is already present in mononucleated myoblasts. However, several features of the complex observed in the myoblasts, such as the total amount of phosphorylase bound to the polysaccharide, and the type of glycogen agglomerates present in the cells, differ from those of the myotubes. It is postulated that at the moment when the myoblasts differentiate, definitive metabolic events take place. At this time the protein-glycogen complex is completed, phosphorylase being bound to small glycogen particles, and the multinucleated myotubes begin to accumulate a reserve of free glycogen. This latter constituent is mobilized first upon glucose starvation.