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.

Differential targeting of components of the dystrophin complex to the postsynaptic membrane.

Accumulating evidence points to the participation of dystroglycan in the clustering of nicotinic acetylcholine receptors at the neuromuscular junction [Côté et al. (1999) Nature Genet., 3, 338--342]. Dystroglycan is part of a multimolecular complex, either associated with dystrophin (the dystrophin-associated protein complex) at the sarcolemma or with utrophin (the utrophin-associated protein complex) at the neuromuscular junction. Understanding the assembly of this complex at the developing synapse led us to investigate, in Torpedo electrocyte, the intracellular routing and the targeting of several of its components, including dystroglycan, syntrophin, dystrophin and dystrobrevin. We previously demonstrated that acetylcholine receptors and rapsyn, the 43-kDa receptor-associated protein at the synapse, are cotargeted to the postsynaptic membrane via the exocytic pathway [Marchand et al. (2000) J. Neurosci., 20, 521--528]. Using cell fractionation, immunopurification and immuno-electron microscope techniques, we show that beta-dystroglycan, an integral glycoprotein that constitutes the core of the dystrophin-associated protein complex localized at the innervated membrane, is transported together with acetylcholine receptor and rapsyn in post-Golgi vesicles en route to the postsynaptic membrane. Syntrophin, a peripheral cytoplasmic protein of the complex, associates initially with these exocytic vesicles. Conversely, dystrophin and dystrobrevin were absent from these post-Golgi vesicles and associate directly with the postsynaptic membrane. This study provides the first evidence for a separate targeting of the various components of the dystrophin-associated protein complex and a step-by-step assembly at the postsynaptic membrane.

Modulation of the oxygen affinity of cobalt-porphyrin by globin.

We have combined two extreme effects which influence the oxygen affinity to obtain a cobalt-based oxygen carrier with an affinity similar to that of human adult hemoglobin (HbA). The goal was to obtain an oxygen transporter with a lower oxidation rate. Exchange of the heme group (Fe-protoporphyrin IX) in Hb with a cobalt-porphyrin leads to a reduction in oxygen affinity by over a factor of 10, an oxygen affinity too low for use as a blood substitute. At the other extreme, certain globin sequences are known to provide a very high oxygen affinity; for example, Hb Ascaris displays an oxygen affinity 1000 times higher than HbA. We demonstrate here that these opposing effects can be additive, yielding an oxygen affinity similar to that of HbA, but with oxygen binding to a cobalt atom. We have tested the effect of substitution of cobalt-porphyrin for heme in normal HbA, sperm whale (SW) Mb (Mb), and high affinity globins for leghemoglobin, two trematode Hbs: Paramphistomum epiclitum (Pe) and Gastrothylax crumenifer (Gc). As for HbA or SW Mb, the transition from heme to cobalt-porphyrin in the trematode Hbs leads to a large decrease in the oxygen affinity, with oxygen partial pressures for half saturation (P(50)) of 5 and 25 mm Hg at 37 degrees C for cobalt-Pe and cobalt-Gc, respectively. A critical parameter for Hb-based blood substitutes is the autoxidation rate; while both metals oxidize to an inactive state, we observed a decrease in the oxidation rate of over an order of magnitude for cobalt versus iron, for similar oxygen affinities. The time constants for autoxidation at 37 degrees C were 250 and 100 h for Pe and Gc, respectively.

The torpedo electrocyte: a model system to study membrane-cytoskeleton interactions at the postsynaptic membrane.

Many aspects of the organization of the electromotor synapse of electric fish resemble the nerve-muscle junction. In particular, the postsynaptic membrane in both systems share most of their proteins. As a remarquable source of cholinergic synapses, the Torpedo electrocyte model has served to identify the most important components involved in synaptic transmission such as the nicotinic acetylcholine receptor and the enzyme acetylcholinesterase, as well as proteins associated with the subsynaptic cytoskeleton and the extracellular matrix involved in the assembly of the postsynaptic membrane, namely the 43-kDa protein-rapsyn, the dystrophin/utrophin complex, agrin, and others. This review encompasses some representative experiments that helped to clarify essential aspects of the supramolecular organization and assembly of the postsynaptic apparatus of cholinergic synapses.

The myristoylated protein rapsyn is cotargeted with the nicotinic acetylcholine receptor to the postsynaptic membrane via the exocytic pathway.

Rapsyn, a 43 kDa protein required to cluster nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction, is tightly associated with the postsynaptic membrane via an N-terminal myristoylated site. Recent studies have shown that some acylated proteins associate with the exocytic pathway to become targeted to their correct destination. In this work, we used Torpedo electrocyte to investigate the intracellular routing of rapsyn compared to those of AChR and Na,K-ATPase, the respective components of the innervated and noninnervated membranes. We previously demonstrated that these latter two proteins are sorted and targeted to plasma membrane via distinct populations of post-Golgi vesicles (). Biochemical and immunoelectron microscopy analyses of various populations of post-Golgi vesicles immunopurified with magnetic beads led us to identify post-Golgi transport vesicles containing both rapsyn and AChR. These data suggest that rapsyn, as for AChR, specifically follows the exocytic pathway. Furthermore, immunogold-labeling experiments provided in situ evidence that AChR and rapsyn are cotransported in the same post-Golgi vesicles. Taken together, our observations suggest that rapsyn and AChR are cotargeted to the postsynaptic membrane.

Targeting of acetylcholine receptor and 43 kDa rapsyn to the postsynaptic membrane in Torpedo marmorata electrocyte.

In this study we have investigated the intracellular routing of two major components of the postsynaptic membrane in Torpedo electrocytes, the nicotinic acetylcholine receptor and the extrinsic 43 kDa protein rapsyn, and of a protein from the non-innervated membrane, the Na+,K+ ATPase. We isolated subpopulations of post-Golgi vesicles (PGVs) enriched either in AChR or in Na+,K+ ATPase. Rapsyn was associated to AChR-containing PGVs suggesting that both AChR and rapsyn are targeted to intracellular organelles in the secretory pathway before delivery to the postsynaptic membrane. In vitro assays further show that rapsyn-containing PVGs do bind more efficiently to microtubules compared to Na+,K+ ATPase-enriched PVGs. These data provide evidence in favor of the contribution of the secretory pathway to the delivery of synaptic components.

Expression of utrophin and its mRNA in denervated mdx mouse muscle.

Utrophin is a large cytoskeletal protein which shows high homology to dystrophin. In contrast to the sarcolemmal distribution of dystrophin, utrophin accumulates at the postsynaptic membrane of the neuromuscular junction. Because of its localization within this compartment of muscle fibers, expression of utrophin may be significantly influenced by the presence of the motor nerve. We tested this hypothesis by denervating muscles of mdx mouse and monitoring levels of utrophin and its mRNA by immunofluorescence, immunoblotting and RT-PCR. A significant increase in the number of utrophin positive fibers was observed by immunofluorescence 3 to 21 days after sectioning of the sciatic nerve. Quantitative analyses of utrophin and its transcripts in hindlimb muscles denervated for two weeks showed only a moderate increase in the levels of both utrophin (approximately 2-fold) and its transcript (approximately 60 to 90%). The present data suggest that although utrophin is a component of the postsynaptic membrane, its neural regulation is distinct from that of the acetylcholine receptor.

Correlation of carbon monoxide association rates and the position of absorption band III in hemoproteins.

We have examined the absorbance of a charge-transfer transition near 760 nm, known as band III, in several hemoproteins and heme complexes. The band III position correlates with the rate of carbon monoxide binding to the heme. A band III present at 760 nm indicates an unfavorable geometry of the heme for carbon monoxide binding; a red-shift of the band III to 765 nm indicates a less-constrained geometry of the heme as evidenced by higher carbon monoxide association rates. The band III position correlates well with the Raman frequency of the Fe-His(F8) bond as suggested previously for normal hemoglobin A [Sassaroli, M. & Rousseau, D. L. (1987) Biochemistry 26, 3092-3098]. Aplysia myoglobin and the chimeric heme protein kinase FixL from Bradyrhizobium japonicum, hemoproteins with an apolar residue in place of the highly conserved polar histidine E7, do not fit the relationship between the band III position and the rate of binding of carbon monoxide to the heme. With these few exceptions, the measurement of band III appears to be a practical means to probe the stretch frequency of the Fe-His(F8) bond.

Identification of dystrophin-binding protein(s) in membranes from Torpedo electrocyte and rat muscle.

Using solubilized dystrophin isolated from torpedo electric tissue and in vitro blot overlay assay, we have identified dystrophin-binding proteins in membranes from Torpedo electrocyte and rat muscle. In acetylcholine receptor-rich membranes from Torpedo marmorata electric tissue, an extrinsic protein of M(r) 52,000, known as the 58-kDa protein (Froehner, S.C. (1984) J. Cell Biol. 99, 88-96), represents the major binding site for dystrophin. When membranes were solubilized by non-ionic detergents, the 52-kDa protein as well as a few proteins of M(r) 200,000, 87,000, and 45,000 co-extract and copurify with dystrophin. In rat sarcolemma, a protein doublet of approximately 58-60 kDa also binds dystrophin in vitro, this protein likely being the DAP 59 characterized by Ervasti and Campbell (Ervasti, J. M., and Campbell, K. P. (1991) Cell 66, 1121-1131). We postulate that the 52- and 59-kDa proteins are functional homologs that play the role of "receptors" for dystrophin in various specialized membrane domains.

Localization of dystrophin and dystrophin-related protein at the electromotor synapse and neuromuscular junction in Torpedo marmorata.

The immunological identification of dystrophin isoforms at the neuromuscular junction and Torpedo marmorata electromotor synapse was attempted using various antibodies. A polyclonal antibody raised against electrophoretically purified dystrophin from T. marmorata electrocyte has been thoroughly investigated. This antibody recognized dystrophin in the electric tissue as well as sarcolemmal and synaptic neuromuscular junction dystrophin in all studies species (T. marmorata, rat, mice and human) at serum dilutions as high as 1:10,000. At variance, no staining of either the sarcolemma or neuromuscular junction was observed in Duchenne muscular dystrophy or mdx mice skeletal muscles. In these muscles, other members of the dystrophin superfamily, in particular the dystrophin-related protein(s) encoded by autosomal genes are present. These data thus demonstrate the specificity of our antibodies for dystrophin. Anti-dystrophin-related protein antibodies [Khurana et al. (1991) Neuromusc. Disorders 1, 185-194] which gave a strong immunostaining of the neuromuscular junction in various species, including T. marmorata, cross-reacted weakly with the postsynaptic membrane of the electrocyte. Taken together, these observations are in favor of the existence of a protein very homologous to dystrophin at the electromotor synapse in T. marmorata, whereas both dystrophin and dystrophin-related protein co-localize at the neuromuscular junction as in all species studied. The electrocyte thus offers the unique opportunity to study the interaction of dystrophin with components of the postsynaptic membrane.

Interaction domains of neurofilament light chain and brain spectrin.

We have previously demonstrated that brain spectrin binds to the low-molecular-mass subunit of neurofilaments (NF-L) [Frappier, Regnouf & Pradel (1987) Eur. J. Biochem. 169, 651-657]. In the present study, we seek to locate their respective binding domains. In the first part we demonstrate that brain spectrin binds to a 20 kDa domain of NF-L. This domain is part of the rod domain of neurofilaments and plays a role in the polymerization process. However, the polymerization state does not seem to have any influence on the interaction. In the second part, we provide evidence that NF-L binds to the beta-subunit of not only brain spectrin but also human and avian erythrocyte spectrins. The microtubule-associated protein, MAP2, which has also been shown to bind to microfilaments and neurofilaments, binds to the same domain of NF-L as spectrin does. Finally, among the tryptic peptides of brain spectrin, we show that some peptides of low molecular mass (35, 25, 20 and 18 kDa) co-sediment with either NF-L or F-actin.

Identification of tubulin-binding proteins of the chicken erythrocyte plasma membrane skeleton which colocalize with the microtubular marginal band.

The plasma membrane of nucleated erythrocytes contains a microtubular marginal band which appears to be associated with the plasma membrane skeleton. In this report, we identify two families of cytoskeletal proteins which may be involved in such an association. These proteins, of molecular mass 78 kDa and 48 kDa on SDS-PAGE, are shown to bind tubulin based on a 125I-labeled tubulin binding assay. Solubilization of isolated chicken erythrocyte plasma membranes in Triton X-100 shows that these proteins centrifuge with the pellet, indicating that they are bound to the membrane skeleton. Finally, immunofluorescence studies using antisera raised against the 78 kDa and 48 kDa proteins show that they colocalize with the marginal band in intact cells. Colocalization of cytoskeletal tubulin-binding proteins with the marginal band favors a hypothesis suggesting that the 78 kDa and 48 kDa proteins are involved in the association of the two molecular superstructures.

A mixing chamber to enucleate avian and fish erythrocytes: preparation of their plasma membrane.

Biochemical studies of the plasma membrane and the cytoskeleton of nucleated erythrocytes are strongly limited by the difficulties encountered in enucleating large quantities of cells. We describe an easily built hydrodynamic system which allows rapid preparation of large amounts of avian and fish erythrocyte plasma membranes. The contents of two 25-ml syringes containing hemolyzed nucleated erythrocytes are forced through four capillaries to a specially designed mixing chamber which fills a collecting syringe. The 50-ml erythrocyte suspension can be processed in 2 s. The high speed flow is achieved with a hand-activated piston. The turbulences in the mixing chamber are carried to an optimal efficiency by the vis-à-vis disposition of the four mixing jets. The enucleated membranes are separated from the nuclei and residual nucleated cells by differential centrifugations. They do not show contamination with nuclear material. Erythrocytes from chicken and trout have been used. They present striking differences in their stability toward hydrodynamic disruption, erythrocytes from chicken being far more stable. Ninety-five percent of trout erythrocytes are enucleated after only one run through the mixing chamber. Two runs performed at the maximal flow rate are necessary to enucleate chicken erythrocytes with a yield of 80%. In the former case most of the purified enucleated plasma membranes are fragmented in small vesicles while they retain a large size in the case of chicken erythrocytes. The proteins of the membranes thus prepared are characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: we found that erythrocyte membranes from trout are remarkable for their small spectrin content compared to those from chicken.

Conservation of tubulin alpha-beta differences in zinc-induced sheets with variations in pH and microtubule-associated protein content.

Zinc-induced tubulin sheets were grown at pH values of 5.7 and 6.4 from porcine brain tubulin purified by phosphocellulose chromatography as well as from microtubule protein containing tubulin plus 20% (w/w) unfractionated microtubule-associated proteins (MAPs). Electron micrographs of negatively stained sheets were analyzed by a combination of real space cross-correlation and Fourier space methods, providing two-dimensional reconstructions to approximately 16 A resolution. The reconstructed images revealed that the protofilaments comprising zinc-induced sheets are composed of two clearly distinguishable alternating subunits, presumably corresponding to the alpha- and beta-tubulin monomers, whose morphologies are not significantly influenced by pH or the presence of MAPs during sheet assembly. Sheets assembled at pH 5.7, either with or without MAPs, were divided into two domains by a protofilament discontinuity which was not present in sheets assembled at pH 6.4, and displayed a 2.4 A reduction of the interprotofilament distance in projection relative to sheets assembled at pH 6.4. We conclude that morphological differences between tubulin subunits represent intrinsic structural features not contributed by MAPs, and that pH is more important than MAP content in influencing the lattice parameters of zinc-induced sheets.

Interactions of actin and tubulin with human deoxyhemoglobin. Their possible occurrence within erythrocytes.

Short actin filaments are an essential component of the red-cell membrane skeleton, and microtubules are also present in nucleated erythrocytes as a marginal band. Actin and tubulin share the property of possessing a very anionic terminal peptide. Since deoxyhemoglobin (Hb) is known to be a strong polyanion-binding protein, we have considered how it may interact with actin and tubulin within the intact cell. Here we demonstrate that actin and tubulin form in vitro a high-affinity complex with Hb. This is shown by measuring, by stopped-flow experiments, the decrease of the binding rate constant of CO to Hb in the presence of increasing amounts of actin and tubulin. One tetramer of Hb is bound by an actin monomer, and about two tetramers by an alpha, beta-tubulin heterodimer. Binding assays in batch experiments with immobilized tubulin give the same stoichiometry. Formation of the complexes involves the 2,3-bisphosphoglycerate-binding site of Hb and a negatively charged domain, most likely the highly acidic N and C-terminal peptides of actin and tubulin. In addition to providing new opportunities to study the structural and functional properties of actin and tubulin, these results support the idea that in the case of partial metabolic depletion of bisphosphoglycerate and ATP in erythrocytes, Hb may interact with oligomeric actin and tubulin present in the cytoskeleton.

Heme protein fluorescence versus pressure.

Fluorescence spectra of several ferric heme proteins have been measured vs. pressure to 6,000 bars. Sperm whale myoglobin (SW Mb), Aplysia myoglobin, leghemoglobin (Lb), and cytochrome P450 all show excitation and emission spectra characteristic of tryptophan in proteins with peak emission at 330-340 nm. At one bar, the fluorescence is weak due to energy transfer to the heme group, which makes the yield a sensitive probe of protein unfolding at high pressure. After an initial decrease of a few percent per kbar, the protein shows a large increase in fluorescence at high pressure. The increase is pH dependent and the results indicate that several high pressure states occur. For SW Mb at 15 degrees C an increase of a factor of 20 occurs with midpoint at 2,000 bars at pH 5 and is only partially reversible, while the increase at pH 7 occurs at 4,000 bars and is only half as large and is completely reversible. Aplysia Mb and Lb show a similar effect, but unfold at a higher pressure than SW Mb. P450 also shows a transition to a state of higher fluorescence, but the transition in this case is irreversible as a stable form, P420, is formed. The fluorescence intensity measurements permit an estimation of the increase in the TRY-heme distance in the high pressure state.