Ultrastructural localization of immunoreactive neurophysins using monoclonal antibodies and protein A-gold

Using three different monoclonal antibodies against rat neurophysins (5), with protein A-gold as immunocytochemical marker (27), the murid hypothalamoneurohy-pophysial system was studied at the ultrastructural level. Postembedding staining was done on epoxy-embedded sections of supraoptic nuclei and posterior pituitaries. Specific immunolabeling of vasopressinergic and oxytocinergic neurosecretory granules was observed in tissues fixed with glutaraldehyde or glutaraldehyde mixtures (containing paraformaldehyde and picric acid), with or without osmium tetroxide postfixation and with or without sodium metaperiodate oxidation. Some autophagic vacuoles containing lysed neurosecretory granules were also neurophysin immunoreactive. Nonspecific background staining was extremely low. An attempt was made to appraise labeling intensities semiquantitatively by counting gold particles in relation to number of secretory granules per axonal varicosity. Immunoreactivity was measurably influenced by the mode of fixation, sodium metaperiodate oxidation, and titer and affinity of the antibody. The protein A-gold technique using monoclonal antibodies against neurophysins provides a superior means of ultrastructural analysis of the hypothalamoneurohypophysial system, both visually and morphometrically.     

Association of the Mr 58,000 postsynaptic protein of electric tissue with Torpedo dystrophin and the Mr 87,000 postsynaptic protein.

Dystrophin was purified by immunoaffinity chromatography from detergent-solubilized Torpedo electric organ postsynaptic membranes using monoclonal antibodies. A major doublet of proteins at Mr 58,000 and minor proteins at Mr 87,000, Mr 45,000, and Mr 30,000 reproducibly copurified with dystrophin. The Mr 58,000 and Mr 87,000 proteins were identical to previously described peripheral membrane proteins (Mr 58,000 protein and 87,000 protein) whose muscle homologs are associated with the sarcolemma (Froehner, S. C., Murnane, A. A., Tobler, M., Peng, H. B., and Sealock, R. (1987) J. Cell Biol. 104, 1633-1646; Carr, C., Fischbach, G. D., and Cohen, J. B. (1989) J. Cell Biol. 109, 1753-1764). The copurification of dystrophin and Mr 58,000 protein was shown to be specific, since dystrophin was also captured with a monoclonal antibody against the Mr 58,000 protein but not by several control antibodies. The Mr 87,000 protein was a major component (along with the Mr 58,000 protein) in material purified on anti-58,000 columns, suggesting that the Mr 58,000 protein forms a distinct complex with the Mr 87,000 protein, as well as with dystrophin. Immunofluorescence staining of skeletal and cardiac muscle from the dystrophin-minus mdx mouse with the anti-58,000 antibody was confined to the sarcolemma as in normal muscle but was much reduced in intensity, even though immunoblotting demonstrated that the contents of Mr 58,000 protein in normal and mdx muscle were comparable. Thus, the Mr 58,000 protein appears to associate inefficiently with the sarcolemmal membrane in the absence of dystrophin. This deficiency may contribute to the membrane abnormalities that lead to muscle necrosis in dystrophic muscle.     

Ultrastructural localization of human kidney antigens using monoclonal antibodies.

A highly sensitive method of ultrastructural-immunoperoxidase staining was developed for use with monoclonal antibodies which have been raised in this laboratory to a variety of antigens of the human kidney. Because of the susceptibility of the antigens to fixation and processing, a four layer, pre-embedding method of staining was used. Results confirmed and clarified previously reported light microscopy results, indicating that an antigen recognized by the PHM5 antibody was found on the podocyte cell membrane within the glomerulus and was not present within the glomerular basement membrane. The antigen was also present on the extraglomerular endothelial cell membrane. The study also demonstrated the presence of an antigen specific to endothelial cells throughout the renal cortex, and gave further insight into the precise localization of glomerular basement membrane components including fibronectin. The method of staining is now being used together with detailed ultrastructural studies to identify the cells produced from isolated glomeruli in tissue culture.     

Determination of the tissue distributions and relative concentrations of the postsynaptic 43-kDa protein and the acetylcholine receptor in Torpedo

A protein of Mr 43,000 (43-kDa protein) occurs on the postsynaptic membrane in close association with the acetylcholine receptor and comprises a major part of the postsynaptic cytoskeletal apparatus. We have devised an immunological assay for the 43-kDa protein to determine if it is confined to receptor-specific sites or if it, like general cytoskeletal proteins, has a more widespread tissue distribution. The assay utilizes monoclonal antibodies (Mab) to the 43-kDa protein that recognize two spatially separate epitopes. One Mab, attached to the well of a microtiter plate, binds the antigen which is then available to bind the biotin-derivatized second Mab. Bound second antibody is detected with either avidin-alkaline phosphatase or a more elaborate system using avidin, rabbit anti-avidin, and anti-rabbit IgG-alkaline phosphatase conjugate. A similar assay was developed for the receptor. The 43-kDa protein and the receptor are found in electric organ and, in 500-fold lower concentrations, in skeletal muscle but are not detectable in heart, liver, pancreas, or brain. In electric organ, the receptor and the 43-kDa protein are present in approximately equimolar concentrations. These results indicate that the 43-kDa protein is not a general membrane-associated cytoskeletal element and that its occurrence, and possibly also its function, is related to the acetylcholine receptor.     

A postsynaptic Mr 58,000 (58K) protein concentrated at acetylcholine receptor-rich sites in Torpedo electroplaques and skeletal muscle

In the study of proteins that may participate in the events responsible for organization of macromolecules in the postsynaptic membrane, we have used a mAb to an Mr 58,000 protein (58K protein) found in purified acetylcholine receptor (AChR)-enriched membranes from Torpedo electrocytes. Immunogold labeling with the mAb shows that the 58K protein is located on the cytoplasmic side of Torpedo postsynaptic membranes and is most concentrated near the crests of the postjunctional folds, i.e., at sites of high AChR concentration. The mAb also recognizes a skeletal muscle protein with biochemical characteristics very similar to the electrocyte 58K protein. In immunofluorescence experiments on adult mammalian skeletal muscle, the 58K protein mAb labels endplates very intensely, but staining of extrasynaptic membrane is also seen. Endplate staining is not due entirely to membrane infoldings since a similar pattern is seen in neonatal rat diaphragm in which postjunctional folds are shallow and rudimentary, and in chicken muscle, which lacks folds entirely. Furthermore, clusters of AChR that occur spontaneously on cultured Xenopus myotomal cells and mouse muscle cells of the C2 line are also stained more intensely than the surrounding membrane with the 58K mAb. Denervation of adult rat diaphragm muscle for relatively long times causes a dramatic decrease in the endplate staining intensity. Thus, the concentration of this evolutionarily conserved protein at postsynaptic sites may be regulated by innervation or by muscle activity.     

Binding of antibodies to acetylcholine receptors in Electrophorus and Torpedo electroplax membranes

Antisera against purified acetylcholine receptors from the electric tissues of Torpedo californica and of Electrophorus electricus were raised in rabbits. The antisera contain antibodies which bind to both autologous and heterologous receptors in solution as shown by an immunoprecipitation assay. Antibodies in both types of antisera bind specifically to the postjunctional membrane on the innervated surface of the intact electroplax from Electrophorus electric tissue as demonstrated by an indirect immunohistochemical procedure using horseradish peroxidase conjugated to anti-rabbit IgG. Only anti-Electrophorus receptor antisera, however, cause inhibition of the receptor-mediated depolarization of the intact Electrophorus electroplax. The lack of inhibition by anti-Torpedo receptor antibodies, which do bind, suggests that the receptor does not undergo extensive movement during activity. The binding of anti-Torpedo antibodies to receptor-rich vesicles prepared by subcellular fractionation of Torpedo electric tissue was demonstrated by both direct and indirect immunohistochemical methods using ferritin conjugates. These vesicles can be conveniently collected and prepared for electron microscopy on Millipore filters, a procedure requiring only 25 micrograms of membrane protein per filter. In addition, it was possible to visualize the binding of anti-Torpedo receptor antibodies directly, without ferritin. These anti-Torpedo receptor antibodies, however, do not inhibit the binding of acetylcholine or of alpha-neurotoxin to receptor in Torpedo microsacs but do inhibit binding of alpha-neurotoxin to Torpedo receptor in Triton X-100 solution. It is likely that the principal antigenic determinants on receptor are at sites other than the acetylcholine-binding sites and that inhibition of receptor function, when it occurs, may be due to a stabilization by antibody binding of an inactive conformational state.     

Identification and purification of a novel Mr 43,000 tropomyosin-binding protein from human erythrocyte membranes

A new Mr 43,000 tropomyosin-binding protein (TMBP) has been identified in erythrocyte membranes by binding of 125I-labeled Bolton-Hunter tropomyosin to nitrocellulose blots of membrane proteins separated by sodium dodecyl sulfate-gel electrophoresis. This protein is not actin, because 125I-tropomyosin does not bind to purified actin on blots. Binding of 125I-tropomyosin to this protein is specific because it is inhibited by excess unlabeled tropomyosin but not by F-actin or muscle troponins. This protein has been purified to 95% homogeneity from a 1 M Tris extract of tropomyosin-depleted erythrocyte membranes by DEAE-cellulose and hydroxylapatite chromatography, followed by gel filtration on Ultrogel AcA 44. The purified protein has a Stokes radius of 3.9 nm and a sedimentation coefficient of 2.8 S, corresponding to a native molecular weight of 43,000. Binding of 125I-tropomyosin to the purified TMBP saturates at one tropomyosin molecule (Mr 60,000) to two Mr 43,000 TMBPs, with an affinity of about 5 X 10(-7) M. The TMBP is associated with the membrane skeleton after extraction of membranes with the non-ionic detergent, Triton X-100, and is present with respect to tropomyosin at a ratio of about one for every two tropomyosin molecules. Because there is enough tropomyosin for two tropomyosin molecules to be associated with each of the short actin filaments in the membrane skeleton, the erythrocyte membrane TMBP, together with tropomyosin, could function to restrict the number of spectrin molecules attached to each of the short actin filaments and thus specify the hexagonal symmetry of the spectrin-actin lattice. Alternatively, this TMBP could be homologous to one of the muscle troponins and might function with tropomyosin to regulate erythrocyte actomyosin-ATPase activity and influence erythrocyte shape.     

Arrangement of the acetylcholine receptor subunits in the resting and desensitized states, determined by cryoelectron microscopy of crystallized Torpedo postsynaptic membranes

Two conformational states of the nicotinic acetylcholine receptor have been investigated by cryoelectron microscopy of flattened vesicular crystals grown from Torpedo marmorata postsynaptic membranes. One was obtained from the vesicles without acetylcholine present, and is presumed to correspond to the native, or resting state; the other was obtained from the vesicles after exposure to 100 microM to 5 mM carbamylcholine (an acetylcholine analogue) and is presumed to correspond to a desensitized state. Both conformations were determined in three-dimensions to a resolution of 18 A, sufficient to reveal the configurations of the five subunits around the central ion channel over most of their length. The subunits of either structure have a similar appearance, consistent with their amino acid homology. They are each aligned almost parallel to the axis of the receptor, conferring a high degree of pentagonal symmetry to the bilayer portion and a contiguous region on the synaptic side. Their external surfaces form a pronounced ridge in the bilayer portion, which broadens toward the synaptic end. Comparison of features in the two three-dimensional maps reveals that carbamylcholine induces a quaternary rearrangement, involving predominantly the delta-subunit. The densities corresponding to this subunit are tilted by approximately 10 degrees tangential to the axis of the receptor over a large fraction of its length, and become misaligned relative to the densities corresponding to the other four subunits. The gamma-subunit is also affected, being displaced slightly away from the axis of the receptor. The alpha- and beta-subunits may be affected on a more localized scale. The overall changes are most pronounced in the synaptic region, where the ligand-binding site is located, and in the cytoplasmic region, which may be closer to the gate of the channel. The physiological process of desensitization appears to be associated with a structural transition in which the subunits switch to a less symmetrical configuration.     

Interaction of the 43K protein with components of Torpedo postsynaptic membranes

Interactions of the major Mr 43 000 peripheral membrane protein (43K protein) with components of Torpedo postsynaptic membranes have been examined. Treatment of membranes with copper o-phenanthroline promotes the polymerization of 43K protein to dimers and higher oligomers. These high molecular weight forms of 43K protein can be converted to monomers by reduction with dithiothreitol and do not contain any of the other major proteins found in these membranes, including the subunits of the acetylcholine receptor, as shown by immunoblotting with monoclonal antibodies. To study directly its interactions with the membrane, the 43K protein was radioiodinated and purified by immunoaffinity chromatography. Purified 43K protein binds tightly to pure liposomes of various compositions in a manner that is not inhibited by KCl concentrations up to 0.75 M. The binding can be reversed by adjusting the pH of the reaction to 11, the same treatment that removes 43K protein from postsynaptic membranes. Unlabeled 43K protein solubilized from Torpedo membranes with cholate can be reconstituted with exogenously added lipids in the absence of the receptor. The results suggest that 43K protein molecules are amphipathic and that they may interact with each other and with the lipid bilayer. These interactions cannot explain the coextensive distribution of 43K proteins with acetylcholine receptors in situ. However, they could account for the association of the 43K protein with the postsynaptic membrane and may contribute to the maintenance of the structure of the cytoplasmic specialization of which this protein is a major component.     

Mapping of surface structures of electrophorus acetylcholine receptor using monoclonal antibodies

Forty monoclonal antibodies to acetylcholine receptor from the electric organs of Electrophorus electricus have been characterized by immunoglobulin isotype, affinity for receptor, and specificity for species, subunit, and determinants within subunits. Using these antibodies, nine immunogenic regions on the receptor molecule were distinguished. Most of these are species specific, and are located on various subunits of the acetylcholine receptor. The least species-specific region forms the "main immunogenic region" (MIR). Most monoclonal antibodies and most antibodies in conventional antisera are directed at this region. The MIR is located on the extracellular surface of the alpha subunits and is homologous to the MIR which we previously described on Torpedo californica receptor. An homologous MIR is also a characteristic feature of receptor from mammalian muscle. The possible immunological and structural significance of the MIR is discussed.     

Ultrastructural localization of rat Clara cell 10 KD secretory protein by the immunogold technique using polyclonal and monoclonal antibodies

Using a monoclonal antibody and affinity-purified polyclonal antiserum against a 10 KD protein isolated from rat pulmonary lavage, we have localized the protein within Clara cells by a post-embedment protein A-gold technique. The gold particles were localized over the secretory granules of rat Clara cells. Ultrastructural immunolocalization was abolished when the primary antibodies were previously absorbed with purified 10 KD protein. Other pulmonary cells, including type II pneumocytes and ciliated cells, were negative with this technique. These results demonstrate the presence of the 10 KD protein in the secretory granules of the Clara cell and support the concept that this protein constitutes a specific and unique secretory product of Clara cells.     

Activation and inactivation kinetics of Torpedo californica acetylcholine receptor in reconstituted membranes

By use of a quench-flow technique to measure tracer ion flux rates in a physiologically significant time domain, the kinetics of activation and inactivation of purified reconstituted acetylcholine receptor (AChR) were investigated. After solubilization in sodium cholate, purification by affinity chromatography, and reconstitution into soybean lipids, the AChR from Torpedo californica displayed a characteristically fast rate of ion influx measured with 86Rb+. At 4 degrees C 1 mM carbamoylcholine (Carb) stimulated a fast (t1/2 = 7 ms) first-order filling of vesicle internal volume that presented a 10(4)-fold stimulation of ion flux rate by Carb. The concentration dependence of activation was sigmoidal with a half-maximal value at 3 X 10(-4) M Carb. In the presence of Carb, the purified AChR also underwent a two-step inactivation (desensitization) process. Inactivation was measured by preincubating AChR with Carb for various times (milliseconds to minutes) and then measuring the 86Rb+ influx rate. The two inactivation processes were each characterized by a distinct maximum rate (5.3 and 0.10 s-1) and by a different dependence on Carb concentration. The slow phase of inactivation gave a half-maximal rate at 2.5 X 10(-4) M Carb, and the fast inactivation was half-maximal at 1.3 X 10(-3) M Carb. The concentration dependence curves for both inactivation processes were approximately hyperbolic. The results are discussed in terms of models that describe the relationship between ligand binding and the processes of channel activation and desensitization.     

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.     

Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies.

Three peptides corresponding to residues 354-367, 364-374, 373-387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP-dependent protein kinase, the tyrosine-specific protein kinase and the calcium/phospholipid-dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase it was shown that only peptides 354-367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP-dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non-phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Antibodies to peptide 354-367 were found to react preferentially with non-phosphorylated receptor whereas the two other anti-peptide antibodies bound equally to phosphorylated and non-phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms.     

Protease effects on the structure of acetylcholine receptor membranes from Torpedo californica

Protease digestion of acetylcholine receptor-rich membranes derived from Torpedo californica electroplaques by homogenization and isopycnic centrifugation results in degradation of all receptor subunits without any significant effect on the appearance in electron micrographs, the toxin binding ability, or the sedimentation value of the receptor molecule. Such treatment does produce dramatic changes in the morphology of the normally 0.5- to 2-microns-diameter spherical vesicles when observed by either negative-stain or freeze-fracture electron microscopy. Removal of peripheral, apparently nonreceptor polypeptides by alkali stripping (Neubig et al. 1979, Proc. Natl. Acad. Sci. U. S. A. 76:690-694) results in increased sensitivity of the acetylcholine receptor membranes to the protease trypsin as indicated by SDS gel electrophoretic patterns and by the extent of morphologic change observed in vesicle structure. Trypsin digestion of alkali- stripped receptor membranes results in a limit degradation pattern of all four receptor subunits, whereupon all the vesicles undergo the morphological transformation to minivesicles. The protein-induced morphological transformation and the limit digestion pattern of receptor membranes are unaffected by whether the membranes are prepared so as to preserve the receptor as a disulfide bridged dimer, or prepared so as to generate monomeric receptor.     

Asynchronous assembly of the acetylcholine receptor and of the 43-kD nu1 protein in the postsynaptic membrane of developing Torpedo marmorata electrocyte

The assembly of the nicotinic acetylcholine receptor (AchR) and the 43-kD protein (v1), the two major components of the post synaptic membrane of the electromotor synapse, was followed in Torpedo marmorata electrocyte during embryonic development by immunocytochemical methods. At the first developmental stage investigated (45-mm embryos), accumulation of AchR at the ventral pole of the newly formed electrocyte was observed within columns before innervation could be detected. No concomitant accumulation of 43-kD immunoreactivity in AchR-rich membrane domains was observed at this stage, but a transient asymmetric distribution of the extracellular protein, laminin, which paralleled that of the AchR, was noticed. At the subsequent stage studied (80-mm embryos), codistribution of the two proteins was noticed on the ventral face of the cell. Intracellular pools of AchR and 43-kD protein were followed at the EM level in 80-mm electrocytes. AchR immunoreactivity was detected within membrane compartments, which include the perinuclear cisternae of the endoplasmic reticulum and the plasma membrane. On the other hand, 43-kD immunoreactivity was not found associated with the AchR in the intracellular compartments of the cell, but codistributed with the AchR at the level of the plasma membrane. The data reported in this study suggest that AchR clustering in vivo is not initially determined by the association of the AchR with the 43-kD protein, but rather relies on AchR interaction with extracellular components, for instance from the basement membrane, laid down in the tissue before the entry of the electromotor nerve endings.     

Ultrastructural immunocytochemical localization of the transferrin receptor using a monoclonal antibody in human KB cells

Using a monoclonal antibody (HB21) against the human transferrin receptor, we have localized this receptor in cultured KB human carcinoma cells by fluorescence and ultrastructural immunocytochemistry. The receptor was found diffusely distributed on the cell surface, concentrated in clathrin-coated pits of the cell surface, in intracellular endocytic vesicles (receptosomes) derived from coated pits, in tubular elements of the trans-reticular Golgi system, and in microtubule-associated membranous elements thought to be part of the constitutive exocytic system. This distribution is the same as that previously shown for labeled transferrin in these same cells (Willingham MC, Hanover JA, Dickson BB, Pastan J: Proc Natl Acad Sci USA 81:175, 1984). No significant amounts of receptor were found in lysosomes. An aggregation of membranous elements containing this receptor was found in the pericentriolar region of cells during mitosis. Together with the previous data on the immunocytochemical localization of transferrin, these results suggest that the transferrin receptor may constitutively enter and exit KB cells by endocytosis and exocytosis, carrying bound transferrin into and out of the cell for the purpose of supplying iron from the extracellular environment for cell growth.