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Clathrin-associated proteins purifies from bovine brain exhibited an ultraviolet spectrum with absorbance maximum at 256 nm and were found to contain tightly bound nucleotide. This nucleotide was identified as AMP and/or ADP by thin-layer and high-performance liquid chromatographic analyses. The phosphorylation state of the bound nucleotide varied with storage conditions, suggesting that exchange with ATP might occur while a molar ratio of two nucleotides per protein molecule is maintained. This nucleotide binding site may play a role in the functions of clathrin-associated proteins.

High-purity bovine brain clathrin elicited antibodies, which were separated by affinity chromatography on clathrin conjugated with CNBr-activated Sepharose 4B. Indirect immunofluorescent labeling on cultured rat fibroblasts showed the characteristic dotted fluorescence throughout the entire cytoplasm and concentrated patches of fluorescence in areas where the Golgi apparatus and GERL (Golgi apparatus-endoplasmic reticulum-lysosome complex) exist next to the nucleus. Cerebellar tissue sections exhibited profuse immunofluorescence in the granular layer; dissociated cultures of neonatal rat cerebellar cells showed intense fluorescence in the cytoplasm and processes of granule cell neurons. The data suggest that clathrin is an abundant protein distinctly localized in the cytoplasm of cells and clearly correlated in its distribution and arrangement with actin stress fibers.

Two monoclonal antibodies (S-8G8 and S-6G7) are characterized that react with an abundant neuronal protein associated with brain clathrin-coated vesicles (CCVs). This 185-kDa polypeptide (NP185) is not a transmembrane cargo molecule and is distinguishable from clathrin by several criteria including neuronal specificity, chymotryptic sensitivity, migration during two-dimensional gel electrophoresis, lack of cross-reactivity of S-8G8 or S-6G7 with purified clathrin, and lack of associated clathrin light chains. When 0.9 M NaCl extracts of CCVs were diluted and immunoprecipitated by either S-8G8 or S-6G7, NP185 precipitated as a complex with a fraction of the CCV assembly polypeptides. Immunofluorescence microscopy of PC12 cells cultured in nerve growth factor (NGF) revealed that NP185 was distributed in a punctate manner throughout the mature neurites. Immunoblot analysis of PC12 cell extracts, taken at various times during NGF-induced differentiation, revealed that steady-state accumulation of NP185 reaches significant levels 3 days after the addition of NGF and returns to undetectable levels when NGF is removed from the cultures. Significantly, the quantity of NP185 detected in differentiated PC12 cells exceeded the quantity of clathrin. These data indicate that while NP185 may be a specialized component of neuronal CCVs, its function in neuronal cells cannot be associated exclusively with these organelles.

Using the Lowicryl K4M embedding technique, together with indirect immunoferritin or immunogold labeling on ultra-thin sections, tubulin, calmodulin and phospholipase A2 were distinctly localized in ejaculated bull spermatozoa. Calmodulin was concentrated on the plasma membrane, nucleus, post-acrosomal substance, and, in lesser amounts, between coarse fibers and axonemal microtubules of the flagellum. Phospholipase A2 was distributed evenly along the plasma membrane, nucleus, acrosome, post-acrosomal substance, and in the flagellum, on mitochondria, fibrous sheath, coarse fibers, between coarse fibers and axonemal microtubules. Antibodies to tubulin labeled only axonemal microtubules, including the central pair of microtubules. Patterns of tubulin labeling were identical when ferritin granule- or gold particle-conjugated antibodies were tested. In agreement with our previous biochemical studies demonstrating calmodulin binding to phospholipase A2, concomitant with enhancement of phospholipase A2 activity (Arch Biochem Biophys 241:413, 1985), the overlapping distribution of calmodulin and phospholipase A2 in several parts of the sperm suggests that these proteins may play a concerted role in male gamete function in preparation for or during fertilization. The distinct distribution of tubulin along flagellum microtubules indicates their special function in sperm mobility.

Affinity-purified monospecific antibodies and indirect immunogold and immunoferritin labeling on ultra-thin sections of low-temperature Lowicryl K4M-embedded samples were used to study the redistribution of calmodulin in ram spermatids and epididymal spermatozoa at the electron microscopic level. Calmodulin appeared as an integral component of well-defined structures or organelles of these cells. In young spermatids, calmodulin was localized in the nucleus, cytoplasm, and developing acrosome. During spermatogenesis and epididymal maturation, calmodulin left the acrosome to reach the perinuclear substance and finally became concentrated in the post-acrosomal area of the head, although some calmodulin remained associated with the tip of the acrosome. Such a redistribution is consistent with the preferential location of Ca2+ in the post-acrosomal cytoplasm of ejaculated spermatozoa. Calmodulin was also observed in the flagellum associated with the plasma membrane and with the motility apparatus, between coarse fibers and axonemal microtubules. These changes in calmodulin distribution may account for the Ca2+-dependent regulation of spermatogenesis and sperm maturation. Calmodulin therefore appears to be a pleiotropic regulator of male gamete development and functions.

By indirect immunofluorescence, the major protein of coated vesicles and coated pits was localized in rat retina. Specific fluorescence was circumscribed to regions containing synaptic endings in high density such as the cell internal plexiform layer (IPL) and external plexiform layer (EPL). Fine dots or patches of fluorescence were seen in the EPL. Weaker fluorescence was observed in the narrow cytoplasmic strips surrounding the large nucleus of the ganglion cell, internal nuclear and external nuclear cell layers. There was no fluorescent staining in the retinal pigment epithelium, a layer where active endocytosis of the outer segment of the rods and cones take place. The high concentration of clathrin found in the IPL and EPL is indicative of active recycling of synaptic membrane concomitant to neurotransmitter release and receptor molecule turnover.

The mechanism by which flat hexagonal lattices of clathrin trimers transform into pentagonal/hexagonal spheres remains a mystery. In light of the geometrical nature of this process we have pursued a mathematical approach to the question. Through the geometrical analysis of flat hexagonal lattices we have discovered three possible forms of transformation to introduce curvature into the centre of the lattice: hub-centre transformation; hub-edge transformation; fringe transformation. Hub-edge and fringe transformations are used first to close the lattice while introducing localized curvature at the edges of the lattice. Hub-centre transformation is used after closure to relax the severely localized curvature generated during closure. This scheme not only maximizes the size of the coated vesicle generated, but also minimizes the number of transformations, thus minimizing the energy expended.

Evidence obtained from quinuclidinylbenzilate binding determinations suggested that muscarinic acetylcholine receptor molecules are present in purified bovine brain coated vesicles. Immunoprecipitates formed from coated vesicles with polyclonal antibodies to clathrin bound on the surface of fixed Staphylococcus aureus cells also showed quinuclidinylbenzilate binding activity. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Muscarinic receptor sites for quinuclidinylbenzilate binding to coated vesicles displayed a Kd of 25 pM and a Bmax of about 191 fmol/mg of protein. Binding competition experiments using atropine, N-methylscopolamine, oxotremorine, and carbamylcholine confirmed the typical muscarinic nature of the binding site. Ranking order of potency for the receptors was: atropine greater than N-methylscopolamine greater than oxotremorine greater than carbachol Analysis of data using a two-site model revealed 13% high-affinity sites for oxotremorine, 66% high-affinity sites for carbachol, and 62% for the antagonist N-methylscopolamine. Heterogeneity of binding affinities for muscarinic drugs detected in the coated vesicles may be related to the presence of coated vesicle subpopulations in brain tissue, (Kohtz, D. S., Kohtz, J. D., Schook, W. J., and Puszkin, S. (1985) J. Cell Biol. 101, 48a; Pfeffer, S. R., and Kelly, R. B. (1985) Cell 40, 949-957).

alpha-Actinin isolated from dog muscle was used to incite antibodies in rabbits, Antibodies, purified by affinity chromatography on CNBr-Sepharose coupled with alpha-actinin and then ferritin-labeled were found to localize on the Z disc of muscle sarcomeres. Molecules of alpha-actinin as an adsorbed monolayer on the surface of polystyrene Lytron particles could bind muscle-actin and tropomyosin from solution. Both the ATPase activity and superprecipitation of an erythrocyte-actin and muscle-myosin hybrid actomyosin complex were altered by alpha-actinin, while tropomyosin diminished these alpha-actinin effects. The binding properties of alpha-actinin are consistent with those of an anchoring protein for microfilaments in nonmuscle cells.

Two molar urea (pH 7.5) and column chromatography on Sepharose 4B were used to separate clathrin (coat protein) from the membrane of coated vesicles from bovine brain. Lytron (polystyrene) particles were used for study of the interaction of clathrin with contractile proteins. Muscle G-actin, F-actin, and alpha-actinin were bound by clathrin-coated Lytron particles, while no interaction was found when muscle tropomyosin and serum albumin were tested. Clathrin molecules dispersed in a solution of 20 mM Tris-HCl (pH 7.5) were found to be elongated. When the pH was adjusted from 7.5 to 6.5, clathrin molecules associated into basketlike or cage structures similar in size and shape to those observed in enriched preparations of coated vesicles. Below pH 6.0, cages or baskets became amorphous aggregates. Raising the pH from 6.5 to 8.0, addition of 5-10 mM ATP or EDTA, or addition of 200 mM KCl resulted in the dissassembly of baskets and the formation of filamentous arrays of various widths. Because of clathrin's biochemical and biophysical properties, its interaction with contractile proteins, and its presence in the membrane of vesicles of various cell types, we classified clathrin in the group of mechanochemical proteins.

Melachronous beating of cilia of epithelial surfaces of most respiratory airways moves the overlying mucous layer in a caudal direction. The molecular mechanisms controlling ciliary beat remain largely unknown. Calcium, an element in its cationic form, is ubiquitous in biological functions and its concentration is critical for ciliary beating. Calmodulin, a calcium-binding protein which regulates the activity of many enzymes and cellular processes, may regulate ciliary beating by controlling enzymes responsible for mechanochemical movement between adjacent peripheral microtubule doublets composing the ciliary axoneme. As a first step in describing a calmodulin-related controlling mechanism for ciliary beating, calmodulin was localized in the ciliated cells lining the respiratory tracts of hamsters by electron microscopy, using an indirect immunoperoxidase technique with anticalmodulin antibodies as the molecular probe. Thin-sections revealed calmodulin located on microtubules and dynein arms of the ciliary shaft, basal body, apical cytoskeletal microtubules, and plasma membranes in specimens fixed with 1 mM Ca+2. Specimens fixed with less Ca+2 (1 microM), Mn+2, Mg+2, and EGTA showed a diffuse pattern of calmodulin with loci of greatest densities on basal body microtubule triplets. Demembranated specimens showed a less specific localization on axonemal microtubules but only on cells fixed with Ca+2. Calmodulin, by binding calcium, may function in ciliary beating in the respiratory tract of mammals either directly or indirectly through its effects on the energy-producing enzymes and by control of Ca+2 flux through plasma membranes.

The N-terminal segment of human interleukin-2 (hIL-2) appears to mediate binding of the beta hIL-2 receptor (R. Robb, C. Rusk, J. Yodoi, and W. Greene, Proc. Natl. Acad. Sci. USA 84:2002-2006, 1987). An affinity-purified antibody prepared against this peptide segment (p81) is shown here to cross-react with a homologous region of the human T-cell leukemia virus type I (HTLV-I) envelope glycoprotein, raising the interesting possibility that the envelope glycoprotein of HTLV-I can interact with the beta hIL-2 receptor.

The two forms of clathrin light chains (LCA and LCB) or clathrin-associated proteins (CAP1 and CAP2) have presented an immunochemical paradox. Biochemically similar, both possess two known functional parameters: binding the clathrin heavy chain and mediating the action of an uncoating ATPase. All previously reported anti-CAP mAbs, however, react specifically with only CAP1 (Brodsky, F. M., 1985, J. Cell Biol., 101:2047-2054; Kirchhausen, T., S. C. Harrison, P. Parham, and F. M. Brodsky, 1983, Proc. Natl. Acad. Sci. USA, 80:2481-2485). Four new anti-CAP mAbs are reported here: two, C-7H12 and C-6C1, react with both forms; two others, C-10B2 and C-4E5, react only with the lower form. Sandwich ELISAs indicated that C-10B2, C-4E5, C-6C1, and C-7H12 react with distinct epitopes. Monoclonal antibodies C-10B2 and C-4E5 immunoprecipitate clathrin-coated vesicles (CCVs) and react with CAP2 epitopes accessible to chymotrypsin on the vesicle. These mAbs inhibit phosphorylation of CAP2 by endogenous CCV casein kinase II. In contrast, C-6C1 and C-7H12 react with epitopes that are relatively insensitive to chymotrypsin. CAP peptide fragments containing these epitopes remain bound to reassembled cages or CCVs after digestion. Immunoprecipitation and ELISAs demonstrate that C-7H12 and C-6C1 react with unbound CAPs but not with CAPs bound to triskelions or CCVs. The data indicate that the CAPs consist of at least two discernible structural domains: a nonconserved, accessible domain that is relevant to the phosphorylation of CAP2 and a conserved, inaccessible domain that mediates the binding of CAPs to CCVs.

The assembly of clathrin into baskets or cages in vitro may depend on formation of complex between clathrin and a polypeptide doublet migrating in the 30000-mol.wt. region. Clathrin with several associated proteins was isolated from coated-vesicle fractions of bovine cerebral cortex. Most associated proteins were separated by Sepharose 4B column chromatograhy. The eluted clathrin retained only the 30000-mol.wt. doublet and assembled into baskets at pH 6.5. Limited proteolysis of coated vesicles or clathrin assembled as baskets removed these clathrin-associated proteins (CAPs) without detectably altering clathrin. Enzyme-treated clathrin assembled into open-lattice structures but no longer formed baskets in vitro. Latex particles with bound enzyme cleaved the CAPs from coated vesicles and clathrin baskets, suggesting that the CAPs protrude from the exterior of the clathrin lattice.

alpha-Actinin isolated from bovine brain migrated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis like muscle alpha-actinin with an apparent mol.wt. of 100000 and cross-reacted with antibodies to muscle alpha-actinin. Brain alpha-actinin modulated actin-myosin Mg2+-activated adenosine triphosphatase activity and, when bound by polystyrene particles, was found to bind muscle actin and tropomyosin from solution. Brain alpha-actinin, in conjunction with the other components of the contractile and relaxing complex, may play a role in the release of neurotransmitters from synaptic vesicles.

A protein kinase activity was observed in coated vesicles, prepared from bovine brain, that had clathrin-associated protein 2 (CAP2, also known as clathrin light chain 2) as its principal substrate. Coated vesicles were purified by sucrose density gradient centrifugation followed by Sephacryl S-1000 column chromatography, and all buffers utilized in these procedures contained a mixture of proteolysis inhibitors to maintain CAP2 kinase activity. Incubation of vesicles with [gamma-32P]ATP in the presence of 7 microM polylysine resulted in an overall increase in the incorporation of phosphate. NaDodSO4/PAGE revealed that the principal recipient of this additional phosphate was CAP2 (Mr 33,000), the faster-migrating component of the clathrin coat-associated proteins, whereas CAP1 (Mr 36,000) was not phosphorylated. A number of other proteins, in the Mr 140,000 and 100,000 regions, were phosphorylated to a lesser extent. Polyarginine and polyethylenimine also supported CAP2 phosphorylation, but arginine and lysine were ineffective. The phosphorylated protein was identified as CAP2 because addition of exogenous CAPs resulted in increased incorporation of label into Mr 33,000 polypeptides and because heat treatment of labeled vesicles followed by ultracentrifugation resulted in recovery of labeled Mr 33,000 protein in the supernatant. Phosphorylation of CAP2 may play a regulatory role in clathrin coat/coated vesicle functions.