Localization of phosphorylase kinase subunits at the sarcoplasmic reticulum of rabbit skeletal muscle by monoclonal and polyclonal antibodies

Molecular structures related to phosphorylase kinase have been localized by light and electron microscopy in tissue sections of rabbit skeletal muscle employing polyclonal antibodies directed against the holoenzyme as well as monoclonal antibodies specific for its alpha-, beta- or gamma-subunits. In frozen sections of prefixed muscle fibres both known major regions of glycogen deposition, the intermyofibrillar space and the perinuclear area, are stained predominantly. In sections of unfixed muscle in which cytosolic phosphorylase kinase was removed by extensive washes prior to immunostaining the immunolabel is mainly associated with the sarcoplasmic reticulum (SR). This membrane location is further confirmed by immunoblot analysis of proteins solubilized from isolated SR with Triton X-114. Employing monoclonal antibodies two membrane proteins are identified as the alpha- and beta-subunits of phosphorylase kinase by Western blots. Immunoprecipitates reveal also the gamma-subunit; the delta-subunit, i.e., calmodulin, is enriched with the solubilized enzyme. It proves that a SR membrane associated form of holophosphorylase kinase exists in muscle. Functionally, this kinase might be involved in phosphorylation of phosphatidylinositol present on the SR Ca2+ transport ATPase and thereby might play a role in regulation of Ca2+ transport.     

Demonstration of immunoreactive forms of erythrocyte protein 4.2 in nonerythroid cells and tissues

Protein 4.2 is a major component of the erythrocyte membrane cytoskeleton. Here we show that immunoreactive forms of human (Mr 72,000) and pig (Mr 75,000) protein 4.2 are also associated with the plasma membrane of various nonerythroid cells and tissues, such as platelets, brain, and kidney. Protein 4.2 can be extracted from platelet membranes under the same conditions (pH 11, 1 M KI, 1 M urea) which are required to extract protein 4.2 from the erythrocyte plasma membrane. The demonstration of protein 4.2 in nucleated cells that contain also several other proteins of the erythrocyte membrane cytoskeleton indicates some general principles underlying the molecular construction of the plasma membrane in erythrocytes and nonerythroid cells.     

Association of kidney and parotid Na+, K(+)-ATPase microsomes with actin and analogs of spectrin and ankyrin

Kidney Na+,K(+)-ATPase has been recently shown to bind erythroid ankyrin and to colocalize with ankyrin at the basolateral cell surface of kidney epithelial cells. These observations suggest that Na+,K(+)-ATPase is linked via ankyrin to the spectrin/actin-based membrane cytoskeleton. In the present study we show that Na+,K(+)-ATPase and analogs of spectrin, ankyrin and actin copurify from detergent extracts of pig kidney and parotid gland membranes. Actin, spectrin and ankyrin were extracted from purified Na+,K(+)-ATPase microsomes at virtually identical conditions as their counterparts from the erythrocyte membrane, i.e., 1 mM EDTA (spectrin, actin) and 1 M KCl (ankyrin). Visualization of the stripped proteins by rotary shadowing revealed numerous elongated spectrin-like dimers (100 nm) and tetramers (215 nm), a fraction of which (17%) was associated with globular (10 nm) ankyrin-like particles. Like erythrocyte ankyrin, kidney ankyrin was cleaved into a soluble 72 kDa fragment and a membrane-bound 90 kDa fragment. Consistent with our previous immunocytochemical findings on the pig kidney, Na+,K(+)-ATPase and ankyrin were found to be colocalized at the basolateral plasma membrane of striated ducts and acini of the pig parotid gland. The present findings confirm and extend the recently proposed concept that in polarized epithelial cells Na+,K(+)-ATPase may serve as major attachment site for the spectrin-based membrane cytoskeleton to the basolateral cell domain. Connections of integral membrane proteins to the cytoskeleton may help to place these proteins at specialized domains of the cell surface and to prevent them from endocytosis.     

Intermediate stages in the disassembly of synaptic ribbons in cone photoreceptors of the crucian carp,Carassius carassius

Synaptic ribbons are trilaminated plate-shaped presynaptic densities of certain types of receptor cells and neurons. In cone photoreceptors, these structures dissassemble and reassemble in response to light and to a variety of other stimuli. We used the lithium-ionenhanced disassembly and reassembly of synaptic ribbons to characterize structural intermediates in these cyclic changes. A few minutes after exposure of isolated retinas from the crucian carp (Carassius carassius) to lithium, ribbons fragmented into 50-nm-sized dense globular structures. These small spheres were concentrically surrounded by synaptic vesicles attached to them by stalk-like fine bridging filaments. Disassembly always started at the free cytoplasmic edges of the ribbons and proceeded toward the membrane-associated edges. As the disassembly process never started at the membraneanchored site, synaptic ribbons appeared to be polarized structures with functionally different ends. Spheres were subjected to further depolymerization. They disintegrated into clusters of small granular material and disappeared after ca. 45 min of lithium treatment. Spheres were not observed during the reassembly of synaptic ribbons, indicating that the assembly of synaptic ribbons proceeds via smaller subunits.     

Distribution of myosin and the glial fibrillary acidic protein (GFA Protein) in rat spinal cord and in the human frontal cortex as revealed by immunofluorescence microscopy

Summary The glial fibrillary acidic (GFA) protein and myosin were localized in rat spinal cord and human frontal cortex using specific antibodies against GFA protein from human spinal cord and highly purified smooth myosin from chicken gizzard by means of an indirect immunofluorescence microscopical approach. A strong GFA protein and myosin immunoreactivity was found in astrocytes of the white and grey matter and in the external glial limitans membrane. The very fine branches of astrocytic processes stained with antiGFA protein, but not with anti-myosin. Similar results were obtained with the human frontal cortex, where myosin antibodies failed to reveal the very fine branches of protoplasmic astrocytes.As a whole, staining with the GFA protein antiserum was more crisp than with the myosin antibody.Thanks are due to Professor J.R. Wolff, Max-Planck Institute for Biophysical Chemistry, Göttingen, for stimulating discussions, to Ursula König, Christa Mahlmeister and Renate Steffens for skilful technical assistance, and to Heidi Waluk for the photographic workSupported by grants from Deutsche Forschungsgemeinschaft (Br 634/1, Dr 91/1, Un 34/4, Ste 105/19)Dedicated to Prof. Dr. med. H. Leonhardt on the occasion of his 60. birthday     

Evidence for the concentration of F-actin and myosin in synapses and in the plasmalemmal zone of axons

Fluorescent staining with phalloidin, a specific probe for F-actin, and antibodies to non-muscle myosin from thymus was used to localize actin and myosin in brain neurons of the rat. Phalloidin and anti-myosin displayed a preferential affinity for synaptic formations in the cerebellum, the brain stem, the spinal cord and the retina. The conclusion that F-actin and myosin are concentrated in synaptic terminals was further established by simultaneous staining of isolated rat brain synaptosomes with phalloidin and anti-thymus myosin as well as by the demonstration of a selective affinity of anti-thymus myosin for a 200 000-Mr protein band in gel electrophoretograms of synaptic fractions. Apart from synaptic areas, phalloidin and anti-thymus myosin reacted also, albeit rather weakly, with a narrow circumferential layer located in the area of the plasma membrane of virtually all axons in the white matter and the spinal roots. The spatial coexistence of myosin and actin in brain synapses and axons is of particular interest in view of various dynamic functions that have been proposed for axonal and synaptic actin.     

Distribution of actin and the actin-associated proteins myosin, tropomyosin, alpha-actinin, vinculin, and villin in rat and bovine exocrine glands

Actin, myosin, and the actin-associated proteins tropomyosin, alpha-actinin, vinculin, and villin were localized in acinar cells of rat and bovine pancreas, parotid, and prostate glands by means of immunofluorescent staining of both frozen tissue sections and semithin sections of quick-frozen, freeze-dried, and plastic-embedded tissues. Antibodies to actin, myosin, tropomyosin, alpha-actinin, and villin reacted strongly with a narrow cytoplasmic band extending beneath the luminal border of acinar cells. The presence of villin, which has so far been demonstrated only in intestinal and kidney brush border, was further confirmed by antibody staining of blotted electrophoresis gels of whole acinar cell extracts. Fluorescently labelled phalloidin, which reacts specifically with F-actin, gave similar staining, within the cell apex to that obtained with antibodies to actin, myosin, tropomyosin, alpha-actinin, and villin. In contrast, immunostaining with antibodies to vinculin was restricted to the area of the junctional complex. Ultrastructurally, the apical immunoreactive band corresponded to a dense web composed of interwoven microfilaments, which could be decorated with heavy meromyosin. Outside this apical terminal web, antibodies to myosin and tropomyosin gave only a weak immunostaining (confined to the lateral cell borders) whereas antibodies to actin and alpha-actinin led to a rather strong bead-like staining along the lateral and basal cell membrane most probably marking microfilament-associated desmosomes. Anti-villin immunofluorescence was confined to the apical terminal web. It is suggested that the apical terminal web is important for the control of transport and access of secretory granules to the luminal plasma membrane and that villin, which is known to bundle or sever actin filaments in a Ca(++)-dependent manner, might participate in the regulation of actin polymerization within this strategically located network of contractile proteins.     

Three different actin filament assemblies occur in every hair cell: each contains a specific actin crosslinking protein

The apex of hair cells of the chicken auditory organ contains three different kinds of assemblies of actin filaments in close spatial proximity. These are (a) paracrystals of actin filaments with identical polarity in stereocilia, (b) a dense gellike meshwork of actin filaments forming the cuticular plate, and (c) a bundle of parallel actin filaments with mixed polarities that constitute the circumferential filament belt attached to the cytoplasmic aspect of the zonula adhaerens (ZA). Each different supramolecular assembly of actin filaments contains a specific actin filament cross-linking protein which is unique to that particular assembly. Thus fimbrin appears to be responsible for paracrystallin packing of actin filaments in stereocillia; an isoform of spectrin resides in the cuticular plate where it forms the whisker-like crossbridges, and alpha actinin is the actin crosslinking protein of the circumferential ZA bundle. Tropomyosin, which stabilizes actin filaments, is present in all the actin filament assemblies except for the stereocilia. Another striking finding was that myosin appears to be absent from the ZA ring and cuticular plate of hair cells although present in the ZA ring of supporting cells. The abundance of myosin in the ZA ring of the surrounding supporting cells means that it may be important in forming a supporting tensile cellular framework in which the hair cells are inserted.     

Structure and assembly of hemagglutinin mutants of fowl plague virus with impaired surface transport.

Five temperature-sensitive mutants of influenza virus A/FPV/Rostock/34 (H7N1), ts206, ts293, ts478, ts482, and ts651, displaying correct hemagglutinin (HA) insertion into the apical plasma membrane of MDCK cells at the permissive temperature but defective transport to the cell surface at the restrictive temperature, have been investigated. Nucleotide sequence analysis of the HA gene of the mutants and their revertants demonstrated that with each mutant a single amino acid change is responsible for the transport block. The amino acid substitutions were compared with those of mutants ts1 and ts227, which have been analyzed previously (W. Schuy, C. Will, K. Kuroda, C. Scholtissek, W. Garten, and H.-D. Klenk, EMBO J. 5:2831-2836, 1986). With the exception of ts206, the changed amino acids of all mutants and revertants accumulate in three distinct areas of the three-dimensional HA model: (i) at the tip of the 80-A (8-nm)-long alpha helix, (ii) at the connection between the globular region and stem, and (iii) in the basal domain of the stem. The concept that these areas are critical for HA assembly and hence for transport is supported by the finding that the mutants that are unable to leave the endoplasmic reticulum at the nonpermissive temperature do not correctly trimerize. Upon analysis by density gradient centrifugation, cross-linking, and digestion with trypsin and endoglucosaminidase H, two groups can be discriminated among these mutants: with ts1, ts227, and ts478, the HA forms large irreversible aggregates, whereas with ts206 and ts293, it is retained in the monomeric form in the endoplasmic reticulum. With a third group, comprising mutants ts482 and ts651 that enter the Golgi apparatus, trimerization was not impaired.     

Freeze-fracture studies of cytoplasmic inclusions occurring in experimental lipidosis as induced by amphiphilic cationic drugs.

The ultrastructure of cytoplasmic inclusions, which characterize experimental lipidosis as induced by several amphiphilic cationic drugs, was studied by means of freeze-fracturing and thin-sectioning. Retinal and adrenal tissues of rats chronically treated with high oral doses of chlorphentermine were used. In thin sections the cytoplasmic inclusions, which were previously shown to represent lysosomes overloaded with polar lipids, exhibit lamellated or lattice-like internal patterns. The present freeze-fracture observations are interpreted as to indicate that the lamellated inclusions contain polar lipids in the lamellar phase, whereas those with lattice-like patterns contain polar lipids in a hexagonal phase.