David W. Pumplin

Obscurin modulates the assembly and organization of sarcomeres and the sarcoplasmic reticulum

Obscurin (~800 kDa) in striated muscle closely surrounds sarcomeres at the level of the M‐band and Z‐disk where, we hypothesize, it participates in the assembly of the contractile apparatus and membrane systems required for Ca2+ homeostasis. In this study, we used small inhibitory RNA (siRNA) technology to reduce the levels of obscurin in primary cultures of skeletal myotubes to study its role in myofibrillogenesis and the organization of the sarcoplasmic reticulum (SR). siRNA‐treated myotubes showed a specific and dramatic reduction in the ~800 kDa form of obscurin by reverse transcription‐polymerase chain reaction, immunoblotting, and immunofluorescence. M‐bands and A‐bands, but not Z‐disks or I‐bands, were disrupted when the synthesis of obscurin was inhibited. Small ankyrin 1, an integral protein of the network SR that binds to obscurin, also failed to align around developing sarcomeres in treated myotubes. Myosin and myomesin levels were significantly reduced in treated myotubes but α‐actinin was not, suggesting that down‐regulation of obscurin destabilizes proteins of the M‐band and A‐band but not of the Z‐disk. Our findings suggest that obscurin is required for the assembly of the M‐band and A‐band and for the regular alignment of the network SR around the contractile apparatus.—Kontrogianni‐Konstantopoulos, A., Catino, D. H., Strong, J. C., Sutter, S., Borisov, A. B., Pumplin, D. W., Russell, M. W., Bloch, R. J. Obscurin modulates the assembly and organization of sarcomeres and the sarcoplasmic reticulum. FASEB J. 20, 2102–2111 (2006)

The membrane skeleton.

One important element that defines cell shape is the membrane skeleton. This filamentous network is closely apposed to the cytoplasmic face of the plasma membrane where it gives mechanical support to the membrane, provides specific attachment sites for cytoskeletal components and helps to organize some integral membrane proteins into domains. The membrane skeleton of erythrocytes has been studied extensively by biochemical and ultrastructural methods, but similar structures in other cell types are just beginning to be defined. In this review, David Pumplin and Robert Bloch draw attention to these nonerythroid skeletons and compare and contrast them with the erythrocyte model.

Normal variations in presynaptic active zones of frog neuromuscular junctions

SummaryNeuromuscular junctions of frog cutaneous pectoris muscles were examined by the complementary-replica freeze-fracture technique. The large number of active zones revealed in this way permitted quantitation of normal and disrupted zones in control muscles and in muscles deprived of external calcium for periods of up to eight hours. Although some 87% of the active zones examined had the usual appearance of two double rows of intramembrane particles flanking a low ridge, several varieties of disrupted active zones appeared in which the rows were interrupted, dispersed, rotated, or reduced to single rows. The frequency of disruptions was not increased by calcium deprivation. Disorganized active zones at tips of neuromuscular junctions may represent normal remodelling of the junctions.

Differential expression of carbohydrate blood‐group antigens on rat taste‐bud cells: Relation to the functional marker α‐gustducin

An afferent nerve fiber supplying a taste bud receives input from several taste receptor cells, yet is predominantly responsive to one of the classic taste qualities (salt, acid, sweet, or bitter). This specificity requires recognition between taste receptor cells and nerve fibers that may be mediated by surface markers correlating with function. In an effort to identify potential markers, we used immunofluorescence and confocal microscopy to examine expression of the oligosaccharide blood‐group antigens Lewisb, A, and H type 2 in taste buds of the rat oral cavity. We compared the distributions of these antigens with that of α‐gustducin, a G‐protein subunit implicated in responses to sweet‐ and bitter‐tasting substances. The A and Lewisb antigens were present only on spindle‐shaped cells whose apical processes reached the taste pore. These antigens were not present on epithelial cells surrounding taste buds, and Lewisb was not found elsewhere in the digestive tract. Lewisb and A were not removed by lipid extraction, suggesting that they are present on glycoproteins rather than glycolipids. All Lewisb‐positive cells expressed α‐gustducin, but only a fraction of α‐gustducin–positive cells expressed Lewisb. The fraction of taste‐bud cells expressing Lewisb decreased in the order: vallate papillae > foliate papillae > nasoincisor duct. The epiglottis had almost no taste‐bud cells that expressed Lewisb. The A antigen appeared on taste‐bud cells that also expressed α‐gustducin in the order: foliate and vallate papillae > nasoincisor duct and epiglottis > fungiform papillae. In addition, the A antigen was present on many cells that lacked α‐gustducin in foliate and vallate papillae. In vallate papillae, cells expressed either A or Lewisb, but not both. Lewisb appears to be restricted to differentiated light cells that also express α‐gustducin and may be involved in intercellular interactions of these cells. J. Comp. Neurol. 415:230–239, 1999.

Sarcolemmal Reorganization in Facioscapulohumeral Muscular Dystrophy

We examined the sarcolemma of skeletal muscle from patients with facioscapulohumeral muscular dystrophy (FSHD1A) to learn if, as in other murine and human muscular dystrophies, its organization and relationship to nearby contractile structures are altered.

Synaptic proteins in rat taste bud cells: Appearance in the Golgi apparatus and relationship to ?-gustducin and the Lewisb and A antigens

Taste receptor cells are continuously replaced during the life of the animal, but many of their sensory axons respond primarily to stimuli belonging to a single taste quality. This suggests that a newly arising taste cell must form a synapse with an appropriate sensory axon, requiring cell recognition that is likely to be mediated by surface markers. As an approach to studying this process, we attempted to locate synapses by immunolabeling taste buds of rats for proteins involved in neurotransmitter release. In taste bud cells of vallate papillae and nasoincisor ducts, double‐labeling experiments showed that syntaxin‐1, SNAP‐25, synaptobrevin, and synaptophysin colocalized with the Golgi marker βCOP in elongated cytoplasmic compartments that extended from the perinuclear region into apical and basal processes of the cells. Labeled cells were spindle‐shaped, identifying them as light cells. Syntaxin‐1 appeared only in taste cells, but SNAP‐25, synaptobrevin, and synaptophysin were also seen in nerve fibers. The synaptic vesicle glycoprotein SV2 appeared only in nerve fibers. Taste cells of fungiform papillae did not show immunoreactivity for presynaptic proteins or Golgi markers, but axonal labeling was similar to that in other regions. Taste cells with α‐gustducin could express either presynaptic proteins or the carbohydrate blood group antigen Lewisb, but not both. Therefore, Lewisb and presynaptic proteins are not expressed during the same period in the life of a taste bud cell. Most taste cells expressing syntaxin‐1 (82%) also expressed the A blood group antigen, whether or not they expressed α‐gustducin. J. Comp. Neurol. 427:171–184, 2000.

A model of spectrin as a concertina in the erythrocyte membrane skeleton

To maintain its distinctive biconcave shape, the erythrocyte has a skeleton composed largely of the protein spectrin, which associates closely and exclusively with the cell membrane. Although the membrane skeleton forms through specific protein-protein interactions of defined stoichiometry, it has a flexible structure and organization due to the unusual molecular properties of spectrin. Here we describe these properties and propose a model to account for the extensibility of spectrin and for its organization in the skeleton.

Intercellular interactions in the mammalian olfactory nerve

The small, unmyelinated axons of olfactory sensory neurons project to the olfactory bulb in densely packed fascicles, an arrangement conducive to axo‐axonal interactions. We recently demonstrated ephaptic interactions between these axons in the olfactory nerve layer, the layer of the olfactory bulb in which the axon fascicles interweave and rearrange extensively. In the present study, we hypothesized that the axons, which express connexins, may have another mode of communication: gap junctions. Previous transmission electron microscopy (TEM) studies have failed to demonstrate such junctions. However, the definitive method for detecting gap junctions, freeze fracture, has not been used to examine the interaxonal connections of the olfactory nerve layer. Here, we apply a combined approach of TEM and freeze fracture to determine if gap junctions are present between the olfactory axons. Gap junctions involving olfactory axons were not found. However, by freeze fracture, P faces of both the axons and ensheathing cells (glia that surround the axon fascicles) contained distinctive linear arrays of particles, aligned along the small columns of extracellular space. In axons, few intramembranous particles were present outside of these arrays. Multi‐helix proteins, including ion channels and connexin hemichannels, have been shown to be visible as particles by freeze fracture. This suggests that the proteins important for signal transmission are confined to the linear arrays. Such an arrangement would facilitate ephaptic transmission, calcium waves, current oscillations, and paracrine communication and may be important for olfactory neural code processing. J. Comp. Neurol. 466:230–239, 2003.

Distinctions between gap junctions and sites of intermediate filament attachment in the leech C.N.S.

SummaryFreeze-fracture studies on the nerve cord of the leechHirudo medicinalis reveal that the plasma membranes of various cells, including glial and muscle cells, contain at least two distinct types of aggregated intramembrane particles, identified as hemidesmosomes and gap junctions. Hemidesmosomes consist of angular particles irregularly arranged in circular or elongate patches in external leaflets (E-faces), and are associated with a bundle of intermediate filaments extending into the cytoplasm. Hemidesmosomes of specific axons abut on extracellular space at openings in the surrounding glial sheath. Gap junctions are patches of rounder particles in cytoplasmic leaflets (P-faces) and are more uniformly spaced; they have a corresponding array of pits in the complementary E-face. Gap junctions connect processes of adjacent smooth muscle cells, and apparently interconnect glial processes. Thus, different types of cells in the leech C.N.S. have similar intramembrane specializations. Moreover, the hemidesmosomes and gap junctions might, on superficial examination, be confused.

Association of acetylcholine receptors with peripheral membrane proteins: evidence from antibody-induced coaggregation.

Acetylcholine receptors (AChR) are associated with several peripheral membrane proteins that are concentrated on the cytoplasmic face of the plasma membrane at the neuromuscular junction, and at aggregates of AChR that form in vitro. We tested the linkage among these proteins by inducing microaggregation of AChR, then determining if a given peripheral membrane protein accumulated with the receptors in microaggregates. In most experiments, we used isolated membrane fragments that are rich in AChR and accessible to antibodies against intracellular antigens. We showed that the 43 kD receptor-associated protein always aggregated with AChR, whether microaggregation was driven by antibodies to the 43 kD protein, or to the receptor itself. Antibodies to the 58 kD receptor-associated protein also always aggregated the 58 kD protein with the receptor. Our results are consistent with a model for AChR-rich membrane in which the 43 kD and 58 kD proteins are both closely associated with the AChR.When we induced microaggregation in intact muscle cells with anti-AChR antibodies, our results were less definitive. The 43 kD receptor-associated protein microaggregated with AChR, but the 58 kD protein was not especially enriched at AChR microaggregates. We discuss the advantages of using isolated AChR-rich membrane fragments to study the association of AChR with peripheral membrane proteins.