Mapping of the calpain proteolysis products of the junctional foot protein of the skeletal muscle triad junction

Summary The Ca²⁺ activated neutral protease calpain II in a concentration-dependent manner sequentially degrades the Junctional foot protein (JFP) of rabbit skeletal muscle triad junctions in either the triad membrane or as the pure protein. This progression is inhibited by calmodulin. Calpain initially cleaves the 565 kDa JFP monomer into peptides of 160 and 410 kDa, which is subsequently cleaved to 70 and 340 kDa. The 340 kDa peptide is finally cleaved to 140 and 200 kDa or its further products. When the JFP was labeled in the triad membrane with the hydrophobic probe 3-(trifuoromethyl) 3-(m) [¹²⁵I]iodophenyl diazirine and then isolated and proteolysed with calpain II, the [¹²⁵I] was traced from the 565 kDa parent to M _r, 410 kDa and then to 340 kDa, implying that these large fragments contain the majority of the transmembrane segments. A 70-kDa frament was also labeled with the hydrophobic probe, although weakly suggesting an additional transmembrane segment in the middle of the molecule. These transmembrane segments have been predicted to be in the C-terminal region of the JFP. Using an ALOM program, we also predict that transmembrane segments may exist in the 70 kDa fragment. The JFP has eight PEDST sequences; this finding together with the calmodulin inhibition of calpain imply that the JFP is a PEDST-type calpain substrate. Calpain usually cleaves such substrates at or near calmodulin binding sites. Assuming such sites for proteolysis, we propose that the fragments of the JFP correspond to the monomer sequence in the following order from the N-terminus: 160, 70, 140 and 200 kDa. For this model, new calmodulin sequences are predicted to exist near 160 and 225 kDa from the N-terminus. When the intact JFP was labeled with azidoATP, label appeared in the 160 and 140 kDa fragments, which according to the above model contain the GXGXXG sequences postulated as ATP binding sites. This transmembrane segment was predicted by the ALOM program. In addition, calpain and calpastatin activities remained associated with triad component organelles throughout their isolation. These findings and the existence of PEDST sequences suggest that the JFP is normally degraded by calpain in vivo and that degradation is regulated by calpastatin and calmodulin     

Purification and properties of the RuvA and RuvB proteins of Escherichia coli

Summary The RuvA and RuvB proteins of Escherichia coli play important roles in the post-replicational repair of damaged DNA, genetic recombination and cell division. In this paper, we describe the construction of over expression vectors for RuvA and RuvB and detail simple purification schemes for each protein. The purified 22 kDa RuvA polypeptide forms a tetrameric protein (M_r ca. 100000) as observed by gel filtration. The tetramer is stabilised by strong disulphide bridges that resist denaturation during SDS-PAGE (in the absence of boiling and -mercaptoethanol). In contrast, purified RuvB polypeptides (37 kDa) weakly associate to form a dimeric protein (M_r ca. 85000). At low protein concentrations, the RuvB dimer dissociates into monomers. The multimeric forms of each protein may be covalently linked by the bifunctional cross-linking reagent dimethyl suberimidate. Addition of purified RuvA and RuvB to a RecA-mediated recombination reaction was found to stimulate the rate of strand exchange leading to the rapid formation of heteroduplex DNA.     

Retinular fine structure in compound eyes of diurnal and nocturnal sphingid moths

Summary Retinular fine structure has been compared in the superposition compound eyes of three sphingid moths, one nocturnal, Cechenena, and two diurnal, Cephonodes and Macroglossum. Cechenena and Cephonodes have tiered retinas with three kinds of retinular cells: two distal, six regular and one basal. The distal retinular cells in Cechenena are special in having a complex partially intracellular rhabdomere not present in Cephonodes. Macroglossum lacks the distal retinular cell. In Cephonodes a unique rhabdom type, formed by the six regular retinular cells in the middle region of the retinula, is divided into three separate longitudinal plates arranged closely parallel to one another. Their constituent microvilli are consequently all nearly unidirectional. The ratio of rhabdom volume to retinular cell volume in the two diurnal sphingids is 10–27%; this is about the same as that (25%) of skipper butterflies, but significantly smaller than in the nocturnal Cechenena (60%). In the diurnal sphingids retinular cell membranes show elongate meandering profiles with septate junctions between adjacent retinular cells. From the comparative fine structure of their eyes the diurnal sphingids and the skippers would appear to be phylogenetically closely related.Supported in part by grants from Ministry of Education Japan (Special Project Research in Animal Behaviors)     

Gap junctions and impulse propagation in embryonic epithelium of Amphibia

Summary Epithelium of amphibian embryos (Cynops orientalis, Xenopus laevis) was found in preceding experiments to generate and conduct impulses during a limited stage (26–37) of development. In order to elucidate the structural basis of impulse propagation, epithelial cells of four stages were examined by the freeze-etching method: (I) before and (II) during acquisition of conductivity; (III) when propagation was fully established, and (IV) when it was no longer present. Only few gap junctions (GJ) of small size were found in groups I and IV. GJ in epithelia of group III were increased in number and size, and appeared morphologically coupled, i.e., with more loosely arranged connexons. The size of gap-junctional particles did not differ significantly between coupled and uncoupled stages. Zonulae occludentes seemed leaky in stage I, and tight in stages II–IV. Thus, the morphological characteristics of specialized junctions between non excitable cells correlated with the opening and closing of low resistance intercellular current pathways during embryonic development.Gap junctions in particular seem to form an essential link in the non-neural stimulus-response system, which may facilitate the mobility of the embryo during early phases of aquatic life before the reflex pathways have been established. Coupling and uncoupling of gap junctions may also play an important role in the regulation of cell differentiation and morphogenetic movement. The experimental model used in this study provides a useful tool for further investigations of structural correlates of gap junctional permeability under physiological conditions.     

Ultrastructural and biochemical identification of con A receptors in the desmosome

Correlated ultrastructural and biochemical methods were used to identify and localize Concanavalin A (Con A) receptors in the desmosomes of bovine epidermis. Specific carbohydrate residues were labeled with ferritin-Con A in thin sections of tissue embedded in a hydrophilic resin. Quantitative mapping of ferritin distribution in labeled desmosomes revealed that Con A receptors are localized in the intercellular zone and concentrated along the desmosomal midline or central dense stratum. Labeling was almost entirely absent when sections were treated with ferritin-Con A in the presence of 0.1 M α-methyl mannoside, a hapten-inhibitor of Con A. “Whole” desmosomes and desmosomal intercellular regions (desmosomal “cores”) were purified from bovine muzzle epidermis. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals a limited number of major desmosomal protein constituents. Certain of these are glycoproteins and are greatly enriched in the core fraction. Almost all the desmosomal glycoproteins are intensely labeled when electrophoretic gels of whole desmosome or core fractions are exposed to fluorescent Concanavalin A.     

Multiple Molecular Forms of Acetylcholine Receptors in Cultured Skeletal Muscle Cells: Subcellular Localization and Characterization

Abstract: Skeletal muscle cells of newborn rats, cultured in the absence of neuronal influence, were found to contain two types of cell surface acetylcholine receptors as demonstrated by isoelectric focusing. The isoelectric points of the two types of receptors were indistinguishable from those of junctional and extrajunctional types of receptors in mature animals. The cultured cells had two classes of intracellular α-bungarotoxin (αBT) binding components; one had the same sedimentation coefficient as that of surface receptors (9S), and the other had much smaller apparent molecular weights. Only a single major component was detected by isoelectric focusing analysis of the 9s intracellular aBT binding component, with a PI value close to that of the extra junctional receptor. These results suggest that the junctional and extrajunctional types of receptors may be synthesized through a common precursor.     

Ultrastructural changes at gap junctions between lesioned crayfish axons

Summary In crayfish, the severed distal segment of single lateral giant axon (SLGA) often survives for at least 10 months after lesioning if this segment retains a septal region of apposition with an adjacent, intact SLGA. In control (unsevered) SLGAs, this septal region usually contains gap junctions and 50–60 nm vesicles near the axolemma of both SLGAs. From 1–14 days after lesioning, the distal segment of a severed SLGA undergoes obvious ultrastructural changes in mitochondria and neurotubular organization compared to control SLGAs or to adjacent, intact SLGAs in the same animal. Gap junctions are very difficult to locate in severed SLGAs within 24 h after lesioning. From two weeks to ten months after lesioning, the surviving stumps of severed SLGAs often appear remarkably normal except that structures normally associated with the presence of gap junctions remain very difficult to find.These and other data suggest that SLGA distal segments receive trophic support from adjacent, intact SLGAs. The mechanism of this support probably could not be via diffusion across gap junctions between intact and severed SLGAs since gap junctions largely disappear after lesioning. However, trophic maintenance could occur via the exocytotic — pinocytotic action of 50–60 nm vesicles which are always present on both sides of the septum between an intact SLGA and a severed SLGA distal segment.This work was supported by NIH research grant NS-14412 and and RCDA 00070 to G.D.B.     

Ultrastructure of the muscles of the dorsal diaphragm in Locusta migratoria

Summary The alary muscles of Locusta migratoria adults make up the major tissue of the dorsal diaphragm which separates pericardial and perivisceral sinuses in the abdomen. The alary muscles are striated with a sarcomere at rest measuring about 9 m. The Z-line has a staggered-beaded arrangement with A-bands and I-bands readily discernable. Thick myofilaments are surrounded by 10 or more thin filaments. The sarcoplasm has few mitochondria near the area of the Z-line, dyads are present and sarcoplasmic reticulum is poorly developed. Axons which innervate the alary muscle are either contained within invaginated folds of the sarcolemma of the muscle cells or the muscle cells send finger-like projections to envelop the axons. The synaptic terminals contain synaptic vesicles between 40 and 45 nm in diameter and a few electron-dense granules near or less than 170 nm in diameter. Away from synaptic terminals the axon profiles show few or no granules. The axons are accompanied everywhere by well-developed glial cells. This then is not typical neurosecretomotor innervation, however, the presence of electron-dense granules suggests the possibility of peptidergic neurotransmission.     

Mesothelial intercellular junctions and pathways

Summary Freeze-etch preparations of mesothelial cells taken from the peritoneum of mouse reveal the presence of vesicles invaginating the apical and the basal cell surfaces. These vesicles are scarcely seen within the cytoplasm. Long tortuous tubular profiles extend for considerable distance within the cytoplasm and are frequently associated with the vesicles. The possible nature and role of the vesicles and the tubules in transport phenomena across the mesothelial barrier, are discussed in relation to the pore theory advanced by physiologists and the stomata concept observed by early German and contemporary anatomists. Occludens junctions of the leaky type are seen though their macular or zonular nature is yet to be established.     

Cell junctions in the epithelium of Bowman's capsule

Summary The intercellular connections between the epithelial cells of Bowman's capsule were investigated. It could be demonstrated that typical zonulae occludentes (tight junctions) are present in the species (rat, hamster, and Tupaia) studied. Freeze-fracturing shows a network of anastomizing strands; some species variations are described. In the rat two strands are common. In the golden hamster mostly two to four and occasionally five strands occur. In Tupaia regularly three tight junction strands are found and also gap junctions associated with the zonulae occludentes. In thin sections the goniometric analysis confirms the freeze-fracturing results and reveals attachment zones of macular shape, which are classified as intermediate junctions and desmosomes. The functional role of these cell junctions observed in the epithelium of Bowman's capsule is discussed.