Localization of anti-clathrin antibody in the sarcomere and sensitivity of myofibril structure to chloroquine suggest a role for clathrin in myofibril assembly

Immunofluorescence microscopy has been used to demonstrate that X22, a monoclonal antibody specific for clathrin heavy chain, localizes in repetitive bands that appear soon after the fusion of skeletal myoblasts into multinucleate fibers. This organization has been found in cultures containing myotubes that develop in vitro from explants of newborn rat hindlimb cells and in myotubes derived from the L8E63 myogenic line. Bands were also prominent in skinned fibers prepared from adult rat soleus muscle and in cardiac myocytes grown in vitro from 4-day heart ventricles. Immunofluorescence banding was localized in the sarcomere as a doublet, with one element on either side of the Z line. Evidence that supports the conclusion that the reaction with X22 antibody is specific and indicative of the localization of clathrin in the sarcomere includes: (1) Identical titration of X22 antibody reactivity with the determinant in coated vesicles and in the sarcomere. (2) Conditions (eg., pH and Tris) that disrupt clathrin baskets or prevent its assembly likewise disrupt the localization of X22 in bands. (3) Chloroquine inhibits both the normal trafficking of clathrin in the cell and X22 banding in the sarcomere. (4) Immunoblot analysis of myotube lysates reveals a single band with an electrophoretic mobility identical to the 180,000-Da clathrin heavy chain. (5) The assembly of clathrin into sarcomeric bands occurs early in the development of the myofibrillar apparatus. Quantitation of the appearance of X22 banding in primary cultures of myotubes indicates that it precedes that of other myofibrillar proteins and that assembly takes place in the following order: X22, titin, myosin heavy chain, actin, and desmin. The assembly of myosin, titin, and actin into sarcomeric bands, as well as X22, is inhibited by chloroquine. Upon prolonged exposure to chloroquine previously assembled proteins are drastically reduced or no longer evident in the sarcomere. On the basis of these results and considering the role of clathrin in intracellular transport and its capacity to interact with actin and alpha-actinin, we suggest that clathrin may have diverse roles in the assembly, integrity, and functioning of the sarcomere and its integration with the sarcolemma. The early organization of X22 into bands further suggests that clathrin may also function early in the assembly of the contractile system.     

Specific binding of sulphated polymers to ram sperm proacrosin

Sulphated polysaccharides and zona pellucida glycoproteins have been shown to bind non-enzymatically to proacrosin, the protein found within the acrosomal vesicle of mammalian spermatozoa. The mechanism of this interaction has been investigated using ¹²⁵I-fucoidan to probe purified ram sperm proacrosin. Results show that (a) binding of' ¹²⁵I-fucoidan to proacrosin is inhibited only by sulphated polymers and (b) recognition is mediated by poly(sulphate) groups and is largely independent of the composition of the polymer chain. It is suggested that a similar mechanism is responsible for the interaction between proacrosin and zona pellueida glycoproteins during the early stages of fertilization in mammals and this process mediates firm binding of spermatozoa to the egg.     

Proton-induced grana formation in chloroplasts. Distribution of chlorophyll-protein complexes and Photosystem II photochemistry

Lowering the pH of the incubation medium to pH 5.4 leads to grana formation morphologically similar to that induced by metal cations. The same phenomenon is observed in EDTA-washed chloroplasts, indicating that it is not due in part to electrostatic ‘masking’ by residual cations associated with the membranes. Digitonin fractionation studies have indicated that the distribution of the major chlorophyll-protein complexes between granal and stromal membrane regions is similar at pH 5.4 in the absence of Mg²⁺, and at pH 7.4 in the presence of Mg²⁺. Chlorophyll fluorescence induction studies have indicated that the primary photochemistry of Photosystem II (PS II) is stimulated by lowering the pH to 5.4, just as it is upon metal cation addition at higher pH values. The failure to observe such an increase at pH 5.4 by measuring electron transport to ferricyanide is attributed to a combination of an inhibition by this pH of electron transport at a site after Q reduction and an increase in the number of PS II centres detached from the plastoquinone pool. We conclude that the stacked configuration of chloroplast membranes leads to increased PS II primary photochemistry, which is most simply explained in terms of a redistribution of excitation energy towards PS II.     

The proteins of the content of the secretory granules of the rat parotid gland

The proteins of the secretory granules of the rat parotid gland were characterized by sodium dodecylsulfate gel electrophoresis, by chromatography of [³H]prolinelabeled proteins on DEAE-cellulose and by amino acid analysis.Sodium dodecylsulfate gel electrophoresis of the secretory granule content showed five principal proteins and a limited number of minor components. Only two of the principal bands could be identified as known secretory enzymes of the parotid gland. One was identified as the α-amylase and one as deoxyribonuclease. Peroxidase and ribonuclease form minor portions of the secretory proteins.The other three major proteins constitute, together, about 60% by weight, of the secretory granule content proteins. Of these, one which represents more than 30% of the total granule protein was found to contain uniquely high amounts of leucine residues (21 mole%). Another one of these principal proteins was relatively rich in cysteine residues (7 mole%).The fifth principal protein was found to contain high amounts of proline (28 mole%) glutamic acid (17 mole%) and glycine (18 mole%) residues. Its amino acid composition was very similar to that of the proline-rich proteins that were previously shown to be present in the membrane isolated from these granules. This protein, however, differed from the “membranous” proline-rich proteins by several criteria.Two minor glycoproteins of the secretory granule content were also found to be rich in proline residues (37 mole%). As with the other proline-rich proteins of the granule, they contained no sulphur-containing amino acids, stained faintly pink with Coomassie Blue and were underestimated by the Lowry method. They differ however, from all the other proline-rich proteins of the granule by having a significantly higher content of threonine, less glycine (9 mole%) and much less glutamic acid (3 mole%).Of the principal proteins, only the deoxyribonuclease and the half-cystine-rich proteins were positively stained by periodic acid Schiff staining.The possible functions of the leucine-rich, the half cystine-rich and the various proline-rich proteins are discussed.     

Basic protein changes during the final stages of sperm maturation in the house cricket

Polyacrylamide gel electrophoresis has been used to analyse basic protein changes during the final stages of spermiogenesis in the house cricket. Mature sperm were obtained from the spermathecae of inseminated females. Their basic protein is electrophoretically heterogeneous, with two major and two minor components, all of unusually high mobilities, as expected ofprotamine. No histones are present. Testis also contains basic protein components of high mobilities, although in small amount relative to the histones present. Testis preparations were centrifuged on a density gradient of colloidal silica to separate nuclei of different stages of spermiogenesis from each other, and it was found that very late spermatids contain relatively large amounts of protamine. At least seven different protamine-like components, each with a different mobility, occur during the final maturation stages. The particular components present, and their abundancies, vary during development. The complement first found in spermatids is different from that of a later spermatid; still another complement is found in sperm from the seminal vesicle; and still another in mature sperm. Components which are abundant in spermatids are progressively eliminated, while components which are barely detectable in them gradually increase in abundance to become the major components of the basic protein complement at maturity.     

The impacts of cytoplasmic incompatibility factor (cifA and cifB) genetic variation on phenotypes

Wolbachia are maternally transmitted, intracellular bacteria that can often selfishly spread through arthropod populations via cytoplasmic incompatibility (CI). CI manifests as embryonic death when males expressing prophage WO genes cifA and cifB mate with uninfected females or females harboring an incompatible Wolbachia strain. Females with a compatible cifA-expressing strain rescue CI. Thus, cif-mediated CI confers a relative fitness advantage to females transmitting Wolbachia. However, whether cif sequence variation underpins incompatibilities between Wolbachia strains and variation in CI penetrance remains unknown. Here, we engineer Drosophila melanogaster to transgenically express cognate and non-cognate cif homologs and assess their CI and rescue capability. Cognate expression revealed that cifA;B native to D. melanogaster causes strong CI, and cognate cifA;B homologs from two other Drosophila-associated Wolbachia cause weak transgenic CI, including the first demonstration of phylogenetic type 2 cifA;B CI. Intriguingly, non-cognate expression of cifA and cifB alleles from different strains revealed that cifA homologs generally contribute to strong transgenic CI and interchangeable rescue despite their evolutionary divergence, and cifB genetic divergence contributes to weak or no transgenic CI. Finally, we find that a type 1 cifA can rescue CI caused by a genetically divergent type 2 cifA;B in a manner consistent with unidirectional incompatibility. By genetically dissecting individual CI functions for type 1 and 2 cifA and cifB, this work illuminates new relationships between cif genotype and CI phenotype. We discuss the relevance of these findings to CI’s genetic basis, phenotypic variation patterns, and mechanism.     

Genetic differentiation and signatures of local adaptation revealed by RADseq for a highly dispersive mud crab Scylla olivacea (Herbst, 1796) in the Sulu Sea

Connectivity of marine populations is shaped by complex interactions between biological and physical processes across the seascape. The influence of environmental features on the genetic structure of populations has key implications for the dynamics and persistence of populations, and an understanding of spatial scales and patterns of connectivity is crucial for management and conservation. This study employed a seascape genomics approach combining larval dispersal modeling and population genomic analysis using single nucleotide polymorphisms (SNPs) obtained from RADseq to examine environmental factors influencing patterns of genetic structure and connectivity for a highly dispersive mud crab Scylla olivacea (Herbst, 1796) in the Sulu Sea. Dispersal simulations reveal widespread but asymmetric larval dispersal influenced by persistent southward and westward surface circulation features in the Sulu Sea. Despite potential for widespread dispersal across the Sulu Sea, significant genetic differentiation was detected among eight populations based on 1,655 SNPs (F_ST = 0.0057, p < .001) and a subset of 1,643 putatively neutral SNP markers (F_ST = 0.0042, p < .001). Oceanography influences genetic structure, with redundancy analysis (RDA) indicating significant contribution of asymmetric ocean currents to neutral genetic variation (Radj2 = 0.133, p = .035). Genetic structure may also reflect demographic factors, with divergent populations characterized by low effective population sizes (N _e < 50). Pronounced latitudinal genetic structure was recovered for loci putatively under selection (F_ST = 0.2390, p < .001), significantly correlated with sea surface temperature variabilities during peak spawning months for S. olivacea (Radj2 = 0.692–0.763; p < .050), suggesting putative signatures of selection and local adaptation to thermal clines. While oceanography and dispersal ability likely shape patterns of gene flow and genetic structure of S. olivacea across the Sulu Sea, the impacts of genetic drift and natural selection influenced by sea surface temperature also appear as likely drivers of population genetic structure. This study contributes to the growing body of literature documenting population genetic structure and local adaptation for highly dispersive marine species, and provides information useful for spatial management of the fishery resource.