Eukaryotic protein production in designed storage organelles

Background  

Protein bodies (PBs) are natural endoplasmic reticulum (ER) or vacuole plant-derived organelles that stably accumulate large amounts of storage proteins in seeds. The proline-rich N-terminal domain derived from the maize storage protein γ zein (Zera) is sufficient to induce PBs in non-seed tissues of Arabidopsis and tobacco. This Zera property opens up new routes for high-level accumulation of recombinant proteins by fusion of Zera with proteins of interest. In this work we extend the advantageous properties of plant seed PBs to recombinant protein production in useful non-plant eukaryotic hosts including cultured fungal, mammalian and insect cells.     

Geographic Variation in Male Sexual Signals in Strawberry Poison Frogs (Dendrobates pumilio)

In this paper, we compare the advertisement calls of 207 neotropical strawberry poison frogs (Dendrobates pumilio) collected in 21 localities along a transect from northern Costa Rica to western Panama. Populations varied most in call duration and call rate, while pulse rate and duty cycle were less variable. Multivariate analyses showed that call variation followed a cline with higher call rates, shorter calls, lower duty cycles and higher pulse rates in the southeast. Body size decreased towards the southeast and explained most variation in dominant frequency, as well as some residual variation in call rate. We conclude that a combination of geography and morphology is largely responsible for call variation within this species. Two inferred bio‐acoustic groups were roughly in accordance with two genetic groups, geographically separated in central Costa Rica. However, genetic distances among populations did not co‐vary with call dissimilarity after correction for geographic distances. Thus, differences in calls between genetic groups are probably mainly a result of clinal variation. These findings agree with the general observation that bio‐acoustic variation is often not (highly) associated with genetic divergence. Moreover, colour polymorphism observed among Panamanian populations was not reflected in a higher variability in call parameters relative to the monomorphic Costa Rican populations.     

“Candidatus Hepatoplasma crinochetorum,” a New, Stalk-Forming Lineage of Mollicutes Colonizing the Midgut Glands of a Terrestrial Isopod

Uncultivated bacteria that densely colonize the midgut glands (hepatopancreas) of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda) were identified by cloning and sequencing of their 16S rRNA genes. Phylogenetic analysis revealed that these symbionts represent a novel lineage of the Mollicutes and are only distantly related (<82% sequence identity) to members of the Mycoplasmatales and Entomoplasmatales. Fluorescence in situ hybridization with a specific oligonucleotide probe confirmed that the amplified 16S rRNA gene sequences indeed originated from a homogeneous population of symbionts intimately associated with the epithelial surface of the hepatopancreas. The same probe also detected morphotypically identical symbionts in other crinochete isopods. Scanning and transmission electron microscopy revealed uniform spherical bacterial cells without a cell wall, sometimes interacting with the microvilli of the brush border by means of stalk-like cytoplasmic appendages, which also appeared to be involved in cell division through budding. Based on the isolated phylogenetic position and unique cytological properties, the provisional name “Candidatus Hepatoplasma crinochetorum” is proposed for this new taxon of Mollicutes colonizing the hepatopancreas of P. scaber.     

Removal of Ca2+ channel beta3 subunit enhances Ca2+ oscillation frequency and insulin exocytosis

An oscillatory increase in pancreatic beta cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel beta3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using beta3 subunit-deficient beta cells. beta3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in beta cells lacking the beta3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the beta3 subunit negatively modulated InsP3-induced Ca2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the beta3 subunit in the beta cell may constitute the basis for a novel diabetes therapy.     

Structural biology: Paper alert

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in structural biology.     

Genetic dissection of sex determinism, inflorescence morphology and downy mildew resistance in grapevine

A genetic linkage map of grapevine was constructed using a pseudo-testcross strategy based upon 138 individuals derived from a cross of Vitis vinifera Cabernet Sauvignon × Vitis riparia Gloire de Montpellier. A total of 212 DNA markers including 199 single sequence repeats (SSRs), 11 single strand conformation polymorphisms (SSCPs) and two morphological markers were mapped onto 19 linkage groups (LG) which covered 1,249 cM with an average of 6.7 cM between markers. The position of SSR loci in the maps presented here is consistent with the genome sequence. Quantitative traits loci (QTLs) for several traits of inflorescence and flower morphology, and downy mildew resistance were investigated. Two novel QTLs for downy mildew resistance were mapped on linkage groups 9 and 12, they explain 26.0–34.4 and 28.9–31.5% of total variance, respectively. QTLs for inflorescence morphology with a large effect (14–70% of total variance explained) were detected close to the Sex locus on LG 2. The gene of the enzyme 1-aminocyclopropane-1-carboxylic acid synthase, involved in melon male organ development and located in the confidence interval of all QTLs detected on the LG 2, could be considered as a putative candidate gene for the control of sexual traits in grapevine. Co-localisations were found between four QTLs, detected on linkage groups 1, 14, 17 and 18, and the position of the floral organ development genes GIBBERELLIN INSENSITIVE1, FRUITFULL, LEAFY and AGAMOUS. Our results demonstrate that the sex determinism locus also determines both flower and inflorescence morphological traits.     

Two calcium‐binding chaperones from the fat body of the Colorado potato beetle,Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) involved in diapause

Molecular chaperones are crucial for the correct folding of newly synthesized polypeptides, in particular, under stress conditions. Various studies have revealed the involvement of molecular chaperones, such as heat shock proteins, in diapause maintenance and starvation; however, the role of other chaperones in diapause and starvation relatively is unknown. In the current study, we identified two lectin‐type chaperones with calcium affinity, a calreticulin (LdCrT) and a calnexin (LdCnX), that were present in the fat body of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) during diapause. Both proteins possessed an N‐globular domain, a P‐arm domain, and a highly charged C‐terminal domain, while an additional transmembrane domain was present in LdCnX. Phylogenetic analysis revealed distinction at the order level. Both genes were expressed in multiple tissues in larval and adult stages, and constitutively throughout development, though a starvation response was detected only for LdCrT. In females, diapause‐related expression analysis in the whole body revealed an upregulation of both genes by post‐diapause, but a downregulation by diapause only for LdCrT. By contrast, males revealed no alteration in their diapause‐related expression pattern in the entire body for both genes. Fat body‐specific expression analysis of both genes in relation to diapause revealed the same expression pattern with no alteration in females and downregulation in males by post‐diapause. This study suggests that calcium‐binding chaperones play similar and possibly gender‐specific roles during diapause.     

Fiber‐type composition in the perivertebral musculature of lizards: Implications for the evolution of the diapsid trunk muscles

The perivertebral musculature of lizards is critical for the stabilization and the mobilization of the trunk during locomotion. Some trunk muscles are also involved in ventilation. This dual function of trunk muscles in locomotion and ventilation leads to a biomechanical conflict in many lizards and constrains their ability to breathe while running (“axial constraint”) which likely is reflected by their high anaerobic scope. Furthermore, different foraging and predator‐escape strategies were shown to correlate with the metabolic profile of locomotor muscles in lizards. Because knowledge of muscle's fiber‐type composition may help to reveal a muscle's functional properties, we investigated the distribution pattern of muscle fiber types in the perivertebral musculature in two small lizard species with a generalized body shape and subjected to the axial constraint (Dipsosaurus dorsalis, Acanthodactylus maculatus) and one species that circumvents the axial constraint by means of gular pumping (Varanus exanthematicus). Additionally, these species differ in their predator‐escape and foraging behaviors. Using refined enzyme‐histochemical protocols, muscle fiber types were differentiated in serial cross‐sections through the trunk, maintaining the anatomical relationships between the skeleton and the musculature. The fiber composition in Dipsosaurus and Acanthodactylus showed a highly glycolytic profile, consistent with their intermittent locomotor style and reliance on anaerobic metabolism during activity. Because early representatives of diapsids resemble these two species in several postcranial characters, we suggest that this glycolytic profile represents the plesiomorphic condition for diapsids. In Varanus, we found a high proportion of oxidative fibers in all muscles, which is in accordance with its high aerobic scope and capability of sustained locomotion. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.