Fast fluorescence laser tracking microrheometry. I: instrument development

To gain insight into cellular mechanotransduction pathways, we have developed a fluorescence laser tracking microrheometer (FLTM) to measure material rheological features on micrometer length scales using fluorescent microspheres as tracer particles. The statistical analysis of the Brownian motion of a particle quantifies the viscoelastic properties of the probe's environment, parameterized by the frequency-dependent complex shear modulus G*(ω). This FLTM has nanometer spatial resolution over a frequency range extending from 1 Hz to 50 kHz. In this work, we first describe the consecutive stages of instrument design, development, and optimization. We subsequently demonstrate the accuracy of the FLTM by reproducing satisfactorily the known rheological characteristics of purely viscous glycerol solutions and cross-linked polyacrylamide polymer networks. An upcoming companion article will illustrate the use of FLTM in studying the solid-like versus liquid-like rheological properties of fibroblast cytoskeletons in living biological samples.     

Oncostatin M receptor-beta mutations underlie familial primary localized cutaneous amyloidosis

Familial primary localized cutaneous amyloidosis (FPLCA) is an autosomal-dominant disorder associated with chronic skin itching and deposition of epidermal keratin filament-associated amyloid material in the dermis. FPLCA has been mapped to 5p13.1–q11.2, and by candidate gene analysis, we identified missense mutations in the OSMR gene, encoding oncostatin M-specific receptor β (OSMRβ), in three families. OSMRβ is a component of the oncostatin M (OSM) type II receptor and the interleukin (IL)-31 receptor, and cultured FPLCA keratinocytes showed reduced activation of Jak/STAT, MAPK, and PI3K/Akt pathways after OSM or IL-31 cytokine stimulation. The pathogenic amino acid substitutions are located within the extracellular fibronectin type III-like (FNIII) domains, regions critical for receptor dimerization and function. OSM and IL-31 signaling have been implicated in keratinocyte cell proliferation, differentiation, apoptosis, and inflammation, but our OSMR data in individuals with FPLCA represent the first human germline mutations in this cytokine receptor complex and provide new insight into mechanisms of skin itching.     

Retrotransposition of Nonviral RNAs in an Avian Packaging Cell Line

Retroviruses produced from the quail packaging cell line SE21Q1b predominantly contain cellular RNAs instead of viral RNAs. These RNAs can be reverse transcribed and integrated into the genomes of newly infected cells and are thereafter referred to as newly formed retrogenes. We investigated whether retrogene formation can occur within SE21Q1b cells themselves and whether this occurs intracellularly or via extracellular reinfection. By using packaging cell line mutants derived from the SE21Q1b provirus and selectable reporter constructs, we found that the process requires envelope glycoproteins and a retroviral packaging signal. Our results suggest that extracellular reinfection is the primary route of retrotransposition of nonviral RNAs.     

The Gag Domains Required for Avian Retroviral RNA Encapsidation Determined by Using Two Independent Assays

The Rous sarcoma virus (RSV) Gag precursor polyprotein is the only viral protein which is necessary for specific packaging of genomic RNA. To map domains within Gag which are important for packaging, we constructed a series of Gag mutations in conjunction with a protease (PR) active-site point mutation in a full-length viral construct. We found that deletion of either the matrix (MA), the capsid (CA), or the protease (PR) domain did not abrogate packaging, although the MA domain is likely to be required for proper assembly. A previously characterized deletion of both Cys-His motifs in RSV nucleocapsid protein (NC) reduced both the efficiency of particle release and specific RNA packaging by 6- to 10-fold, consistent with previous observations that the NC Cys-His motifs played a role in assembly and RNA packaging. Most strikingly, when amino acid changes at Arg 549 and 551 immediately downstream of the distal NC Cys-His box were made, RNA packaging was reduced by more than 25-fold with no defect in particle release, demonstrating the importance of this basic amino acid region in packaging. We also used the yeast three-hybrid system to study avian retroviral RNA-Gag interactions. Using this assay, we found that the interactions of the minimal packaging region (Mpsi) with Gag are of high affinity and specificity. Using a number of Mpsi and Gag mutants, we have found a clear correlation between a reporter gene activation in a yeast three-hybrid binding system and an in vivo packaging assay. Our results showed that the binding assay provides a rapid genetic assay of both RNA and protein components for specific encapsidation.     

Male morph predicts investment in larval immune function in the dung beetle, Onthophagus taurus

Investment in immunity is costly, so that resource-based trade-offsbetween immunity and sexually selected ornaments might be expected.The amount of resources that an individual can invest in eachtrait will be limited by the total resources available to them.It would therefore be informative to investigate how investmentin immune function changes during growth or production of thesexual trait as resources are diverted to it. Using the dungbeetle, Onthophagus taurus, which displays both sexual and maledimorphism in horn size, we examined changes in one measureof immune function, phenoloxidase (PO) activity, in the hemolymphof larvae prior to and during horn growth. We found that POlevels differed between small- and large-horned males throughoutthe final instar prior to the point where investment in horngrowth was taking place. PO levels in females were intermediateto the 2 male morphs. These differences could not be accountedfor by differences in condition, measured as hemolymph proteinlevels and weight. We suggest that the observed differencesmight be associated with sex- and morph-specific variation injuvenile hormone levels.     

Tyrosine and serine phosphorylation regulate the conformation and subsequent threonine phosphorylation of the L1 cytoplasmic domain

Previously we identified threonine-1172 (T1172) in the cytoplasmic domain of the cell adhesion molecule L1 as phosphorylated in pancreatic cancer cells. Although both CKII- and PKC-blockade suppressed this modification, only CKII was capable of phosphorylating T1172 of a recombinant L1 cytoplasmic domain, suggesting the requirement for additional events to facilitate availability of T1172 to PKC. In this study, we demonstrate that the region around T1172 exists in distinct conformations based on both T1172 phosphorylation and the integrity of surrounding residues. We further demonstrate the role of membrane-proximal and membrane-distal residues in regulating cytoplasmic domain conformation, and that modification of 3 of the 4 tyrosines in the L1 cytoplasmic domain promote conformational changes that facilitate other events. In particular, phenylalanine-substitution of tyrosine-1151 or tyrosine-1229 promote opening up of the cytoplasmic domain in a manner that facilitates phosphorylation of the other 3 tyrosines, as well as phosphorylation of T1172 by PKCα. Importantly, we show that phosphorylation of serine-1181 is required for T1172 phosphorylation by CKII. These data define a specific role for secondary structure in regulating the availability of T1172 that facilitates phosphorylation by PKC.     

Genetic breeding system and investment patterns within nests of Dawson's burrowing bee (Amegilla dawsoni) (Hymenoptera: Anthophorini)

Dawson's burrowing bee is a large solitary ground-nesting bee endemic to the arid zone of Western Australia. In this study, we use microsatellite markers to analyse the genotypes of offspring from individual nests to determine the number of effective mates for each female. From these data we have determined that females almost certainly mate only once which is consistent with male reproductive tactics that include protandry and intense male-male competition for access to virgin females. We also use the molecular data to show that the nesting female is the mother of all the offspring of her nest and that brood parasitism is unlikely in this species. The data indicate that females make daughters at the beginning of the season followed by large sons in the middle, and then small sons at the end. Females often place one brood cell directly above another. The distribution of sex and morph in these doublets follows a pattern with most containing a female on the bottom and a minor male on the top, followed by almost equal numbers of female on top of female and minor male on top of major male. This pattern is likely favoured by emergence patterns, with males emerging before females and minor males emerging before major males. We suggest that although minor males have low reproductive success, their production may nonetheless be beneficial in that minor males open up emergence tunnels for their larger and reproductively more valuable siblings. In addition, minor males may be a best of a bad job product arising from changes in the costs to nesting females of gathering brood provisions over the course of the flight season.     

Comparative thermal adaptation in cicadas (Hemiptera: Cicadidae) inhabiting Mediterranean ecosystems

The thermal responses of cicadas inhabiting the Mediterranean ecosystems in Europe, North America, South Africa, and Australia are investigated. A total of 37 species and two subspecies from 17 genera representing eight tribes and three subfamilies of cicadas are investigated. The analysis includes species that are restricted to the Mediterranean ecosystem as well as those which also inhabit additional environments. The data suggest that cicadas adapt to the climate type regardless of particular types of plants within the various Mediterranean communities. Similarly, cicada thermal responses are independent of body size or taxonomic affinities. There is a wider range of body temperatures for the maximum voluntary tolerance temperature than for heat torpor or minimum flight temperatures. This diversity seems to be determined by the subdivision of the habitat used and the behavior of the species. All species possess relatively elevated heat torpor temperatures adapting to the general thermal characteristics of the Mediterranean ecosystem. The data suggest that cicadas adapt to the Mediterranean climate type regardless of the diversity of particular types of plants within the various communities, of body size or of taxonomic position.