Clarin-1, Encoded by the Usher Syndrome III Causative Gene, Forms a Membranous Microdomain: POSSIBLE ROLE OF CLARIN-1 IN ORGANIZING THE ACTIN CYTOSKELETON*

Clarin-1 is the protein product encoded by the gene mutated in Usher syndrome III. Although the molecular function of clarin-1 is unknown, its primary structure predicts four transmembrane domains similar to a large family of membrane proteins that include tetraspanins. Here we investigated the role of clarin-1 by using heterologous expression and in vivo model systems. When expressed in HEK293 cells, clarin-1 localized to the plasma membrane and concentrated in low density compartments distinct from lipid rafts. Clarin-1 reorganized actin filament structures and induced lamellipodia. This actin-reorganizing function was absent in the modified protein encoded by the most prevalent North American Usher syndrome III mutation, the N48K form of clarin-1 deficient in N-linked glycosylation. Proteomics analyses revealed a number of clarin-1-interacting proteins involved in cell-cell adhesion, focal adhesions, cell migration, tight junctions, and regulation of the actin cytoskeleton. Consistent with the hypothesized role of clarin-1 in actin organization, F-actin-enriched stereocilia of auditory hair cells evidenced structural disorganization in Clrn1^−/− mice. These observations suggest a possible role for clarin-1 in the regulation and homeostasis of actin filaments, and link clarin-1 to the interactive network of Usher syndrome gene products.Usher syndrome is the most common cause of human inherited deafness and blindness, accounting for ∼50% of all cases (1). There are three clinical types of Usher syndrome, types I, II, and III (1–3). Usher type I is characterized by profound congenital deafness and vestibular dysfunction, and Usher type II is characterized by moderate to severe deafness. Usher type III is distinguished from types I and II by progressive (non-congenital) deafness together with variable impairment of vestibular function. All Usher types lead to progressive retinal degeneration with a retinitis pigmentosa-like appearance. Five causative genes have been identified for Usher syndrome type I, and three genes for type II (3). The protein products of Usher type I and II genes are functionally heterogeneous, including an unconventional myosin, scaffold proteins, G-protein-coupled receptor, and cadherins. Adding to this heterogeneity, the Usher syndrome type III gene encodes a novel transmembrane protein named clarin-1 (CLRN1)³ (4–6) with an unknown function. The heterogeneity of genes involved in Usher syndrome makes it extremely challenging to elucidate shared and distinctive disease mechanisms.CLRN1 belongs to a superfamily of four-transmembrane proteins that includes the tetraspanin and claudin families. CLRN1 and its paralogues, CLRN2 and CLRN3, form the Clarin family, which is conserved throughout vertebrate species and shows limited sequence homology to the tetraspanins (4). Tetraspanins are considered to be structural proteins that interact laterally with other membrane proteins such as ion channels, integrins, and other tetraspanins (7, 8) to form tetraspanin-enriched microdomains. Tetraspanin-enriched microdomains embody other proteins to allow localized transmission of signals, cell-cell adhesion/fusion, cell-matrix interactions, and/or formation of diffusion barriers against small molecules. Similar to tetraspanins, CLRN1 retains only limited hydrophilic regions exposed to cytoplasmic or extracellular aqueous phases (Fig. 1A) and, apparently, lacks any functional domains. Although CLRN1 is structurally related and similar to tetraspanins, it is currently unknown whether CLRN1 can form specific microdomains. The question also remains as to what one or more functions CLRN1 microdomains serve if indeed they do exist.Open in a separate windowFIGURE 1.CLRN1 is a plasma membrane protein localized at F-actin-enriched protrusions. A, the topology and transmembrane domains shown were predicted with the HMMTOP transmembrane topology prediction server (55). The possible N-linked glycosylation site is indicated. Also shown (red circle) is the previously predicted motif near the CLRN1 C-terminal tail that may serve as a PDZ-binding site (4). B, immunolocalization of Human WT CLRN1. C, immunolocalization of Na/K ATPase in HEK293 cells stably expressing CLRN1. D, merged image of B and C indicates that CLRN1 and Na/K ATPase co-localize. Images B–D are single optical sections of HEK293 cells. E, cell surface biotinylation was performed to separate cell surface proteins (avidin-bound) (AB) from intracellular proteins (flow-through) (FT). Immunoblots of both fractions reveal that most of the CLRN1 protein localized to the plasma membrane. HEK293 cells alone and HEK293 cells expressing CLRN1 were preincubated for 30 min with Sulfo-NHS-SS-Biotin to label cell surface proteins. After cells were harvested, biotin-labeled CLRN1 protein levels were measured by immunoblotting. F, localization of human WT CLRN1 in HEK293 cells stably expressing CLRN1. G, F-actin in HEK293 cells stably expressing CLRN1. F-actins were labeled with phalloidin-Alexa 488. H, merged image of F and G. CLRN1 localized at both microvilli (arrows) and lamellipodia (arrowheads). I–K, CLRN1 localization studied by immunofluorescence confocal microscopy after disruption of F-actin by cytochalasin D treatment. I, CLRN1 localized diffusely on the plasma membrane. J, F-actin localization is shown. K, merged image of I and J. After disruption of F-actin, CLRN1 and F-actin no longer co-localize. Images F–K were generated from multiple optical sections by a maximum intensity projection. Scale bars, 50 μm.CLRN1 is expressed in sensory hair cells (4) where it may interact with other co-existing Usher gene products or cellular machinery essential for the maintenance of these cells. Increasing evidence suggests that products of Usher type I and II genes form large networks of interacting proteins, and that F-actin plays a major role in organizing these networks (reviewed in Refs. 2, 9). The core of these networks is the Usher type IC gene product, Harmonin, which interacts directly with F-actin in vitro and stabilizes F-actin when it is expressed heterologously in HeLa cells (10). Harmonins retain multiple PDZ domains dedicated to interacting with products of Usher type I and type II genes (reviewed in Refs. 2, 9) and also serve as PDZ domain-based scaffolds to anchor Usher proteins to F-actin. A link between Usher gene products and actin-based organelles also has been established in vivo. In Usher syndrome I and II mouse models, the actin-enriched stereocilia are morphologically and functionally defective (11–14). Because the causative gene for Usher type III was identified more recently than those of Usher types I and II, little is known about the pathogenesis of Usher syndrome III. Epistatic interactions between Usher syndrome type IB and Usher syndrome III may suggest linkage among CLRN1, Myosin VIIa, and F-actin (15). Clinically, patients with the N48K CLRN1 mutation have a rod and cone degenerative phenotype similar to Usher type IIA patients (16), suggesting a common pathological pathway for Usher types IIA and III. Despite the genetic and phenotypic characterization in humans, the molecular function of CLRN1 remains elusive, as well as its relationship and interaction with other Usher gene products. Therefore, identifying possible interactive partners of CLRN1 should improve understanding of the function of CLRN1 and the common pathological pathways of progressive hearing and vision loss in the Usher syndromes.Here we investigated whether CLRN1 can form microdomains similar to the tetraspanin-enriched microdomain, and if so, what the function of such microdomains might be. Our studies indicate that CLRN1 forms membranous cholesterol-rich compartments on plasma membranes and interacts with and regulates the machinery involved in actin filament organization. To understand the pathogenesis of Usher syndrome, we asked whether and how the Usher syndrome III causative mutation, N48K, results in dysfunction of the clarin-1-enriched microdomains involved in organizing actin. To determine whether Clrn1 is involved in the regulation of actin cytoskeleton in vivo, we studied the structure of F-actin-enriched stereocilia bundles in Clrn1^−/− mouse. Because actin provides important scaffolds in Usher interactome, the observations described herein provide a novel molecular link between CLRN1 and the identified gene products of Usher types I and II.     

The effects of diet quality and quantity on plumage colour and growth of great tit Parus major nestlings: a food manipulation experiment along a pollution gradient

The yellow carotenoid-based plumage coloration of great tit Parus major nestlings is found to be paler in polluted and urban environments. Because carotenoid pigmentation is often considered to be a condition dependent trait in birds we wanted to find out whether food-limitation and poor nestling condition could explain the pale plumage colour in a polluted area. P. major nestlings were supplemented with variable diets along a well known heavy metal pollution gradient around a copper smelter: two food treatments with carotenoids, one food treatment with little carotenoid and one unsupplemented control. Our field experiment showed that nestlings in the polluted area grew better with carotenoid rich diets, while such effect was not found in the unpolluted area. Nestlings showed higher plasma carotenoid (lutein) levels and higher plumage carotenoid chroma values in the unpolluted area than in the polluted area. However, plasma lutein levels or plumage colour were not associated with heavy metal levels in nestling faeces (a proxy for dietary exposure). Our results provide only weak evidence for carotenoid-based colouration to be condition-dependent in great tit nestlings as we found a positive relationship between body mass and carotenoid chroma in the non-supplemented control group only. The positive relationship between body mass and plumage colour intensity is more likely to be produced by the fact that good availability of caterpillars, an important food source for P. major, also means a good availability of carotenoids to nestlings. Our results suggest that main reason for pale nestling plumage in the polluted area is lower quality invertebrate food, and not nutrition-related oxidative stress.     

Harmonization is more important than experience—results of the first Nordic–Baltic diatom intercalibration exercise 2007 (stream monitoring)

The goal of this study was a harmonization of diatom identification and counting among diatomists from the Scandinavian and Baltic countries to improve the comparison of diatom studies in this geographical area. An analysis of the results of 25 diatomists following the European standard EN 14407 during an intercalibration exercise showed that a high similarity was achieved by harmonization and not because of a long experience with diatoms. Sources of error were wrong calibration scales, overlooking of small taxa, especially small Navicula s.l., misidentifications (Eunotia rhomboidea was mistaken for Eunotia incisa) and unclear separation between certain taxa in the identification literature. The latter was discussed during a workshop with focus on the Achnanthes minutissima group, the separation of Fragilaria capucina var. gracilis from F. capucina var. rumpens, and Nitzschia palea var. palea from N. palea var. debilis. The exercise showed also that the Swedish standard diatom method tested here worked fine with acceptable error for the indices IPS (Indice de Polluo-sensibilité Spécifique) and ACID (ACidity Index for Diatoms) when diatomists with a low similarity (Bray–Curtis <60%) with the auditor in at least one of the samples are excluded.     

EVOLUTION OF GIGANTISM IN NINE‐SPINED STICKLEBACKS

The relaxation of predation and interspecific competition are hypothesized to allow evolution toward “optimal” body size in island environments, resulting in the gigantism of small organisms. We tested this hypothesis by studying a small teleost (nine‐spined stickleback, Pungitius pungitius) from four marine and five lake (diverse fish community) and nine pond (impoverished fish community) populations. In line with theory, pond fish tended to be larger than their marine or lake conspecifics, sometimes reaching giant sizes. In two geographically independent cases when predatory fish had been introduced into ponds, fish were smaller than those in nearby ponds lacking predators. Pond fish were also smaller when found in sympatry with three‐spined stickleback (Gasterosteus aculeatus) than those in ponds lacking competitors. Size‐at‐age analyses demonstrated that larger size in ponds was achieved by both increased growth rates and extended longevity of pond fish. Results from a common garden experiment indicate that the growth differences had a genetic basis: pond fish developed two to three times higher body mass than marine fish during 36 weeks of growth under similar conditions. Hence, reduced risk of predation and interspecific competition appear to be chief forces driving insular body size evolution toward gigantism.     

Worldwide Prevalence of Class 2 Integrases outside the Clinical Setting Is Associated with Human Impact

An intI-targeted PCR assay was optimized to evaluate the frequency of partial class 2-like integrases relative to putative, environmental IntI elements in clone libraries generated from 17 samples that included various terrestrial, marine, and deep-sea habitats with different exposures to human influence. We identified 169 unique IntI phylotypes (≤98% amino acid identity) relative to themselves and with respect to those previously described. Among these, six variants showed an undescribed, extended, IntI-specific additional domain. A connection between human influence and the dominance of IntI-2-like variants was also observed. IntI phylotypes 80 to 99% identical to class 2 integrases comprised ∼70 to 100% (n = 65 to 87) of the IntI elements detected in samples with a high input of fecal waste, whereas IntI2-like sequences were undetected in undisturbed settings and poorly represented (1 to 10%; n = 40 to 79) in environments with moderate or no recent fecal or anthropogenic impact. Eleven partial IntI2-like sequences lacking the signature ochre 179 codon were found among samples of biosolids and agricultural soil supplemented with swine manure, indicating a wider distribution of potentially functional IntI2 variants than previously reported. To evaluate IntI2 distribution patterns beyond the usual hosts, namely, the Enterobacteriaceae, we coupled PCR assays targeted at intI and 16S rRNA loci to G+C fractionation of total DNA extracted from manured cropland. IntI2-like sequences and 16S rRNA phylotypes related to Firmicutes (Clostridium and Bacillus) and Bacteroidetes (Chitinophaga and Sphingobacterium) dominated a low-G+C fraction (∼40 to 45%), suggesting that these groups could be important IntI2 hosts in manured soil. Moreover, G+G fractionation uncovered an additional set of 36 novel IntI phylotypes (≤98% amino acid identity) undetected in bulk DNA and revealed the prevalence of potentially functional IntI2 variants in the low-G+C fraction.Integrons are genetic modules described in pathogenic and commensal bacteria that confer the ability to capture and express promoterless DNA units, called gene cassettes, which encode a variety of adaptive functions including antibiotic resistance (9, 42, 64). The acquisition of gene cassettes occurs through a site-specific recombination mechanism catalyzed by an integron-encoded integrase (IntI). The integrative recombination reaction occurs primarily between an integron receptor site (attI) and a cassette-associated sequence known as the attC site or 59-base element (11). However, integron integrases are able to recognize and process nonspecific secondary targets as well as attI and attC sites with a high degree of sequence variation (20, 25). This versatility facilitates the exchange of exogenous genes between different integrons through various recombination reactions (attI × attC, attI × attI, and attC × attC) that propel the adaptability and evolution of bacterial genomes (8, 11, 31, 38, 55, 58). Although integrons can be chromosomally encoded, they also may be horizontally transferred via transduction or by transposons associated with conjugative plasmids (42, 61). Three major groups (classes 1 to 3) are known to be associated with laterally transferred elements and highly prevalent in the clinical scene. In most of the cases, these have also been reported to harbor almost exclusively gene cassettes encoding antibiotic resistance functions (42). All together, these traits have led to their designation as “mobile” (9) or “clinical” (22) integrons. Although integrons have been traditionally classified according to the percent identity of the nucleotide or predicted amino acid sequence of their respective intI genes (9, 43, 71), several structural features and differences in abundance patterns have been identified which distinguish classes 1 to 3 (9, 42).Class 1 integrons are the most widely studied variant and are typically linked to replicative Tn21 transposons, which appears to contribute to their extensive distribution (48). A key feature commonly reported within the class 1 module is the presence of a highly conserved 3′ region comprised of a qacEΔ gene and a sul1 gene, which provide protection against quaternary ammonium compounds and sulfa drugs, respectively. In contrast, class 2 integrons are routinely associated with nonreplicative Tn7 transposons, are less frequently detected and, hence, remain an understudied group relative to their class 1 counterparts (42, 48, 65). Even less is known about the class 3 variants, which so far have been described in only three instances (71).Except for the identical IntI2 elements recently reported in Providencia stuartii and Escherichia coli strains isolated from beef cattle sources and the human urinary tract, respectively, all known integrases encoded by class 2 integrons are considered nonfunctional due to the presence of the ochre 179 codon (6, 40, 42). Nevertheless, it has been argued that integrons with truncated class 2 integrases might be implicated in the transfer and high prevalence of antibiotic resistance genes among clinical isolates, possibly via the in trans activities of other functional integrases or the suppression of the stop codon (27). So far, class 2 integrons have been described in association with isolates affiliated to the gamma, beta, and epsilon subdivisions of the Proteobacteria but have been more frequently reported among members of the Gammaproteobacteria group, particularly the Enterobacteriaceae (1, 14, 19, 56, 57). However, most of these studies have focused on easily culturable, aerobic bacteria or those of clinical importance, leading to the exclusion of unculturable or difficult-to-grow commensals that could be inconspicuous but important reservoirs of class 2 elements in the environment. Although the occurrence and quantification of integrons and integron-associated genes by means of molecular, culture-independent methods are being increasingly documented outside the clinical scene (18, 22, 28, 48, 49, 51, 65, 70), the estimates of the extant diversity of the integron platform in nature are still rudimentary. Likewise, further work is needed for the identification of environmental hosts of integrons commonly found in clinical strains without the bias associated with culture techniques (48).In order to provide a comprehensive view of integron integrase variation and prevalence patterns of IntI2 elements in the environment, we PCR amplified partial intI sequences from metagenomic DNA isolated from various terrestrial, marine, and deep-sea habitats exposed to various degrees of anthropogenic or fecal impact. Amplification conditions were optimized to facilitate the assessment of the frequency of IntI2-like sequences relative to that of environmental integron integrases. Additionally, since the guanine-plus-cytosine content of DNA corresponds to taxonomy (68), we coupled G+C fractionation of total DNA (4, 5, 29, 30) with PCR assays targeted at intI and 16S rRNA genes to identify potential, unconventional hosts of class 2 integrons in soil that had received swine manure.     

Extrapair Paternity and Maternity in the Three-Toed Woodpecker,Picoides tridactylus: Insights from Microsatellite-Based Parentage Analysis

Molecular techniques have revealed that avian mating systems are more diverse and complex than previously thought. We used microsatellite markers to determine genetic parentage, the prevalence of extrapair paternity and quasi-parasitism (i.e. situations where a male''s extrapair mate lay in his nest) in a socially monogamous population of three-toed woodpeckers (Picoides tridactylus) in southern Finland. A total of 129 adults and nestlings, representing 5–9 families annually from 2004–2007, were genotyped at up to ten microsatellite loci. The results of genetic assignment tests confirmed that monogamous parentage characterized the majority (84.6%, 22/26) of broods, and that most (93.8%, 75/80) nestlings were the offspring of their social parents. Two of 80 nestlings (2.5%) in two of 26 broods (7.7%) were sired by extrapair males and quasi-parasitism occurred in 3.8% (3/80) of nestlings and 7.7% (2/26) of broods. Hence, the levels of extrapair parentage were low, possibly because both genetic polygyny and polyandry are constrained by the high paternal effort required for parental care. The co-occurrence of low levels of extrapair paternity and quasi-parasitism are discussed in light of ecological and behavioural factors characterizing the species biology.     

Refuge availability and sequence of predators determine the seasonal succession of crustacean zooplankton in a clay-turbid lake

The contribution of predators and abiotic factors to the regulation of the biomass and seasonal succession of crustacean zooplankton was studied in Lake Rehtijärvi (southern Finland). Field data in combination with bioenergetics modeling indicated that invertebrate planktivory by Chaoborus depressed cladoceran populations during early summer. In particular, bosminids that generally form the spring biomass peak of cladocerans in stratified temperate lakes did not appear in the samples until July. In July, predation pressure by chaoborids was relaxed due to their emergence period and cladoceran population growth appeared to be limited by predation by planktivorous fish. The effect of fish predation was amplified by reduced refuge availability for cladocerans. The concentration of dissolved oxygen below the epilimnion was depleted, forcing cladocerans to move upward to less turbid and thus more dangerous water layers. The effect of size selective predation by fish resulted in reduced mean size of cladocerans during the period when refuge thickness (thickness of the water layer with oxygen concentration <1 mg l^?1 and water turbidity >30 NTU) was lowest. The results confirmed that in clay-turbid lakes, invertebrate predators could be the main regulators of herbivorous zooplankton even when cyprinid fish are abundant.     

Predation leads to assembly rules in fragmented fish communities

Diamond [Assembly of species communities. In: Ecology and Evolution of Communities (eds Cody, M.L. & Diamond, J.M.). Harvard University Press, Cambridge, pp. 342–444] proposed that resource competition leads to checkerboard-like distributions of competing species. This proposal prompted research that revealed checkerboard patterns within a wide range of communities, but the mechanisms that generate such patterns are still poorly understood. Here we present whole-lake natural experiments and analyses of species–environment relationships in small coastal lake fish communities that were fragmented when land uplift isolated these lakes from the Baltic Sea, showing that a combination of predation and habitat suitability generated checkerboard distributions. Checkerboard patterns developed because two piscivores, northern pike and Eurasian perch, caused the extinction of several prey species in deep lakes. Conversely, low oxygen levels in shallow lakes caused extinction of the piscivores, and these areas served as a refuge for tolerant prey species. Based on these findings, we suggest that habitat suitability and biotic interactions should be viewed simultaneously in null models of assembly rules.     

Synthesis, cannabinoid receptor activity, and enzymatic stability of reversed amide derivatives of arachidonoyl ethanolamide

Retroanandamide (2f) and its 10 analogues (1a-e, 2a-e) were synthesized and evaluated for the cannabinoid receptor activation by a [35S]GTPgammaS binding assay using rat cerebellar membranes, and Chinese hamster ovary cell membranes expressing human CB2 receptors. The primary goal of the study was to develop cannabinoid receptor agonists having improved enzymatic stability compared to endogenous N-arachidonoyl ethanolamide (AEA). Furthermore, by reversing the amide bond of AEA, the formation of arachidonic acid would be prevented. Finally, an effect of the carbonyl carbon position on the cannabinoid receptor activity was explored by synthesizing retroanandamide analogues having different chain lengths (1a-e, C19; 2a-f, C20). All the synthesized compounds, except 2c, behaved as partial agonists for the both cannabinoid receptors. In rat brain homogenate, the reversed amides possessed significantly higher stability against FAAH induced degradation than AEA. Therefore, the reversed amide analogues of AEA may serve as enzymatically stable structural basis for the drug design based on the endogenous cannabinoids.     

Temperature-induced structural transition in-situ in porcine lens — Changes observed in void size distribution

The function of mammalian ocular lens is to provide a sharp image to the retina. Accordingly, the lens needs to be transparent and minimize light scattering. To do so the lens fiber cells first loose intracellular organelles, organize the cytoplasm and arrange the fiber cell membranes. Because the fiber cells are metabolically inactive, the plasma membrane becomes the only cellular organelle and consequently, the phase behavior of these membranes determines the physiological state of the lens. Previous studies have shown that lipids extracted from the nuclear and cortical region of human lens show a temperature-induced phase transition close to the body temperature. Yet, the physiological function of this phase transition is not known, and even the presence of the phase transition in intact lenses is unknown. Positron annihilation lifetime spectroscopy (PALS) was used to characterize the sub-nanometer-sized local structure of intact porcine lens and these studies were complemented with differential scanning calorimeter and mass spectrometric analysis in extracted porcine lens lipids. Using PALS, we present evidence for the presence of a temperature-dependent structural transition centered at 35.5 °C in-situ in clear extracted porcine lenses. Further studies employing extracted lens lipids and purified egg-yolk sphingomyelin and cholesterol mixtures suggest that the nano-scale transition emerges from the phase behavior of lens lipids. Based on our results, PALS seems to be a viable method for gaining additional information on biological tissues, especially since it enables non-destructive studies on intact tissues.