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.     

shRNA Expression Plasmids Generated by a Novel Method Efficiently Induce Gene-Specific Knockdown in a Silkworm Cell Line

RNAi knockdown by using shRNA expression plasmids is widely used to determine the function of individual genes in mammals. Here we developed a simple method to create an IR DNA in a U6 small nuclear RNA promoter-based parent vector using a single-stranded IR DNA with short hairpin structure and Bst DNA polymerase. Furthermore, we demonstrated that the shRNA expression plasmids constructed by our method effectively induced target-specific RNAi in the silkworm cell line. We also found that sequence preference in the silkworm cell line was much lower than in mammalian cells and shRNA-induced RNAi was influenced by the length of the stem region.     

T cell activation in abnormal perinatal events

The aim of this study was to determine the percentage of CD45RO⁺ T cells in umbilical cord blood from neonates born at less than 37 weeks of gestation. Fifty-nine patients were enrolled in this study, including 49 with preterm and 10 with term deliveries. Preterm deliveries were divided into two categories; spontaneous (Group A, n = 31) and indicated (Group B, n = 18). Perinatal infection was categorized as C-CAM, H-CAM and neonatal infection. The percentage of CD45RO⁺ T cells in the umbilical cord was assessed using flow cytometry. IL-6 was measured using ELISA. In Group A, the percentage of CD45RO⁺ T cells and concentrations of IL-6 in patients with perinatal infection ( n = 18) were significantly higher than in those without perinatal infection ( n = 13). A significant correlation between percentage of CD45RO⁺ T cells and IL-6 concentrations was observed in the cord blood ( r = 0.62, P = 0.001). In Group B, pink–tinged amniotic fluid was observed in seven cases. In these cases, an increase in the percentage of CD45RO⁺ T cells (>10%) was noted. In the cases without perinatal infection, which included all those delivered at term ( n = 32), no correlation was observed between the percentage of CD45RO⁺ T cells and gestational age at delivery ( r =−0.139, P = 0.448). We concluded that a high percentage of CD45RO⁺ cord blood T cells is observed not only in perinatal infection, but also in the presence of abnormal perinatal events such as maternal bleeding in preterm gestation.     

Fast track to a phosphoprotein sketch - MALDI-TOF characterization of TLC-based tryptic phosphopeptide maps at femtomolar detection sensitivity

Tryptic phosphopeptide mapping by TLC on microcrystalline cellulose has been a convenient method to get a fast and highly reproducible overview of the number of phosphopeptides present in any given (32)P-labeled phosphoprotein. This method also provides an immediate presentation of the relative phosphorylation stoichiometry between individual phosphopeptides. However, so far, traditional tryptic phosphopeptide maps have not been useful for phosphoproteomics applications, as the S/N has been very poor, due to the large number of quenching substances and contaminants present on cellulose plates. In this study, we present a rapid and easy method for phosphopeptides identification from 2-D phosphopeptide maps (2-D-PPMs). We obtain improved sensitivity (femtomole levels) upon MALDI-TOF MS analysis of phosphopeptides extracted from 2-D-PPMs. Using this approach we could confidently characterize the major phosphorylation sites of in vivo and in vitro (32)P-labeled proteins.     

Interleukin-21 triggers both cellular and humoral immune responses leading to therapeutic antitumor effects against head and neck squamous cell carcinoma

BACKGROUND: Interleukin-21 (IL-21) plays important roles in the regulation of T, B, and natural killer (NK) cells. We hypothesized that the cytokine may provide a novel immunotherapy strategy for cancer by stimulating both Th1 and Th2 immune responses. In this context, antitumor immunity induced by IL-21 was examined in mice bearing subcutaneous head and neck squamous cell carcinomas (HNSCC). METHODS: A plasmid vector encoding murine IL-21 was injected intravenously into mice with pre-established HNSCC tumors, either alone or in combination with a vector construct expressing IL-15. Cytotoxic T lymphocyte (CTL) and NK killing activities were evaluated by chrome release assays, while HNSCC-specific antibody was examined by flow cytometry and ELISA. RESULTS: Significant antitumor effects were obtained by repeated transfection with either the IL-21 or the IL-15 gene. Co-administration of both cytokine genes resulted in increased suppression of tumor growth, significantly prolonging the survival periods of the animals. Thirty percent of the tumor-bearing mice that received the combination therapy survived for more than 300 days, completely rejecting rechallenge with the tumor at a distant site. IL-21 induced significant elevation of HNSCC-specific CTL activity, while IL-21 and IL-15 augmented NK activity in an additive manner. IL-21 gene transfer also promoted the production of tumor-specific IgG. CONCLUSIONS: In vivo transduction of the IL-21 gene elicits powerful antitumor immunity, including both humoral and cellular arms of the immune response, and results in significant suppression of pre-established HNSCC. Co-transfer of the IL-15 gene further improved the therapeutic outcome, mainly by augmenting NK tumoricidal activity. The biological effects of IL-21 may be in sharp contrast to those of conventional Th1 and Th2 cytokines, suggesting intriguing implications of this cytokine for the classical concept of Th1 vs. Th2 paradigm.     

Expression patterns of two genes for the delta-subunit of mitochondrial F1-ATP synthase from sweet potato in transgenic tobacco plants and cells.

Two nuclear genes, F1 delta-1 and F1 delta-2, coding for the delta-subunit of mitochondrial F1-ATP synthase, which corresponds to oligomycin-sensitivity conferring protein in animal and yeast mitochondria, were isolated from sweet potato. The gene for the delta-subunit was composed of 6 exons and these two genes shared high sequence similarities to each other not only in exons but also in introns and in the 5'-upstream regions. However, the 5'-upstream regions of F1 delta-1 and F1 delta-2 were distinguishable by the presence of novel sequences, designated Ins-1 and Ins-2, respectively. Ins-1 and Ins-2 contained a terminal direct repeat of 10 bp and 12 bp, respectively, and various forms of repeat sequences. The promoter fusion of both F1 delta-1 and F1 delta-2 with the GUS coding sequence gave expression of GUS activity in transformed tobacco BY-2 cells, although the levels of GUS activity and the patterns of expression during the growth of cells were different between the two. In transgenic tobacco plants, the two fusion genes showed similar levels of expression in leaves and stems, while F1 delta-2:GUS gave significantly higher levels of expression in roots than F1 delta-1:GUS. Deletion of Ins-1 from the 5'-upstream region of F1 delta-1:GUS did not affect the expression of the fusion gene in various organs of transgenic plants. However, it caused significant enhancement of expression in transformed tobacco BY-2 cells.     

Gene transfer mediated by YKS-220 cationic particles: convenient and efficient gene delivery reagent.

A monocationic lipid, YKS-220, with a symmetrical and biodegradable structure can be used as an effective gene transfer vector in a cationic particle form (not a cationic liposome form), and is obtained by diluting an ethanol solution of YKS-220 and DOPE (1:5, molar ratio) with an aqueous medium. This preparation method is more convenient than that for cationic liposomes. YKS-220 cationic particles showed a heterogeneous large mean diameter of 4.4 microm. An obvious size change was not observed when plasmid DNA was added. The transfection activity of YKS-220 cationic particles was comparable to those of YKS-220 liposomes and DOSPA liposomes (LipofectAMINE), and even higher than that of DOGS (TRNSFECTAM). Interestingly, the YKS-220 cationic particle/DNA complexes were resistant to the neutralizing effect of serum. All of these findings indicate that YKS-220 cationic particles are a convenient and efficient gene delivery reagent.     

Association between Poor Glycemic Control,Impaired Sleep Quality,and Increased Arterial Thickening in Type 2 Diabetic Patients

Objective

Poor sleep quality is an independent predictor of cardiovascular events. However, little is known about the association between glycemic control and objective sleep architecture and its influence on arteriosclerosis in patients with type-2 diabetes mellitus (DM). The present study examined the association of objective sleep architecture with both glycemic control and arteriosclerosis in type-2 DM patients.

Design

Cross-sectional study in vascular laboratory.

Methods

The subjects were 63 type-2 DM inpatients (M/F, 32/31; age, 57.5±13.1) without taking any sleeping promoting drug and chronic kidney disease. We examined objective sleep architecture by single-channel electroencephalography and arteriosclerosis by carotid-artery intima-media thickness (CA-IMT).

Results

HbA1c was associated significantly in a negative manner with REM sleep latency (interval between sleep-onset and the first REM period) (β=-0.280, p=0.033), but not with other measurements of sleep quality. REM sleep latency associated significantly in a positive manner with log delta power (the marker of deep sleep) during that period (β=0.544, p=0.001). In the model including variables univariately correlated with CA-IMT (REM sleep latency, age, DM duration, systolic blood pressure, and HbA1c) as independent variables, REM sleep latency (β=-0.232, p=0.038), but not HbA1c were significantly associated with CA-IMT. When log delta power was included in place of REM sleep latency, log delta power (β=-0.257, p=0.023) emerged as a significant factor associated with CA-IMT.

Conclusions

In type-2 DM patients, poor glycemic control was independently associated with poor quality of sleep as represented by decrease of REM sleep latency which might be responsible for increased CA-IMT, a relevant marker for arterial wall thickening.     

2',4'-BNA(NC): a novel bridged nucleic acid analogue with excellent hybridizing and nuclease resistance profiles

Oligonucleotides modified with 2 ',4 '-BNA(NC) (N-H)/(N-Me) monomers exhibited excellent hybridizing and nuclease resistance properties. Duplex and triplex thermal stabilities were greatly enhanced by incorporating 2',4'-BNA(NC) (N-H) and (N-Me) monomers and nuclease resistance was tremendously higher than that of natural oligonucleotide.