ABSTRACT
BACKGROUND: Inherited cardiac conduction disease is a rare bradyarrhythmia associated with mutations in various genes that affect action potential propagation. It is often characterized by isolated conduction disturbance of the His-Purkinje system, but it is rarely described as a syndromic form. OBJECTIVES: The authors sought to identify the genetic defect in families with a novel bradyarrhythmia syndrome associated with bone malformation. METHODS: The authors genetically screened 15 European cases with genotype-negative de novo atrioventricular (AV) block and their parents by trio whole-exome sequencing, plus 31 Japanese cases with genotype-negative familial AV block or sick sinus syndrome by targeted exon sequencing of 457 susceptibility genes. Functional consequences of the mutation were evaluated using an in vitro cell expression system and in vivo knockout mice. RESULTS: The authors identified a connexin-45 (Cx45) mutation (p.R75H) in 2 unrelated families (a de novo French case and a 3-generation Japanese family) who presented with progressive AV block, which resulted in atrial standstill without ventricular conduction abnormalities. Affected individuals shared a common extracardiac phenotype: a brachyfacial pattern, finger deformity, and dental dysplasia. Mutant Cx45 expressed in Neuro-2a cells showed normal hemichannel assembly and plaque formation. However, Lucifer yellow dye transfer and gap junction conductance between cell pairs were severely impaired, which suggested that mutant Cx45 impedes gap junction communication in a dominant-negative manner. Tamoxifen-induced, cardiac-specific Cx45 knockout mice showed sinus node dysfunction and atrial arrhythmia, recapitulating the intra-atrial disturbance. CONCLUSIONS: Altogether, the authors showed that Cx45 mutant p.R75H is responsible for a novel disease entity of progressive atrial conduction system defects associated with craniofacial and dentodigital malformation.
Subject(s)
Atrioventricular Block/etiology , Connexins/genetics , DNA/genetics , Dentofacial Deformities/complications , Mutation , Adolescent , Adult , Animals , Atrioventricular Block/genetics , Atrioventricular Block/physiopathology , Child , Child, Preschool , Connexins/metabolism , DNA Mutational Analysis , Dentofacial Deformities/genetics , Dentofacial Deformities/metabolism , Disease Models, Animal , Disease Progression , Electrocardiography , Female , Humans , Male , Mice , Mice, Transgenic , Middle Aged , Pedigree , Phenotype , Young AdultABSTRACT
Gap junctional intercellular communication is assumed to play an important role during pre- and peri-implantation development. In this study, we eliminated connexin43 (Cx43) and connexin45 (Cx45), major gap junctional proteins in the pre- and peri-implantation embryo. We generated Cx43 -/- Cx45 -/- embryos by Cx43 +/- Cx45 +/- intercrossing, because mice deficient in Cx43 (Cx43 -/-) exhibit perinatal lethality and those deficient in Cx45 (Cx45 -/-) exhibit early embryonic lethality. Wild-type, Cx43 -/-, Cx45 -/-, and Cx43 -/- Cx45 -/- blastocysts all showed similar outgrowths in in vitro culture. Moreover, Cx43 -/- Cx45 -/- embryos were obtained at the expected Mendelian ratio up to embryonic day 9.5, when the Cx45 -/- mutation proved lethal. The Cx43 -/- Cx45 -/- embryos seemed to have no additional developmental abnormalities in comparison with the single knockout strains. Thus, pre- and peri-implantation development does not require Cx43 and Cx45. Other gap junctional proteins are expressed around these stages and these may compensate for the lack of Cx43 and Cx45.
Subject(s)
Blastocyst/metabolism , Connexin 43/genetics , Connexins/genetics , Embryo, Mammalian/metabolism , Animals , Connexin 43/deficiency , Connexins/deficiency , Embryo Culture Techniques , Embryo Implantation , Embryo, Mammalian/embryology , Genotype , Immunohistochemistry , Mice, Knockout , Time FactorsABSTRACT
Among gap junction-encoding genes, the loss of connexin (Cx) 45 most profoundly obstructs embryogenesis through an endocardial cushion defect and conduction block. However, the interdependence of these defects is not known, and the details of conduction block have not been elucidated. Here, we examined mouse embryos with a region-specific deletion of Cx45 in the myocardium (CA-Cre; Cx45(flox/flox)) or endothelium (Tie2-Cre; Cx45(flox/flox)). Although the deletion of Cx45 in the myocardium was heterogeneous, the CA-Cre; Cx45(flox/flox) embryos were lethal at the same stage as the constitutive Cx45-deficient (Cx45(-/-)) embryos. We determined the onset and patterns of their conduction block through point-tracking in video recordings of embryonic heart contractions. An incomplete conduction block at the atrioventricular canal appeared at embryonic day (E) 8.5 and was predominant around the lethal E9.5 stage in both the Cx45(-/-) and CA-Cre; Cx45(flox/flox) embryos. Although the Cx45(-/-) hearts showed a consistently severe reduction in atrioventricular conduction velocity, the CA-Cre; Cx45(flox/flox) hearts had delay times within the normal range and showed frequent retrograde conduction. As previously reported, the Cx45(-/-) endocardial cushion was consistently defective, and nuclear factor of activated T-cells cytoplasmic (NFATc)1 within the endocardium showed inactive cytoplasmic distribution. In CA-Cre; Cx45(flox/flox), however, the endocardial cushion was partially formed, with active NFATc1 within the endocardium. There was no developmental abnormality in the Tie2-Cre; Cx45(flox/flox) embryos. These results indicate that myocardial dysfunction is responsible for most of the reported defects in Cx45(-/-), which are alleviated by sporadic Cx45 expression in the CA-Cre; Cx45(flox/flox) myocardium.
Subject(s)
Connexins/metabolism , Endocardium/embryology , Endocardium/metabolism , Heart/embryology , Myocardium/metabolism , Animals , Embryonic Development/physiology , Endothelium/embryology , Endothelium/metabolism , Gap Junctions/metabolism , Mice , NFATC Transcription Factors/metabolismABSTRACT
Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin (Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells (ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.
ABSTRACT
Tektins are evolutionarily conserved filament-forming proteins localized in flagella and cilia, and have been reported to be involved in the stability and structural complexity of axonemal microtubules. Five mammalian Tektins (Tektin1-5) have been reported. Of these, Tektin2 (TEKT2) has been found to be required for normal flagellum structure and function. Tekt2-null sperm display flagellum bending and reduced motility, probably due to disruption of the dynein inner arm. However, the subcellular localization of TEKT2 in spermatozoa has not been clarified at the ultrastructural level. To elucidate the molecular localization of TEKT2 in flagella of rat spermatozoa, we performed confocal laser scanning microscopy, extraction of flagella followed by immunoblot analysis, and immunogold electron microscopy. Extraction of sperm flagella by SDS-EDTA resulted in complete extraction of axonemal tubulins, while TEKT2 was only partially released from flagella, suggesting that TEKT2 might be present in the peri-axonemal component, not directly associated with axonemal tubulins. Confocal laser scanning microscopy and pre-embedding immunoelectron microscopy revealed that TEKT2 is associated with the surface of outer dense fibers (ODFs). TEKT2 may function as an ODF-affiliated molecule required for flagellum stability and sperm motility.
Subject(s)
Flagella/metabolism , Microtubule Proteins/metabolism , Spermatozoa/metabolism , Animals , Gene Expression Regulation/physiology , Male , Microtubule Proteins/genetics , RatsSubject(s)
Connexins/analysis , Gap Junctions/physiology , Heart Conduction System/physiology , Animals , HumansABSTRACT
Claudins constitute tight junction (TJ) strands and regulate paracellular permeability, which varies in the epithelial cells of various organs. Heterotypic claudin compatibility and/or the association of TJ particles to either the protoplasmic (P) or exoplasmic (E) face may be related to paracellular permeability. This study examined the relationship between the TJ morphology, heterotypic claudin compatibility and paracellular permeability using claudin-10b- or claudin-15-expressing HEK293 cells and MDCK I cells. Claudin-10b or -15 expressed in TJ-free HEK293 cells formed E-face- or P-face-associated TJ particles, respectively. The coculture of claudin-1-expressing HEK293 cells and either claudin-10b- or claudin-15-expressing HEK293 cells showed that claudin-10b and -15 were not compatible with claudin-1. The expression of claudin-10b or -15 in high-resistance MDCK I cells did not alter the expression of endogenous claudins except for claudin-3 and dramatically reduced transepithelial electrical resistance by increasing the permeability of Na(+) but it did not change that of Cl(-). The expression of claudin-10b or -15 in MDCK I cells either decreased or increased the flux of 4 kDa dextran, respectively. The coculture of MDCK I cells and either claudin-10b- or claudin-15-expressing MDCK I cells showed claudin-10b to be partly compatible, while claudin-15 was incompatible with the endogenous claudins in MDCK I cells. These results indicate that the TJ morphology cannot predict the properties of either paracellular permeability or heterotypic claudin compatibility.
Subject(s)
Membrane Proteins/metabolism , Tight Junctions/metabolism , Animals , Cell Line , Claudin-1 , Claudins , Dogs , Electrophoresis , Freeze Fracturing , Humans , Immunoblotting , Mice , Microscopy, ConfocalABSTRACT
Patch-clamp experiments were performed to investigate the behavior of voltage-activated inward currents in vas deferens myocytes from Na(V)1.6-null mice (Na(V)1.6(-/-)) lacking the expression of the Na(+) channel gene, Scn8a, and their wild-type littermates (Na(V)1.6(+/+)). Immunohistochemistry confirmed expression of Na(V)1.6 in the muscle of Na(V)1.6(+/+), but not Na(V)1.6(-/-), vas deferens. PCR analysis revealed that the only beta(1)-subunit gene expressed in Na(V)1.6(+/+) vas deferens was Scn1b. In Na(V)1.6(+/+) myocytes, the threshold for membrane currents evoked by 20 msec voltage ramps (-100 mV to 60 mV) was -38.5 +/- 4.6 mV and this was shifted to a more positive potential (-31.2 +/- 4.9 mV) by tetrodotoxin (TTX). In Na(V)1.6(-/-) myocytes, the threshold was -30.4 +/- 3.4 mV and there was no TTX-sensitive current. The Na(+) current (I(Na)) in Na(V)1.6(+/+) myocytes had a bell-shaped current-voltage relationship that peaked at approximately -10 mV. Increasing the duration of the voltage ramps beyond 20 msec reduced the peak amplitude of I(Na). I(Na) displayed both fast (tau approximately 10 msec) and slow (tau approximately 1 sec) recovery from inactivation, the magnitude of the slow component increasing with the duration of the conditioning pulse (5-40 msec). During repetitive activation (5-40 msec pulses), I(Na) declined at stimulation frequencies > 0.5 Hz and at 10 Hz Subject(s)
Muscle, Smooth/metabolism
, Myocytes, Smooth Muscle/metabolism
, Nerve Tissue Proteins/metabolism
, Sodium Channels/metabolism
, Sodium/metabolism
, Vas Deferens/metabolism
, Animals
, Cell Separation
, Electric Stimulation
, Immunohistochemistry
, Ion Channel Gating
, Kinetics
, Male
, Membrane Potentials
, Mice
, Mice, Inbred C3H
, Mice, Knockout
, Muscle, Smooth/cytology
, Muscle, Smooth/drug effects
, Myocytes, Smooth Muscle/drug effects
, NAV1.6 Voltage-Gated Sodium Channel
, Nerve Tissue Proteins/antagonists & inhibitors
, Nerve Tissue Proteins/deficiency
, Nerve Tissue Proteins/genetics
, Patch-Clamp Techniques
, RNA, Messenger/metabolism
, Reverse Transcriptase Polymerase Chain Reaction
, Sodium Channel Blockers/pharmacology
, Sodium Channels/deficiency
, Sodium Channels/genetics
, Tetrodotoxin/pharmacology
, Vas Deferens/cytology
, Vas Deferens/drug effects
, Voltage-Gated Sodium Channel beta-1 Subunit
ABSTRACT
In Japan, dissection of human body is generally prohibited by the Penal Code, i.e. the criminal law. However, the Postmortem Examination and Corpse Preservation Act allows for the dissection of the body in very limited situations, that include gross anatomy dissection and pathological and forensic autopsy in medical and dental schools. Growing numbers of co-medical schools have been founded more recently in Japan, and not a small number of co-medical schools try to adopt human body dissection in the course of anatomy education. The present short communication reminds us of the ways of thinking of the Postmortem Examination and Corpse Preservation Act and the Act on Body Donation for Medical and Dental Education in order that anatomy education in medical as well as co-medical schools takes place under the regulation by these two laws.
Subject(s)
Anatomy/education , Anatomy/legislation & jurisprudence , Cadaver , Dissection/education , Dissection/legislation & jurisprudence , Education, Medical/legislation & jurisprudence , Autopsy/legislation & jurisprudence , Forensic Pathology/education , Forensic Pathology/legislation & jurisprudence , Humans , Japan , Schools, Dental , Schools, MedicalABSTRACT
Gap junctions are clusters of transmembrane protein channels for intercellular communication and are composed of connexin (Cx). The vascular endothelial cells express Cx37, Cx40, and Cx43. We herein examined the spatial distribution of the endothelial connexins Cx37, Cx40, and Cx43 in rat large veins including the cranial vena cava, thoracic section of the caudal vena cava, and abdominal section of the caudal vena cava. We also examined the mean size of the endothelial cells and quantified the protein expression levels of the endothelial connexins. We found that the large veins heterogeneously expressed Cx37, Cx40, and Cx43 as follows: Cx40 > Cx37 > > Cx43 in the cranial vena cava, Cx37 > Cx43 > > Cx40 in the thoracic section of the caudal vena cava, and Cx40 > Cx43 > > Cx37 in the abdominal section of the caudal vena cava. Double immunostaining of two of the endothelial connexins revealed that the gap-junction plaques were composed of various combinations of endothelial connexins. The mean size of the endothelial cells was large, moderate, or small in the cranial vena cava, the abdominal section of the caudal vena cava, or the thoracic section of the caudal vena cava, respectively. The heterogeneity of the endothelial cells of the rat large veins in terms of the connexin expression suggests that the endothelial cells are differently coupled in the large veins. The present data are useful for investigating, for example, disease-related alterations in expression of endothelial connexins in large veins.
Subject(s)
Connexin 43/metabolism , Connexins/metabolism , Endothelial Cells/cytology , Venae Cavae/metabolism , Animals , Male , Rats , Rats, Wistar , Venae Cavae/cytology , Gap Junction alpha-5 Protein , Gap Junction alpha-4 ProteinABSTRACT
Claudins constitute tight junction (TJ) strands. In order to examine the function of the second extracellular loop (ECL2), we constructed 1CLDeltaFY and 1CLDeltaPL in which highly conserved amino acids, FY or PL, in the ECL2 of mouse claudin-1 were deleted. They were then tagged with either EGFP at the NH(2)-terminus (EGFP1CLDeltaFY and EGFP1CLDeltaPL) or the myc-epitope at the COOH-terminus (1CLDeltaFYmyc and 1CLDeltaPLmyc). The expression of EGFP1CLDeltaFY and EGFP1CLDeltaPL in TJ-free HEK293 cells formed TJ strands resembling those formed by wild-type claudin-1. The expression of 1CLDeltaPLmyc in TJ-bearing MDCK II cells induced aberrant TJ strands in the lateral plasma membranes whose intramembranous particles were almost equally distributed in the P- and E-face. In contrast, 1CLDeltaFYmyc formed aggregates of short continuous strands which were frequently associated with vesicle-like structures. Coculture experiments with MDCK II cells showed that 1CLDeltaPLmyc was localized at heterotypic cell-cell junctions but 1CLDeltaFYmyc was not. These results suggest that changes in the TJ morphology due to the expression of either 1CLDeltaFYmyc or 1CLDeltaPLmyc may be caused by some factors specific to epithelial MDCK II cells including endogenous claudins.
Subject(s)
Epithelial Cells/ultrastructure , Fibroblasts/ultrastructure , Membrane Proteins/metabolism , Tight Junctions/ultrastructure , Animals , Cell Line , Claudin-1 , Dogs , Epithelial Cells/metabolism , Fibroblasts/metabolism , Freeze Fracturing , Humans , Membrane Proteins/genetics , Mice , Microscopy, Electron , Mutation , Tight Junctions/metabolism , TransfectionABSTRACT
Cardiac troponin T (cTnT) is a component of the troponin (Tn) complex in cardiac myocytes, and plays a regulatory role in cardiac muscle contraction by anchoring two other Tn components, troponin I (TnI) and troponin C, to tropomyosin (Tm) on the thin filaments. In order to determine the in vivo function of cTnT, we created a null cTnT allele in the mouse TNNT2 locus. In cTnT-deficient (cTnT(-/-)) cardiac myocytes, the thick and thin filaments and alpha-actinin-positive Z-disk-like structures were not assembled into sarcomere, causing early embryonic lethality due to a lack of heartbeats. TnI was dissociated from Tm in the thin filaments without cTnT. In spite of loss of Tn on the thin filaments, the cTnT(-/-) cardiac myocytes showed regular Ca(2+)-transients. These findings indicate that cTnT plays a critical role in sarcomere assembly during myofibrillogenesis in the embryonic heart, and also indicate that the membrane excitation and intracellular Ca(2+) handling systems develop independently of the contractile system. In contrast, heterozygous cTnT(+/-) mice had a normal life span with no structural and functional abnormalities in their hearts, suggesting that haploinsufficiency could not be a potential cause of cardiomyopathies, known to be associated with a variety of mutations in the TNNT2 locus.
Subject(s)
Arrhythmias, Cardiac/genetics , Heart Rate, Fetal , Heart/embryology , Myocytes, Cardiac/metabolism , Sarcomeres/metabolism , Troponin T/physiology , Actinin/metabolism , Actins/metabolism , Animals , Arrhythmias, Cardiac/physiopathology , Calcium Signaling/genetics , Echocardiography , Embryo, Mammalian , Gene Targeting , Genes, Lethal , Heart/physiopathology , Heart Rate, Fetal/genetics , Heart Ventricles/cytology , Heart Ventricles/embryology , Heart Ventricles/physiopathology , Immunohistochemistry , Mice , Mice, Knockout , Muscle Fibers, Skeletal/physiology , Myocytes, Cardiac/ultrastructure , Organ Culture Techniques , Sarcomeres/genetics , Tropomyosin/metabolism , Troponin T/geneticsABSTRACT
By differential display technique followed by RT-PCR and DNA sequence analyses, we isolated carcinoembryonic antigen-related cell adhesion molecule 6 (Ceacam6) and its novel spliced variant Ceacam6-Long (Ceacam6-L) from rat testis. Ceacam6-L mRNA was generated by retention of 67 nucleotide-length third intron in Ceacam6 gene. Ceacam6-L is a member of an immunoglobulin superfamily and encodes a protein of 50 kDa with a signal sequence at the N-terminus, one immunoglobulin (Ig)-like domain, three IgCAM domains, a transmembrane region, and a short intracellular region. Expression analyses by RT-PCR and Northern blot showed that Ceacam6-L was exclusively expressed in rat testis and first detectable at 5 wk during postnatal development of testis. We performed immunoblot analyses and immunohistochemistry using the anti-CEACAM6-L antibody. Confocal laser scanning microscopy revealed that CEACAM6-L was not present at blood-testis barrier junctions between Sertoli cells but localized at the interface between Sertoli cells and germ cells, possibly to work as an adhesion molecule in the apical compartment of the seminiferous epithelium. At stages VII-VIII, at which all of the elongated spermatids migrated to the luminal surface of the seminiferous tubules, CEACAM6-L was found to locate at the concave side of elongated spermatid heads, following the curvature of their sickle-shaped nuclei, suggesting that CEACAM6-L might be involved in the anchoring of spermatids to Sertoli cells and spermiation. We concluded that CEACAM6-L might be a novel adhesion molecule constructing the apical ectoplasmic specialization in testis.
Subject(s)
Antigens, CD/physiology , Cell Adhesion Molecules/physiology , Introns/physiology , Sertoli Cells/physiology , Spermatids/physiology , Testis/cytology , Alkylating Agents/pharmacology , Amino Acid Sequence , Animals , Antigens, CD/genetics , Blotting, Northern , Blotting, Western , Busulfan/pharmacology , Cell Adhesion Molecules/genetics , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Gene Expression Profiling , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Immunoglobulins/physiology , Introns/genetics , Male , Microscopy, Confocal , Molecular Sequence Data , Rats , Reverse Transcriptase Polymerase Chain Reaction , Seminiferous Epithelium/physiologyABSTRACT
Tight junctions (TJs) create a paracellular permeability barrier to restrict the passage of ions, small solutes, and water. Ameloblasts are enamel-forming cells that sequentially differentiate into preameloblasts, secretory, transition, and ruffle-ended and smooth-ended maturation ameloblasts (RAs and SAs). TJs are located at the proximal and distal ends of ameloblasts. TJs at the distal ends of secretory ameloblasts and RAs are well-developed zonula occludens, but other TJs are moderately developed but incomplete zonula occludens (ZO) or less-developed macula occludens. We herein examined the immunofluorescence localization of TJ proteins, 10 claudin isoforms, occludin, ZO-1, and PAR3, a cell polarity-related protein, in ameloblasts of rat upper incisors. ZO-1 and claudin-1 were detected at both ends of all ameloblasts except for the distal ends of SAs. Claudin-4 and occludin were detected at both ends of transition and maturation ameloblasts except for the distal ends of SAs. PAR3 was detected at the proximal TJs of all ameloblasts and faintly at the distal TJs of early RAs. These results indicate that functional zonula occludens formed at the distal ends of the secretory ameloblasts and RAs consisted of different TJ proteins. Therefore, the distal TJs of secretory ameloblasts and RAs may differentially regulate the paracellular permeability to create a microenvironment suitable for enamel deposition and enamel maturation, respectively. In addition, PAR3 may be principally involved in the formation and maintenance of the proximal, but not distal, TJs.
Subject(s)
Ameloblasts/metabolism , Carrier Proteins/metabolism , Incisor/metabolism , Membrane Proteins/metabolism , Tight Junctions/metabolism , Animals , Claudin-1 , Claudin-4 , Nerve Tissue Proteins , Occludin , Phosphoproteins/metabolism , Rats , Rats, Wistar , Zonula Occludens-1 ProteinABSTRACT
Recent studies suggest that the morphological and physiological properties of tight junctions (TJs) are determined by the combination and mixing ratios of claudin isoforms. In this study, we tried to characterize mouse cell lines by expression of claudin isoforms to use for studying epithelial TJs by overexpression or suppression of claudin(s) in the cells and found that claudin-2 was expressed in a few mouse rectum carcinoma cells, CMT93 cells. We have isolated CMT93-I and -II cells from CMT93 cells by immunohistochemical screening for the presence or absence of claudin-2 expression. Immunofluorescence and RT-PCR analyses showed that expression of claudin-4, -6, -7 and -12 was detected in both cell lines, but claudin-2 was only expressed in CMT93-II cells. There were no differences in paracellular permeability between CMT93-I and -II cells examined by 4 kDa FITC-dextran and fluorescein sodium, or in the number of TJ strands examined by freeze-fracture electron microscopy. However, the transepithelial electrical resistance (TER) of CMT93-I cells was approximately 6.5 times higher than that of CMT93-II cells, suggesting that expression of claudin-2 may be related to decreased TER. Comparative examinations of CMT93-I and -II cells provide a clue how the combination and mixing ratios of claudin isoforms regulate the paracellular permeability.
Subject(s)
Carcinoma/metabolism , Carcinoma/ultrastructure , Membrane Proteins/biosynthesis , Rectal Neoplasms/metabolism , Rectal Neoplasms/ultrastructure , Tight Junctions/metabolism , Tight Junctions/ultrastructure , Animals , Cell Line , Cell Membrane/physiology , Claudins , Cryoelectron Microscopy , Electric Impedance , Fibroblasts/metabolism , Fibroblasts/ultrastructure , Freeze Fracturing , Immunoblotting , Immunohistochemistry , Membrane Proteins/genetics , Mice , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Tektins are composed of a family of filament-forming proteins localized in cilia and flagella. Four types of mammalian Tektins have been reported, and at least two types of Tektins, Tektin2 and Tektin4, have been verified to be present in sperm flagella. A new member of the TEKTIN gene family, which was designated as rat Tektin5, was obtained by PCR technique. Rat Tektin5 cDNA consists of 1,674 bp encoding a 62.8 kDa protein of 558 amino acids. Tektin5 protein contains a Tektin domain as well as a nonapeptide signature sequence that is a prominent feature of Tektin proteins. RT-PCR analysis indicated that Tektin5 was predominantly expressed in testis and that its expression was up-regulated during testis development. Immunoblot analyses revealed that Tektin5 is present in sperm flagella but not in heads and that it is completely released from rat spermatozoa by 6 M urea treatment, but not extracted by 1% Triton X-100 and 0.6 M potassium thiocyanate. Confocal laser scanning microscopy revealed that Tektin5 was located in the middle piece of flagella in rat spermatozoa with no immunolabeling in the heads and the principal piece. Immunogold electron microscopy adopting pre-embedding method discovered that Tektin5 is predominantly associated with the inner side of the mitochondrial sheath. Tektin5 might work as a middle piece component requisite for flagellar stability and sperm motility.
Subject(s)
Flagella/chemistry , Microtubule Proteins/genetics , Spermatozoa/chemistry , Amino Acid Sequence , Animals , Antibodies/chemistry , Antibody Specificity , Antigen-Antibody Reactions , Cloning, Molecular , Gene Expression Profiling , Humans , Immunohistochemistry , Male , Mice , Microscopy, Confocal , Microscopy, Immunoelectron , Microtubule Proteins/immunology , Microtubule Proteins/isolation & purification , Molecular Sequence Data , Phylogeny , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Spermatozoa/ultrastructure , Tissue Embedding , Urea/chemistryABSTRACT
Claudins are one of the transmembrane proteins found at tight junctions (TJs); they constitute the backbone of TJ strands and comprise a multigene family. Claudins share a YV sequence at the COOH-termini, which is considered to be a ZO-binding motif. Overexpression of claudin-15 (15CL) or claudin-15 tagged with enhanced green fluorescent protein at the NH2-terminus (EGFP-15CL) induced aberrant strands in MDCK II cells, even though claudin-15 has the ZO-binding motif. Morphometric analysis by freeze-fracture electron microscopy revealed that the mean number of apical TJ strands increased by 47% in EGFP-1CL-expressing cells, 21% in EGFP-15CL-expressing cells, and 28% in 15CL-expressing cells. The number of free-ended apical strands increased remarkably in EGFP-15CL- and 15CL-expressing cells, but not in EGFP-1CL-expressing cells. When MDCK cells expressing EGFP-1CL, EGFP-15CL or 15CL were co-cultured with parent MDCK cells, which express claudin-1 but not claudin-15, EGFP-15CL and 15CL could not be concentrated at the apical junctional region between the heterotypic cells, though EGFP-1CL could. These results suggest that not only binding to ZO-1, but also head-to-head compatibility between claudin species, is involved in organizing claudin proteins at the apical junctional region.
Subject(s)
Fibroblasts/metabolism , Membrane Proteins/metabolism , Tight Junctions/metabolism , Animals , Base Sequence , Cell Line , Claudin-1 , Claudins , Dogs , Fluorescent Dyes/metabolism , Green Fluorescent Proteins/genetics , Humans , Kidney/cytology , Membrane Proteins/genetics , Mice , Microscopy, Confocal , Molecular Sequence Data , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tight Junctions/ultrastructure , Up-Regulation/geneticsABSTRACT
We have analyzed the expression profiles of ten genes in terms of testis development and organ specificity in rat, which were selected from 215 round spermatid-specific transcripts listed in a database. Out of the ten genes, we directed our attention to one gene, a germ cell-less like-2 gene (gcl-2), a homolog of Drosophila gcl gene (gcl), which is a component of the germ plasma and required for primordial germ cell formation. Rat genome contains duplicate rat gcl-2 (rgcl-2) genes, rgcl-2A and rgcl-2B, both of which are located at Xq13. RT-PCR analysis showed that the expression of the two genes was up-regulated during testis development and that they were predominantly expressed in the testis. Both rgcl-2A and rgcl-2B encode a protein of 498 amino acid residues, showing 90.56% identity at the amino acid level. Confocal laser scanning microscopy revealed that rgcl-2 protein was synthesized in the cytoplasm of elongating spermatids and at least a part of it was integrated into the middle piece of spermatozoa during spermiogenesis. Immunogold electron microscopy uncovered that rgcl-2 was localized at the abaxial (convex) surface of outer dense fibers (ODF) of rat sperm flagella. Therefore, we concluded that rgcl-2 is a new component of ODF in sperm flagella.
Subject(s)
Cytoplasm/chemistry , Gene Expression Profiling , Oligonucleotide Array Sequence Analysis , Proteins/analysis , Sperm Tail/chemistry , Amino Acid Sequence , Animals , Base Sequence , Cytoplasm/ultrastructure , Male , Microscopy, Confocal , Microscopy, Immunoelectron , Molecular Sequence Data , Proteins/genetics , Proteins/metabolism , Rats , Rats, Wistar , Sperm Tail/ultrastructure , Spermatids/chemistry , Spermatids/ultrastructure , Spermatogenesis/physiology , Testis/growth & developmentABSTRACT
Claudins are integral membrane proteins at tight junctions (TJs) and form TJ strands. In the present study, we found that claudin-7 was localized along the entire lateral membranes of epididymal epithelium, including the apical junctional region throughout the epididymis, but claudin-8 was restricted to the apical junctional region. This finding raises the possibility that aberrant TJ strands may be formed on lateral membranes. Thus, we focused on examining whether TJ strands exist on lateral membranes of epididymal epithelium. Freeze-fracture electron microscopy showed that aberrant TJ strands were observed in only a few principal cells in all segments of the epididymis except for the initial segment, indicating that the occurrence of aberrant strands is very rare. Aberrant TJ strands were smooth and not subdivided into individual particles in the protoplasmic face, and complementary grooves in the extracellular face were almost free of particles. Aberrant TJ strands in the distal caput and corpus epididymis were accompanied by many vesicle-like structures but those in the proximal caput and cauda epididymis were not. These results suggest that most of claudin-7 in lateral membranes may exist in a nonpolymerized form and may play some different roles other than the formation of TJ strands, for example, in the formation of a pool of claudin proteins or in the reinforcement of cell adhesion.