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Find video protocols related to scientific articles indexed in Pubmed.
Deletion of small ankyrin 1 (sAnk1) isoforms results in structural and functional alterations in aging skeletal muscles fibers.
Am. J. Physiol., Cell Physiol.
PUBLISHED: 10-31-2014
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Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4-6 month old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4-6 months of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12-15 months) EDL skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contractile performance of muscles from sAnk1 KO mice and a reduced exercise tolerance in an endurance test on treadmill compared to control mice. Moreover, reduced Q? charge and L-type Ca(2+)current, that are indexes of affected e-c coupling, were observed in diaphragm fibers from 12-15 month old mice, but not in other skeletal muscles from sAnk1 KO mice. Altogether, these findings show that the ablation of sAnk1, by altering the organization of the SR, renders skeletal muscles susceptible to undergo structural and functional alterations more evident with age, and point to an important contribution of sAnk1 to the maintenance of the longitudinal SR architecture.
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TMEM14C is required for erythroid mitochondrial heme metabolism.
J. Clin. Invest.
PUBLISHED: 08-26-2014
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The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.
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Genetic Regulation of Female Sexual Maturation and Longevity Through Circulating IGF1.
J. Gerontol. A Biol. Sci. Med. Sci.
PUBLISHED: 07-30-2014
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We previously reported that insulin-like growth factor 1 (IGF1) was involved in coregulating female sexual maturation and longevity. To understand the underlying genetic mechanisms, based on the strain survey assays of development and aging traits, we crossed two mouse strains, KK/HIJ and PL/J, and produced 307 female F2 mice. We observed the age of vaginal patency (AVP) and the life span of these females. We also measured circulating IGF1 level at 7, 16, 24, 52, and 76 weeks. IGF1 level at 7 weeks significantly correlated with AVP. IGF1 levels at ages of 52 and 76 weeks negatively correlated with longevity (p ? .05). A gene mapping study found 22, 4 ,and 3 quantitative trait loci for IGF1, AVP, and life span, respectively. Importantly, the colocalization of IGF1, AVP, and life span quantitative trait loci in the distal region of chromosome 2 suggests this locus carries gene(s) that could regulate IGF1, AVP, and life span. In this region, proprotein convertase subtilisin/kexin type 2 has been found to be associated with female sexual maturation in a human genome-wide association study. We verified the roles of proprotein convertase subtilisin/kexin type 2 in regulating IGF1 and AVP by showing that depletion of proprotein convertase subtilisin/kexin type 2 significantly reduced IGF1 and delayed AVP in mice, suggesting that it also might be involved in the regulation of aging.
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Iron regulatory protein-1 protects against mitoferrin-1-deficient porphyria.
J. Biol. Chem.
PUBLISHED: 02-07-2014
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Mitochondrial iron is essential for the biosynthesis of heme and iron-sulfur ([Fe-S]) clusters in mammalian cells. In developing erythrocytes, iron is imported into the mitochondria by MFRN1 (mitoferrin-1, SLC25A37). Although loss of MFRN1 in zebrafish and mice leads to profound anemia, mutant animals showed no overt signs of porphyria, suggesting that mitochondrial iron deficiency does not result in an accumulation of protoporphyrins. Here, we developed a gene trap model to provide in vitro and in vivo evidence that iron regulatory protein-1 (IRP1) inhibits protoporphyrin accumulation. Mfrn1(+/gt);Irp1(-/-) erythroid cells exhibit a significant increase in protoporphyrin levels. IRP1 attenuates protoporphyrin biosynthesis by binding to the 5'-iron response element (IRE) of alas2 mRNA, inhibiting its translation. Ectopic expression of alas2 harboring a mutant IRE, preventing IRP1 binding, in Mfrn1(gt/gt) cells mimics Irp1 deficiency. Together, our data support a model whereby impaired mitochondrial [Fe-S] cluster biogenesis in Mfrn1(gt/gt) cells results in elevated IRP1 RNA-binding that attenuates ALAS2 mRNA translation and protoporphyrin accumulation.
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Alterations in thin filament length during postnatal skeletal muscle development and aging in mice.
Front Physiol
PUBLISHED: 01-01-2014
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The lengths of the sarcomeric thin filaments vary in a skeletal muscle-specific manner and help specify the physiological properties of skeletal muscle. Since the extent of overlap between the thin and thick filaments determines the amount of contractile force that a sarcomere can actively produce, thin filament lengths are accurate predictors of muscle-specific sarcomere length-tension relationships and sarcomere operating length ranges. However, the striking uniformity of thin filament lengths within sarcomeres, specified during myofibril assembly, has led to the widely held assumption that thin filament lengths remain constant throughout an organism's lifespan. Here, we rigorously tested this assumption by using computational super-resolution image analysis of confocal fluorescence images to explore the effects of postnatal development and aging on thin filament length in mice. We found that thin filaments shorten in postnatal tibialis anterior (TA) and gastrocnemius muscles between postnatal days 7 and 21, consistent with the developmental program of myosin heavy chain (MHC) gene expression in this interval. By contrast, thin filament lengths in TA and extensor digitorum longus (EDL) muscles remained constant between 2 mo and 2 yr of age, while thin filament lengths in soleus muscle became shorter, suggestive of a slow-muscle-specific mechanism of thin filament destabilization associated with aging. Collectively, these data are the first to show that thin filament lengths change as part of normal skeletal muscle development and aging, motivating future investigations into the cellular and molecular mechanisms underlying thin filament adaptation across the lifespan.
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Effects of housing density in five inbred strains of mice.
PLoS ONE
PUBLISHED: 01-01-2014
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To evaluate the effect of increased mouse density in a cage, mice were housed at the density recommended by the 1996 Guide for the Care and Use of Laboratory Animals and at densities that were approximately 2, 2.6, and 3 times greater. Five strains of mice (129S1/SvImJ, A/J, BALB/cByJ, C57BL/6J, and DBA/2J) were evaluated throughout 3- and 8-month timeframes for health and well-being, including mortality, cardiac measures, plasma cholesterol, body weight, bone mineral density, organ weights, hematology, behavioral observations, and open field and light-dark tests. For 22 of the 27 traits measured, increased housing density had no significant effect. Kidney weight, adrenal weight, and heart rate decreased as mice were housed more densely, and some of the decreases were statistically significant. Reduced kidney weight, adrenal weight, and heart rate are not considered to be negative outcomes and may even indicate reduced stress. However, all measurements of these three traits were within normal physiological ranges. Percent fat increased slightly in strains 129S1/SvImJ, A/J, and DBA/2J, but did not increase in strains BALB/cByJ, and C57BL/6J. These results indicate that mice can be housed at higher densities than those currently recommended.
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Strain-specific variations in cation content and transport in mouse erythrocytes.
Physiol. Genomics
PUBLISHED: 03-12-2013
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Studies of ion transport pathophysiology in hematological disorders and tests of possible new therapeutic agents for these disorders have been carried out in various mouse models because of close functional similarities between mouse and human red cells. We have explored strain-specific differences in erythrocyte membrane physiology in 10 inbred mouse strains by determining erythrocyte contents of Na(+), K(+), and Mg(2+), and erythrocyte transport of ions via the ouabain-sensitive Na-K pump, the amiloride-sensitive Na-H exchanger (NHE1), the volume and chloride-dependent K-Cl cotransporter (KCC), and the charybdotoxin-sensitive Gardos channel (KCNN4). Our data reveal substantial strain-specific and sex-specific differences in both ion content and trans-membrane ion transport in mouse erythrocytes. These differences demonstrate the feasibility of identifying specific quantitative trait loci for erythroid ion transport and content in genetically standardized inbred mouse strains.
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Analysis of the mobilities of band 3 populations associated with ankyrin protein and junctional complexes in intact murine erythrocytes.
J. Biol. Chem.
PUBLISHED: 12-06-2011
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Current models of the erythrocyte membrane depict three populations of band 3: (i) a population tethered to spectrin via ankyrin, (ii) a fraction attached to the spectrin-actin junctional complex via adducin, and (iii) a freely diffusing population. Because many studies of band 3 diffusion also distinguish three populations of the polypeptide, it has been speculated that the three populations envisioned in membrane models correspond to the three fractions observed in diffusion analyses. To test this hypothesis, we characterized band 3 diffusion by single-particle tracking in wild-type and ankyrin- and adducin-deficient erythrocytes. We report that ?40% of total band 3 in wild-type murine erythrocytes is attached to ankyrin, whereas ?33% is immobilized by adducin, and ?27% is not attached to any cytoskeletal anchor. More detailed analyses reveal that mobilities of individual ankyrin- and adducin-tethered band 3 molecules are heterogeneous, varying by nearly 2 orders of magnitude and that there is considerable overlap in diffusion coefficients for adducin and ankyrin-tethered populations. Taken together, the data suggest that although the ankyrin- and adducin-immobilized band 3 can be monitored separately, significant heterogeneity still exists within each population, suggesting that structural and compositional properties likely vary considerably within each band 3 complex.
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Comparative proteomics reveals deficiency of SLC9A1 (sodium/hydrogen exchanger NHE1) in ?-adducin null red cells.
Br. J. Haematol.
PUBLISHED: 06-21-2011
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Spherocytosis is one of the most common inherited disorders, yet presents with a wide range of clinical severity. While several genes have been found mutated in patients with spherocytosis, the molecular basis for the variability in severity of haemolytic anaemia is not entirely understood. To identify candidate proteins involved in haemolytic anaemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in red blood cells (RBCs) from normal and ?-adducin (Add2) knock-out mice. We detected seven proteins that were decreased and 48 proteins that were increased in ?-adducin null RBC ghosts. Since haemolytic anaemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Among the 48 proteins increased in Add2 knockout RBCs, only 11 were also increased in reticulocytes. Of the proteins decreased in Add2 knockout RBCs, ?-adducin showed the greatest intensity difference, followed by SLC9A1, the sodium-hydrogen exchanger previously termed NHE1. We verified these mass spectrometry results by immunoblot. This is the first example of SLC9A1deficiency in haemolytic anaemia and suggests new insights into the mechanisms leading to fragile RBCs.
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Lack of protein 4.1G causes altered expression and localization of the cell adhesion molecule nectin-like 4 in testis and can cause male infertility.
Mol. Cell. Biol.
PUBLISHED: 04-11-2011
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Protein 4.1G is a member of the protein 4.1 family, which in general serves as adaptors linking transmembrane proteins to the cytoskeleton. 4.1G is thought to be widely expressed in many cells and tissues, but its function remains largely unknown. To explore the function of 4.1G in vivo, we generated 4.1G(-/-) mice and bred the mice in two backgrounds: C57BL/6 (B6) and 129/Sv (129) hybrids (B6-129) and inbred B6. Although the B6 4.1G(-/-) mice showed no obvious abnormalities, deficiency of 4.1G in B6-129 hybrids was associated with male infertility. Histological examinations of these 4.1G(-/-) mice revealed atrophy, impaired cell-cell contact and sloughing off of spermatogenic cells in seminiferous epithelium, and lack of mature spermatids in the epididymis. Ultrastructural examination revealed enlarged intercellular spaces between spermatogenic and Sertoli cells as well as the spermatid deformities. At the molecular level, 4.1G is associated with the nectin-like 4 (NECL4) adhesion molecule. Importantly, the expression of NECL4 was decreased, and the localization of NECL4 was altered in 4.1G(-/-) testis. Thus, our findings imply that 4.1G plays a role in spermatogenesis by mediating cell-cell adhesion between spermatogenic and Sertoli cells through its interaction with NECL4 on Sertoli cells. Additionally, the finding that infertility is present in B6-129 but not on the B6 background suggests the presence of a major modifier gene(s) that influences 4.1G function and is associated with male infertility.
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Mice as a mammalian model for research on the genetics of aging.
ILAR J
PUBLISHED: 03-18-2011
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Mice are an ideal mammalian model for studying the genetics of aging: considerable resources are available, the generation time is short, and the environment can be easily controlled, an important consideration when performing mapping studies to identify genes that influence lifespan and age-related diseases. In this review we highlight some salient contributions of the mouse in aging research: lifespan intervention studies in the Interventions Testing Program of the National Institute on Aging; identification of the genetic underpinnings of the effects of calorie restriction on lifespan; the Aging Phenome Project at the Jackson Laboratory, which has submitted multiple large, freely available phenotyping datasets to the Mouse Phenome Database; insights from spontaneous and engineered mouse mutants; and complex traits analyses identifying quantitative trait loci that affect lifespan. We also show that genomewide association peaks for lifespan in humans and lifespan quantitative loci for mice map to homologous locations in the genome. Thus, the vast bioinformatic and genetic resources of the mouse can be used to screen candidate genes identified in both mouse and human mapping studies, followed by functional testing, often not possible in humans, to determine their influence on aging.
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Comparative proteomics reveals deficiency of NHE-1 (Slc9a1) in RBCs from the beta-adducin knockout mouse model of hemolytic anemia.
Blood Cells Mol. Dis.
PUBLISHED: 02-21-2011
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Hemolytic anemia is one of the most common inherited disorders. To identify candidate proteins involved in hemolytic anemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in RBCs from normal and beta-adducin (Add2) knock-out mice. We detected 7 proteins that were decreased and 48 proteins that were increased in the beta-adducin knock-out RBC ghost. Since hemolytic anemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Label-free analysis identified 47 proteins that were increased in the reticulocyte-enriched samples and 21 proteins that were decreased. Among the proteins increased in Add2 knockout RBCs, only 11 were also found increased in reticulocytes. Among the proteins decreased in Add2 knockout RBCs, beta- and alpha-adducin showed the greatest intensity difference, followed by NHE-1 (Slc9a1), the sodium-hydrogen exchanger. We verified these mass spectrometry results by immunoblot. This is the first example of a deficiency of NHE-1 in hemolytic anemia and suggests new insights into the mechanisms leading to fragile RBCs. Our use of label-free comparative proteomics to make this discovery demonstrates the usefulness of this approach as opposed to metabolic or chemical isotopic labeling of mice.
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Identification of distal cis-regulatory elements at mouse mitoferrin loci using zebrafish transgenesis.
Mol. Cell. Biol.
PUBLISHED: 01-19-2011
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Mitoferrin 1 (Mfrn1; Slc25a37) and mitoferrin 2 (Mfrn2; Slc25a28) function as essential mitochondrial iron importers for heme and Fe/S cluster biogenesis. A genetic deficiency of Mfrn1 results in a profound hypochromic anemia in vertebrate species. To map the cis-regulatory modules (CRMs) that control expression of the Mfrn genes, we utilized genome-wide chromatin immunoprecipitation (ChIP) datasets for the major erythroid transcription factor GATA-1. We identified the CRMs that faithfully drive the expression of Mfrn1 during blood and heart development and Mfrn2 ubiquitously. Through in vivo analyses of the Mfrn-CRMs in zebrafish and mouse, we demonstrate their functional and evolutionary conservation. Using knockdowns with morpholinos and cell sorting analysis in transgenic zebrafish embryos, we show that GATA-1 directly regulates the expression of Mfrn1. Mutagenesis of individual GATA-1 binding cis elements (GBE) demonstrated that at least two of the three GBE within this CRM are functionally required for GATA-mediated transcription of Mfrn1. Furthermore, ChIP assays demonstrate switching from GATA-2 to GATA-1 at these elements during erythroid maturation. Our results provide new insights into the genetic regulation of mitochondrial function and iron homeostasis and, more generally, illustrate the utility of genome-wide ChIP analysis combined with zebrafish transgenesis for identifying long-range transcriptional enhancers that regulate tissue development.
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A novel ENU-generated truncation mutation lacking the spectrin-binding and C-terminal regulatory domains of Ank1 models severe hemolytic hereditary spherocytosis.
Exp. Hematol.
PUBLISHED: 09-28-2010
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Hereditary spherocytosis (HS) is a heterogeneous group of spontaneously arising and inherited red blood cell disorders ranging from very mild subclinical cases to severe and life-threatening cases, with symptoms linked directly to the severity of the mutation at the molecular level. We investigated a novel mouse model in which the heterozygotes present with the diagnostic hallmarks of mild HS and surviving homozygotes phenocopy severe hemolytic HS.
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Sequence variation at multiple loci influences red cell hemoglobin concentration.
Blood
PUBLISHED: 09-10-2010
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A substantial genetic contribution underlies variation in baseline peripheral blood counts. We performed quantitative trait locus/loci analyses to identify chromosome regions harboring genes influencing red cell hemoglobin concentration using the cell hemoglobin concentration mean (CHCM), a directly measured parameter analogous to the mean cell hemoglobin concentration. Fourteen significant loci (gene symbols Chcmq1-Chcmq14) were detected. Seven of these influenced CHCM in a sex-specific fashion, and 2 showed significant interactive effects (epistasis). For quantitative trait locus/loci detected in multiple crosses, confidence intervals were narrowed using statistical and bioinformatic approaches. Two strong candidate genes emerged and were further analyzed: adult ?-globin (Hbb) for Chcmq3 on Chr 7, and transferrin (Trf) for Chcmq2 on Chr 9. High and low allele parental strains in crosses detecting Chcmq3 segregate 100% with the known ancestral haplotype blocks, hemoglobin (Hb) diffuse (Hbb(d)) and Hb single (Hbb(s)), respectively. Hbb(d) consists of nonidentical major and minor polypeptides and exhibits an increased positive charge relative to Hbb(s) due to the net loss of 2 negative residues in the Hbb(dminor) polypeptide, resulting in a pI of 7.85 versus 7.13. Thus, as shown in human erythrocytes, positively charged Hbs are associated with cell dehydration and increased CHCM in mouse erythrocytes.
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Comparison of unrestrained plethysmography and forced oscillation for identifying genetic variability of airway responsiveness in inbred mice.
Physiol. Genomics
PUBLISHED: 09-07-2010
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Lung function detection in mice is currently most accurately measured by invasive techniques, which are costly, labor intensive, and terminal. This limits their use for large-scale or longitudinal studies. Noninvasive assays are often used instead, but their accuracy for measuring lung function parameters such as resistance and elastance has been questioned in studies involving small numbers of mouse strains. Here we compared parameters detected by two different methods using 29 inbred mouse strains: enhanced pause (Penh), detected by unrestrained plethysmography, and central airway resistance and lung elastance, detected by a forced oscillation technique. We further tested whether the phenotypic variations were determined by the same genomic location in genome-wide association studies using a linear mixed model algorithm. Penh, resistance, and elastance were measured in nonexposed mice or mice exposed to saline and increasing doses of aerosolized methacholine. Because Penh differed from airway resistance in several strains and because the peak genetic associations found for Penh, resistance, or elastance were located at different genomic regions, we conclude that using Penh as an indicator for lung function changes in high-throughput genetic studies (i.e., genome-wide association studies or quantitative trait locus studies) measures something fundamentally different than airway resistance and lung elastance.
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Severe anemia in the Nan mutant mouse caused by sequence-selective disruption of erythroid Kruppel-like factor.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 08-09-2010
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Studies of mouse models of anemia have long provided fundamental insights into red blood cell formation and function. Here we show that the semidominant mouse mutation Nan ("neonatal anemia") carries a single amino acid change (E339D) within the second zinc finger of the erythroid Krüppel-like factor (EKLF), a critical erythroid regulatory transcription factor. The mutation alters the DNA-binding specificity of EKLF so that it no longer binds promoters of a subset of its DNA targets. Remarkably, even when mutant Nan and wild-type EKLF alleles are expressed at equivalent levels, the mutant form selectively interferes with expression of EKLF target genes whose promoter elements it no longer binds. This interference yields a distorted genetic output and selective protein deficiencies that differ from those seen in EKLF-heterozygous and EKLF-null red blood cells and presents a unique and unexpected mechanism of inherited disease.
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Tropomodulin 1-null mice have a mild spherocytic elliptocytosis with appearance of tropomodulin 3 in red blood cells and disruption of the membrane skeleton.
Blood
PUBLISHED: 06-28-2010
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The short actin filaments in the red blood cell (RBC) membrane skeleton are capped at their pointed ends by tropomodulin 1 (Tmod1) and coated with tropomyosin (TM) along their length. Tmod1-TM control of actin filament length is hypothesized to regulate spectrin-actin lattice organization and membrane stability. We used a Tmod1 knockout mouse to investigate the in vivo role of Tmod1 in the RBC membrane skeleton. Western blots of Tmod1-null RBCs confirm the absence of Tmod1 and show the presence of Tmod3, which is normally not present in RBCs. Tmod3 is present at only one-fifth levels of Tmod1 present on wild-type membranes, but levels of actin, TMs, adducins, and other membrane skeleton proteins remain unchanged. Electron microscopy shows that actin filament lengths are more variable with spectrin-actin lattices displaying abnormally large and more variable pore sizes. Tmod1-null mice display a mild anemia with features resembling hereditary spherocytic elliptocytosis, including decreased RBC mean corpuscular volume, cellular dehydration, increased osmotic fragility, reduced deformability, and heterogeneity in osmotic ektacytometry. Insufficient capping of actin filaments by Tmod3 may allow greater actin dynamics at pointed ends, resulting in filament length redistribution, leading to irregular and attenuated spectrin-actin lattice connectivity, and concomitant RBC membrane instability.
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Targeted deletion of betaIII spectrin impairs synaptogenesis and generates ataxic and seizure phenotypes.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-15-2010
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The spectrin membrane skeleton controls the disposition of selected membrane channels, receptors, and transporters. In the brain betaIII spectrin binds directly to the excitatory amino acid transporter (EAAT4), the glutamate receptor delta, and other proteins. Mutations in betaIII spectrin link strongly to human spinocerebellar ataxia type 5 (SCA5), correlating with alterations in EAAT4. We have explored the mechanistic basis of this phenotype by targeted gene disruption of Spnb3. Mice lacking intact betaIII spectrin develop normally. By 6 months they display a mild nonprogressive ataxia. By 1 year most Spnb3(-/-) animals develop a myoclonic seizure disorder with significant reductions of EAAT4, EAAT1, GluRdelta, IP3R, and NCAM140. Other synaptic proteins are normal. The cerebellum displays increased dark Purkinje cells (PC), a thin molecular layer, fewer synapses, a loss of dendritic spines, and a 2-fold expansion of the PC dendrite diameter. Membrane and expanded Golgi profiles fill the PC dendrite and soma, and both regions accumulate EAAT4. Correlating with the seizure disorder are enhanced hippocampal levels of neuropeptide Y and EAAT3 and increased calpain proteolysis of alphaII spectrin. It appears that betaIII spectrin disruption impairs synaptogenesis by disturbing the intracellular pathways selectively regulating protein trafficking to the synapse. The mislocalization of these proteins secondarily disrupts glutamate transport dynamics, leading to seizures, neuronal damage, and compensatory changes in EAAT3 and neuropeptide Y.
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Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in alpha-spectrin-deficient red cells.
Blood
PUBLISHED: 01-07-2010
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Five spontaneous, allelic mutations in the alpha-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph(1J), sph(2J), sph(2BC), sph(Dem)). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph(3J), a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sph(Ihj), a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent beta-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph(4J), a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, beta-adducin. The severity of anemia in sph(4J) indicates that the highly conserved cysteine residue at the C-terminus of alpha-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.
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Protein 4.2 binds to the carboxyl-terminal EF-hands of erythroid alpha-spectrin in a calcium- and calmodulin-dependent manner.
J. Biol. Chem.
PUBLISHED: 12-11-2009
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Spectrin and protein 4.1 cross-link F-actin protofilaments into a network called the membrane skeleton. Actin and 4.1 bind to one end of beta-spectrin. The adjacent end of alpha-spectrin, called the EF-domain, is calmodulin-like, with calcium-dependent and calcium-independent EF-hands. It has no known function. However, the sph(1J)/sph(1J) mouse has very fragile red cells and lacks the last 13 amino acids in the EF-domain, suggesting the domain is critical for skeletal integrity. Using pulldown binding assays, we find the alpha-spectrin EF-domain either alone or incorporated into a mini-spectrin binds native and recombinant protein 4.2 at a previously identified region of 4.2 (G(3) peptide). Native 4.2 binds with an affinity comparable with other membrane skeletal interactions (K(d) = 0.30 microM). EF-domains bearing the sph(1J) mutation are inactive. Binding of protein 4.2 to band 3 (K(d) = 0.45 microM) does not interfere with the spectrin-4.2 interaction. Spectrin-4.2 binding is amplified by micromolar concentrations of Ca(2+) (but not Mg(2+)) by three to five times. Calmodulin also binds to the EF-domain (K(d) = 17 microM), and Ca(2+)-calmodulin blocks Ca(2+)-dependent binding of protein 4.2 but not Ca(2+)-independent binding. The data suggest that protein 4.2 is located near protein 4.1 at the spectrin-actin junctions. Because proteins 4.1 and 4.2 also bind to band 3, the erythrocyte anion channel, we suggest that one or both of these proteins cause a portion of band 3 to localize near the spectrin-actin junctions and provide another point of attachment between the membrane skeleton and the lipid bilayer.
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alpha- and beta-Adducin polymorphisms affect podocyte proteins and proteinuria in rodents and decline of renal function in human IgA nephropathy.
J. Mol. Med.
PUBLISHED: 09-25-2009
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Adducins are cytoskeletal actin-binding proteins (alpha, beta, gamma) that function as heterodimers and heterotetramers and are encoded by distinct genes. Experimental and clinical evidence implicates alpha- and beta-adducin variants in hypertension and renal dysfunction. Here, we have addressed the role of alpha- and beta-adducin on glomerular function and disease using beta-adducin null mice, congenic substrains for alpha- and beta-adducin from the Milan hypertensive (MHS) and Milan normotensive (MNS) rats and patients with IgA nephropathy. Targeted deletion of beta-adducin in mice reduced urinary protein excretion, preceded by an increase of podocyte protein expression (phospho-nephrin, synaptopodin, alpha-actinin, ZO-1, Fyn). The introgression of polymorphic MHS beta-adducin locus into MNS (Add2, 529R) rats was associated with an early reduction of podocyte protein expression (nephrin, synaptopodin, alpha-actinin, ZO-1, podocin, Fyn), followed by severe glomerular and interstitial lesions and increased urinary protein excretion. These alterations were markedly attenuated when the polymorphic MHS alpha-adducin locus was also present (Add1, 316Y). In patients with IgA nephropathy, the rate of decline of renal function over time was associated to polymorphic beta-adducin (ADD2, 1797T, rs4984) with a significant interaction with alpha-adducin (ADD1, 460W, rs4961). These findings suggest that adducin genetic variants participate in the development of glomerular lesions by modulating the expression of specific podocyte proteins.
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Targeted deletion of the gamma-adducin gene (Add3) in mice reveals differences in alpha-adducin interactions in erythroid and nonerythroid cells.
Am. J. Hematol.
PUBLISHED: 05-09-2009
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In red blood cells (RBCs) adducin heterotetramers localize to the spectrin-actin junction of the peripheral membrane skeleton. We previously reported that deletion of beta-adducin results in osmotically fragile, microcytic RBCs and a phenotype of hereditary spherocytosis (HS). Notably, alpha-adducin was significantly reduced, while gamma-adducin, normally present in limited amounts, was increased approximately 5-fold, suggesting that alpha-adducin requires a heterologous binding partner for stability and function, and that gamma-adducin can partially substitute for the absence of beta-adducin. To test these assumptions we generated gamma-adducin null mice. gamma-adducin null RBCs appear normal on Wrights stained peripheral blood smears and by scanning electron microscopy. All membrane skeleton proteins examined are present in normal amounts, and all hematological parameters measured are normal. Despite a loss of approximately 70% of alpha-adducin in gamma-adducin null platelets, no bleeding defect is observed and platelet structure appears normal. Moreover, systemic blood pressure and pulse are normal in gamma-adducin null mice. gamma- and beta-adducin null mice were intercrossed to generate double null mice. Loss of gamma-adducin does not exacerbate the beta-adducin null HS phenotype although the amount alpha-adducin is reduced to barely detectable levels. The stability of alpha-adducin in the absence of a heterologous binding partner varies considerably in various tissues. The amount of alpha-adducin is modestly reduced ( approximately 15%) in the kidney, while in the spleen and brain is reduced by approximately 50% with the loss of a heterologous beta- or gamma-adducin binding partner. These results suggest that the structural properties of adducin differ significantly between erythroid and various nonerythroid cell types.
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Aging in inbred strains of mice: study design and interim report on median lifespans and circulating IGF1 levels.
Aging Cell
PUBLISHED: 04-09-2009
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To better characterize aging in mice, the Jackson Aging Center carried out a lifespan study of 31 genetically-diverse inbred mouse strains housed in a specific pathogen-free facility. Clinical assessments were carried out every 6 months, measuring multiple age-related phenotypes including neuromuscular, kidney and heart function, body composition, bone density, hematology, hormonal levels, and immune system parameters. In a concurrent cross-sectional study of the same 31 strains at 6, 12, and 20 months, more invasive measurements were carried out followed by necropsy to assess apoptosis, DNA repair, chromosome fragility, and histopathology. In this report, which is the initial paper of a series, the study design, median lifespans, and circulating insulin-like growth factor 1 (IGF1) levels at 6, 12, and 18 months are described for the first cohort of 32 females and 32 males of each strain. Survival curves varied dramatically among strains with the median lifespans ranging from 251 to 964 days. Plasma IGF1 levels, which also varied considerably at each time point, showed an inverse correlation with a median lifespan at 6 months (R = -0.33, P = 0.01). This correlation became stronger if the short-lived strains with a median lifespan < 600 days were removed from the analysis (R = -0.53, P < 0.01). These results support the hypothesis that the IGF1 pathway plays a key role in regulating longevity in mice and indicates that common genetic mechanisms may exist for regulating IGF1 levels and lifespan.
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The scat mouse model highlights RASA3, a GTPase activating protein, as a key regulator of vertebrate erythropoiesis and megakaryopoiesis.
Small GTPases
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Although significant progress has been made in the past decades in our understanding of bone marrow failure syndromes and anemia, many pathological conditions of unknown origin remain. Mouse models have significantly contributed to our understanding of normal erythropoiesis and the pathogenesis of erythroid disorders. Recently, we identified in the scat (severe combined anemia and thrombocytopenia) mouse model a missense mutation (G125V) in the Rasa3 gene, encoding a Ras GTPase activating protein (GAP). RASA3 is lost during reticulocyte maturation through the exosomal pathway and is therefore absent in mature erythrocytes. In wild-type reticulocytes, RASA3 is bound to the plasma membrane, a prerequisite for its GAP activity, but is mislocalized to the cytosol in scat. This mislocalization leads to RASA3 loss of function and higher levels of Ras-GTP, the active form of Ras, are consistently found in scat mature red cells. Finally, RASA3 function is conserved among vertebrates, since erythropoiesis and thrombopoiesis are impaired in zebrafish in which rasa3 is knocked-down by morpholinos, and RASA3 is expressed in human erythroleukemia cells as well as in primary cells. In this commentary, we highlight the critical, conserved and non-redundant function of RASA3 in the context of vertebrate erythropoiesis and megakaryopoiesis. We notably discuss the mechanism of RASA3 downregulation and speculate on the most intriguing part of the phenotype observed in scat; the transient remission period.
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Strain-specific hyperkyphosis and megaesophagus in Add1 null mice.
Genesis
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The three adducin proteins (?, ?, and ?) share extensive sequence, structural, and functional homology. Heterodimers of ?- and ?-adducin are vital components of the red cell membrane skeleton, which is required to maintain red cell elasticity and structural integrity. In addition to anemia, targeted deletion of the ?-adducin gene (Add1) reveals unexpected, strain-dependent non-erythroid phenotypes. On an inbred 129 genetic background, Add1 null mice show abnormal inward curvature of the cervicothoracic spine with complete penetrance. More surprisingly, a subset of 129-Add1 null mice develop severe megaesophagus, while examination of peripheral nerves reveals a reduced number of axons in 129-Add1 null mice at four months of age. These unforeseen phenotypes, described here, reveal new functions for adducin and provide new models of mammalian disease.
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Critical function for the Ras-GTPase activating protein RASA3 in vertebrate erythropoiesis and megakaryopoiesis.
Proc. Natl. Acad. Sci. U.S.A.
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Phenotype-driven approaches to gene discovery using inbred mice have been instrumental in identifying genetic determinants of inherited blood dyscrasias. The recessive mutant scat (severe combined anemia and thrombocytopenia) alternates between crisis and remission episodes, indicating an aberrant regulatory feedback mechanism common to erythrocyte and platelet formation. Here, we identify a missense mutation (G125V) in the scat Rasa3 gene, encoding a Ras GTPase activating protein (RasGAP), and elucidate the mechanism producing crisis episodes. The mutation causes mislocalization of RASA3 to the cytosol in scat red cells where it is inactive, leading to increased GTP-bound Ras. Erythropoiesis is severely blocked in scat crisis mice, and ~94% succumb during the second crisis (~30 d of age) from catastrophic hematopoietic failure in the spleen and bone marrow. Megakaryopoiesis is also defective during crisis. Notably, the scat phenotype is recapitulated in zebrafish when rasa3 is silenced. These results highlight a critical, conserved, and nonredundant role for RASA3 in vertebrate hematopoiesis.
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