Identification of quantitative trait loci that modify the severity of hereditary spherocytosis in wan, a new mouse model of band-3 deficiency

GATA1 plays essential roles in erythroid gene expression. The N-terminal finger of GATA1 (GATA1-Nf) is important for association with FOG1. Substitution mutations in GATA1-Nf, such as GATA1 V205M that diminish the GATA1-FOG1 association, have been identified in human thrombocytopenia and anemia cases. A mouse model of human thrombocytopenia has been established using a transgenic complementation rescue approach; GATA1-deficient mice were successfully rescued from embryonic lethality by excess expression of GATA1 V205G , but rescued adult mice suffered from severe thrombocytopenia. In this study, we examined GATA1-deficient mice rescued with GATA1 V205G at a comparable level to endogenous GATA1. Mice rescued with this level of GATA1 V205G rarely survive to adulthood. Rescued newborns suffered from severe anemia and jaundice accompanied with anisocytosis and spherocytosis. Expression of Slc4a1, Spna1, and Aqp1 genes (encoding the membrane proteins band-3, ␣-spectrin, and aquaporin-1, respectively) were strikingly diminished, whereas expression of other canonical GATA1-target genes, such as Alas2, were little affected. Lack of these membrane proteins provoked perturbation of membrane skeleton. Importantly, the red cells exhibited increased reactive oxygen species accumulation. These results thus demonstrate that the loss of the GATA1-FOG1 interaction causes a unique combination of membrane protein deficiency and disturbs the function of GATA1 in maintaining erythroid homeostasis. IntroductionGATA1 is a founding member of the GATA family of transcription factors. Because GATA1, GATA2, and GATA3 are highly expressed in hematopoietic lineages, these factors are collectively referred to as hematopoietic GATA factors. 1 The expression of hematopoietic GATA factor genes are strictly regulated through distinct molecular mechanisms, consequently each GATA molecule shows specificity in its expression profile. 1 In particular, GATA1 is expressed in lineage-committed cells, such as erythroid, megakaryocytic, eosinophilic, mast, and dendritic cells. 2,3 GATA1 plays key roles in regulating the differentiation and survival of these hematopoietic lineage cells. Targeted knockout of the Gata1 gene leads to in utero lethality of the mutant embryos because of defects in primitive and definitive hematopoiesis. 4,5 Conditional Gata1 gene knockout gives rise to red cell aplasia and severe thrombocytopenia. 6 In addition, GATA1 dysfunction is predictive of specific leukemias in mice and humans. 2 GATA1 contains 3 functional domains, that is an N-terminal transactivation (NT) domain, an N-terminal zinc finger (Nf) domain, and a C-terminal zinc finger (cf) domain. 7 These 3 domains have been shown to be important for GATA1 function. 7 In transgenic complementation rescue analyses, GATA1 lacking either the Nf or cf domain poorly rescues GATA1-deficient mice from embryonic lethality, which indicates the importance of both Nf and cf domains in GATA1 function in vivo. 7 The Nf domain interacts with FOG1 (friend of GATA1), and also stabilizes...

Section: Discussionmentioning

confidence: 99%

Mature erythrocyte membrane homeostasis is compromised by loss of the GATA1-FOG1 interaction

Hasegawa¹,

Shimizu

Mohandas

et al. 2012

“…23 Antibodies to TMs were an affinity-purified rabbit polyclonal to purified human RBC TMs 19,24 that recognizes both the upper ␥-TM (hTM5, TM5NM1) and lower ␣-TM (TM5b) bands, 25,26 and a mouse monoclonal from the Developmental Studies Hybridoma Bank (CH1 that recognizes only the upper, TM5NM1 band. Other antibodies were rabbit antisera to the cytoplasmic domain of band 3 27 and to full-length protein 4.1R (a gift from Cathy Korsgren, Harvard Medical School), and affinity-purified chicken antibodies against full-length recombinant ␣1 and ␤1 adducins (gifts from Diana Gilligan, Puget Sound Blood Bank). Mouse monoclonals were 17C7 to ␣I-spectrin (AbCAM), 3F2.3 to the ␤2 subunit of CapZ (Developmental Studies Hybridoma Bank), and C4 to actin (a gift from Dr J. Lessard, University of Cincinnati).…”

Section: Ghost Preparation Sodium Dodecyl Sulfate-polyacrylamide Gelmentioning

confidence: 99%

Tropomodulin 1-null mice have a mild spherocytic elliptocytosis with appearance of Tropomodulin 3 in red blood cells and disruption of the membrane skeleton

Moyer

Nowak

Kim

et al. 2010

Self Cite

150

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.(Blood. 2010;116(14): 2590-2599) IntroductionThe membrane skeleton is composed of a highly cross-linked network of spectrin, actin filaments, and accessory proteins that underlies the plasma membrane of differentiated cells. This network creates membrane domains by anchoring and restricting the long-range distribution of membrane proteins and plays an important role in determining cell shapes, membrane contours, and mechanical properties. 1,2 The organization of the membrane skeleton is best known in red blood cells (RBCs), where it is organized as a quasi-hexagonal network with connecting strands formed by long, flexible spectrin molecules and vertices formed by short actin filaments. 3-5 Each short actin filament forms the core of a junctional complex with 2 rod-shaped tropomyosin (TM) molecules along the filament, 2 tropomodulin 1 (Tmod1) molecules capping the pointed filament end and an ␣/␤-adducin heterodimer capping the barbed filament end. 6,7 Dematin (protein 4.9) is also associated with the short actin filaments as are ␤1-spectrin protein 4.1R complexes that extend from the sides of the filaments to form the extended spectrin-actin network. The network is connected to membrane macromolecular complexes by multiple linkages: from ␤-spectrin by ankryin to band 3, and from the junctional complex by protein 4.1, ␣/␤-adducin and dematin to band 3, glycophorin C, the glucose transporter, and other components. 8 Disruptions either of attachments of the spectrin-actin lattice to membrane macromolecular complexes ("vertical" connections), or linkages within the plane of the spectrin-actin lattice ("horizontal" connections) lead to ...

“…31,32 Consequently, to obtain sufficient band 3-null erythrocytes for adducin studies, a new band 3 knockout mouse that would survive to adulthood had to be created. Band 3 knockout mice were generated through homologous recombination of embryonic stem cells.…”

Section: Generation Of Band 3 Knockout Mice That Survive To Adulthoodmentioning

confidence: 99%

Adducin forms a bridge between the erythrocyte membrane and its cytoskeleton and regulates membrane cohesion

Anong

Franco

Chu

et al. 2009

131

153

The erythrocyte membrane skeleton is the best understood cytoskeleton. Because its protein components have homologs in virtually all other cells, the membrane serves as a fundamental model of biologic membranes. Modern textbooks portray the membrane as a 2-dimensional spectrin-based membrane skeleton attached to a lipid bilayer through 2 linkages: band 3-ankyrin-␤-spectrin and glycophorin C-protein 4.1-␤-spectrin. 1-7 Although evidence supports an essential role for the first bridge in regulating membrane cohesion, rupture of the glycophorin C-protein 4.1 interaction has little effect on membrane stability. 8 We demonstrate the existence of a novel band 3-adducin-spectrin bridge that connects the spectrin/actin/protein 4.1 junctional complex to the bilayer. As rupture of this bridge leads to spontaneous membrane fragmentation, we conclude that the band 3-adducinspectrin bridge is important to membrane stability. The required relocation of part of the band 3 population to the spectrin/actin junctional complex and its formation of a new bridge with adducin necessitates a significant revision of accepted models of the erythrocyte membrane. (Blood. 2009;114: 1904-1912 IntroductionThe model of the erythrocyte membrane presented in cell biology, hematology, and biochemistry textbooks shows 2 major protein bridges that span between the phospholipid bilayer and the spectrin/actin skeleton. [1][2][3][4][5][6][7] The more prominent bridge, a linkage from the integral membrane protein, band 3, to spectrin via ankyrin, is composed of multiple high-affinity protein-protein interactions. [9][10][11] Defects or deficiencies in either band 3 or ankyrin lead to a decrease in cohesion between the lipid bilayer and membrane skeleton, resulting in loss of membrane surface area and a pathology termed hereditary spherocytosis. [12][13][14] Manual rupture of this bridge by addition of competing fragments of either band 3 or ankyrin, or by addition of competing monoclonal antibodies, or mutation of the ankyrin binding site on band 3 induces spontaneous membrane vesiculation and fragmentation. [14][15][16] Spontaneous mutations in the ankyrin-bridging function in other cells can also lead to serious pathologies. [17][18][19][20] Taken together, these data support the importance of the ankyrin-spectrin bridge in maintaining membrane integrity.The second bridge connecting the membrane bilayer to the spectrinactin skeleton consists of the membrane-spanning protein, glycophorin C (GPC), tethered to spectrin via the adapter protein 4.1. [21][22][23] The complex of cytoskeletal proteins at this nexus (primarily actin, dematin, tropomyosin, adducin, protein 4.1, and tropomodulin) forms a junctional complex from which spectrin tetramers extend radially into a 2-dimensional lattice that provides mechanical stability to the overlying membrane. Based on the finding that GPC-deficient red cells exhibit decreased membrane mechanical stability, it has been inferred that the GPC-protein 4.1 bridge is essential to erythrocyte integrity. 24,25 However...