ALBERT

Description: The adducins are a family of three closely related proteins (α, β, γ) encoded by distinct genes. α- and γ-adducin are expressed ubiquitously, while β expression is restricted to hematopoietic cells and the brain. In red blood cells (RBCs) adducin localizes to spectrin-actin junctions in the membrane skeleton as αβ heterotetramers. Previously (Gilligan et. al., PNAS, 1999) we showed that deletion of β-adducin results in osmotically fragile, microcytic RBCs and an overall phenotype of hereditary spherocytosis (HS). Notably, α-adducin was significantly reduced in β-adducin null RBCs. We also demonstrated that γ-adducin is present in low amounts in normal mouse RBCs and is upregulated ∼5-fold in β-adducin null RBCs. The increase in γ-adducin suggests that αγ heterotetramers may be compensating for the absence of β-adducin. In an effort to analyze γ-adducin function in RBCs in greater detail, we generated a conditional γ-adducin knockout allele in mice using a Cre-loxP strategy to delete exon 2 containing the start codon. All mice were maintained on a segregating B6.129 genetic background. Western blotting confirmed the absence of γ-adducin in spleen homogenates and RBC ghost preparations from γ-adducin null mice. All other membrane skeleton proteins examined by a combination of SDS-PAGE and western blotting, including α- and β-adducin, are normal in γ-adducin null RBCs (spectrin, ankyrin, band 3, protein 4.1, protein 4.2, dematin). Phenotypically, γ-adducin null mice display normal growth curves and show no overt defects. γ-adducin null RBCs appear normal on Wright’s stained peripheral blood smears and by scanning electron microscopy (SEM). The RBC count, hemoglobin content, hematocrit, MCV, reticulocyte %, osmotic fragility, and all other hematopoietic parameters are normal in γ-adducin null mice vs. wildtype. The apparent compensation by γ-adducin in β-adducin null red cells previously observed was tested by intercrossing mice null for γ- and β-adducin to produce βγ null double homozygotes. The additional loss of γ-adducin did not exacerbate the β-adducin null RBC phenotype as judged by examination of peripheral blood smears and SEM. Moreover, RBC osmotic fragility and complete blood counts in βγ-adducin null mice did not differ from β-adducin null mice. Western blotting of RBC ghost proteins confirmed reduction of α-adducin to ∼20% of normal in β-adducin null mice, as previously described. Strikingly, α-adducin in βγ-null RBC ghosts is reduced to barely detectable levels (

Description: The membrane skeleton underlies the lipid bilayer and imparts strength and deformability to the red blood cell (RBC). Spectrin, the major component of the membrane skeleton, is crosslinked by short actin filaments into a two-dimensional array at junctional complexes, which are composed of multiple additional proteins including adducin. Three mammalian adducins (α, β, γ) exist that are encoded by distinct genes (Add1, Add2, Add3). α- and γ-adducin are ubiquitously expressed, with the highest expression of α-adducin seen in erythroid cells, brain, kidney, and heart. Expression of β-adducin is restricted to erythroid cells and brain. In RBCs heterotetramers of α- and β-adducin regulate actin filament length via barbed end capping. Previously, we deleted β-adducin in mice. Loss of β-adducin resulted in decreased α-adducin and up-regulated γ-adducin (5-fold), producing fragile, microcytic RBCs and an overall phenotype of hereditary spherocytosis (HS). Here, we report on recently generated α-adducin null mice. We inactivated the α-adducin gene (Add1) by targeted deletion of exons 10–12, and confirmed by western blotting the complete loss of α-adducin in RBC ghosts and in the brain. All mice were maintained on a segregating B6.129 genetic background. Heterozygous mice are normal in all parameters examined to date. Notably, although β-adducin gene expression is normal, western blotting revealed a complete absence of β-adducin protein in α-adducin null RBC ghosts. γ-adducin, present at low levels in normal mouse RBCs, was unchanged, as were all other membrane skeleton proteins examined by SDS-PAGE and/or western blotting (spectrin, ankyrin, band 3, protein 4.1, protein 4.2, dematin). α-adducin null mice display characteristics of mild compensated hemolytic anemia. The hematocrit is significantly decreased (43 vs. 46% in wildtype). RBCs are microcytic (MCV 41.9 vs. 46.4 fL) and osmotically fragile (50% lysis at 215 mOsm NaCl vs. 190 mOsm). The MCH is significantly decreased while the MCHC is significantly elevated, suggestive of RBC dehydration. The percentage of circulating reticulocytes is significantly increased (5.0 vs. 2.7%). Spleen weights are normal. Examination of peripheral blood smears and scanning electron microscopy confirms microcytic, anisocytotic RBCs with spherocytes and elliptocytes present. α-adducin null mice show postnatal growth retardation and approximately 66% develop critical hydrocephalus with marked expansion of the lateral and third ventricles by the fourth month of age. Normal littermates never show hydrocephalus. We conclude: α-adducin null mice have mild, compensated hemolytic anemia; β-adducin is unstable in RBCs in the absence of α-adducin; loss of α-adducin in the brain is associated with a high incidence of lethal hydrocephalus. Additional studies of the α-adducin null mouse model will be useful in defining protein functions and interactions in RBCs, the requirement for adducin in platelet function, the role of adducin in the brain, and its role in the regulation of systemic blood pressure.

Description: Abstract 776 The spontaneous recessive mutation scat (severe combined anemia and thrombocytopenia) arose on the inbred BALB/cBy (BALB) mouse strain. The phenotype of scat is cyclic. All homozygotes are severely anemic and thrombocytopenic at birth. Leukocytes are also significantly depleted (Table). Approximately 13% die during this first “crisis” episode that lasts, on average, until the 9th postnatal day. Remarkably, a spontaneous remission ensues in those surviving the neonatal crisis period wherein all peripheral blood values revert to normal. A second crisis follows, and 94% of the mice die by 30 days of age. The recessive ENU (N-ethyl-N-nitrosourea)-induced mutation, hlb381, on the C57BL/6J (B6) background, is characterized by severe thrombocytopenia and leukopenia with mild anemia. Unlike scat, hlb381 is not cyclical. The phenotype is present at birth and persists throughout life. Despite the phenotypic differences, scat and hlb381 interact genetically; double heterozygotes show non-cyclical severe thrombocytopenia and leukopenia, and mild anemia (Table). This interaction implies that the scat and hlb381 genetic defects affect the same gene or distinct genes within the same pathway. Both scat and hlb381 mapped to overlapping intervals on mouse chromosome 8. Sequence analysis of genes within the interval identified Rasa3 (GAPIII, GAP1IP4BP) as a strong candidate gene for both scat and hlb381. In scat, Rasa3 carries a missense mutation near the N-terminus (G125V, exon 5) and, in hlb381, a missense mutation near the C-terminus (H794L, exon 23). RASA3 is a GTPase activating protein (GAP) that negatively regulates p21 Ras function by accelerating GTP hydrolysis and converting Ras to the inactive GDP bound form. Analysis in Panther and SIFT predicts that both residues are highly conserved and substitutions are likely to be deleterious. Rasa3 is widely expressed throughout embryonic and fetal development in mice, and is ubiquitously expressed in zebrafish 24 hours post fertilization (hpf). RASA3 protein is detected in erythroid tissues and platelets in the adult mouse. Analysis of scat spleen and bone marrow erythrocyte populations by FACS (dual staining for Ter119 and CD71 followed by forward scatter of the Ter119 high population) reveals a severe block in erythropoiesis during crisis periods. The proerythroblast, EryA (basophilic erythroblasts), and EryB (late basophilic and polychromatophilic erythroblasts) populations are significantly increased in frequency vs. wild type, and the EryC (orthochromatophilic erythroblasts and reticulocytes) population is markedly decreased. Annexin V staining revealed no significant differences in any of these populations. Notably, a similar delay in erythroid maturation, albeit much milder, is also seen in hlb381. In pull-down assays using the Ras-binding domain of Raf1 to affinity purify active GTP-bound Ras followed by detection by western blotting with pan-Ras antibody, active GTP-bound Ras is deficient in scat crisis red cells but recovers during remission. Finally, injection of two independent splice-blocking morpholinos designed to disrupt exon 5 and induce disruption of rasa3 mRNA processing resulted in a major decrease in the number of hemoglobinized cells when stained with o-dianisidine at 48 and 72 hpf in zebrafish. Over 90% of morphants showed no hemoglobinized cells at all, or vastly reduced numbers (

Description: In the red blood cell (RBC), adducin is present primarily as tetramers of α- and β-subunits at spectrin-actin junctions, or junctional complexes. Mouse RBCs also contain small amounts of γ-adducin. Platelets contain α- and γ-adducin only. Adducin functions as a barbed-end actin capping protein to regulate actin filament length and recruits spectrin to the ends of actin filaments. To further define adducin's role in vivo, we generated α-adducin knockout mice. α-Adducin is absent in all tissues examined in homozygous null mice. In RBCs, β- and γ-adducin are also absent, indicating that α-adducin is the limiting subunit in tetramer formation at the spectrin-actin junction. Similarly, γ-adducin is absent in α-null platelets. α-Adducin–null mice display compensated hemolytic anemia with features characteristic of RBCs in hereditary spherocytosis (HS), including spherocytes with significant loss of surface area, decreased mean corpuscular volume (MCV), cell dehydration, and increased osmotic fragility. Platelets maintain their normal discoid shape, and bleeding times are normal. α-Adducin–null mice show growth retardation at birth and throughout adulthood. Approximately 50% develop lethal communicating hydrocephalus with striking dilation of the lateral, third, and fourth ventricles. These data indicate that adducin plays a role in RBC membrane stability and in cerebrospinal fluid homeostasis.