Targeted disruption of the beta adducin gene (Add2) causes red blood cell spherocytosis in mice

Abstract

Adducins are a family of cytoskeleton proteins encoded by three genes (alpha, beta, gamma). In a comprehensive assay of gene expression, we show the ubiquitous expression of alpha- and gamma-adducins in contrast to the restricted expression of beta-adducin. beta-adducin is expressed at high levels in brain and hematopoietic tissues (bone marrow in humans, spleen in mice). To elucidate adducin's role in vivo, we created beta-adducin null mice by gene targeting, deleting exons 9-13. A 55-kDa chimeric polypeptide is produced from the first eight exons of beta-adducin and part of the neo cassette in spleen but is not detected in peripheral RBCs or brain. beta-adducin null RBCs are osmotically fragile, spherocytic, and dehydrated compared with the wild type, resembling RBCs from patients with hereditary spherocytosis. The lack of beta-adducin in RBCs leads to decreased membrane incorporation of alpha-adducin (30% of normal) and unexpectedly promotes a 5-fold increase in gamma-adducin incorporation into the RBC membrane skeleton. This study demonstrates adducin's importance to RBC membrane stability in vivo.

Figure 1

A multiple-tissue expression array (CLONTECH) of polyA RNA from 76 human tissues and cell lines probed with human cDNAs for α-, β-1, β-2, and γ-adducins. α- and γ-adducins show expression in all samples whereas β-1 and β-2 show restricted expression, mainly in brain and hematopoietic tissues. Probes for β-1 and β-2 were generated by PCR to encompass their distinct carboxy termini and 3′UTRs (β-1, exons 14–17; β2, exon 13b). Subcloned PCR products were labeled by random priming with ³²P-dCTP.

Figure 2

A multiple-tissue expression Northern blot (CLONTECH) of polyA RNA from seven mouse tissues probed with mouse cDNAs for α, β-1, β-2, and γ-adducins. H, heart; B, brain; S, spleen; Lu, lung; L, liver; K, kidney; T, testis (- is a blank lane). α- and γ-adducin show expression in all lanes whereas β-1 and β-2 show restricted expression, mainly in brain and spleen.

Figure 3

(A) Strategy for targeted deletion of exons 9–13 from Add2, the gene encoding β-adducin. (B) Southern blot of EcoRV-restricted DNA from embryonic stem cell clones electroporated with the vector from A and selected for G418 resistance. The probe is indicated in A and detects the targeted allele in the clone on the right.

Figure 4

(A) Coomassie blue-stained Steck gel shows no difference in −/− RBC ghosts. (B–D) Western blotting of RBC ghosts and platelets. Lanes: 1, human RBC ghosts; 2, +/+ RBC ghosts; 3, +/− RBC ghosts; 4, −/− RBC ghosts; 5, +/+ platelets. Antibodies used for detection are indicated to the right.

Figure 5

(A) Bright field light microscopy. Arrows indicate rounded elliptocytes; arrowheads indicate microspherocytes. (B) Scanning electron microscopy demonstrates that −/− RBCs are smaller and spherocytic compared with +/+ RBCs. (Bar = 1 μm.) (C) Increased iron deposition (dark blue color) in spleen from −/− mice compared with +/+ mice.

Figure 6

(A) Osmotic fragility curve demonstrates right shift for −/− RBCs. (B) Osmotic gradient deformability profiles demonstrate markedly reduced deformability for −/− RBCs; duplicate measurements are shown for each genotype. (C) Measurements of cation content show normal Na⁺ but decreased K⁺ and Na⁺ plus K⁺ in −/− RBCs.

Figure 7

Northern blotting of total RNA from mouse tissues. B, brain; H, heart; S, spleen; K, kidney. (A) Using a full-length β-1 cDNA as probe, 9- and 4-kb mRNAs are detected in brain and spleen. (B) Using a probe from the neoR cassette, a 1.8-kb mRNA is seen in all tissues from targeted mice and also hybridizes to the 9- and 4-kb mRNAs from targeted mice. (C) Using a probe for the deleted exons 9–12, no mRNA is detected in −/− mice, indicating that the 9- and 4-kb mRNAs are aberrant transcripts inserting antisense neoR sequence in the place of deleted β-adducin exons.

Figure 8

Western blotting of spleen homogenates. Lanes: 1, human RBC ghosts; 2, human platelets; 3–8, spleen homogenates, separated into 30,000 × g pellet (P) and supernatant (S) fractions: 3, +/+ P; 4, +/+ S; 5, +/− P; 6, +/− S; 7, −/− P; 8, −/− S. Gel in A is Coomassie blue-stained to demonstrate protein loading. Gels in B–E are stained with the antibodies indicated on the right of the panels. The arrow marks the 55-kDa band in lanes 6 and 8 that is immunoreactive to NH₂-β antibody. β-1 is not detected in −/− samples, but α and γ are detected in all pellet samples.

Figure 9

Western blotting of brain homogenates. Lanes: 1, human RBC ghosts; 2, human platelets; 3–8, brain homogenates, separated into 30,000 × g pellet (P) and supernatant (S) fractions: 3, +/+ P; 4, +/+ S; 5, +/− P; 6, +/− S; 7, −/− P; 8, −/− S. β-1 is not detected in −/− samples, but α and γ are detected in all samples.