Targeted disruption of the β adducin gene ( Add 2 ) causes red blood cell spherocytosis in mice

Abstract

Adducins are a family of cytoskeleton proteins encoded by three genes (␣, ␤, ␥). In a comprehensive assay of gene expression, we show the ubiquitous expression of ␣-and ␥-adducins in contrast to the restricted expression of ␤-adducin. ␤-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 ␤-adducin null mice by gene targeting, deleting exons 9-13. A 55-kDa chimeric polypeptide is produced from the first eight exons of ␤-adducin and part of the neo cassette in spleen but is not detected in peripheral RBCs or brain. ␤-adducin null RBCs are osmotically fragile, spherocytic, and dehydrated compared with the wild type, resembling RBCs from patients with hereditary spherocytosis. The lack of ␤-adducin in RBCs leads to decreased membrane incorporation of ␣-adducin (30% of normal) and unexpectedly promotes a 5-fold increase in ␥-adducin incorporation into the RBC membrane skeleton. This study demonstrates adducin's importance to RBC membrane stability in vivo.Adducin was originally described as a protein kinase C substrate in RBCs (1). Purified human RBC adducin consists of two similar polypeptides, ␣ (M r of 103,000) and ␤ (M r of 97,000) (2). In vitro, RBC adducin crosslinks actin filaments with spectrin in a Ca 2ϩ -calmodulin-dependent manner (3), and bundles (4) and caps (5) actin filaments. Adducin is also a substrate for kinase (6, 7) and protein kinase A (8). The third member of the adducin family, ␥-adducin, was discovered as a protein kinase C binding protein in kidney (9). ␥-adducin, a doublet of 84,000 and 86,000 M r , interacts with ␣-adducin in kidney cell extracts (9). Alternatively spliced mRNAs have been described from all three adducin genes (10, 11). Most encode truncated isoforms compared with the originally described isoforms, and their functions are not yet known. To determine the function of ␤-adducin in vivo, we created a null mutation in mice. ␤-adducin null RBCs are osmotically fragile and demonstrate properties similar to RBCs from patients with hereditary spherocytosis. This study demonstrates adducin's importance in RBC structure in vivo. MATERIALS AND METHODSTargeted Disruption of the ␤-Adducin Gene (Add2). The targeting vector was constructed in the pPNT (12) plasmid by using a 3.9-kilobase (kb) EcoRI-BamHI fragment as the 5Ј homology segment and a 1.9-kb EcoRI-NotI fragment as the 3Ј homology segment (Fig. 3A). Transfected 129͞Sv-derived J1 embryonic stem cells (12) were cultured and selected in G418 and gancyclovir. Genomic DNA was digested with EcoRV and was analyzed by Southern blotting using a flanking EcoRVEcoRI fragment as the hybridization probe (Fig. 3B). Blastocyst injection and embryo transfer were performed by using standard techniques (13). Male chimeras were mated to C57BL͞6J females to generate heterozygotes. Progeny were genotyped by using PCR on tail biopsies.Red Blood Cell Analysis. Blood counts were determined by using a Technicon H3 analyzer (Bayer Diagnosti...

Adducin Promotes Micrometer-Scale Organization of β2-Spectrin in Lateral Membranes of Bronchial Epithelial Cells

“…This strategy eliminates the possibility of compensation through up-regulation of ␤-adducin in HBE cells, which is a concern based on the finding that ␥-adducin is up-regulated in RBCs of the ␤-adducin knockout mouse (Gilligan et al, 1999). We first identified a shRNA that was effective in knocking down ␣-adducin after screening six potential candidates through cotransfection of human GFP-␣-adducin cDNA and pSuper shRNA plasmids into mouse NIH3T3 fibroblasts.…”

“…Adducin is a key assembly protein that recruits both erythroid and nonerythroid spectrins to the fast growing ends of actin filaments Bennett, 1986, 1987;Bennett et al, 1988;Kuhlman et al, 1996;Li and Bennett, 1996;Li et al, 1998). Loss of ␤-adducin results in increased fragility of erythrocyte membranes (Gilligan et al, 1999;Chen et al, 2007). Adducin is widely expressed in most cell types, including epithelial cells where it is localized at the lateral membrane (Kaiser et al, 1989).…”

Rostafuroxin Protects from Podocyte Injury and Proteinuria Induced by Adducin Genetic Variants and Ouabain

“…The b-adducin-null mouse model (official strain designation 129-Add2tm1Llp/Llp) has been previously described (Gilligan et al, 1999). Wild-type 129/Sv mice, carrying the same b-adducin polymorphism (Q529R) described in the MHS rats (Tripodi et al, 1996), were used as controls.…”

TDP-43 regulatesβ-adducin(Add2) transcript stability

“…45 Briefly, 0.6-1.2 mg of total RNA (prepared from transfected HeLa cells, treated with siRNA for Luciferase or TDP-43, as described above, were incubated with 20 ng of posphorylated dT (12)(13)(14)(15)(16)(17)(18) oligos. Samples were denatured at 65 C, renatured at 42 C, incubated for 60 min in the presence of T4-DNA ligase.…”

“…While Add1 and Add3 are expressed ubiquitously, Add2 is restricted to erythroid tissues and the nervous system. [17][18][19] In the brain, the adducin heterodimers are present in the growth cones of axons, presynaptic nerve terminals and post-synaptic dendritic spines. 20,21 It is very abundant in hippocampal dendritic spines.…”