Targeted Disruption of the Adducin Gene ( Add2) Causes Red Blood Cell

Proc. Natl. Acad. Sci. USA Vol. 96, pp. 10717–10722, September 1999 Cell Biology Targeted disruption of the ␤ adducin gene (Add2) causes red blood cell spherocytosis in mice DIANA M. GILLIGAN*†,LARISSA LOZOVATSKY*, BABETTE GWYNN‡,CARLO BRUGNARA§,NARLA MOHANDAS¶, AND LUANNE L. PETERS‡ *Department of Internal Medicine (Hematology), Yale University School of Medicine, New Haven, CT 06510; ‡The Jackson Laboratory, Bar Harbor, ME 04609; §Department of Laboratory Medicine, Children’s Hospital, Boston, MA 02115; and ¶Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720 Communicated by Edward A. Adelberg, Yale University, New Haven, CT, July 8, 1999 (received for review May 25, 1999) ABSTRACT Adducins are a family of cytoskeleton pro- was analyzed by Southern blotting using a flanking EcoRV- teins encoded by three genes (␣, ␤, ␥). In a comprehensive EcoRI fragment as the hybridization probe (Fig. 3B). Blasto- assay of gene expression, we show the ubiquitous expression cyst injection and embryo transfer were performed by using of ␣- and ␥-adducins in contrast to the restricted expression standard techniques (13). Male chimeras were mated to of ␤-adducin. ␤-adducin is expressed at high levels in brain C57BL͞6J females to generate heterozygotes. Progeny were and hematopoietic tissues (bone marrow in humans, spleen in genotyped by using PCR on tail biopsies. mice). To elucidate adducin’s role in vivo, we created ␤-ad- Red Blood Cell Analysis. Blood counts were determined by ducin null mice by gene targeting, deleting exons 9–13. A using a Technicon H3 analyzer (Bayer Diagnostics, Tarrytown, 55-kDa chimeric polypeptide is produced from the first eight NY). Smears were stained with Wright-Giemsa (Sigma). Re- exons of ␤-adducin and part of the neo cassette in spleen but ticulocytes were counted manually after staining with new is not detected in peripheral RBCs or brain. ␤-adducin null methylene blue. RBCs are osmotically fragile, spherocytic, and dehydrated Osmotic Gradient Ektacytometry. Fresh RBCs were mixed compared with the wild type, resembling RBCs from patients continuously with a 4% polyvinylpyrrolidone solution of in- with hereditary spherocytosis. The lack of ␤-adducin in RBCs creasing osmolality (60–600 mosM). The deformability index leads to decreased membrane incorporation of ␣-adducin was recorded as a function of osmolality at a constant applied (30% of normal) and unexpectedly promotes a 5-fold increase shear stress of 170 dynes͞cm2 by using an ektacytometer in ␥-adducin incorporation into the RBC membrane skeleton. (Bayer Diagnostics) (14). This study demonstrates adducin’s importance to RBC mem- Osmotic Fragility. Osmotic fragility was measured in cells brane stability in vivo. washed three times in mouse isotonic saline (340 mosM) and resuspended at a final hematocrit of Ϸ5%. Ten microliters was Adducin was originally described as a protein kinase C sub- added to two-hundred and ninety microliters of lysis medium strate in RBCs (1). Purified human RBC adducin consists of in microplates and was incubated at room temperature for 20 ␣ ␤ two similar polypeptides, (Mr of 103,000) and (Mr of min. After gentle centrifugation, supernatant was removed, 97,000) (2). In vitro, RBC adducin crosslinks actin filaments and percent lysis was calculated from the absorbance at 540 ϩ ⅐ with spectrin in a Ca2 -calmodulin-dependent manner (3), and nm. Lysis medium contained either 2 mM Tris Hepes or 159 ⅐ bundles (4) and caps (5) actin filaments. Adducin is also a mM NaCl and 2 mM Tris Hepes, or combinations of these two ␳ media (range 120–325 mosM, pH 7.5 at 20°C). RBC Naϩ and substrate for kinase (6, 7) and protein kinase A (8). The third ϩ member of the adducin family, ␥-adducin, was discovered as a K content were measured as described (15). ͞ protein kinase C binding protein in kidney (9). ␥-adducin, a SDS PAGE and Western Blotting. Whole blood was col- ␣ lected in acid citrate dextrose by retroorbital phlebotomy or doublet of 84,000 and 86,000 Mr, interacts with -adducin in kidney cell extracts (9). Alternatively spliced mRNAs have cardiac puncture of anesthetized mice. RBCs and platelet-rich ϫ been described from all three adducin genes (10, 11). Most plasma were separated by centrifugation at 120 g for 20 min. encode truncated isoforms compared with the originally de- RBCs and platelets were washed with murine PBS (10 mM ͞ ͞ ͞ ͞ scribed isoforms, and their functions are not yet known. To NaCl 155 mM KCl 10 mM glucose 1 mM MgCl2 2.5 mM determine the function of ␤-adducin in vivo, we created a null KHPO4, pH 7.4) containing 1–4 mM pefabloc protease inhib- mutation in mice. ␤-adducin null RBCs are osmotically fragile itor (Boehringer Mannheim). Washed platelets were dissolved and demonstrate properties similar to RBCs from patients with Laemmli sample buffer and were boiled for 10 min. Hemoglobin-depleted RBC ghosts were prepared as described with hereditary spherocytosis. This study demonstrates addu- ͞ cin’s importance in RBC structure in vivo. (2). SDS PAGE was performed by the method of Fairbanks as modified by Steck (16) or by the method of Laemmli (17). Tissue samples were dissected from mice after perfusion MATERIALS AND METHODS with PBS, were placed immediately in liquid nitrogen, and Ϫ Targeted Disruption of the ␤-Adducin Gene (Add2). The were stored at 80°C until use. Samples were homogenized in 0.32 M sucrose in PBS containing 1–4 mM pefabloc. A low targeting vector was constructed in the pPNT (12) plasmid by ϫ Eco Bam Ј speed (2,500 rpm 5 min) pellet of nuclei and debris was using a 3.9-kilobase (kb) RI- HI fragment as the 5 ϫ homology segment and a 1.9-kb EcoRI-NotI fragment as the discarded. Samples then were fractionated into 30,000 g 3Ј homology segment (Fig. 3A). Transfected 129͞Sv-derived J1 pellet and supernatant fractions, consisting of crude mem- embryonic stem cells (12) were cultured and selected in G418 branes and cytoplasm, respectively. Samples were dissolved in and gancyclovir. Genomic DNA was digested with EcoRV and Laemmli buffer and were run on 4–15% gradient SDS- The publication costs of this article were defrayed in part by page charge Abbreviation: kb, kilobase. †To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked ‘‘advertisement’’ in Internal Medicine (Hematology), Yale University School of Medi- accordance with 18 U.S.C. §1734 solely to indicate this fact. cine, WWW 403, 333 Cedar Street, New Haven, CT 06510. E-mail: PNAS is available online at www.pnas.org. [email protected]. 10717 Downloaded by guest on September 30, 2021 10718 Cell Biology: Gilligan et al. Proc. Natl. Acad. Sci. USA 96 (1999) polyacrylamide gels. Samples on duplicate gels were stained by placed in fresh fixative overnight and were processed to Coomassie blue or were transferred to nitrocellulose. Blots paraffin. Sections (5 ␮m) were stained with hematoxylin and were blocked in PBS-Triton-BSA [0.15 M NaCl, 0.1 mM eosin for routine examination and with Prussian blue to detect EDTA, and 10 mM NaHPO4 (pH 7.4) with 0.2% Triton X-100 iron. Scanning electron microscopy of fixed, whole red blood and 3% BSA), were incubated with primary antibody in cells was performed as described (21). PBS-Triton-BSA overnight at 4°C, were washed, and were incubated with peroxidase-protein A (Bio-Rad) in PBS- RESULTS Triton-BSA at 22°C for 1 hour. Blots were washed and incubated with chemiluminescence substrate (Amersham The Adducins Are Widely Expressed in Mouse and Human Pharmacia) and were exposed to x-ray film (Kodak). Films Tissues. We examined adducin expression by using a multiple were scanned by using a Molecular Dynamics Computing tissue expression array with mRNA from 76 human tissues and Densitometer. cell lines. Human ␣ - and ␥- adducin cDNA probes hybridize Antibodies. All antibodies were affinity-purified from rabbit to every sample in the array, with decreased expression of serum. ␣- and ␤-adducin antibodies, a gift from H. Clive ␣-adducin in the tumor cell lines in column 10 (Fig. 1). Palfrey (University of Chicago), were raised against full-length ␤-adducin cDNA probes were designed to detect sequences human RBC adducin and have been described previously (18). encoding the alternative carboxy termini designated ␤-1 and The ␥-adducin antibody, a gift from Susan Jaken (University ␤-2 (10). The ␤-1 probe hybridizes to all neuronal tissues (Fig. of Vermont), was raised against recombinant peptide com- 1, columns 1–3) with highest expression in the cerebellum. The prising amino acids 467–674, the carboxy terminus (9). The ␤-1 probe also shows high expression in bone marrow (Fig. 1, ␤ NH2-terminal -adducin antibody was raised against a mixture 7G), fetal brain (11A), and fetal liver (11D) (the site of fetal of six synthetic peptides (a gift from Vann Bennett, Duke hematopoiesis). A weak signal is detected in testis (Fig. 1, 8F), University) corresponding to the following amino acids from fetal spleen (11E), and the K562 cell line (10C). Similarly, the human ␤-1 adducin (19): 126–145, 336–362, 422–445, 446– ␤-2 probe hybridizes to all neuronal and hematopoietic tissues, 471, 463–480, 696–721. An antibody to the carboxy terminus although the signal is weaker than ␤-1 (film exposed 7ϫ of ␤-1 was made by using a recombinant polypeptide compris- longer). Surprisingly, the ␤-2 probe shows expression in lung ing amino acids 538–726 from human ␤-1 adducin. (Fig. 1, both adult 8A and fetal 11G) whereas ␤-1 is not Northern Blotting. Total RNA was isolated by using the expressed in lung. guanidinium thiocyanate-phenol-chloroform method (20). ␣- and ␥-adducin expression also occur in all mouse tissues Northern blotting was performed by standard techniques. examined (Fig. 2). Expression of ␤-adducin in the mouse is Hybridization was carried out at 42°C in formamide and similar to human expression with high levels in brain and washing at 65°C.

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