Correspondence to: Vann Bennett, Department of Biochemistry, Duke University Medical Center, 363 Carl Bldg., Res Drive, Box 3892, Durham, NC 27710-0001. Tel:(919) 684-3538 Fax:(919) 684-3590 E-mail:benne012{at}mc.duke.edu.
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Abstract |
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This report describes a congenital myopathy and major loss of thymic lymphocytes in ankyrin-B (-/-) mice as well as dramatic alterations in intracellular localization of key components of the Ca2+ homeostasis machinery in ankyrin-B (-/-) striated muscle and thymus. The sacoplasmic reticulum (SR) and SR/T-tubule junctions are apparently preserved in a normal distribution in ankyrin-B (-/-) skeletal muscle based on electron microscopy and the presence of a normal pattern of triadin and dihydropyridine receptor. Therefore, the abnormal localization of SR/ER Ca ATPase (SERCA) and ryanodine receptors represents a defect in intracellular sorting of these proteins in skeletal muscle. Extrapolation of these observations suggests defective targeting as the basis for abnormal localization of ryanodine receptors, IP3 receptors and SERCA in heart, and of IP3 receptors in the thymus of ankyrin-B (-/-) mice. Mis-sorting of SERCA 2 and ryanodine receptor 2 in ankyrin-B (-/-) cardiomyocytes is rescued by expression of 220-kD ankyrin-B, demonstrating that lack of the 220-kD ankyrin-B polypeptide is the primary defect in these cells. Ankyrin-B is associated with intracellular vesicles, but is not colocalized with the bulk of SERCA 1 or ryanodine receptor type 1 in skeletal muscle. These data provide the first evidence of a physiological requirement for ankyrin-B in intracellular targeting of the calcium homeostasis machinery of striated muscle and immune system, and moreover, support a catalytic role that does not involve permanent stoichiometric complexes between ankyrin-B and targeted proteins. Ankyrin-B is a member of a family of adapter proteins implicated in restriction of diverse proteins to specialized plasma membrane domains. Similar mechanisms involving ankyrins may be essential for segregation of functionally defined proteins within specialized regions of the plasma membrane and within the Ca2+ homeostasis compartment of the ER.
Key Words: calcium homeostasis, IP3 receptor, ankyrin, sarcoplasmic reticulum, gene knockout
THE ER of metazoan cells is a continuous system of intracellular membranes segregated into subdomains with specialized functions (
Little is known about how Ca2+ homeostasis proteins are targeted to specialized sites within the ER. Presumably, accessory proteins are required for segregation of functionally related but structurally distinct Ca2+ compartment proteins within the ER, for delivery of some of these proteins to the Golgi, and for subsequent sorting of proteins to specialized regions of the sarcoplasmic reticulum. Ankyrins are a family of membrane-associated adapter proteins (
Gene knockouts in mice have provided insight into physiological functions of the ankyrin family. Disruption of ankyrin-G expression in the cerebellum prevents restriction of voltage-gated sodium channels and L1CAM cell adhesion molecules to axon initial segments (
This study reports that ankyrin-B is expressed in striated muscle and the thymus in addition to the nervous system of normal mice. Ankyrin-B (-/-) mice exhibit musculoskeletal defects, a major reduction in thymic lymphocytes, and the majority die on the first day after birth. Moreover, ryanodine receptors and SERCA are not distributed normally in ankyrin-B (-/-) striated muscle, and IP3 receptors are abnormally localized in ankyrin-B (-/-) thymic lymphocytes. These findings considered together with the requirement for ankyrin-G in targeting voltage-gated sodium channels to axon initial segments (
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Materials and Methods |
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Materials
The following primary antibodies were used: affinity-purified rabbit anti-ankyrin-B (COOH terminus specific) polyclonal antibody (2 subunit, (Affinity Bioreagents); SERCA, type 1 and 2 (Affinity Bioreagents); ryanodine receptor type 1 and 2 (Affinity Bioreagents); triadin (Affinity Bioreagents); IP3R (type 1, 2, and 3; Accurate Biochemicals); and sarcomere
-actinin (Sigma). Secondary antibodies used were: rhodamine- or fluorescein-conjugated goat antimouse IgG (Pierce and Jackson ImmunoResearch Laboratories) at 5 µg/ml and Cy-5- or rhodamine-conjugated goat antirabbit IgG (Jackson ImmunoResearch Laboratories) at 5 µg/ml. F-actin was visualized using biotin-phalloidin (5 U/ml; Molecular Probes) and Cy5-labeled streptavidin (Jackson ImmunoResearch Laboratories).
Procedures
Mouse genotypes were established by Southern blot analysis (
Neonatal Cardiomyocyte Culture and Transfection
Hearts were dissected from neonatal (13-d-old) mice under sterile conditions by bathing the animal in 70% alcohol. Each heart was then placed in a 12-well flat-bottom tissue culture plate containing 2 ml of Ham's F10 nutrient media. The atrium and major blood vessels were removed and the ventricle was rinsed by squeezing with forceps to wash away the blood. The ventricle was minced finely in 1.5 ml of 0.05% trypsin, 0.2 mM NaEDTA, and placed in a humidified incubator (37°C, 95% air-5% CO2) for 15 min. Ventricles were then resuspended several times in a 1-ml Eppendorf pipette and incubated for another 15 min. After incubation, 0.2 ml of 2-mg/ml soybean trypsin inhibitor was added, followed by the addition of 0.2 ml of 0.2-mg/ml collagenase (type VII; Sigma). The homogenate was resuspended several times and incubated further for 3050 min until dissociation of cells was complete. Then 2 ml of complete media (DME/Ham's F10, 10% FBS, and 10% HS) was added, and cells were pelleted by centrifugation at 500 g for 510 min. Cells were resuspended in 2 ml complete media and transferred to a 35-mm petri dish to remove fibroblasts by differential adherence (2 h at 37°C). Cardiomyocytes in the supernatant were collected by centrifugation at 500 g for 510 min, and resuspended in 1 ml complete media. 0.25 ml of cell suspension were plated on a fibronectin-coated plate at a density of 1 x 106/ml and washed 24 h later with complete media to remove dead cells and debris. To minimize growth of nonmuscle cells, complete media was replaced with defined growth medium (DMEM/F10) with additions of insulin (1 µg/ml), transferrin (5 µg/ml), LiCl (1 nM), NaSeO4 (1 nM), ascorbic acid (25 µg/ml), thyroxine (1 nm), or with serum-free medium (DMEM/F10). Cardiomyocytes were transfected using Effectene (Qiagen) with 0.1 µg of cDNA encoding 220-kD ankyrin-B or 190-kD ankyrin-G (
Electron Microscopy
Quadriceps muscles from 7-d-old littermates were placed directly in Karnovsky glutaraldehyde/formaldehyde/cacodylate fixative plus tannic acid (pH 7.4) and sliced into smaller pieces (
Ca2+ Dynamics
Cytosolic Ca2+ levels were viewed in spontaneously contracting neonatal cardiomyocytes loaded with 10 µM fluo-3/AM (Molecular Probes) by ratioing fluo-3 images (excitation 488 nm, emission 510 nm) against a reference image acquired at rest (I/I0) as described (
Creatine Kinase Activity
Creatine kinase activity in mouse serum was measured using a Sigma Diagnostics Creatine Kinase kit.
Fractionation of Skeletal Muscle
Fresh rat skeletal muscle samples were homogenized in a buffer (1 g/15 ml) containing 0.25 M sucrose, 100 mM Tris, pH 7.4, 5 mM sodium EDTA, 5 mM sodium EGTA, PMSF (200 µg/ml), leupeptin (10 µg/ml), AEBSF (0.5 mM), and pepstatin (10 µg/ml), centrifuged at 1,300 g for 10 min, rehomogenized and spun again. The supernatant was spun at 9,000 g for 10 min. The second supernatant was spun at 190,000 g for 1 h. Pellets were resuspended in 3 ml buffer and loaded on a 2050% sucrose gradient, and then centrifuged at 150,000 g for 16 h. Samples were treated as described above.
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Results |
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Musculoskeletal Defects and Neonatal Myopathy in Ankyrin-B (-/-) Mice
Ankyrin-B (-/-) mice display abnormal posture with kyphosis and winged scapulae (Fig 1 A). Creatine kinase activity also is elevated about fourfold in sera from ankyrin-B (-/-) mice compared with normal littermates, based on determinations of seven litters at ages ranging from postnatal day 1 to 13 (Fig 1 B). Measurements of neonatal heterozygotes revealed some mice (8 out of 26) with a two- to threefold increase and other mice with normal levels of serum creatine kinase activity. For comparison, patients with congenital myopathies or mild muscular dystrophy frequently have normal levels or small elevation in levels of creatine kinase activity.
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Transmission electron micrographs of ankyrin-B (-/-) heart (not shown) and skeletal muscle (Fig 1 C) demonstrate overall normal sarcomere organization. However, occasional localized sites of severely disorganized sarcomeres and loss of striations encompassing 36 sarcomeres were observed in ankyrin-B (-/-) skeletal muscle (Fig 1 C, yellow asterisk). Disorganized sarcomeres were not observed in skeletal muscle from normal littermates, and also were not evident in heart sections of either wild-type or ankyrin-B (-/-) mice (not shown).
Sites of disorganized sarcomeres, combined with elevation of serum creatine kinase activity and reduced myofibril size (not shown), support the conclusion that ankyrin-B (-/-) mice suffer from a neonatal myopathy and suggest a physiological role of ankyrin-B in muscle. Ankyrin-B polypeptides of 220 and 150 kD are expressed in skeletal muscle and heart of normal mice, and are not detectable in ankyrin-B (-/-) mice (
Abnormal Ca2+ Waves and Mis-Sorting of Ca2+ Homeostasis Proteins in Ankyrin-B (-/-) Cardiomyocytes
Previous reports that ankyrin associates with IP3 receptors and ryanodine receptors (-actinin (Fig 3 A1), F-actin (Fig 4, AH, left panels), and voltage-gated calcium channels (Fig 3 C). Normal cardiomyocytes exhibit a regular sinusoidal oscillation of cytosolic Ca2+ levels with a frequency of about 1 Hz and an increase in Ca2+ elevation over the basal level of
2.5-fold (Fig 2 A). Ankyrin-B (-/-) cardiomyocytes exhibit an irregular pattern of Ca2+ release with periods of prolonged elevation (average 2.5 times longer than normal) combined with a threefold reduction in frequency (Fig 2 B). The spatial pattern of Ca2+ release also was abnormal in ankyrin-B (-/-) cells (Fig 2). Wild-type cardiomyocytes exhibited one or two well resolved sites of elevated Ca2+ which then propagated along the cell (Fig 2 A, top), while mutant cells exhibited multiple simultaneous foci of elevated calcium (Fig 2 B, top).
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The distribution of SR proteins involved in Ca2+ release and uptake was examined in ankyrin4-B (+/+) and (-/-) cardiomyocytes maintained in culture for 46 d (Fig 3 and Fig 4) and in sections of skeletal muscle (Fig 5). SERCA 2 (Fig 3 A2, left) is distributed in normal cardiomyocytes in a longitudinal and cross-striated pattern coinciding with the network SR and sarcomeres, which are identified by labeling of -actinin, a component of the Z-line (Fig 3 A1, left). In striking contrast, SERCA 2 of ankyrin-B (-/-) cardiomyocytes is restricted to a perinuclear distribution (Fig 3 A2, right) and is completely absent from striations associated with sarcomeres (Fig 3 A1, right panel). The absence of SERCA 2 from contractile units of ankyrin-B (-/-) cardiomyocytes could contribute to the prolonged time of decay of Ca2+ transients observed in these cells (Fig 2). SERCA 1 also is missing from sarcomeres in sections of skeletal muscle of 7-d-old ankyrin-B (-/-) mice (Fig 5 B). Labeling for SERCA does occur along muscle fibers, but this may represent nonspecific interactions of antibodies with components of connective tissue (Fig 5B).
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Ryanodine receptors are abnormally localized in ankyrin-B (-/-) cardiomyocytes (Fig 3 and Fig 4), and skeletal muscle (Fig 5). Ryanodine receptor type 2 of normal cardiomyocytes is present in a single periodic pattern (Fig 3 B, left) that overlays the Z-line (not shown) and corresponds to the site of T-tubules in heart cells. Ryanodine receptor type 1 in normal skeletal muscle is distributed in a periodic pattern as a double row (Fig 5 A, left), as expected from a distribution flanking T-tubules in these cells. Ryanodine receptor type 2 in ankyrin-B (-/-) cardiomyocytes is localized in 0.51-µm structures dispersed throughout the cell and in a pattern distinct from striations observed in normal cells (Fig 3 B, right). Ryanodine receptor type 1 in ankyrin-B (-/-) skeletal muscle exhibits only occasional sites of label within muscle fibers (Fig 5 A, right). Fluorescent label for the ryanodine receptor type 1 is localized adjacent to the plasma membrane in ankyrin-B (-/-) fibers (Fig 5 A, right), but this may represent nonspecific interactions of antibodies as noted above with SERCA.
Rescue of Abnormal Localization of SERCA and Ryanodine Receptors in Ankyrin-B (-/-) Cardiomyocytes by Transfection with cDNA Encoding 220-kD Ankyrin-BGFP
Transfection of ankyrin-B (-/-) cardiomyocytes with cDNA encoding 220-kD ankyrin-B with a COOH-terminal green fluorescent protein (GFP) tag restored normal striated patterns of distribution of SERCA 2 (Fig 4 C, middle) as well as ryanodine receptor type 2 (Fig 4 G, middle). 220-kD ankyrin-BGFP in rescued cardiomyocytes was visualized with antibody against GFP, and exhibited a striated distribution similar to that of native ankyrin-B (Fig 4; normal in A and E; rescued in C and G). The presence of sarcomeres in these cardiomyocytes was established by labeling F-actin with biotin-phalloidin/Cy5-streptavidin (Fig 4, AH, left panels). Ability to restore normal organization of both SERCA 2 and ryanodine receptor type 2 with 220-kD ankyrin-B demonstrates that other ankyrin-B spliceoforms, such as the 150-kD polypeptide, are not required for this activity. Transfection with cDNA encoding 190-kD ankyrin-GGFP did not restore SERCA 2 or ryanodine receptor type 2 patterns (Fig 4D and Fig H). Moreover, 190-kD ankyrin-GGFP was not localized in a striated pattern but instead was distributed more or less evenly throughout the cytoplasm of transfected cardiomyocytes (Fig 4D and Fig H, right panels). 190-kD ankyrin-GGFP polypeptide contains the same domains as 220-kD ankyrin-B, but has a shorter COOH-terminal domain (
SRT Tubule Junctions Are Preserved in Ankyrin-B (-/-) Skeletal Muscle
The abnormal localization of ryanodine receptors and SERCA in cardiomyocytes (Fig 3 and Fig 4) and skeletal muscle (Fig 5) could be due either to defects in targeting of these protein to otherwise normal SR and junctional SR sites, or to abnormal localization of the entire SR and junctional SR. Immunofluorescence using antibodies against established components of SR/T-tubule junctions and electron microscopy were used to distinguish between these possibilities.
The pattern of labeling for triadin, a SR protein associated with ryanodine receptors at SR/T-tubule junctions (
Direct evidence for apparently normal SR/T-tubule junctions and SR in ankyrin-B (-/-) skeletal muscle is provided by electron micrographs (Fig 6 and Fig 7). Wild-type and ankyrin-B null skeletal muscles both have small T-tubules (small arrows) positioned about midway between the Z-bands (Z) and the M-lines of well-ordered sarcomeres (Fig 6). Where the section plane is favorably oriented, the network of membranes of the longitudinal sarcoplasmic reticulum (SR) can be visualized as well-organized around the myofibrils and sarcomeres in both normal and ankyrin (-/-) fibers (Fig 6). Junction (triads) between the T-tubule (lumen marked by t) and the SR membranes (arrows) also appear similarly well-organized overall, with the density between the SR and T-tubule membranes of the triads (arrows) equally evident in normal and ankyrin-B (-/-) fibers.
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A gallery of skeletal muscle triads from normal (AD) and ankyrin-B (-/-) (EH) animals visualized at higher magnification are presented in Fig 7. In both wild-type and ankyrin-B (-/-) skeletal muscle, the junctional SR (JSR) membrane forms a sac (arrowhead) abutting the T-tubule, where the JSR is seen en face. The JSR membrane is usually marked by a row of particles parallel to the junction and is frequently associated with filamentous densities extending away from the junction at right angles. The particles evident in ankyrin-B (-/-) muscle presumably are comprised of the voltage-gated calcium channel, triadin, and perhaps other SR and/or T-tubule proteins, but not ryanodine receptor 1, which is absent based on immunofluorescence (Fig 5 A). Measurements across 9 examples of well-oriented T-tubuleJSR membrane junctions in normal and 9 in ankyrin-B (-/-) skeletal fibers visualized at the same magnification indicated a ten percent reduction in thickness of the ankyrin-B (-/-) junctions. (The range in normal fibers was 0.91.1 mm [average 1.01 mm]; the range in mutant was 0.81.0 mm [average 0.92 mm].)
Complete absence of ryanodine receptor type 1 in the RYR1 (-/-) mouse results in loss of particles in SRT-tubule junctions and a reduction in thickness of 40% (
The combined observations of normal immunofluorescent patterns of labeling of triadin and DHPR (Fig 5) and electron microscopy (Fig 6 and Fig 7) strongly support the interpretation that the SR and SRT-tubule junctions are normal overall in localization in ankyrin-B (-/-) skeletal muscle.
The reduced amount of ryanodine receptors and absence of SERCA from a normal striated pattern in ankyrin-B (-/-) skeletal muscle therefore reflects a defect in targeting of these proteins to their cellular sites. A similar conclusion also is true for heart based on electron microscopy (not shown), and the normal appearance of DHPR in cardiomyocytes (Fig 3).
Ankyrin-B Is Not Associated in Permanent Stoichiometric Complexes with the Majority of Ryanodine Receptors or SERCA
Ankyrin-B in skeletal muscle is located in two sites that can be resolved in longitudinal (Fig 8 A) and in transverse (Fig 8B and Fig C) sections of muscle fibers. One site, visualized in an optical section along the surface of the plasma membrane, is in a costamere pattern (-actinin (Fig 8 A, a4a6, green). The other location, visualized with an optical section through the interior of the fiber, is in intracellular punctate structures aligned with the A-band (Fig 8 A, a1a3). Transverse sections also reveal ankyrin-B staining at the sarcolemma, and in a punctate intracellular pattern (Fig 8 B, b2 and C, c2). The pattern of ankyrin-B labeling in costameres and in intracellular sites over the A-band is distinct from the localization of ankyrin noted at T-tubules (
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Double immunofluorescence labeling of transverse sections of skeletal muscle reveals that ankyrin-B is localized at sites distinct from both SERCA 1 (Fig 8 B) and from ryanodine receptor type 1 (Fig 8 C). The majority of SERCA1 and ryanodine receptor type 1 are clearly not in close contact with ankyrin-B (Fig 8). Therefore, ankyrin-B cannot participate as a common structural component of the SRT-tubule junction. Possible mechanisms involving a catalytic role of ankyrin-B in localization of ryanodine receptors and SERCA are discussed below.
Subcellular fractionation of skeletal muscle supports the idea that the punctate intracellular staining of ankyrin-B represents vesicles (Fig 9). The majority of 220-kD ankyrin-B pelleted after centrifugation for 10 min at 2,000 g, and presumably is associated with myofibrils (data not shown). However, some 220-kD ankyrin-B sedimented only at high speeds (at least 30 min at 100,000 g). Particulate 220-kD ankyrin-B sediments with a similar density to SERCA and ryanodine receptors after sedimentation to equilibrium in sucrose density gradients, and therefore most likely is associated with membranes (Fig 9 B). Visualization of membrane-associated ankyrin-B by immunofluorescence microscopy revealed small structures <1 µm in diameter that presumably represent small vesicles and are distinct from vesicles labeled for SERCA (not shown).
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Mislocalization of IP3 Receptors in Cardiomyocytes and Thymus of Ankyrin-B (-/-) Mice
IP3 receptors are widely expressed channels responsible for intracellular Ca2+ release regulated by IP3 and Ca2+, and are coexpressed with ryanodine receptors in striated muscle as well as other tissues. IP3 receptors visualized by immunofluorescence are mis-sorted in ankyrin-B neonatal cardiomyocytes (Fig 10 A). IP3 receptors in normal cardiomyocytes are distributed in a striated pattern, while in mutant cells IP3 receptors are in an irregular punctate distribution (Fig 10 A). IP3 receptors in normal skeletal muscle are distributed in a SR pattern distinct from the pattern of ryanodine receptors, while IP3 receptors in ankyrin-B (-/-) skeletal muscle are distributed throughout the cytoplasm (not shown). Levels of accumulation of IP3 receptors are reduced by at least 50% in ankyrin-B (-/-) heart tissue (Fig 10 C).
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The neonatal thymus is an active site of T cell differentiation, a process that is regulated by intracellular calcium transients mediated in part by IP3 receptors and ryanodine receptors (50% (Fig 8 C). IP3 receptors in ankyrin-B (-/-) lymphocytes also exhibit an altered pattern of localization (Fig 10 B, right). IP3 receptors in these lymphocytes are confined adjacent to the plasma membrane, and are generally not present in the perinuclear pattern observed in normal cells (Fig 10 B).
Reduced accumulation and abnormal localization of IP3 receptors in ankyrin-B (-/-) thymus would be anticipated to interrupt normal calcium signaling and differentiation of T cells. Consistent with such a loss of signaling, Toluidine bluestained sections of ankyrin-B (-/-) neonatal thymus reveal cell death of a major fraction of T cells (Fig 11). Epithelial cells are present in equivalent numbers and organization, while the majority of T cells are missing or exhibit pyknotic nuclei. In contrast, sections of normal thymus are densely populated with T cells containing normal nuclei.
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Discussion |
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This report describes a congenital myopathy and major loss of thymic lymphocytes in ankyrin-B (-/-) mice, as well as dramatic alterations in intracellular localization of key components of the Ca2+ homeostasis machinery in ankyrin-B (-/-) striated muscle and thymus. The SR and SRT-tubule junctions are apparently preserved in a normal distribution in ankyrin-B (-/-) skeletal muscle based on electron microscopy and the presence of a normal pattern of triadin and DHPR. The abnormal localization of SERCA and ryanodine receptors therefore represents a defect in intracellular sorting of these proteins in skeletal muscle. Extrapolation of these observations suggests defective targeting as the basis for abnormal localization of ryanodine receptors, IP3 receptors and SERCA in heart, and of IP3 receptors in the thymus of ankyrin-B (-/-) mice. Mis-sorting of SERCA 2 and ryanodine receptor 2 in ankyrin-B (-/-) cardiomyocytes is rescued by expression of the 220-kD ankyrin-B, demonstrating that lack of the 220-kD ankyrin-B polypeptide is the primary defect in these cells. Ankyrin-B is associated with intracellular vesicles, but is not colocalized with the bulk of SERCA 1 or ryanodine receptor type 1 in skeletal muscle. These data provide the first evidence for a physiological requirement for ankyrin-B in intracellular targeting of the calcium homeostasis machinery of striated muscle and immune system, and moreover support a catalytic role that does not involve permanent stoichiometric complexes between ankyrin-B and targeted proteins.
Evidence in support of some form of direct interactions of ankyrin-B with ryanodine receptors and IP3 receptors is provided by in vitro binding assays (
Direct but transient interaction of Ca2+ homeostasis proteins with ankyrin-B could occur during transit of these proteins between the ER and the SR. Exchange of proteins between ER and Golgi is well known to involve a machinery for recruitment of specific cargo proteins into vesicles, and transfer of these vesicles between organelle compartments. A comparable system may also mediate transfer of functionally defined proteins from the ER to specialized sites within the SR. In this case, the basic defect in ankyrin-B (-/-) cells underlying mis-sorting of multiple Ca2+ homeostasis proteins could be a failure in some step required for segregation of these proteins and/or their transport between the ER and SR. According to this hypothesis, ankyrin-B would function as a SR-specific guide or escort using the touring metaphor for protein sorting (
Ankyrin-B is a multifunctional protein that could participate in ER protein sorting at several levels. The multivalent ankyrin membrane-binding domain could bind to and laterally segregate selected proteins within the plane of the ER membrane (
Coordinated assembly and spatial organization of Ca2+ release channels and SERCA within the ER is essential in cells of the immune system as well as in striated muscle. Lymphocytes encode information in the amplitude and frequency of intracellular waves of Ca2+ (
The specialized neuronal ER involved in calcium homeostasis is located in dendritic spines and growth cones of axons and resembles the SR found in muscle as visualized by electron microscopy (
Complete ankyrin-B deficiency in humans would be anticipated to involve defective development of the nervous system, as well as severe dysfunction of striated muscle and immune system that would not be compatible with prolonged postnatal life. However, individuals with weak alleles or partial deficiency of ankyrin-B may be viable. Ankyrin-B (+/-) mice have reduced expression of ankyrin-B and survive to adulthood, although with musculoskeletal defects that remain to be characterized (not shown). These animals may provide models for certain autosomal dominant human channelopathies involving Ca2+ release channels and SERCA, with loss of function due to mis-sorting rather than direct mutations in these proteins. For example, malignant hyperthermia, a leading cause of anesthesia-associated mortality, can be caused by mutations in the ryanodine receptor (
Ankyrin-G and ankyrin-R polypeptides are still expressed in the ankyrin-B (-/-) cardiomyocytes (not shown), and 190-kD ankyrin-G cannot rescue ankyrin-B (-/-) cardiomyocytes (Fig 4). These data indicate that ankyrins, unlike many other multigene families, do not compensate for each other, and presumably have gene-specific functions. The lack of complementation between ankyrins suggests the presence of binding partners specific for each ankyrin, which have not yet been identified. Therefore, the activities proposed above for ankyrin-B in ER protein sorting are likely to involve protein interactions unique for ankyrin-B and not accessible to ankyrin-G. It will be of interest in future experiments to use chimeric ankyrins to define key domains involved in rescue of SERCA and ryanodine receptor sorting as well as cellular targeting of ankyrin-B.
Ankyrin-G and ankyrin-B both have been implicated in segregating diverse proteins within functionally defined membrane domains. However, ankyrin-Gdependent proteins are associated with specialized regions of the plasma membrane (
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Footnotes |
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S. Tuvia and M. Buhusi contributed equally to this paper.
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Acknowledgements |
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This work was funded in part by a grant from the National Institutes of Health (RO1 DK29808). Harold Erickson is thanked for his useful comments. Susan Hester is gratefully acknowledged for preparation of plastic-embedded sections of the thymus.
Submitted: 30 June 1999
Revised: 21 September 1999
Accepted: 20 October 1999
GFP, green fluorescent protein; SERCA, SR/ER Ca ATPase; SR, sarcoplasmic reticulum
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References |
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