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Ankyrin-B is required for intracellular sorting of structurally diverse Ca2+ homeostasis proteins.

Tuvia S, Buhusi M, Davis L, Reedy M, Bennett V - J. Cell Biol. (1999)

Bottom Line: 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.Similar mechanisms involving ankyrins may be essential for segregation of functionally defined proteins within specialized regions of the plasma membrane and within the Ca(2+) homeostasis compartment of the ER.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Departments of Cell Biology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
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 Ca(2+) homeostasis machinery in ankyrin-B (-/-) striated muscle and thymus. The sarcoplasmic 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 Ca(2+) homeostasis compartment of the ER.

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Abnormal spatial and temporal patterns of intracellular calcium release during spontaneous contractions of ankyrin-B (−/−) neonatal cardiomyocytes. Cytosolic Ca2+ levels were measured as a function of time (A and B, bottom) in cultured neonatal cardiomyocytes of littermate ankyrin-B (+/+) (A) and ankyrin-B (−/−) mice (B) using Fluo-3 as a calcium indicator (see Materials and Methods). Top panels of A and B show images of intracellular Ca2+ levels in cells at times indicated by lines, where the color scale on the right shows pseudocolored Ca2+ intensity levels, increasing from black (low Ca2+) to white (high Ca2+). Note that the ankyrin B (−/−) cardiomyocyte shows several distinct foci of increased Ca2+ levels during Ca2+ release (B, top left and right).
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Figure 2: Abnormal spatial and temporal patterns of intracellular calcium release during spontaneous contractions of ankyrin-B (−/−) neonatal cardiomyocytes. Cytosolic Ca2+ levels were measured as a function of time (A and B, bottom) in cultured neonatal cardiomyocytes of littermate ankyrin-B (+/+) (A) and ankyrin-B (−/−) mice (B) using Fluo-3 as a calcium indicator (see Materials and Methods). Top panels of A and B show images of intracellular Ca2+ levels in cells at times indicated by lines, where the color scale on the right shows pseudocolored Ca2+ intensity levels, increasing from black (low Ca2+) to white (high Ca2+). Note that the ankyrin B (−/−) cardiomyocyte shows several distinct foci of increased Ca2+ levels during Ca2+ release (B, top left and right).

Mentions: Previous reports that ankyrin associates with IP3 receptors and ryanodine receptors (Bourguignon et al. 1993; Bourguignon et al. 1995) suggested the possibility that Ca2+ homeostasis might be abnormal in ankyrin-B (−/−) mice. Dynamic patterns of intracellular Ca2+ release and uptake were measured in cardiomyocytes cultured from 1-d-old ankyrin-B (+/+) and (−/−) mice and maintained for 96 h, using Fluo-3 as an indicator (Fig. 2). Ankyrin-B (+/+) and (−/−) cardiomyocytes in these cultures spontaneously contract, and have assembled sarcomeres and T-tubules, based on patterns of fluorescent labeling of α-actinin (Fig. 3 A1), F-actin (Fig. 4, A–H, 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).


Ankyrin-B is required for intracellular sorting of structurally diverse Ca2+ homeostasis proteins.

Tuvia S, Buhusi M, Davis L, Reedy M, Bennett V - J. Cell Biol. (1999)

Abnormal spatial and temporal patterns of intracellular calcium release during spontaneous contractions of ankyrin-B (−/−) neonatal cardiomyocytes. Cytosolic Ca2+ levels were measured as a function of time (A and B, bottom) in cultured neonatal cardiomyocytes of littermate ankyrin-B (+/+) (A) and ankyrin-B (−/−) mice (B) using Fluo-3 as a calcium indicator (see Materials and Methods). Top panels of A and B show images of intracellular Ca2+ levels in cells at times indicated by lines, where the color scale on the right shows pseudocolored Ca2+ intensity levels, increasing from black (low Ca2+) to white (high Ca2+). Note that the ankyrin B (−/−) cardiomyocyte shows several distinct foci of increased Ca2+ levels during Ca2+ release (B, top left and right).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169334&req=5

Figure 2: Abnormal spatial and temporal patterns of intracellular calcium release during spontaneous contractions of ankyrin-B (−/−) neonatal cardiomyocytes. Cytosolic Ca2+ levels were measured as a function of time (A and B, bottom) in cultured neonatal cardiomyocytes of littermate ankyrin-B (+/+) (A) and ankyrin-B (−/−) mice (B) using Fluo-3 as a calcium indicator (see Materials and Methods). Top panels of A and B show images of intracellular Ca2+ levels in cells at times indicated by lines, where the color scale on the right shows pseudocolored Ca2+ intensity levels, increasing from black (low Ca2+) to white (high Ca2+). Note that the ankyrin B (−/−) cardiomyocyte shows several distinct foci of increased Ca2+ levels during Ca2+ release (B, top left and right).
Mentions: Previous reports that ankyrin associates with IP3 receptors and ryanodine receptors (Bourguignon et al. 1993; Bourguignon et al. 1995) suggested the possibility that Ca2+ homeostasis might be abnormal in ankyrin-B (−/−) mice. Dynamic patterns of intracellular Ca2+ release and uptake were measured in cardiomyocytes cultured from 1-d-old ankyrin-B (+/+) and (−/−) mice and maintained for 96 h, using Fluo-3 as an indicator (Fig. 2). Ankyrin-B (+/+) and (−/−) cardiomyocytes in these cultures spontaneously contract, and have assembled sarcomeres and T-tubules, based on patterns of fluorescent labeling of α-actinin (Fig. 3 A1), F-actin (Fig. 4, A–H, 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).

Bottom Line: 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.Similar mechanisms involving ankyrins may be essential for segregation of functionally defined proteins within specialized regions of the plasma membrane and within the Ca(2+) homeostasis compartment of the ER.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Departments of Cell Biology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
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 Ca(2+) homeostasis machinery in ankyrin-B (-/-) striated muscle and thymus. The sarcoplasmic 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 Ca(2+) homeostasis compartment of the ER.

Show MeSH
Related in: MedlinePlus