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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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Ankyrin-B in skeletal muscle is localized at costameres and intracellular sites that are distinct from localization of SERCA 1 and ryanodine receptor type 1. (A). Immunolabeling of a longitudinal section of skeletal muscle with rabbit antibody against ankyrin (a1, a4, red) and a mouse monoclonal antibody against α-actinin (a2, a5, green) with a composite image (a3 and b3). Optical sections were taken at two different Z-heights: at the top, tangential to the cell surface (Z = 0 μm; a4–a6) and in the mid-region of the same cell (Z = −5 μm; a1–a3). Note that arrowheads point to location of Z line (α-actinin) and long arrows to the A-band. Note that ankyrin B labeling aligned with the Z-line appears just at the plasma membrane level (right). B and C show immunofluorescence labeling of cross-sections of skeletal muscle with rabbit antibody against the ankyrin-B (red, b1, b3, c1, and c3) and antibody against either SERCA 1 (B, green, b2 and b3) or ryanodine receptor type 1 and 2 (C, green, c2 and c3). Bars, 10 μm.
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Figure 8: Ankyrin-B in skeletal muscle is localized at costameres and intracellular sites that are distinct from localization of SERCA 1 and ryanodine receptor type 1. (A). Immunolabeling of a longitudinal section of skeletal muscle with rabbit antibody against ankyrin (a1, a4, red) and a mouse monoclonal antibody against α-actinin (a2, a5, green) with a composite image (a3 and b3). Optical sections were taken at two different Z-heights: at the top, tangential to the cell surface (Z = 0 μm; a4–a6) and in the mid-region of the same cell (Z = −5 μm; a1–a3). Note that arrowheads point to location of Z line (α-actinin) and long arrows to the A-band. Note that ankyrin B labeling aligned with the Z-line appears just at the plasma membrane level (right). B and C show immunofluorescence labeling of cross-sections of skeletal muscle with rabbit antibody against the ankyrin-B (red, b1, b3, c1, and c3) and antibody against either SERCA 1 (B, green, b2 and b3) or ryanodine receptor type 1 and 2 (C, green, c2 and c3). Bars, 10 μm.

Mentions: 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 (Craig and Pardo 1983) at the sarcolemma, and is aligned with the Z-lines that are labeled by α-actinin (Fig. 8 A, a4–a6, 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, a1–a3). 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 (Flucher et al. 1990). However, ankyrin-B localization does closely resemble the labeling obtained by Nelson and Lazarides 1984 with antibody raised against chicken erythrocyte ankyrin.


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)

Ankyrin-B in skeletal muscle is localized at costameres and intracellular sites that are distinct from localization of SERCA 1 and ryanodine receptor type 1. (A). Immunolabeling of a longitudinal section of skeletal muscle with rabbit antibody against ankyrin (a1, a4, red) and a mouse monoclonal antibody against α-actinin (a2, a5, green) with a composite image (a3 and b3). Optical sections were taken at two different Z-heights: at the top, tangential to the cell surface (Z = 0 μm; a4–a6) and in the mid-region of the same cell (Z = −5 μm; a1–a3). Note that arrowheads point to location of Z line (α-actinin) and long arrows to the A-band. Note that ankyrin B labeling aligned with the Z-line appears just at the plasma membrane level (right). B and C show immunofluorescence labeling of cross-sections of skeletal muscle with rabbit antibody against the ankyrin-B (red, b1, b3, c1, and c3) and antibody against either SERCA 1 (B, green, b2 and b3) or ryanodine receptor type 1 and 2 (C, green, c2 and c3). Bars, 10 μm.
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Figure 8: Ankyrin-B in skeletal muscle is localized at costameres and intracellular sites that are distinct from localization of SERCA 1 and ryanodine receptor type 1. (A). Immunolabeling of a longitudinal section of skeletal muscle with rabbit antibody against ankyrin (a1, a4, red) and a mouse monoclonal antibody against α-actinin (a2, a5, green) with a composite image (a3 and b3). Optical sections were taken at two different Z-heights: at the top, tangential to the cell surface (Z = 0 μm; a4–a6) and in the mid-region of the same cell (Z = −5 μm; a1–a3). Note that arrowheads point to location of Z line (α-actinin) and long arrows to the A-band. Note that ankyrin B labeling aligned with the Z-line appears just at the plasma membrane level (right). B and C show immunofluorescence labeling of cross-sections of skeletal muscle with rabbit antibody against the ankyrin-B (red, b1, b3, c1, and c3) and antibody against either SERCA 1 (B, green, b2 and b3) or ryanodine receptor type 1 and 2 (C, green, c2 and c3). Bars, 10 μm.
Mentions: 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 (Craig and Pardo 1983) at the sarcolemma, and is aligned with the Z-lines that are labeled by α-actinin (Fig. 8 A, a4–a6, 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, a1–a3). 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 (Flucher et al. 1990). However, ankyrin-B localization does closely resemble the labeling obtained by Nelson and Lazarides 1984 with antibody raised against chicken erythrocyte ankyrin.

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