Sources/Clones
Accurate (SB-SP1, SB-SP2), American Qualex (polyclonal), Biodesign (polyclonal), Biogenesis (B12G3, D4D7, 2C5, polyclonal), Calbiochem (polyclonal), Chemicon (polyclonal), ICN Immunologicals (AA6), Locus Genex, Helsinki (101AA6), Finland, Novocastra (RBC1.5B1, RPC2.3D5), Serotec (D7A3, D4D7), Sigma (polyclonal) and Zymed (Z068).
Fixation/Preparation
The antibody is immunoreactive in fresh-frozen tissue sections and in fixed paraffin-embedded sections following HIER.
Background
Spectrin is a flexible rod-shaped molecule of 200 nm length found in mammalian and avian erythrocytes. It is composed of two non-identical subunits,a and b, and linked to the plasma membrane by the protein ankyrin. Along with actin, ankyrin and band 4.1, spectrin forms a network or membrane skeleton that lies immediately beneath the plasma membrane. The main function of the spectrin cytoskeleton is that of structural support for the bilipid layer of the cell membrane and the spectrin-based membrane skeleton also controls lateral mobility of the erythrocyte membrane proteins (Bennett, 1989, 1990a/b). Thermal denaturation of spectrin leads to disintegration of the erythrocytes into vesicles and deficiencies or structural abnormalities of the membrane skeleton proteins lead to loss of shape or tensile strength of the erythrocytes, resulting in fragmentation and destruction as they pass through the spleen. Defects of spectrin are associated with fragile erythrocytes in hemolytic anemias such as hereditary elliptocytosis, pyropoikilocytosis and spherocytosis (Bennett & Gilligan, 1993).
Non-erythroid cells also show a membrane skeleton which contains spectrin, although the molecular organization in such cells is less understood. Non-erythroid spectrin, known as fodrin with a molecular weight of 240 kD, exhibits many similarities to spectrin, including immunochemical crossreactivity, and is found in virtually all nonerythroid cells. Besides the function of maintaining some specialized membrane domains, fodrin appears to be redistributed in a variety of cell surface events, suggesting that it acts as a dynamic mediator between the cell membrane, membrane skeleton and cytoskeleton. For example, there is significant reorganization of the spectrin network in cells treated with growth factors. In chromaffin cells, stimulation with a calcium ionophore results in secretion and a relocation of spectrin as cytoplasmic aggregates, antibody-induced capping of B lymphocyte surface immunoglobulin leads to redistribution of spectrin similar to the surface proteins and in A-431, an epidermoid carcinoma cell line, EGF induces cell surface remodeling and the accumulation of spectrin in membrane ruffles coincident with its phosphorylation. It is thought that calcium ions influence membrane skeleton assembly and maintenance by binding to spectrin, by calcium-regulated, calmodulin-mediated influence of the interactions between spectrin and other proteins, or by calcium-dependent protease cleavage of spectrin (Harris & Morrow, 1990; Wallis et al 1992; Davis & Bennett, 1994).
Applications
Until recently, the antibody to spectrin/fodrin was employed only on fresh-frozen tissue sections; however, with the use of microwave antigen retrieval, we were able to demonstrate immunoreactivity in fixed paraffin-embedded sections (Sormunen et al, 1997). The interest in fodrin lies in its role in cell adhesion during embryogenesis and in neoplasms. In comparison to their non-neoplastic counterparts, neoplastic epithelial cells show elevated levels of fodrin immunostaining regardless of tumor type. There was strong fragmented and circumferential staining for fodrin which often became accentuated with increasing grades of anaplasia and loss of membrane staining corresponded with loss of tumor cell cohesiveness (Sormunen et al, 1997). More recent work suggests that fodrin is linked to E-cadherin andb-catenin, together having a role in cell-to-cell adhesion. The breakage of this complex is heralded by detachment ofb-catenin and associated with change in cell shape and cell adhesion in breast carcinoma (Sormunen et al, 1998).
References
•Bennett V 1989. The spectrin-actin junction of erythrocyte membrane skeletons. Biochemia Biophysics Acta 988: 107-122.
•Bennett V 1990a. Spectrin: a structural mediator between diverse plasma membrane proteins and the cytoplasm. Current Opinion in Cell Biology 2: 51-56.
•Bennett V 1990b. Spectrin-based membrane skeleton: a multipotential adaptor between plasma membrane and cytoplasm. Physiology Reviews 70: 1029-1065.
•Bennett V, Gilligan DM 1993. The spectrin-based membrane skeleton and micron-scale organization of the plasma membrane. Annual Reviews of Cell Biology 9: 27-66.
•Davis LH, Bennett V 1994. Identification of two regions ofbG spectrin that bind to distinct sites in brain membranes. Journal of Biology and Chemistry 269: 4409-4416.
•Harris AS, Morrow JS 1990. Calmodulin and calcium-dependent protease I coordinately regulate the interaction of fodrin with actin. Proceedings of the National Academy of Sciences USA 87: 3009-3013.
•Sormunen RT, Eskelinen S, Leong AS-Y 1997. Fodrin immunolocalization in epithelial tumors. Applied Immunohistochemistry 5: 179-184.
•Sormunen RT, Leong AS-Y, Vaaraniemi JP et al 1998. Fodrin, E-cadherin and beta-catenin immunolocalization in infiltrating ductal carcinoma of the breast correlated with selected prognostic indices. Journal of Pathology (submitted).
•Wallis CJ, Wenegieme EF, Babitch JA 1992. Characterisation of calcium binding to brain spectrin. Journal of Biology and Chemistry 267: 4333-4337.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
Accurate (SB-SP1, SB-SP2), American Qualex (polyclonal), Biodesign (polyclonal), Biogenesis (B12G3, D4D7, 2C5, polyclonal), Calbiochem (polyclonal), Chemicon (polyclonal), ICN Immunologicals (AA6), Locus Genex, Helsinki (101AA6), Finland, Novocastra (RBC1.5B1, RPC2.3D5), Serotec (D7A3, D4D7), Sigma (polyclonal) and Zymed (Z068).
Fixation/Preparation
The antibody is immunoreactive in fresh-frozen tissue sections and in fixed paraffin-embedded sections following HIER.
Background
Spectrin is a flexible rod-shaped molecule of 200 nm length found in mammalian and avian erythrocytes. It is composed of two non-identical subunits,a and b, and linked to the plasma membrane by the protein ankyrin. Along with actin, ankyrin and band 4.1, spectrin forms a network or membrane skeleton that lies immediately beneath the plasma membrane. The main function of the spectrin cytoskeleton is that of structural support for the bilipid layer of the cell membrane and the spectrin-based membrane skeleton also controls lateral mobility of the erythrocyte membrane proteins (Bennett, 1989, 1990a/b). Thermal denaturation of spectrin leads to disintegration of the erythrocytes into vesicles and deficiencies or structural abnormalities of the membrane skeleton proteins lead to loss of shape or tensile strength of the erythrocytes, resulting in fragmentation and destruction as they pass through the spleen. Defects of spectrin are associated with fragile erythrocytes in hemolytic anemias such as hereditary elliptocytosis, pyropoikilocytosis and spherocytosis (Bennett & Gilligan, 1993).
Non-erythroid cells also show a membrane skeleton which contains spectrin, although the molecular organization in such cells is less understood. Non-erythroid spectrin, known as fodrin with a molecular weight of 240 kD, exhibits many similarities to spectrin, including immunochemical crossreactivity, and is found in virtually all nonerythroid cells. Besides the function of maintaining some specialized membrane domains, fodrin appears to be redistributed in a variety of cell surface events, suggesting that it acts as a dynamic mediator between the cell membrane, membrane skeleton and cytoskeleton. For example, there is significant reorganization of the spectrin network in cells treated with growth factors. In chromaffin cells, stimulation with a calcium ionophore results in secretion and a relocation of spectrin as cytoplasmic aggregates, antibody-induced capping of B lymphocyte surface immunoglobulin leads to redistribution of spectrin similar to the surface proteins and in A-431, an epidermoid carcinoma cell line, EGF induces cell surface remodeling and the accumulation of spectrin in membrane ruffles coincident with its phosphorylation. It is thought that calcium ions influence membrane skeleton assembly and maintenance by binding to spectrin, by calcium-regulated, calmodulin-mediated influence of the interactions between spectrin and other proteins, or by calcium-dependent protease cleavage of spectrin (Harris & Morrow, 1990; Wallis et al 1992; Davis & Bennett, 1994).
Applications
Until recently, the antibody to spectrin/fodrin was employed only on fresh-frozen tissue sections; however, with the use of microwave antigen retrieval, we were able to demonstrate immunoreactivity in fixed paraffin-embedded sections (Sormunen et al, 1997). The interest in fodrin lies in its role in cell adhesion during embryogenesis and in neoplasms. In comparison to their non-neoplastic counterparts, neoplastic epithelial cells show elevated levels of fodrin immunostaining regardless of tumor type. There was strong fragmented and circumferential staining for fodrin which often became accentuated with increasing grades of anaplasia and loss of membrane staining corresponded with loss of tumor cell cohesiveness (Sormunen et al, 1997). More recent work suggests that fodrin is linked to E-cadherin andb-catenin, together having a role in cell-to-cell adhesion. The breakage of this complex is heralded by detachment ofb-catenin and associated with change in cell shape and cell adhesion in breast carcinoma (Sormunen et al, 1998).
References
•Bennett V 1989. The spectrin-actin junction of erythrocyte membrane skeletons. Biochemia Biophysics Acta 988: 107-122.
•Bennett V 1990a. Spectrin: a structural mediator between diverse plasma membrane proteins and the cytoplasm. Current Opinion in Cell Biology 2: 51-56.
•Bennett V 1990b. Spectrin-based membrane skeleton: a multipotential adaptor between plasma membrane and cytoplasm. Physiology Reviews 70: 1029-1065.
•Bennett V, Gilligan DM 1993. The spectrin-based membrane skeleton and micron-scale organization of the plasma membrane. Annual Reviews of Cell Biology 9: 27-66.
•Davis LH, Bennett V 1994. Identification of two regions ofbG spectrin that bind to distinct sites in brain membranes. Journal of Biology and Chemistry 269: 4409-4416.
•Harris AS, Morrow JS 1990. Calmodulin and calcium-dependent protease I coordinately regulate the interaction of fodrin with actin. Proceedings of the National Academy of Sciences USA 87: 3009-3013.
•Sormunen RT, Eskelinen S, Leong AS-Y 1997. Fodrin immunolocalization in epithelial tumors. Applied Immunohistochemistry 5: 179-184.
•Sormunen RT, Leong AS-Y, Vaaraniemi JP et al 1998. Fodrin, E-cadherin and beta-catenin immunolocalization in infiltrating ductal carcinoma of the breast correlated with selected prognostic indices. Journal of Pathology (submitted).
•Wallis CJ, Wenegieme EF, Babitch JA 1992. Characterisation of calcium binding to brain spectrin. Journal of Biology and Chemistry 267: 4333-4337.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
