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Gene name - alpha Spectrin Synonyms - Cytological map position - 62B2--62B7 Function - Docking and structural protein Keyword(s) - cytoskeleton |
Symbol - alpha-Spec FlyBase ID:FBgn0003470 Genetic map position - 3-[1.4] Classification - spectrin Cellular location - cytoplasm |
Spectrin is an elongated molecule that is a constituent of the submembrane cytoskeleton of epithelial cells, making up many tissues of the fly. Drosophila has a single 278 kDa form of alpha-Spectrin that can heterodimerize with either of two ß-subunits, each one the product of a distinct gene: a conventional 265 kDa ß form (Byers, 1989) and a larger 430 kDa form known as ßHeavy or ßH (Dubreuil, 1990). A good example of the subcellular distribution of the three spectrin proteins is illustrated by the follicular epithelium surrounding the egg. alpha-Spectrin is distributed along the lateral and apical domains (the apical domain is the side of the cell facing the oocyte) of the follicle cell plasma membrane whereas the conventional ß-subunit is localized prominently to the lateral follicle cell membrane at all stages. In contrast, the ßH subunit is concentrated on the apical surface of the follicle cell, not unlike the arrangement seen in cellularizing embryos. Thus, a common alpha-subunit forms heterodimers with ß-spectrin on the lateral membrane or with ßH-spectrin on the apical membrane (Lee, 1997).
Spectrin influences cells in one of two ways. It can be thought of as part of an infrastructure that functions to stabilize cell shape and/or cell-cell contacts, or as a scaffold for the proper (stable) positioning of membrane bound proteins and other cytoskeletal elements or proteins involved in cell signaling (Lee, 1997). Three aspects of spectrin are considered below:
1. Spectrin protein-protein interactions
Mammalian spectrin interacts with the the adherens junction, associating directly with the cadherin complex via alpha-catenin (Lombardo, 1994) or indirectly via a F-actin-alpha-catenin interaction (Rimm, 1995). The spectrin network is in a perfect position to link cell adhesion and cell polarity to the polarization of the Na/K-ATPase, the cell's Sodium pump. In fact, Na/K-ATPase requires a membrane skeleton to anchor it to sites of cell adhesion. Ankyrin and adducin associate with spectrin and are colocalized with spectrin at sites of cell-cell contact in epithelial cells. Na/K-ATPase interacts with ankyrin and is colocalized with spectrin and Ankyrin in epithelial cells (Hu, 1995). E-cadherin and F-actin, in contrast to ankyrin, adducin, and the Na/K-ATPase, exhibit unaltered distribution in beta-spectrin-deficient mammalian cells. In Drosophila, adherens junction associated proteins include Shotgun (E-cadherin), Hu-li tao shao (an adducin like protein), Actin, Ankyrin, and Armadillo. In mammalian erythrocytes membrane attachment of the cytoskeleton is provided by ankyrin, which associates with ß-spectrin, and by protein 4.1 In turn, these proteins associate with integral membrane proteins, including the anion exchanger and glycophorin C. Protein 4.1, as well as adducin, protein 4.9 dematin, tropomyosin and tropomodulin, may also mediate the association of spectrin with actin, based on immunolocalization and in vitro binding experiments (Bennett, 1993).
2. Involvement of spectrin in oogenesis
Ovarian stem cells, located in the germarium of the ovary, produce one cystoblast at a time, (the precursor cell of the egg). Cystoblasts divide synchronously four times with incomplete cytokinesis to eventually form 16 cell cysts connected by ring canals that serve as cytoplasmic bridges between the 16 cystocytes. During cyst formation, a region of specialized, spectrin-rich cytoplasm, called the fusome, traverses the intercellular connections (the ring canals) that link the individual cystocytes. Subsequently, 15 cystocytes begin to transport specific RNAs and other components into the remaining cell, the future oocyte.
The fusome contains four membrane cytoskeletal proteins: alpha-Spectrin, ß-Spectrin, the adducin-like Hu-li tai shao and Ankyrin. Stem cells and cystocytes contain a large sphere of fusomal material, termed a spectrosome. During the four cystocyte mitoses, one pole of each spindle associates with the fusome, and following each mitosis, as the spindles disaggregate, additional fusomal material accumulates in their place. Thus, by the fourth division, the fusome forms one large branched structure that extends though the ring canals into all the cells in a cyst. alpha-Spectrin deficient cells were generated in fly ovaries and the effects on cyst fomation and oocyte differentiation were observed (reviewed in McKearin, 1997). In alpha-Spectrin mutant ovarioles, cyst formation is inevitably disrupted. Mutant egg chambers almost always contained fewer than 16 cells and often lack an oocyte; most appear to degenerate before completing oogenesis. alpha-Spectrin staining is completely gone from fusomes and cell membranes in these ovarioles. HTS protein and ß-Spectrin are also lacking in mutant egg chambers. Ring canals, however, are normal in mutant egg chambers. It is concluded that although fusomes are not required to block cytokinesis or to initiate ring canal formation (Lin, 1994), fusomes are nevertheless involved in cyst formation and oocytye determination. It is thought that in the absence of a fusome cystocyte cell cycles are synchronized only between cell pairs (as only an even number of cells is present in mutant cysts), rather than throughout the cyst. These findings suggest that the fusome has a function in coordinating the cell cycles of cystocytes (de Cuevas, 1996).
Of particular interest is the association of fusomes with the pole of the mitotic spindle (Lin, 1995). During the first cystoblast division, fusome material is associated with only one pole of the mitotic spindle, demonstrating that this division is asymmetric. During the subsequent three divisions, the growing fusome always associates with the pole of each mitotic spindle that remains in the mother cell, and only extends through the newly formed ring canals after each division is completed. The protein Inscuteable is thought to link cytoskeleton to spindle-orientation and subcellular distribution of Prospero and Numb. Prospero and Numb are directly involved in determining alternate cell fates in asymmetric cell division. It is likely that the interaction of the fusome constituents with the mitotic spindle is relevent to cell fate determination in asymmetric division during embryogenesis.
3. The involvement of spectrin in epithelial cell polarity
alpha-Spectrin is required for ovarian follicle monolayer integrity. To examine the role of alpha-Spectrin, transgenic flies were created from alpha-Spectrin null flies. The Spectrin transgene contained the cDNA for alpha-Spectrin flanked by FLP recombination target sequences. Heat-shock induction of the FLP recombinase during the first larval instar ensured that mutant clones in adult flies lacked the alpha-Spectrin protein. Because of protein turnover and dilution, no alpha-Spectin could be detected on the plasma membrane of follicle epithelial cells in alpha-Spectrin minus clones of three day old females following recombinase induction during the first instar period. Eyes mutant for alpha-Spectrin show a roughened appearance. Ovarioles composed of alpha-Spectrin mutant cysts contain malformed stage 9 egg chambers and a lack of later stage egg chambers, suggesting that alpha-Spectrin plays an integral role during oogenesis. However, normal follicle cell monolayers were observed surrounding mutant cysts.
Follicle cell clones in stage 9 egg chambers show an alteration in cell shape changes in both the anterior and posterior ends of the egg. In complete alpha-Spectrin mutant egg chambers the follicle monolayer is disorganized, with multiple layers of cuboidal-shaped follicle cells at the posterior end of the egg chamber and on occasion at the anterior pole. All other aspects of egg chamber morphology appear normal, including proper formation of interfollicular stalks. It was concluded that cell division rather than cell migration accounts for the excess number of cells at the posterior end of egg chambers. ßHeavy-Spectrin fails to localize on the apical plasma membrane of mutant cells, suggesting that heterodimerization with alpha-Spectrin is needed for the proper localization of ßH-spectrin on the apical plasma membrane. The total amount of ß-spectrin associated with the lateral plasma membrane is diminished in the absence of alpha-Spectrin. Ankyrin distribution is also altered in alpha-Spectrin minus clones, but the assembly of conventional ß-spectrin and ankyrin at the lateral domain of the follicle cell plasma membrane is not altogether prevented. Alpha-catenin localization to the adherens junction is completely lost in the hyperplastic posterior follicle cells. Surprisingly, Na/K-ATPase distribution is unaltered (Lee, 1997).
Thus, as a consequence of disrupting the apical membrane skeleton, a distinct sub-population of follicle cells undergoes unregulated proliferation, which leads to the loss of monolayer organization and disruption of the anterior-posterior axis of the oocyte. These results suggest that the spectrin-based membrane skeleton is required in a developmental pathway that controls follicle cell monolayer and proliferation (Lee, 1997).
The alpha subunit of spectrin consists of two large domains of repetitive sequence (segments 1-9 and 11-19) separated by a short nonrepetitive sequence (segment 10). The 106-residue repetitive segments are defined by a consensus sequence of 54 residues. Chicken alpha-Spectrin shares 50 of these consensus positions. Through comparision of spectrin and alpha-actinin sequences, a second lineage of spectrin segments (20 and 21) is described that differs from the 106-residue segments by an 8-residue insertion and by a lack of many of the consensus residues (Dubreuil, 1989).
Two sites were localized at which calcium may regulate spectrin function. First, a site responsible for calmodulin binding to Drosophila alpha-Spectrin is present near the junction of repetitive segments 14 and 15. Second, a domain that includes two EF hand Ca2+ binding sequences binds radioactive Ca2+ in blot overlay assays. EF hand sequences from a homologous domain of Drosophila alpha Actinin does not bind calcium under the same conditions (Dubreuil, 1991).
The alpha and beta chains of spectrin are homologous, yet they have acquired different structural features that work in synergy to give the multimer its overall properties. The primary amino acid sequence of each spectrin subunit is dominated by tandemly repeated 106-residue motifs. By comparing the complete Drosophila beta-spectrin sequence with other spectrins evidence was found that a higher-order, 848-amino acid supra-motif is tandemly repeated in both alpha- and beta-spectrin. These data argue that alpha- and beta-spectrin, rather than evolving independently from sequences encoding the ancestral 106-residue motifs, must have arisen after the establishment of a large supra-motif composed of eight of the 106-residue motifs. These data suggest the segment structure of a progenitor gene that gave rise to both alpha- and beta-spectrin as well as dystrophin. The structural differences that evolved after the split between the alpha- and beta-spectrin genes confer the independent functions that exist in their products today (Byers, 1992).
The pleckstrin homology (PH) domain, which is approximately 100 amino acids long, has been found in about 70 proteins involved in signal transduction and cytoskeletal function, a frequency comparable to SH2 (src homology 2) and SH3 domains. PH domains have been shown to bind the beta gamma-subunits of G-proteins and phosphatidylinositol 4,5-bisphosphate (PIP2). It is conceivable that the PH domain of beta-spectrin plays a part in the association of spectrin with the plasma membrane of cells. The solution structure of the 122-residue PH domain of Drosophila beta-spectrin has the following attributes: the overall fold consists of two antiparallel beta-sheets packing against each other at an angle of approximately 60 degrees to form a beta-sandwich, a two-turn alpha-helix unique to spectrin PH domains, and a four-turn C-terminal alpha-helix. One of the major insertions in beta-spectrin PH domains forms a long, basic surface loop and appears to undergo slow conformational exchange in solution. This loop shows big spectral changes upon addition of D-myo-inositol 1,4,5-trisphosphate (IP3). It is proposed that the groove at the outer surface of the second beta-sheet is an important site of association with other proteins. This site and the possible lipid-binding site can serve to localize the spectrin network under the plasma membrane. It should be kept in mind that the common fold observed for the PH domain structures solved so far does not necessarily mean that all PH domains have similar functions. In fact, the residues constituting potential binding sites for ligands or other proteins are only slightly conserved between different PH domains (Zhang, 1995).
date revised: 6 MAR 97
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