alpha-Spectrin

Protein Interactions

The head-end associations of spectrin give rise to tetramers and make it possible for the molecule to form networks. The head-end associations of Drosophila spectrin were analyzed in vitro and in vivo. Immunoprecipitation assays using protein fragments synthesized in vitro from recombinant DNA show that interchain binding at the head end is mediated by segment 0-1 of alpha Spectrin and segment 18 of beta Spectrin. Point mutations equivalent to erythroid spectrin mutations, responsible for human hemolytic anemias, diminish Drosophila spectrin head-end interchain binding in vitro. To test the in vivo consequence of deficient head-end interchain binding, constructs expressing head-end interchain binding mutant alpha Spectrin were introduced into the Drosophila genome and tested for rescue of an alpha Spectrin null mutation. An alpha-Spectrin minigene lacking the codons for head-end interchain binding fails to rescue the lethality of the null mutant, whereas a minigene with a point mutation in these codons overcomes the lethality of the null mutant in a temperature-dependent manner. The rescued flies are viable and fertile at 25 degrees C, but when shifted to 29 degrees C they become sterile because of defects in oogenesis. At 29 degrees C, egg chamber tissue disruption and cell shape changes are evident, even though the mutant spectrin remains stably associated with cell membranes. These results show that spectrin's capacity to form a network is a crucial aspect of its function in nonerythroid cells (Deng, 1995).

Spectrin's function as an actin-crosslinking protein and membrane skeleton component involves the tail end of the molecule, where multiple interactions between two spectrin chains and between these chains and other proteins give rise to complexes that form membrane skeleton network junctions. To determine whether the sequences that contribute to interchain binding can be distinguished from sequences that are involved in other spectrin tail end functions, the regions in each Drosophila spectrin chain that are required for interchain binding in vitro were mapped. Segments 20 and 21 of the alpha chain and 2 and 3 of the beta chain are required for binding. Binding appears to be very dependent on the lateral register of segments in the two apposed chains. Domains of the nonrepetitive segments, 22 of alpha chain and 1 of beta chain, are also involved in associating the two chains. Required sequences within these nonrepetitive segments are interspersed within domains that are known to be involved in associations with other structural proteins, such as actin, and regulatory components, such as protein 4.1 and calcium (Viel, 1994).

The self-association behavior was examined in solution of one of the repeating conformational segments of Drosophila spectrin (D-alpha-14) as well as of the two-segment unit, D-alpha-14,15. In both polypeptides, there is a reversible, moderate affinity dimerization reaction. Equilibration between monomer and dimer is kinetically limited near 5 degrees C, but occurs at a measurable rate at temperatures > or = 20 degrees C. The temperature dependence for equilibration is consistent with the requirement for extensive disruption of helix-helix packing as the reaction proceeds in either direction. Hydrodynamic studies by means of sedimentation velocity confirm that in solution the C helix in the monomer of D-alpha-14 is folded back to interact with the A and B helices, and that the form of monomeric subunit observed in the crystal structure, in which the A and B helices are continuous, does not persist in the monomer in solution. Both the dimer of D-alpha-14 and the monomer of D-alpha-14,15 appear to be twice the length of the D-alpha-14 monomer, while the frictional ration of the D-alpha-14,15 dimer is consistent with four end-to-end triple alpha-helical domains (Ralston, 1996).

A spectrin isoform termed beta H is present in Drosophila that consists of a conventional alpha spectrin subunit complexed with a novel high molecular weight beta subunit (430 kD). The native alpha beta H molecule binds actin filaments with high affinity and has a typical spectrin morphology except that it is longer than most other spectrin isoforms and includes two knoblike structures, attributed to a unique domain of the beta H subunit. Beta H is encoded by a different gene than the previously described Drosophila beta-spectrin subunit, but it shows sequence similarity to beta-spectrin as well as vertebrate dystrophin, a component of the membrane skeleton in muscle. By size and sequence similarity, dystrophin is more similar to this newly described beta-spectrin isoform (beta H) than to other members of the spectrin gene family, such as alpha-Spectrin and alpha-actinin (Dubreuil, 1990).

ß Heavy-spectrin is a unique beta-spectrin from Drosophila melanogaster that is closer in size to dystrophin than to other beta-spectrin members of the spectrin/alpha-actinin/dystrophin gene super-family. Both the subcellular localization of the beta Heavy-spectrin protein and the tissue distribution of beta Heavy-spectrin transcript accumulation change dramatically during embryonic development. Maternally loaded protein is uniformly distributed around the plasma membrane of the egg. During cellularization it is associated with the invaginating furrow canals and also with a region of the lateral membranes at the apices of the forming cells (apicolateral). During gastrulation the apicolateral staining remains and is joined by a new apical cap (or plate) of ß Heavy-spectrin in areas where morphogenetic movements occur. These locations include the ventral and cephalic furrows and the posterior midgut invagination. Thus, dynamic rearrangement of the subcellular distribution of the protein is precisely coordinated with changes in cell shape. In the developing embryo, after the germ band is fully extended, zygotic message and protein accumulate in the musculature, epidermis, hindgut, and trachea. ß Heavy-spectrin in the epidermis, hindgut, and trachea is apically localized, while the protein in the somatic and visceral musculature is not obviously polarized. The distribution of beta Heavy-spectrin suggests roles in establishing an apicolateral membrane domain that is known to be rich in intercellular junctions and in establishing a unique membrane domain associated with contractile processes (Thomas, 1994).

The Crumbs protein of Drosophila is an integral membrane protein, with 30 EGF-like and 4 laminin A G domain-like repeats in its extracellular segment, which is expressed on the apical plasma membrane of all ectodermally derived epithelia. The insertion of Crumbs into the plasma membrane is necessary and sufficient to confer apical character on a membrane domain. Overexpression of crumbs results in an enormous expansion of the apical plasma membrane and the concomitant reduction of the basolateral domain. This is followed by the redistribution of beta Heavy-spectrin, a component of the membrane cytoskeleton, and by the ectopic deposition of cuticle and other apical components into these areas. Strikingly, overexpression of the membrane-bound cytoplasmic portion of Crumbs alone is sufficient to produce this dominant phenotype. These results suggest that crumbs plays a key role in specifying the apical plasma membrane domain of ectodermal epithelial cells of Drosophila (Wodarz, A., 1995).

Oogenesis in Drosophila takes place within germline cysts that support polarized transport through ring canals interconnecting their 15 nurse cells and single oocyte. Developing cystocytes are spanned by a large cytoplasmic structure (known as the fusome) that has been postulated to help form ring canals and determine the pattern of nurse cell-oocyte interconnections. The adducin-like HTS product and alpha-Spectrin are molecular components of fusomes. A related structure has been discovered in germline stem cells, and associations between fusomes and cystocyte centrosomes have also been documented. hts mutations completely eliminate fusomes, causing the formation of abnormal cysts containing a reduced number of cells. These results imply that Drosophila fusomes are required for ovarian cyst formation and suggest that membrane skeletal proteins regulate cystocyte divisions (Lin, 1994).

The proteins encoded by polar-localized mRNAs play an important role in cell fate specification along the anteroposterior axis of the Drosophila embryo. The only maternally synthesized mRNA known previously to be localized to the anterior cortex of both the oocyte and the early embryo is the Bicoid mRNA. A second mRNA is localized to the anterior pole of the oocyte and early embryo. This mRNA encodes a Drosophila homolog of mammalian adducin, a membrane-cytoskeleton-associated protein that promotes the assembly of the spectrin-actin network. A comparison of the spatial distribution of Bicoid and Adducin-like transcripts in the maternal-effect RNA-localization mutants exuperantia, swallow, and staufen indicates different genetic requirements for proper localization of these two mRNAs to the anterior pole of the oocyte and early embryo (Ding, 1993).

Protein 4.1 functions to link transmembrane proteins with the underlying spectrin/actin cytoskeleton. Drosophila 4.1 is localized to the septate junctions of epithelial cells and is encoded by the coracle gene, a new locus whose primary mutant phenotype is a failure in dorsal closure. In addition, coracle mutations dominantly suppress Ellipse, a hypermorphic allele of the Drosophila EGF-receptor homolog. These data indicate that D4.1 is associated with the septate junction, and suggest that it may play a role in cell-cell interactions that are essential for normal development (Fehon, 1994).

Distribution of two family 4.1 proteins, Expanded and Coracle, are disrupted in discs large mutants. Loss of Discs large also affects the distribution of Fasciclin III and neuroglian, two transmembrane proteins thought to be involved in cell adhesion. These results suggest that DLG serves as a binding protein, linking cell surface receptors with the cytoskeleton via family 4.1 proteins (Woods, 1996)

Neuroglian can transmit positional information directly to Ankyrin and thereby polarize its distribution in Drosophila tissue culture cells. Ankyrin is not normally associated with the plasma membrane of S2 tissue culture cells. Upon expression of an inducible neuroglian minigene, however, cells aggregate into large clusters and Ankyrin becomes concentrated at sites of cell-cell contact. Spectrin is also recruited to sites of cell contact in response to neuroglian expression. The accumulation of Ankyrin at cell contacts requires the presence of the cytoplasmic domain of Neuroglian. Whereas Ankyrin is strictly associated with sites of cell-cell contact, Neuroglian is more broadly distributed over the cell surface. A direct interaction between Neuroglian and Ankyrin can be demonstrated using yeast two-hybrid analysis. Thus, Neuroglian appears to be activated by extracellular adhesion so that ankyrin and the membrane skeleton selectively associate with sites of contact and not with other regions of the plasma membrane (Dubreuil, 1996).

Spectrin has been proposed to function as a sorting machine that concentrates interacting proteins such as the Na,K ATPase within specialized plasma membrane domains of polarized cells. However, little direct evidence to support this model has been obtained. A genetic approach has been used to directly test the requirement for the ß subunit of the alphaß spectrin molecule in morphogenesis and function of epithelial cells in Drosophila. ß Spectrin mutations are lethal during late embryonic/early larval development and they produced subtle defects in midgut morphology and stomach acid secretion. The polarized distributions of alphaßH spectrin and ankyrin are not significantly altered in ß spectrin mutants, indicating that the two isoforms of Drosophila spectrin assemble independent of one another, and that ankyrin is upstream of alphaß spectrin in the spectrin assembly pathway. In contrast, ß spectrin mutations have a striking effect on the basolateral accumulation of the Na,K ATPase. The results establish a role for ß spectrin in determining the subcellular distribution of the Na,K ATPase and, unexpectedly, this role is independent of alpha spectrin (Dubreuil, 2000).

The cellular consequences of the ß spectrin mutations were analyzed in epithelial cells of the larval middle midgut. The copper cells in particular require alpha spectrin for their normal differentiation and function in stomach acid secretion. These cells have a peculiar invaginated morphology in which the apical cell surface is tucked within the cell body. The invagination is connected to the gut lumen through a pore formed by neighboring interstitial cells. Smooth septate junctions occupy the apicolateral contact region between copper cells and interstitial cells, forming a collar that surrounds the pore. Despite their unusual morphology, copper cells exhibit many of the properties of conventional epithelia. The apical surface, extending inward from the collar, displays densely packed microvilli toward the gut lumen. The basolateral domain, including the apicolateral collar, is the site of contact with neighboring cells in the epithelial sheet. All plasma membrane markers that have been examined so far are segregated within either the apical or the basolateral domain (Dubreuil, 2000).

Double-label immunofluorescent staining was used to compare the relative distributions of ankyrin and ß spectrin within the basolateral membrane domain of copper cells. ß spectrin, encoded in this study by an epitope-tagged transgene, is detected throughout the basolateral region in first instar larvae, and is especially concentrated in the apicolateral collar. Ankyrin is also concentrated at the collar, with only faint staining visible in the rest of the basolateral domain. Ankyrin staining appears as comma shapes on either side of the entrance to the apical invagination in favorable optical sections. As larvae grow and copper cells increase in size, ankyrin staining becomes visible throughout the basolateral domain, although ankyrin remains relatively concentrated at the apicolateral contacts. These results are consistent with a role for ankyrin in attaching alphaß spectrin to the plasma membrane, both at the apicolateral contact region and throughout the rest of the basolateral domain of copper cells. However, ankyrin staining outside of the apicolateral collar is relatively weak and near the threshold of detection in first instar larvae (Dubreuil, 2000).

Ankyrin staining was used to monitor the effect of ß spectrin mutations on cell pattern in the middle midgut epithelium. The en face pattern of ankyrin staining in the first instar middle midgut provides a convenient map of cell outlines in the epithelial sheet. The apicolateral contacts between wild-type copper cells and interstitial cells appear as small rings interconnected by lines that represent contacts between adjacent interstitial cells. Ankyrin staining reveals the same overall pattern of cell contacts in ß-spec^em6 and ß-spec^em21 male first instar larvae, indicating that development of the cell pattern is normal in the mutants and that the association of ankyrin with the plasma membrane is independent of ß spectrin. However, whereas the ring-shaped profiles are consistently small in the posterior region of the wild-type middle midgut, the rings from ß spectrin mutants are large and irregular. In some cases, the diameter of the pore remains relatively small, whereas the zone of ankyrin staining is broadened into a wide collar. In other cases, the thickness of the ring of ankyrin staining remains narrow, but the pore size is expanded as in most anterior copper cells of the wild-type middle midgut. Thus, the size and shape of the apicolateral contact between copper cells and interstitial cells is dependent on ß spectrin function (Dubreuil, 2000).

The effects of ß spectrin mutations on alpha and ß_H spectrin assembly were also examined by immunofluorescence. ß spectrin appears to be required for efficient basolateral targeting of the alpha subunit, but not for the apical assembly of alphaß_H spectrin (Dubreuil, 2000).

The effect of ß spectrin mutations on plasma membrane polarity was monitored by staining for the Na,K ATPase, which is normally concentrated in the basolateral membrane domain of copper cells. In wild-type larvae, the en face Na,K ATPase pattern appears as rings representing the copper cell basolateral domain. Optical sections through the central region of the gut have revealed that the basolateral staining of copper cells extends up to the point of apicolateral contact with interstitial cells. A fine reticular pattern of cytoplasmic staining is also observed, but most of the signal is associated with the plasma membrane. A striking change in the distribution of Na,K ATPase staining is observed in ß-spec^em6 mutants. The nature of the change is dependent on the region of the gut examined. The most anterior copper cells exhibit occasional plasma membrane staining, although in most cells the Na,K ATPase is associated with intracellular compartments. In the most posterior cells, Na,K ATPase staining is typically punctate and irregular. The large puncta of staining are often closely apposed to the nucleus, indicating that the Na,K ATPase is intracellular rather than clumped at the plasma membrane. Copper cells in between these two regions exhibit very weak staining that is not obviously associated with the plasma membrane. Thus, it appears that there are different fates of the Na,K ATPase within copper cell subpopulations in the ß spectrin mutants. However, in all cases, the normal accumulation of Na,K ATPase at the plasma membrane is severely perturbed by the loss of ß spectrin function (Dubreuil, 2000).

The physiological role of copper cells is to secrete stomach acid. Acid secretion is easily monitored by feeding larvae with yeast paste containing bromphenol blue. The dye changes from a brilliant blue color (pH > 4) to a bright yellow color (pH < 2.35) in the copper cell region of wild-type larvae. In between these pH ranges, the dye exhibits a variable green color. alpha Spectrin mutants lack detectable midgut acidification, presumably because of defects within the apical or basolateral domain, or both, of copper cells. Most control larvae exhibit strong (pH < 2.3) midgut acidification. A significant fraction of the ß-spec^em6 and ß-spec^em15 mutant larvae also exhibit acidification below pH 2.3. Interestingly, the ß-spec^em12 and ß-spec^em21 mutants, which express large truncated fragments of ß spectrin, are less efficient in acid secretion than the mutants that altogether lack detectable ß spectrin. Nevertheless, the effect on midgut acidification in ß spectrin mutants is small compared with the previously described null alpha spectrin mutants. Based on these results it is concluded that the ß spectrin mutations have little effect on plasma membrane integrity or the activity of copper cells, despite their effects on cell morphology and Na,K ATPase localization (Dubreuil, 2000).

Lava lamp (Lva) is nostalgically named for the apical/basal movements observed in the Golgi bodies of Lva mutants during the process of cellularization, reminiscent of the motion of droplets in a lava lamp (Sisson, 2000). Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, a biochemical screen was conducted for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. A novel MMAP, Lava Lamp is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Functional analysis shows that cellularization is dramatically inhibited upon injecting embryos with anti-Lva antibodies (IgG and Fab). In addition, brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. It is suggested that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. These results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion (Sisson, 2000).

Blastoderm embryos were treated with fixatives that efficiently preserve both cortical F-actin and MTs and prepared for immunofluorescence. In syncytial and cellularizing blastoderms, Lva, alpha-Spectrin, and CLIP190 colocalize to large cytoplasmic puncta, some of which are found closely apposed to the furrow front in cellularizing blastoderms. Additional CLIP190-specific puncta are also observed at furrow tips. While most Lva is associated with puncta, low levels are seen throughout the cortical cytoplasm. alpha-Spectrin and CLIP190 are also observed elsewhere within the cortical cytoplasm. Although alpha-Spectrin forms stable complexes with ß- and ß_H-Spectrin, punctate ß- or ß_H-Spectrin localization is not observed. Instead, these proteins are found throughout the cortical cytoplasm. Minimal Spectrin localization is seen along the PM (Sisson, 2000).

Because the punctate colocalization pattern of Lva, alpha-Spectrin, and CLIP190 is reminiscent of that observed for two cis-Golgi markers, ß-coatomer (ß-COP) and a 120-kD integral membrane protein (p120), their distribution was examined relative to the three MMAPs. Indeed, double immunofluorescence shows that p120 and Lva colocalize. alpha-Spectrin and CLIP190 colocalize with the Golgi markers, as with Lva (Sisson, 2000).

Although a special isoform of mammalian ß-Spectrin (ßIsigma-Spectrin) has been shown to associate with Golgi, Golgi-associated Spectrin has not been previously described in Drosophila, where Spectrins have been shown primarily at the PM. To rule out fixation artifacts, three different preparative conditions were tested. In each case, the punctate localization for alpha-Spectrin, Lva, and the Golgi markers was clearly observed, while very weak Spectrin localization was found at the PM. Together, these results suggest that Spectrins reside on both the PM and Golgi bodies of cellularizing blastoderms, and that each Spectrin population is differentially sensitive to the immunofluorescence preparative conditions used (Sisson, 2000).

To assess whether Lva interacts with other proteins, the native size of Lva was compared with other microfilament/microtubule-associated proteins (MMAPs). The S100 and the final protein (MMAP) fraction were passed separately over a gel filtration column, and fractions were assayed by immunoblot. alpha-Spectrin has been previously shown to copurify with ß- and ß_H-Spectrin in two stable heterotetrameric complexes (alpha2ß2 and alpha2ß_H2, respectively) and coimmunoprecipitates with ß- and ß_H-Spectrin, but information on association of Lva with alpha-Spectrin only is presented for simplicity. Immunoblots show that in both the S100 and the MMAP fraction, Lva, CLIP190, alphaß-, and alphaß_H-Spectrin coelute from the column with native molecular weights larger then their predicted molecular weights, indicating that each protein exists in large, stable complexes (Sisson, 2000).

Lva, CLIP190, alphaß-, and alphaß_H-Spectrin also cofractionate over two consecutive F-actin affinity columns, indicating that each protein is associated with a stable F-actin-binding activity. S100 was passed over an F-actin column; ABPs were eluted as before, dialyzed against F-actin-binding buffer, and the soluble protein was passed over a second F-actin column. Immunoblots show that Lva, alphaß_H-Spectrin, CLIP190, and Anillin each efficiently rebind the second column, while KLP3A does not. The initial binding and subsequent rebinding of alphaß-Spectrin to F-actin is relatively weak. Lva, Spectrins, and CLIP190 elute with a common peak in fractions (Sisson, 2000).

Because Lva, CLIP190, and Spectrins, cofractionate in the above experiments, an assessment was made of whether they interact by immunoprecipitation (IP). Anti-Lva antibody efficiently precipitates Lva protein, and co-IPs alphaß_H- and alphaß-Spectrin, as well as CLIP190. Although the anti-alpha-Spectrin and anti-CLIP190 antibodies are inefficient at precipitating their respective antigens, both corroborate the co-IPs obtained with the anti-Lva antibody. Because antibodies to alpha-Spectrin and CLIP190 do not co-IP one another, it is likely that Lva associates with Spectrins and CLIP190 separately (Sisson, 2000).

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