alpha Spectrin
The alpha-spectrin gene is essential for larval survival and development. P-element minigene rescue and sequence analysis were used to identify the alpha-spectrin gene as the l(3)dre3 complementation group of the Dras-Roughened-ecdysoneless region of chromosome 3. Germ line transformants carrying an alpha-spectrin cDNA, whose expression is driven by the ubiquitin promoter, fully rescues the first to second instar lethality characteristic of the l(3)dre3 alleles. The molecular defects in two gamma-ray-induced alleles were identified. One of these mutations, which results in second instar lethality, contains a 73-bp deletion in alpha-Spectrin segment 22 (starting at amino acid residue 2312), producing a premature stop codon between the two EF hands found in this segment. The second mutation, which results in first instar lethality, contains a 20 base pair deletion in the middle of segment 1 (at amino acid residue 92), resulting in a premature stop codon. Examination of the spectrin-deficient larvae reveals a loss of contact between epithelial cells of the gut and disruption of cell-substratum interactions. The most pronounced morphological change is seen in tissues of complex cellular architecture such as the middle midgut where a loss of cell contact between cup-shaped cuprophilic cells and neighboring interstitial cells is accompanied by disorganization of the cuprophilic cell brush borders. This examination of spectrin deficient larvae suggests that an important role of non-erythroid spectrin is to stabilize cell to cell interactions that are critical for the maintenance of cell shape and subcellular organization within tissues (Lee, 1993).
During Drosophila oogenesis, developing germline cysts are spanned by a large cytoplasmic structure called a fusome, containing alpha-Spectrin and the adducin-like product of the hu-li tai shao (hts) gene. Fusomes contain two additional membrane skeletal proteins: beta-spectrin and ankyrin. hts is required for cyst formation and oocyte differentiation; the role of the fusome itself, however, and the organization and function of its other components, remains unclear. By generating clones of alpha Spectrin-deficient cells in the ovary, it can be shown that alpha-Spectrin is also required for cyst formation and oocyte differentiation. Its role in each process is distinct from that of HTS protein. Furthermore, alpha-Spectrin is required for these processes in germline cells, but not in the follicle cells that surround each cyst. The organization of membrane skeletal proteins is more dependent on alpha-Spectrin in the fusome than at the plasma membrane in other cells. These results suggest that the fusome and its associated membrane skeleton play a central role in regulating the divisions and differentiation of cyst cells (de Cuevas, 1996).
To understand the role of the spectrin-based membrane skeleton in generating epithelial polarity, the distribution of membrane skeletal components was characterized in Drosophila ovarian follicle cells and in somatic clones of mutant cells that lack alpha Spectrin. Immunolocalization data reveal that wild-type follicle cells contain two populations of spectrin heterodimers: a network of alphabeta heterodimers concentrated on the lateral plasma membrane and an alphabetaH population targeted to the apical surface. Induction of somatic clones lacking alpha-Spectrin leads to follicle cell hyperplasia. Surprisingly, elimination of alpha-Spectrin from follicle cells does not appear to prevent the assembly of conventional beta-spectrin and ankyrin at the lateral domain of the follicle cell plasma membrane. However, the alpha-subunit is essential for the correct localization of betaH-spectrin to the apical surface. 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 integrity and proliferation (Lee, 1997).
Oligonucleotide primers based on the spectrin-binding domain of human brain ankyrin were used to amplify Drosophila genomic DNA. A cloned 184-bp PCR product was used to isolate Drosophila ankyrin cDNAs. Ankyrin cDNA probes detect a 5.5-kb transcript from embryonic poly(A)+ RNA and a single polytene chromosome locus (101F-102A). The cDNA sequence encodes a 170-kDa protein that is 53% identical to human brain ankyrin (Ank2). Antibodies directed against a recombinant fragment of Drosophila ankyrin react with a 170-kDa polypeptide from Drosophila embryos, larvae, S2 cells, and adult flies. The ankyrin antibody coimmunoprecipitated alpha- and beta-spectrin with ankyrin in detergent extracts of Drosophila embryo membranes. Antibodies against Drosophila ankyrin, alpha-Spectrin and beta-spectrin were used to detect these proteins in wild-type and alpha-Spectrin-mutant larvae. alpha-Spectrin levels are greatly diminished in mutant larvae, but levels of ankyrin and beta-spectrin are indistinguishable from wild type. The persistence of ankyrin and beta-spectrin may explain the relatively mild phenotype of alpha-Spectrin mutants during early Drosophila development (Dubreuil, 1994).
Mutations in Drosophila alpha spectrin cause larval lethality and defects in cell shape and adhesion. An examination was made of the effects on development and function of the larval midgut for two lethal alpha spectrin alleles (alpha-specrg41 and alpha-specrg35). Homozygous null alpha-specrg41-mutant larvae exhibit a striking defect in middle midgut acidification. Domains of acidification and alkalinization in the gut were identified by feeding yeast, containing pH-sensitive dyes, to wild-type instar larve. In contrast, many homozygous alpha-specrg35 mutants are capable of acidification, indicating partial function of the truncated alpha-specrg35 product. Acidification is also blocked by a mutation in the labial gene, which is required for differentiation of cuprophilic cells in the midgut, suggesting that these cells secrete acid. Two isoforms of spectrin (alphabeta and alphabetaH) are segregated within the basolateral and apical domains of cuprophilic cells, respectively. The most conspicuous defect in cuprophilic cells from labial and alpha spectrin mutants is in morphogenesis of the invaginated apical domain, although basolateral defects may also contribute to the acidification phenotype. Acid secretion in vertebrate systems is thought to involve the polarized activities of apical proton pumps and basolateral anion exchangers, both of which interact with spectrin. It is proposed that the alpha-specrg41 mutation in Drosophila interferes with the polarized activities of homologous molecules that drive acid secretion in cuprophilic cells. Three possible explanations for the acid secretion defect in alpha-spectrin mutant larvae are proposed: (1) Spectrin may serve a support role in the apical domain of cuprophilic cells. Loss of alphabetaH spectrin function may lead to a collapse of the apical domain, thereby compromising all apical membrane activities, including proton pumps. (2) The alphabetaH isoform of spectrin may interact directly with proton pumps in the apical domain. (3) Spectrin may interact with and stabilize a basolateral activity, such as an anion-exchange protein, that is indirectly required for apical proton secretion (Dubreuil, 1998).
Spectrins are plasma membrane-associated cytoskeletal proteins
implicated in several aspects of synaptic development and function, including presynaptic vesicle tethering and postsynaptic receptor aggregation. To test these hypotheses, Drosophila mutants lacking either alpha- or ß-spectrin were characterized. The Drosophila genome contains only one
alpha-spectrin and one conventional ß-spectrin
gene, making it an ideal system to genetically manipulate
spectrin levels and examine the resulting synaptic alterations. Both
spectrin proteins are strongly expressed in the Drosophila neuromusculature and highly enriched at the glutamatergic neuromuscular junction. Protein null alpha- and ß-spectrin mutants are embryonic lethal and display severely disrupted neurotransmission without altered morphological synaptogenesis. Contrary to current models, the absence of spectrins does not alter postsynaptic glutamate receptor field function or the ultrastructural localization of presynaptic vesicles. However, the subcellular localization of numerous synaptic proteins is disrupted, suggesting that the defects in presynaptic neurotransmitter release may be attributable to inappropriate assembly, transport, or
localization of proteins required for synaptic function (Featherstone, 2001).
Using antibodies specific for Drosophila spectrins, the
neuromuscular localization of both alpha- and ß-spectrin was examined. Both
alpha-spectrin and ß-spectrin are found in
presynaptic axons proximal to the NMJ and in the periphery of
presynaptic boutons. Although alpha-spectrin staining is typically
weaker, most of the alpha- and ß-spectrin staining in the NMJ appears to be
colocalized. alpha- and ß-spectrin immunoreactivity is also strong throughout muscle. Double-labeling with antibodies against ß-spectrin and the presynaptic protein CSP was examined. Much of the
ß-spectrin and CSP staining is not colocalized, suggesting that the
majority of spectrin protein is associated with the periphery of the
presynaptic membrane and/or dense membrane foldings of the postsynaptic
subsynaptic reticulum. It is concluded from this immunohistochemistry that
both alpha- and ß-spectrin are present at the wild-type
Drosophila NMJ, in both presynaptic and postsynaptic cells (Featherstone, 2001).
NMJ morphology in the spectrin mutants is normal by light and
electron microscopy, yet neurotransmitter release is severely disrupted. In other (non-neuronal) cell types, spectrins have been
proposed to capture and maintain proteins in distinct
membrane-associated domains, especially at sites of cell-cell
interaction. At synapses, proper function requires precise assembly and alignment of the molecular machinery required for synaptic vesicle fusion and
recycling. If this machinery is mislocalized or incorrectly assembled,
it would not be surprising to find a synaptic defect such as is observed in alpha- and ß-spectrin mutants. Although there is no
method by which to test whether the in vivo
submicrometer assembly of proteins is appropriate in spectrin
mutants, it was determined whether synaptic proteins are polarized and
properly localized to the NMJ. In epithelial cells, disruption of
protein polarization attributable to the absence of spectrin is visible
by immunohistochemistry and confocal light microscopy. The same techniques were used to determine whether spectrins play
a similar role in protein compartmentalization at synapses (Featherstone, 2001).
CSP is present in both vesicular membrane-associated and cytosolic fractions of presynaptic boutons; CSP staining normally appears as tightly localized presynaptic puncta. DLG is a plasma membrane-associated PDZ [postsynaptic density-95(PSD-95)/DLG/zona occludens-1] domain protein with 60% homology to PSD-95 that is tightly localized to both presynaptic and postsynaptic membranes. In wild-type embryos, CSP and DLG staining in the body wall neuromusculature is restricted to tightly defined puncta at the NMJ; little or no staining is visible in
either the presynaptic nerve axon or nonsynaptic muscle membrane. In
both alpha- and ß-spectrin mutants, the synaptic
localization of both presynaptic CSP and postsynaptic DLG is
dramatically perturbed. It is concluded that, in both alpha- and
ß-spectrin mutants, CSP is distributed abnormally throughout presynaptic axons, and DLG is distributed abnormally throughout muscle cells. Neither protein appears properly polarized and
localized to the NMJ boutons in spectrin mutants (Featherstone, 2001).
In addition to CSP and DLG, the staining patterns of
several other synaptic proteins, including synaptotagmin, synapsin, and
syntaxin, were examined. Synaptotagmin is a transmembrane protein normally restricted to synaptic vesicles. Synapsin is a spectrin-interacting phosphoprotein that is associated with the presynaptic actin cytoskeleton at synaptic boutons. Syntaxin is a transmembrane protein normally present in
presynaptic membrane, including both axons and synaptic boutons. All of these proteins, like CSP and DLG, show severely disrupted subcellular localization in both alpha- and ß-spectrin mutant embryos (Featherstone, 2001).
Protein distribution was quantified by
comparing staining intensity in NMJ boutons with staining intensity
outside the synapse. In wild-type embryos, fluorescence intensity from each synaptic marker was significantly higher in NMJ boutons than elsewhere. Wild-type embryos showed anti-CSP fluorescence that was 30.75 times higher in boutons than in nerve. In contrast, anti-syntaxin fluorescence in wild-type embryos was only 3.41 times higher in boutons than in nerve. These observations are consistent with the fact that CSP is strongly restricted to synaptic boutons, whereas syntaxin is present throughout the neuronal membrane and only weakly polarized to boutons. This raw ratio represents a measure of both protein localization and antibody quality because poor antibodies might be expected to lower the ratio because of high nonspecific immunoreactivity (high background) and/or reduced specific immunoreactivity. The synaptic/nonsynaptic immunoreactivity ratios in both alpha- and ß-spectrin mutants for CSP, DLG, synaptotagmin, synapsin, and syntaxin were each significantly reduced compared with wild type. It is concluded
from these results that synaptic proteins are improperly polarized and
localized in both alpha- and ß-spectrin mutants (Featherstone, 2001).
Prevailing models place spectrin downstream of ankyrin in a pathway of assembly and function in polarized cells. A transgene rescue strategy was used in Drosophila to test contributions of four specific functional sites in beta spectrin to its assembly and function. (1) Removal of the pleckstrin homology domain blocked polarized spectrin assembly in midgut epithelial cells and was usually lethal. (2) A point mutation in the tetramer formation site, modeled after a hereditary elliptocytosis mutation in human erythrocyte spectrin, had no detectable effect on function. (3) Replacement of repetitive segments 4-11 of beta spectrin with repeats 2-9 of alpha spectrin abolished function but did not prevent polarized assembly. (4) Removal of the putative ankyrin-binding site had an unexpectedly mild phenotype with no detectable effect on spectrin targeting to the plasma membrane. The results suggest an alternate pathway in which spectrin directs ankyrin assembly and in which some important functions of spectrin are independent of ankyrin (Das, 2006; full text of article).
The results presented here provide several novel insights into the assembly and function of spectrin. There are two general models to explain the assembly of the spectrin cytoskeleton in polarized cells. Both models incorporate ankyrin as an adaptor that couples integral membrane proteins to the spectrin cytoskeleton. In the first case, assembly begins with a protein receptor that recruits ankyrin to a specific region of the plasma membrane (Das, 2006).
In this model, ankyrin serves two distinct roles: (1) as an adaptor that couples spectrin to a cue for assembly and (2) as an adaptor that links interacting proteins such as the Na,K ATPase and voltage-dependent sodium channels to the preassembled spectrin cytoskeleton. In the 'spectrin first' model, ankyrin functions as an adaptor that couples interacting membrane proteins to a preassembled spectrin cytoskeleton. In this model, the site of assembly is determined directly by spectrin and the role of ankyrin is to couple the diverse membrane proteins that interact with ankyrin to that site (Das, 2006).
The results of the present study provide the first direct evidence supporting the spectrin-first model. Ankyrin assembly at the basolateral membrane domain of copper cells was dependent on spectrin. Spectrin in turn was dependent on the PH domain of the ß subunit in copper cells and on an as-yet-unidentified signal in salivary gland cells. There are examples of ankyrin-independent assembly of spectrin in other systems: (1) During erythrocyte differentiation, ankyrin assembly occurs after the stable assembly of spectrin. (2) A related observation is that spectrin assembly appears remarkably normal in erythrocytes that lack band 3, the major membrane receptor for ankyrin in the erythrocyte. Thus, even in the best-characterized membrane model, it has been difficult to ascertain the sequence of events that leads to spectrin assembly. (3) Targeting of the alphaßH isoforms of spectrin is thought to occur by an ankyrin-independent mechanism. These spectrins have unusually large and divergent ß subunits and are targeted to the apical membrane domain of polarized epithelia in D. melanogaster. Together with the current results, it appears that targeting to the plasma membrane is a shared property of spectrins, whether or not they interact with ankyrin (Das, 2006).
PH domains have been detected in hundreds of different proteins and in many cases they have physiological roles in binding to phosphoinositides. Structures have been determined for spectrin PH domains from mammals and Drosophila, and binding to phosphoinositides has been demonstrated. The PH domain of spectrins does not have the expected lipid specificity of a protein that mediates phosphatidylinositol (PtdIns)-3-kinase signaling. Although the structure of the spectrin PH domain appears to be compatible with binding to PtdIns(3,4,5)P3, a more likely binding partner in vivo is PtdIns(4,5)P2. The six residues that contact PtdIns(4,5)P2 are conserved in the Drosophila PH domain and out of 37 amino acid identities among PH domains from mammalian ßI, ßII, ßIII, and ßIV spectrins, 31 are conserved in Drosophila. Overall, there was 44% identity between the fly PH domain and mammalian PH domains. For comparison, there was a mean of 62% identity between the PH domains of the four mammalian ß spectrin isoforms. Interestingly, in comparisons of full-length sequences, there was greater identity between Drosophila ß spectrin and human ßII spectrin (57%) than between human ßII and ßIV (53%) or between ßI and ßIV (49%). Therefore, it appears likely that the functions of ß spectrin, including the lipid-binding function of the PH domain, are conserved between Drosophila and mammals (Das, 2006).
The PH domain of spectrin may also mediate membrane targeting through interactions with protein receptors. For example, the membrane-binding activity originally described for brain ß spectrin was protease sensitive. Interactions between PH domains and protein ligands such as protein kinase C and G protein ßgamma subunits have been reported. However, the interaction between the PH domain of a mammalian ß spectrin and ßgamma subunits was tested and found to be comparatively weak. The reason for differential targeting of the ßspecDeltaPH product in a mutant versus a wild-type background is not known. Further experiments will be necessary to determine whether mixed tetramers form and whether other sites in the molecule affect their targeting (Das, 2006).
Although the PH domain was required for the targeting of spectrin in copper cells, neither the PH domain nor ankyrin binding were required for targeting in the salivary gland. One obvious candidate to explain the recruitment observed in salivary gland is the ankyrin-independent membrane-binding site identified near the N terminus of mammalian ß spectrin. It is also possible that multiple membrane-binding sites contribute to targeting in some cells, even though the PH domain alone appears to be critical in copper cells. To help resolve these questions, it will be important in future studies to identify sites that are sufficient for membrane targeting in different cell types and to produce mutant transgenes in which multiple candidate targeting activities have been knocked out simultaneously (Das, 2006).
It was expected that loss of ankyrin binding would severely compromise the function of ß spectrin. Although there was a relatively low viability of flies rescued by the ßspecalpha13 transgene, and they often had wing phenotypes and appeared less healthy than their wild-type siblings, a surprising number of these flies survived as fertile adults. In contrast, the four loss-of-function mutations that have been characterized all exhibited embryonic lethality. The rescue result reinforces the conclusion that spectrin targeting is independent of its interaction with ankyrin and further suggests that some important aspects of spectrin function are independent of its association with ankyrin (Das, 2006).
There are several possible interpretations of ßspecalpha13 rescue that will require further experiments to resolve. (1) There may be redundant cellular mechanisms that can partially compensate for loss of the adaptor function of ankyrin. (2) The modification of the ankyrin-binding domain of ßspecalpha13 may have selectively blocked its association with DAnk1 but left Dank2 binding intact. Once appropriate tools become available it will be important to test whether DAnk2 has an essential function and whether that function depends on the ankyrin-binding site defined here. (3) There may be residual ankyrin-binding activity in ßspecalpha13 that was below the threshold of detection in these experiments. One reason to consider this possibility emerged from sequence comparisons between fly and human ß spectrins. Current structural models indicate that part of the putative ankyrin-binding site may include part of segment 15 (repeat 14), where there is striking sequence conservation. Future experiments will address whether this conservation represents part of the ankyrin-binding site or whether it is an as-yet-unidentified functional site in the ß spectrin molecule. In any case, it's apparent that most of the ankyrin 1-binding activity of spectrin was removed in ßspecalpha13. (4) Finally, it is also formally possible, although unlikely given the degree of sequence conservation, that spectrins and ankyrins in vertebrates and invertebrates have fundamentally different roles in plasma membrane organization and function (Das, 2006).
Another surprising result in the present study was the finding that the behavior of the Na,K ATPase was more closely linked to the behavior of spectrin than to ankyrin. Based on biochemical evidence showing that purified mammalian ankyrin and Na,K ATPase directly interact with one another in vitro, it has been assumed that any effects of spectrin mutations on the behavior of the Na,K ATPase in vivo were likely to be mediated through ankyrin. Current evidence raises the possibility that the Na,K ATPase may be linked to spectrin either directly or perhaps by some other indirect mechanism. It is possible that vertebrates and invertebrates evolved independent mechanisms to link the Na,K ATPase to the spectrin cytoskeleton. It was recently suggested that mammalian KCNQ potassium channels and voltage-dependent sodium channels acquired their functional interaction with ankyrin through a process of convergent molecular evolution, after the split between vertebrates and invertebrates. The conserved ankyrin-binding sequence found in these mammalian proteins is not present in their Drosophila homologues. That does not appear to be the case with the Na,K ATPase, as the amino acid sequence that mediates interaction with ankyrins in vertebrates is remarkably conserved in the Drosophila Na,K ATPase. Future studies should address the possibility that, even though there is a direct interaction between ankyrin and the Na,K ATPase in vitro, there may be an important functional interaction with mammalian spectrin in vivo that is ankyrin independent (Das, 2006).
The greatest sequence identity between Drosophila and mammalian ß spectrins occurs in the first three segments of the protein, which includes the actin-binding site and tail-end subunit interaction sites in segments 2 and 3. Another site of striking sequence conservation among ß spectrins is the partial repeat 18, where alpha and ß spectrin interact to form tetramers. Spectrin is thought to have evolved from a large single-subunit ancestor by fracturing of the coding sequence at a site within an ancestral structural repeat. A point mutation in the N-terminal partial repeat of alpha spectrin (R22S) produced a temperature-sensitive defect in spectrin tetramer formation. This study tested the effect of a comparable mutation in the ß subunit that was also identified by its role in human anemia. The W2033R mutation corresponds to the W2024 position of human erythroid ß spectrin, and that residue is also conserved in human ßII, ßIII, and ßIV, but not in ßV spectrin. This tryptophan residue was dispensable for ß spectrin function in Drosophila, presumably because it does not affect tetramer formation. Polarized targeting of the transgene product was also unaffected by the mutation. Another tryptophan at position 2061 of human erythroid ß spectrin that has been implicated in hereditary elliptocytosis is conserved in human ßII, ßIII, and ßIV spectrin, but not in Drosophila ß or in human ßV spectrin. The importance of these tryptophan residues in nonerythroid spectrin tetramer formation and function has not been tested (Das, 2006).
Relatively little is known about the function of repetitive segments 4-14 in ß spectrin. This region has more limited sequence conservation than segments near the ends of the molecule. Further studies using smaller scale modifications will be required to identify the functional sites that explain the mutant phenotype of ßspecalpha2-9. For now, this transgene helps to establish that not all functional defects in ß spectrin result in mislocalization. Thus, gene modifications that do affect protein targeting, such as ßspecDeltaPH, are probably identifying functional sites that are responsible for targeting (Das, 2006).
The domain swap strategy described in this study takes advantage of the powerful genetic tools available in Drosophila to study protein function. This approach is especially well suited to studies of a modular protein such as spectrin and has provided valuable insights into both the function and the targeting of the protein in vivo. Combined with the fact that Drosophila is a simpler system, having only three spectrin genes and two ankyrin genes, this approach should continue to provide valuable information that will be more difficult to obtain in mammalian systems. One intriguing observation in this study was the effect of the DeltaPH mutation on the size of the rare flies that survived to adulthood. This phenotype was reminiscent of Drosophila mutations that affect PH domain-containing components of the insulin/insulin-like growth factor signaling pathway. Given the importance of a phosphoinositide-binding PH domain to ß spectrin function, it will be interesting to genetically test the possibility that spectrin has a functional interaction with growth factor signaling pathways (Das, 2006).
Synaptic connections are established with characteristic, cell type-specific size and spacing. This study documents a role for the postsynaptic Spectrin skeleton in this process. Transgenic double-stranded RNA was used to selectively eliminate α-Spectrin, β-Spectrin, or Ankyrin. In the absence of postsynaptic α- or β-Spectrin, active zone size is increased and spacing is perturbed. In addition, subsynaptic muscle membranes are significantly altered. However, despite these changes, the subdivision of the synapse into active zone and periactive zone domains remains intact, both pre- and postsynaptically. Functionally, altered active zone dimensions correlate with an increase in quantal size without a change in presynaptic vesicle size. Mechanistically, β-Spectrin is required for the localization of α-Spectrin and Ankyrin to the postsynaptic membrane. Although Ankyrin is not required for the localization of the Spectrin skeleton to the neuromuscular junction, it contributes to Spectrin-mediated synapse development. A model is proposed in which a postsynaptic Spectrin-actin lattice acts as an organizing scaffold upon which pre- and postsynaptic development are arranged (Pielage, 2007).
α- and ß-Spectrin heterotetramers have a length of ~180-265 nm when purified from membrane preparations of D. melanogaster or from preparations of vertebrate erythrocytes or brain tissue. These heterotetramers can bind to short actin fragments and form a stereotypic hexagonal lattice that is linked to the plasma membrane. Interestingly, the dimension of one of these hexagonal Spectrin-actin structures closely corresponds to the size of an average active zone at the D. melanogaster NMJ. The diameter of an average active zone is ~500-600 nm, which corresponds to the size of two Spectrin heterotetramers linked by actin filaments. Therefore, the organization of a postsynaptic Spectrin-actin network into a hexagonal lattice could provide a framework upon which synapse development is organized (Pielage, 2007).
There are several possible scenarios for how a Spectrin-actin lattice could restrict synapse size. In one model, the Spectrin-actin network could stabilize glutamate receptors through direct or indirect interactions. In a second model, the lateral expansion of active zones might be constrained by the dimension of the postsynaptic, hexagonal Spectrin-actin lattice. This lattice could be used to organize the scaffolding protein Dlg, which, in turn, could confine the distribution of integral periactive zone proteins to limit the dimensions of the postsynaptic density. In support of this model, ultrastructural reconstructions revealed a significant increase in the size of active zones at the NMJ of dlg mutants. Finally, the changes in glutamate receptor cluster size and spacing could be a secondary consequence of the disruption of the postsynaptic membrane network that composes the SSR. However, other mutations that affect SSR density and organization do not cause an increase in active zone size (Pielage, 2007).
In the absence of postsynaptic α- or ß-Spectrin, the postsynaptic membrane folds (SSR) no longer tightly surround the presynaptic bouton, but are spread laterally and are severely thinned above and below the synaptic bouton. There are two possible explanations for this phenotype. One possibility is that disorganized Dlg directs the inappropriate formation of SSR. Previous studies have demonstrated that Dlg is necessary and sufficient for SSR formation in D. melanogaster, and the analysis of α- and ß-spectrin-null mutant embryos suggest that Spectrin is required for the localization of Dlg to the synapse. Alternatively, the SSR organization could be disrupted in response to muscle contractions in the absence of postsynaptic α- or ß-Spectrin. The Spectrin skeleton could serve as a protective network because of its elastic properties. The loss of mechanoprotection might explain the lateral stretch of SSR and the reduction of SSR orthogonal to the muscle surface (Pielage, 2007).
This analysis reveals interesting insights into the assembly of the Spectrin skeleton at the postsynaptic membrane. ß-Spectrin is required for the localization of α-Spectrin and Ank to the postsynaptic plasma membrane, whereas α-Spectrin is only required for the appropriate localization of ß-Spectrin and Ank within the subsynaptic reticulum (SSR). Interestingly, the knock down of postsynaptic Ank does not significantly influence the localization of α- or ß-Spectrin. This differs from previous observations in other D. melanogaster tissues and in vertebrates where Ank is required for the localization of ß-Spectrin to specific membrane domains and Spectrin then stabilizes these complexes. Thus, either the membrane localization of ß-Spectrin depends on interactions with other adaptor or transmembrane molecules or ß-Spectrin binds directly to phospholipid components of the postsynaptic membrane through its membrane association domains (Pielage, 2007).
It is interesting to speculate that Spectrin may function to determine active zone dimensions in the vertebrate nervous system. At central synapses, active zone dimensions and spacing are precisely controlled, implying molecular regulation. The importance of controlling active zone dimensions is underscored by a correlation between presynaptic release probability and active zone dimension at hippocampal synapses. A remaining question is whether the Spectrin skeleton could be involved in the activity-dependent regulation of active zone size and efficacy. It is interesting to note that the increase in active zone size at the D. melanogaster NMJ after the knock down of postsynaptic Spectrin does not lead to an apparent increase in release probability. However, given the developmental time frame of these manipulations, it is possible that homeostatic mechanisms readjust presynaptic release to baseline levels (Pielage, 2007).
The importance of the Spectrin skeleton in the vertebrate nervous system has been underscored by the recent discovery that mutations in human ß-III spectrin cause spinocerebellar ataxia type 5. Spinocerebellar ataxia type 5 results in Purkinje cell loss, cerebellar cortical atrophy, and neuromuscular defects. Importantly, one of the human mutations in ß-III spectrin is a point-mutation within the actin-binding domain that is essential for the formation of an intact Spectrin-actin network. Since the human mutations are dominant, it indicates that even small changes in the robustness of the Spectrin-actin network might result in severe neurological defects and disease (Pielage, 2007).
Cell-shape changes during development require a precise coupling of the
cytoskeleton with proteins situated in the plasma membrane. Important elements
controlling the shape of cells are the Spectrin proteins that are expressed as
a subcortical cytoskeletal meshwork linking specific membrane receptors with
F-actin fibers. Drosophila karussell
mutations affect ß-spectrin and lead to distinct axonal
patterning defects in the embryonic CNS. karussell mutants (β-spectrinkus) display a slit-sensitive axonal phenotype characterized by axonal looping in stage-13 embryos. Further analyses of individual, labeled neuroblast lineages revealed abnormally structured growth cones in these animals.
Cell-type-specific rescue experiments demonstrate that ß-Spectrin is
required autonomously and non-autonomously in cortical neurons to allow normal
axonal patterning. Within the cell, ß-Spectrin is associated with
α-Spectrin. Expression of the two genes is tightly
regulated by post-translational mechanisms. Loss of ß-Spectrin
significantly reduces levels of neuronal α-Spectrin expression, whereas
gain of ß-Spectrin leads to an increase in α-Spectrin protein
expression. Because the loss of α-spectrin does not result in an embryonic nervous system phenotype, ß-Spectrin appears to act at least partially independent of α-Spectrin to control axonal patterning (Hülsmeier, 2007; full text of article).
Rescue experiments and direct sequence analysis demonstrate that
kus encodes the Drosophila ß-Spectrin protein.
kus mutants were initially isolated due to a distinct axonal
phenotype, including ectopic CNS-midline crossing of Fasciclin II-positive
axons. To further analyze this phenotype, the progeny of single
neuroblasts were labeled in kus-mutant embryos but, despite the large number of
labeled clones, it was not possible to detect any aberrant midline crossings.
Similarly, when cell-type-specific Gal4 drivers were employed, no clear pathfinding defects could be seen across the midline. It is therefore likely that the
observed phenotype is a result of inappropriate contact between medial
Fasciclin II-expressing axons from both sides of the midline mimicking ectopic
midline crossings. Because wild-type Slit levels are required to position the
longitudinal fascicles, a reduction of slit gene dosage results, as
expected, in a further medial positioning of the longitudinal connectives,
explaining the increase in the number of ectopic midline crosses in these
animals. Similar phenotypes have also been observed by Garbe (2007) (Hülsmeier, 2007).
Interestingly, defects were found in the architecture of the neuronal growth
cones in ß-spectrinkus-mutant animals, which may
explain the general sensitivity of ß-spectrinkus-mutant neurons to guidance signals such as Slit. The enlarged growth cones detected in
ß-spectrinkus mutants correlate nicely with data on
growth cone formation after axotomy; axonal injury leads to an increased activity of the protease calpain, which cleaves Spectrin and results in the removal of the submembranous Spectrin meshwork prior to the regeneration and growth of the growth cone. In
secretory cells, the submembranous Spectrin cytoskeleton prevents the
premature fusion of vesicles with the plasma membrane.
Similarly, Spectrins may function to regulate the fusion of intracellular membrane vesicles needed to enlarge and advance the growth cone, which could explain the enlarged growth cones that were detected in ß-spectrin mutants (Hülsmeier, 2007).
Within the Drosophila nervous system, α- and ß-Spectrin
are the only Spectrins that are expressed. These two proteins form a
heterodimer in which ß-Spectrin appears to be the key determinant,
because α-Spectrin protein is only stable in the presence of
ß-Spectrin and ectopic expression of ß-Spectrin leads to a
concomitant increase in the level of α-Spectrin protein. To test whether
this regulation occurs at the level of RNA or protein, the
expression of the corresponding transcripts was determined, but no alteration was noted. It is possible that the association of α- and ß-Spectrin
blocks ubiquitination of α-Spectrin and its subsequent degradation via
the proteasome. Ubiquitination has been previously reported for α-Spectrin and may thus help to define the correct protein-expression levels (Hülsmeier, 2007).
Despite the intimate coupling of the two expression profiles, it has been
demonstrated that ß-spectrin can function independently of
α-Spectrin. During the development of the midgut, the correct
localization of the Na+/K+-ATPase requires only
ß-spectrin, but not α-spectrin, function.
Similarly, the phenotypes associated with the different spectrin mutants
isolated in this study are distinct. Whereas ß-spectrin leads to
a typical looping of CNS axons during stage 13, no abnormal axonal phenotypes
could be detected for α-spectrin alleles. Similarly, no midline phenotypes were detected for Fasciclin II-positive axons in
α-spectrinE2-26-null mutants. A possible explanation
to this phenotypic discrepancy may be the maternal contribution of
α-spectrin; however, a similarly strong maternal component has
been described for ß-spectrin. Attempts to generate
α-spectrin germline clones using the null allele rg41
failed because of an essential function of α-spectrin during
oogenesis. To circumvent this maternal α-spectrin
function, the hypomorphic α-spectrin-mutant alleles
α-specN-2, α-specP-2 or
a-spec1.3 were employed to generate germline clones using the
ovoD/FRT system. However, embryos with both impaired
maternal and zygotic α-spectrin expression displayed no nervous
system phenotype, supporting the notion that ß-spectrin acts
independent of α-Spectrin protein (Hülsmeier, 2007).
α-spectrin mutations turned out to be sensitive to
background mutations and temperature effects. In addition to the phenotypic
effects of uncharacterized background mutations, a temperature
dependence of the spectrin-mutant phenotypes and temperature-dependent
intragenic complementation of hypomorphic α-spectrin alleles were detected.
It is well-known that microtubule dynamics depend on temperature and that
microtubules depolymerize in the cold. Since microtubule stability is already compromised in α-spectrin mutants, any further destabilization might have significant effects on (neuronal) development. Alternatively, temperature sensitivity might
reflect differences in the efficacy of endocytosis. Although the molecular mechanism underlying the temperature sensitivity of spectrin mutants cannot be
pinpointed, it can be can concluded that Spectrins act as a global stabilizing protein network that coordinates a large variety of membrane receptors, including the Robo receptor that is needed to sense the Slit protein (Hülsmeier, 2007).
α- and ß-Spectrin are major components of a submembrane
cytoskeletal network connecting actin filaments to integral plasma membrane
proteins. Besides its structural role in red blood cells, the Spectrin network
is thought to function in non-erythroid cells during protein targeting and
membrane domain formation. This study demonstrates that ß-Spectrin is
required in neurons for proper midline axon guidance in the
Drosophila embryonic CNS. In ß-spectrin mutants many
axons inappropriately cross the CNS midline, suggesting a role for
ß-Spectrin in midline repulsion. Surprisingly, neither the
Ankyrin-binding nor the pleckstrin homology (PH) domains of ß-Spectrin
are required for accurate guidance decisions. α-Spectrin is dependent
upon ß-Spectrin for its normal subcellular localization and/or
maintenance, whereas α-spectrin mutants exhibit a
redistribution of ß-Spectrin to the axon scaffold. ß-spectrin mutants show specific dose-dependent genetic interactions with the midline repellent slit and its neuronal receptor roundabout (robo), but not with other guidance
molecules. The results suggest that ß-Spectrin contributes to midline
repulsion through the regulation of Slit-Robo pathway components. It is proposed
that the Spectrin network is playing a role independently of Ankyrin in the
establishment and/or maintenance of specialized membrane domains containing guidance molecules that ensure the fidelity of axon repulsion at the midline (Garbe, 2007; full text of article).
The midline guidance defects observed in embryos stained with BP102 and
Fas2 antibodies suggest that ß-Spectrin normally contributes to axonal
migration and more specifically to axon repulsion. Considering that
ß-Spectrin has been shown to modulate the behavior of interacting
membrane proteins within sub-membrane microdomains, and given that
specific dose-dependent genetic interactions with the Slit-Robo pathway were observed, these data support the idea that the Spectrin cytoskeleton modulates the behavior of
molecules that contribute to Robo repulsion. Many important signaling
molecules must be coordinated downstream of guidance receptors so that
navigating growth cones make appropriate decisions. For example, Dock, Pak and
Rac contribute to midline repulsion by forming a complex with the Robo
receptor upon Slit stimulation. Similarly, Drosophila Ena, a member of a
protein family implicated in actin cytoskeleton regulation, functions
cooperatively with Robo at a level downstream of the receptor.
Additionally, the microtubule-associated protein CLASP (Chb - FlyBase) and the
Abelson tyrosine kinase are required to induce restricted cytoskeletal events
at the leading edge of growth cones. Along these lines, ß-Spectrin may affect the
ability of comparable proteins to signal effectively in the Slit-Robo pathway.
Clearly, the Spectrin network cannot account for all Robo function, as many
ipsilateral CNS neurons are still guided properly in ß-spectrin
mutants. Mutations in ß-spectrin also enhance a robo1
null loss-of-function mutation, suggesting that the Spectrin
network probably plays an additional role in repulsion outside of Robo1
signaling. It is important to note that these data do not rule out the alternative possibility that ß-spectrin mutations may have general effects on growth cone migration. If so, then the specific interactions seen with slit and robo might reflect the fact that the Slit-Robo pathway is more susceptible to subtle perturbations than are the other guidance pathways tested (Garbe, 2007).
This analysis of the effect of ß-Spectrin mutations on the
guidance of Ap neurons suggests that ß-Spectrin is required not
for the initial pathfinding of these axons, but rather for maintaining
correctly established connections. Moreover, genetic analysis suggests
that in this context the Slit and Robo system also contributes to this
maintenance function. The observations support the idea that some level of
Slit/Robo repulsion is required continuously to keep ipsilateral axons on
their own side of the midline. A more dramatic example of pathway maintenance
has been described in C. elegans, where mutations in the Zig genes
lead to a 'flipping' of axon pathways over the midline. Additionally, in Drosophila, it has been shown that ß-Spectrin is essential for synapse stabilization. It will be interesting in the future to determine whether the maintenance
function of slit, robo and ß-Spectrin represents a
repulsive mechanism distinct from the mechanism operating during pathway
formation (Garbe, 2007).
This work demonstrates that in the absence of ß-Spectrin the stability
of α-Spectrin is decreased. Thus, it appears that hetero-tetramer
formation is required to maintain proper levels of α-Spectrin in the
nervous system. By contrast, cell body plasma membrane localization of
ß-Spectrin is unaffected in α-spectrin mutants. Consistent
with this result, it has been shown that ß-Spectrin accumulates
independently of α-Spectrin in Drosophila larvae. This
suggests that ß-Spectrin recruitment to and stability at the cell body
plasma membrane are independent of α-Spectrin, and supports the idea of
an α-Spectrin-independent role for ß-Spectrin in neurons (Garbe, 2007).
If α- and ß-Spectrin function together as a hetero-tetramer, why
then are only mild axon guidance defects observed in
α-spectrin single mutants? One explanation is that perhaps
α-Spectrin has a higher maternal component that is able to compensate
for a lack of zygotic expression. Indeed, α-spectrin mutants
survive to a later developmental stage than do ß-spectrin
mutants. However, α-spectrin mutants also exhibit increased
levels of axonally localized ß-Spectrin. Therefore, an
alternative hypothesis could be that the preferential distribution of
ß-Spectrin to axons somehow compensates for a reduction of
α-Spectrin, allowing neurons to make precise steering decisions. Lastly, and perhaps most interesting, ß-Spectrin may function independently of α-Spectrin in neurons. This idea seems plausible given that ß-Spectrin remains properly localized to the cell body plasma membrane in α-spectrin mutants (Garbe, 2007).
α-spectrin mutant embryos show an increase
in the levels of axonal ß-Spectrin, suggesting that α-Spectrin
regulates the accumulation of ß-Spectrin in axons. What does this shift
in ß-Spectrin distribution mean for the neuron? What are the signals that
target ß-Spectrin to axons and why does the localization change in
α-spectrin mutants? One possibility is that the SH3 domain of
α-Spectrin targets ß-Spectrin to the cell body plasma membrane via
a direct interaction with another protein. Another possibility is that, in the
absence of α-Spectrin, ß-Spectrin binds other axonally localized
proteins to a greater degree, thus shifting the overall distribution (Garbe, 2007).
It was originally thought that Ankyrin-G is assembled upstream of
ßIV-Spectrin at axon initial segments;
however, later reports suggested that both Ankyrin-G and ßIV-Spectrin are
required for the localization and stability of one another, as well as for the
stability of VGSCs at axon initial segments and nodes of Ranvier.
Yet, in neonatal cardiomyocytes, Ankyrin-B is required for the proper
distribution and levels of ßII-Spectrin, and an Ankyrin-B protein lacking
the Spectrin-binding domain still localizes properly. These data suggest that
Ankyrin-B localization is independent of ßII-Spectrin (Garbe, 2007).
A mutant form of ß-Spectrin missing the
Drosophila Ankyrin-binding domain (ßSpecΔank) remains
properly localized to cell bodies and axons. Importantly, the same ßSpecΔank protein used in this study correctly accumulates at the plasma membrane of copper cells yet fails to accurately target an Ank-GFP fusion protein in epithelial cells, suggesting
that Ankyrin localization depends on ß-Spectrin but not vice versa. In
addition, ßSpecΔank rescues the guidance errors seen in ß-spectrin mutants. Taken together, these data suggest that Ankyrin-binding is not essential for ß-Spectrin localization and guidance function in neurons, and that Drosophila ß-Spectrin may be assembled upstream of Ankyrin (Garbe, 2007).
It is important to note that Drosophila has another Ankyrin gene,
dank2 (ank2 -- FlyBase), which is expressed specifically in
neurons. Therefore, perhaps accurate ß-Spectrin targeting
occurs via a direct interaction with Dank2. Keeping in mind that
ßSpecΔank is missing the conserved Ankyrin1-binding site, this
logic would imply that ß-Spectrin associates with Dank2 using a different
binding site than that used for Dank1. There does appear to be an intimate
relationship between Dank2 and ß-Spectrin, since Dank2 is mislocalized and
levels are reduced in ß-spectrin mutants. Future experiments will help to establish whether Dank2 can bind ß-Spectrin at a location different than that used by Dank1. If the mutant ßSpecΔank does indeed bind to Dank2, it will be interesting to determine the functional significance of this interaction (Garbe, 2007).
It was also demonstrated that, although the PH domain of Drosophila
ß-Spectrin is not necessary for axonal localization and/or guidance
function, it is required for appropriate localization to the cell body plasma
membrane. From these experiments, it appears that axonally localized
ß-Spectrin is important for making accurate guidance decisions. In the future, it will be important to determine the region of ß-Spectrin that is essential for axonal targeting and whether this domain is important for growth cone migration (Garbe, 2007).
At nodes of Ranvier and axon initial segments, there is an intimate
relationship between the Spectrin network, Ankyrin and CAMs. For
example, in Purkinje and granule cells, Ankyrin-G localization precedes that
of neural CAMs, and is required for proper CAM cluster assembly at axon
initial segments. Other studies of CAMs have revealed that they can commonly
act as contact-mediated attractive and repulsive signals for growing axons. In
Drosophila, pre-synaptically targeted ß-spectrin RNAi
disrupts the stability and organization of CAMs such as Fas2 and Neuroglian, and
neuroglian mutants exhibit defects in motoneuron pathfinding.
Furthermore, neurotactin mutant embryos exhibit midline axon guidance
errors that appear to be similar to those observed in ß-spectrin
mutants, including ectopic midline crossing and longitudinal breaks.
Future studies should investigate whether there are functional links between
CAMs and the Spectrin network in the context of midline axon guidance (Garbe, 2007).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
alpha Spectrin: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References
Society for Developmental Biology's Web server.