alpha Spectrin: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - alpha Spectrin

Synonyms -

Cytological map position - 62B2--62B7

Function - Docking and structural protein

Keyword(s) - cytoskeleton

Symbol - alpha-Spec

FlyBase ID:FBgn0250789

Genetic map position - 3-[1.4]

Classification - spectrin

Cellular location - cytoplasm



NCBI links: Precomputed BLAST | Entrez Gene | UniGene
BIOLOGICAL OVERVIEW

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
  2. The involvement of spectrin in oogenesis (Spectrin is involved in germline cell division and differentiation in the Drosophila ovary).
  3. The involvement of spectrin in epithelial cell polarity and integrity

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).

Alpha-Spectrin and integrins act together to regulate actomyosin and columnarization, and to maintain a mono-layered follicular epithelium

This study reports the role of Spectrins during epithelia morphogenesis using the Drosophila follicular epithelium (FE). α-Spectrin and β-Spectrin are shown to be are essential to maintain a mono-layered FE, but, contrary to previous work, Spectrins are not required to control proliferation. Furthermore, spectrin mutant cells show differentiation and polarity defects only in the ectopic layers of stratified epithelia, similar to integrin mutants. These results identify α-Spectrin and integrins as novel regulators of apical constriction-independent cell elongation, as α-spectrin and integrin cells fail to columnarize. Finally, increasing and reducing the activity of the Rho1-myosin-II pathway enhances and decreases multi-layering of α-spectrin cells, respectively. Similarly, higher myosin-II activity enhances the integrin multi-layering phenotype. This work identifies a primary role for α-Spectrin in controlling cell shape, perhaps by modulating actomyosin. All together, it is suggested that a functional Spectrin-Integrin complex is essential to balance adequate forces, in order to maintain a mono-layered epithelium (Ng, 2016).

This study found that in the germline α-Spec is not a major regulator of the Hippo pathway. Mutations in hippo, β-Spec or α-Spec result in a stratified FE, but contrary to previous interpretations, and unlike Hippo, spectrins are not required for the FCs to exit mitosis. The suggestion that Spec mutant FCs over-proliferate is thought to be an over-interpretation from the multilayering phenotype, as α-Spec cells were not checked for mitotic markers in a previous report. Again unlike hippo, α-Spec mutant PFCs only show defects in differentiation when they are located in the ectopic layers of the stratified FE, and oocyte polarity is largely unaffected in mutant egg chambers. It was recently shown that a β-Spec allele with a premature stop codon at amino-acid 1046 partially phenocopies hippo, with strong defects in FE integrity, actin organization and oocyte polarity. The null β-SpecG113 mutant allele behaves similarly to α-Spec mutants, showing Hnt defects mainly in ectopic layers, but Fas3 mislocalization in monolayers. More importantly, β-SpecG113 FCCs exit mitosis properly. The differences observed between the two β-Spec alleles are likely to be due to the fact that β-SpecG113 is a null allele (Ng, 2016).

In conclusion, α-Spec and β-Spec FCCs do not phenocopy hippo mutants when the cells are part of a monolayer, and they seem to adopt a partial hippo-like differentiation phenotype only when positioned at ectopic layers, even though α-Spec and β-Spec cells never divide after S6. Thus, the main function of the spectrin cytoskeleton in FCs is not proliferation control or regulation of the Hippo pathway, although an interaction between spectrins and Hippo might occur once the FCs are within an aberrantly organized FE. The function of spectrins in FCs is in contrast with other tissues, where α- and β-Spec appear to regulate growth through Hippo (Ng, 2016).

Similar to Hippo, α-Spec and β-Spec are required for the FE to maintain a monolayer. There is an increase in the multilayering phenotype in egg chambers with large clones from S3/6 to S7/8 and 100%, respectively. Also, the presence of control cells in α-Spec mosaic epithelia aids the mutant cells to maintain a monolayer from S6, as there is a higher percentage of S7-9 egg chambers with multilayers when the FE contains large α-Spec clones than when the mutant clone is only at the posterior end. The control of FE architecture appears to be mediated by the lateral spectrin network. Loss of α-Spec seems to disrupt both lateral (α/β) and apical (α/βH) spectrin-based membrane skeleton (SBMS) in the FE, as β and βH subunits are no longer localized laterally and apically in α-Spec cells, but no multilayering was reported for βH-Spec egg chambers, in which a loss of apical α-Spec was observed, suggesting that the loss of the lateral α/β is responsible for the FE stratification. Also, βH-Spec is mislocalized in sosie mutants, but the FE architecture is maintained (Ng, 2016).

Incipient SJs are first detected between the FCs with the completion of proliferation at S6. This study shows that the localization of several SJ components is affected in α-Spec FCCs, suggesting that spectrins are required for proper SJ formation. This is further supported by other observations. (1) Fas3 localization is affected in β-Spec FCCs. (2) Neuroglian (an SJ component) is required for maintaining the stability of the FE. (3) The reduction of both α- and β-Spec leads to mislocalization of Dlg, Neuroglian and Fas2 in neuromuscular junctions. (4) It has been suggested that the SBMS and ankyrin associate with SJ components (Ng, 2016).

As the mislocalization of SJ components in Spec mutant FCCs is observed in monolayers, and thus prior to the onset of stratification, it is speculated that Spec-dependent distribution of SJ components might contribute to the Spec function in the epithelium. This idea is supported by Crumbs overexpression, which leads to defects in SJs and ZA, and multilayering of the ectoderm cells, and by dpak (Pak - FlyBase) FCs, which mislocalize Fas3 and show multilayering and columnarization defects. Furthermore, the aberrant accumulation of Fas2 at the lateral membrane of Tao FCs prevented membrane shrinking in the cuboidal-to-squamous transition. However, fas3, fas2 and cora mutant cells do not show shape defects or multilayering. Thus, if SJ components contribute to the α-Spec phenotype at all, it might be not because they are absent in α-Spec mutant cells, but because they are not properly distributed (Ng, 2016).

Transitions between squamous, cuboidal and columnar epithelial cell shapes are common during development, and contribute to the morphogenesis of tissues. This study demonstrates a cell-autonomous role for α-Spec in promoting the cuboidal-to-columnar shape transition of the FCs. It is important to point out that the FE undergoes lateral elongation without apical constriction, which might allow phenotypes to be interpreted in a simpler manner. This morphogenetic FC behavior is similar to that of vertebrate neuroepithelia, where cell elongation precedes apical constriction, and it would be interesting to study the function of Spec in the columnarization of these cells (Ng, 2016).

Although the molecular mechanism of apical constriction-independent cell elongation is unknown, a primary role for the SBMS is thought to lie in facilitating changes in cell shape, which is further supported by the cell shape defects in α-Spec gut epithelia, perhaps by contributing to the proper distribution of adhesion molecules. This function of the SBMS in membrane biology is conserved in other cells, as spectrins stabilize the plasma membrane during blastoderm cellularization, and control photoreceptor morphogenesis through the modulation of membrane domains. The spectrin cytoskeleton might also impact on FE columnarization by interacting with the actomyosin cytoskeleton. It is known that apical-basal elongation in cytoplasmic actin-binding protein drebrin E (drebrin 1) depleted human Caco2 cells is impaired, as a possible consequence of the lack of interaction between drebrin E with spectrins and actomyosin. Also, the elongation of neuroepithelial cells depends on the assembly of an actomyosin network in the apical junctional complex, regardless of whether cells are constricting or not . In Drosophila wing discs, the Rho1-Myosin II pathway at the apicolateral membrane seem to regulate the cuboidal-to-columnar shape transition, whereas in the germline, Rok and sqh mutant FCs fail to adopt a normal shape. Finally, SBMS seems to modulate cortical actomyosin contractility in the eye, and possibly in the FE. Together, these data suggest that Myosin II activity is aberrant in α-Spec mutant FCs, contributing to defects in columnarization and FE architecture (Ng, 2016).

Increasing Rho1 and Sqh activities enhances the Spec multilayering phenotype, whereas reducing Myosin II activity decreases it. In addition to this functional link between the SBMS and the Rho-Myosin pathway, this study also shows that mys cells fail to columnarize, and that an extra copy of sqh increases the mys multilayering phenotype. It has been shown that integrins regulate the Rho-Myosin pathway to induce actomyosin-generated forces. Thus, as is the case for spectrins, integrins might also control cell shape and epithelia morphogenesis by modulating the actomyosin activity (Ng, 2016).

How the SBMS and integrins might modulate actomyosin is unknown, and one possible mechanism is by regulating Myosin II activity directly. However, an alternative mechanism is proposed. Spectrins can bind F-actin, and integrins and spectrins interact with proteins involved in the association of F-actin with the membrane. Furthermore, α-Spec and integrins regulate the actin cytoskeleton through Rac. Previous studies have shown that both β-Spec and mys mutant FCs display similar defects in the basal level of F-actin, which are recapitulated in α-Spec mutant cells. Thus, any defects in actin organization in mys and Spec mutant FCs could in turn result in defects in the activity of Myosin II (Ng, 2016).

Regardless of whether integrins and spectrins regulate F-actin or myosin, or both, spectrins and integrins might act together. The SH3 domain of α-Spec interacts with Testin ortholog (Tes), a component of integrin-dependent focal adhesions, and mammalian αII-Spec stabilizes β3-integrin anchorage, suggesting α-Spec as a physical link between focal adhesions and F-actin. In the FE, this study observed that α-Spec and αPS1 colocalize in the lateral, and possibly apical, membrane. In addition, it was shown that the localization of α-Spec in mys clones, and the localization of βPS in α-Spec mutant clones, is majorly unaffected. Furthermore, expression of a constitutively active integrin that reduces multilayering of mys FCCs, failed to rescue α-Spec multilayers. Thus, it is proposed that α-Spec and integrins act independently of each other, but as part of the same functional complex regulating the actomyosin cytoskeleton and tissue architecture (Ng, 2016).

An early event following oncogenic mutations in an epithelium is the escape of the daughter cells from the monolayered epithelium, forming disorganized masses. Spindle orientation has been linked to tumor-like growth in various tissues, and this study found that there is a good correlation between spindle misorientation and 'tumor-like masses' at the FE: hippo, mys and α-Spec FCCs show misaligned spindles and severe multilayering, whereas Notch FCCs, which overproliferate, do not show multilayering or spindle orientation defects. However, perpendicular divisions alone are insufficient to promote stratification, and a mechanism, depending on lateral cell-cell adhesions, is in place to avoid multilayering as a sole consequence of spindle misorientation. It is proposed that spindle misorientation contributes to FE disorganization, but that this 'safeguard' mechanism is somehow inactive in hippo, mys and Spec mutant FCCs. What other aspect of the mutant phenotypes might then be linked to multilayering? A clue might come from the Spec mutant and mys FCCs. First, there is an increase in the α-Spec multilayers after S6, when both FCs and egg chambers undergo various morphogenetic changes. Second, the volume of the germline surrounded by large α-Spec FCCs appears smaller. And third, Myosin II activity is increased in α-Spec and mys mutant cells. In this interpretation of the results, a proper distribution of Myosin II activity in a Spec- and integrin-dependent manner allows the right amount of forces to be distributed across the membrane and the epithelium. Thus, it is possible that proper cell-cell interactions, adequate force balance and precise spindle orientation are key to maintaining a monolayered epithelium, especially upon the mechanical stress induced by morphogenesis (Ng, 2016).


PROTEIN STRUCTURE

Amino Acids - 2415

Structural Domains

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).

β-Spectrin functions independently of Ankyrin to regulate the establishment and maintenance of axon connections in the Drosophila embryonic CNS

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).


alpha Spectrin: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 6 MAR 97 

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