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 link: Entrez Gene
alpha-Spec orthologs: Biolitmine
Recent literature
Deng, H., Yang, L., Wen, P., Lei, H., Blount, P. and Pan, D. (2020). Spectrin couples cell shape, cortical tension, and Hippo signaling in retinal epithelial morphogenesis. J Cell Biol 219(4). PubMed ID: 32328630
Although extracellular force has a profound effect on cell shape, cytoskeleton tension, and cell proliferation through the Hippo signaling effector Yki/YAP/TAZ, how intracellular force regulates these processes remains poorly understood. This study reports an essential role for spectrin in specifying cell shape by transmitting intracellular actomyosin force to cell membrane. While activation of myosin II in Drosophila melanogaster pupal retina leads to increased cortical tension, apical constriction, and Yki-mediated hyperplasia, spectrin mutant cells, despite showing myosin II activation and Yki-mediated hyperplasia, paradoxically display decreased cortical tension and expanded apical area. Mechanistically, this study shows that spectrin is required for tethering cortical F-actin to cell membrane domains outside the adherens junctions (AJs). Thus, in the absence of spectrin, the weakened attachment of cortical F-actin to plasma membrane results in a failure to transmit actomyosin force to cell membrane, causing an expansion of apical surfaces. These results uncover an essential mechanism that couples cell shape, cortical tension, and Hippo signaling and highlight the importance of non-AJ membrane domains in dictating cell shape in tissue morphogenesis.
Bouska, M. J. and Bai, H. (2021). Long noncoding RNA regulation of spermatogenesis via the spectrin cytoskeleton in Drosophila. G3 (Bethesda) 11(5). PubMed ID: 33720346
The spectrin cytoskeleton has been shown to be critical in diverse processes such as axon development and degeneration, myoblast fusion, and spermatogenesis. Spectrin can be modulated in a tissue specific manner through junctional protein complexes, however, it has not been shown that long noncoding RNAs (lncRNAs) interact with and modulate spectrin. This study provides evidence of a lncRNA CR45362 that interacts with α-Spectrin, is required for spermatid nuclear bundling during Drosophila spermatogenesis. It was observed that CR45362 showed high expression in the cyst cells at the basal testis, and CRISPR-mediated knockout of CR45362 led to sterile male, unbundled spermatid nuclei, and disrupted actin cones. Through chromatin isolation by RNA precipitation-mass spectrometry (ChIRP-MS), actin-spectrin cytoskeletal components were observed tophysically interact with the lncRNA CR45362. Genetic screening on identified cytoskeletal factors revealed that cyst cell-specific knockdown of α-Spectrin phenocopied CR45362 mutants and resulted in spermatid nuclear bundle defects. Consistently, CR45362 knockout disrupted the co-localization of α-Spectrin and spermatid nuclear bundles in the head cyst cells at the basal testis. Thus, this study uncovered a novel lncRNA CR45362 that interacts with α-Spectrin to stabilize spermatid nuclear bundles during spermatid maturation.
Molina, E., Cataldo, V. F., Eggers, C., Munoz-Madrid, V. and Glavic, A. (2022). p53 Related Protein Kinase is Required for Arp2/3-Dependent Actin Dynamics of Hemocytes in Drosophila melanogaster. Front Cell Dev Biol 10: 859105. PubMed ID: 35721516
Cells extend membrane protrusions like lamellipodia and filopodia from the leading edge to sense, to move and to form new contacts. The Arp2/3 complex sustains lamellipodia formation, and in conjunction with the actomyosin contractile system, provides mechanical strength to the cell. Drosophila p53-related protein kinase (Prpk), a Tsc5p ortholog, has been described as essential for cell growth and proliferation. In addition, Prpk interacts with proteins associated to actin filament dynamics such as α-spectrin and the Arp2/3 complex subunit Arpc4. This study investigated the role of Prpk in cell shape changes, specifically regarding actin filament dynamics and membrane protrusion formation. Reductions in Prpk alter cell shape and the structure of lamellipodia, mimicking the phenotypes evoked by Arp2/3 complex deficiencies. Prpk co-localize and co-immunoprecipitates with the Arp2/3 complex subunit Arpc1 and with the small GTPase Rab35. Importantly, expression of Rab35, known by its ability to recruit upstream regulators of the Arp2/3 complex, could rescue the Prpk knockdown phenotypes. Finally, the requirement of Prpk was evaluated in different developmental contexts, where it was shown to be essential for correct Arp2/3 complex distribution and actin dynamics required for hemocytes migration, recruitment, and phagocytosis during immune response.

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).

beta-III-spectrin spinocerebellar ataxia type 5 mutation reveals a dominant cytoskeletal mechanism that underlies dendritic arborization

A spinocerebellar ataxia type 5 (SCA5) L253P mutation in the actin-binding domain (ABD) of β-III-spectrin causes high-affinity actin binding and decreased thermal stability in vitro. This study shows in mammalian cells, at physiological temperature, that the mutant ABD retains high-affinity actin binding. Significantly, this study provides evidence that the mutation alters the mobility and recruitment of β-III-spectrin in mammalian cells, pointing to a potential disease mechanism. To explore this mechanism, a Drosophila SCA5 model was developed in which an equivalent mutant Drosophila β-spectrin is expressed in neurons that extend complex dendritic arbors, such as Purkinje cells, targeted in SCA5 pathogenesis. The mutation causes a proximal shift in arborization coincident with decreased β-spectrin localization in distal dendrites. SCA5 β-spectrin dominantly mislocalizes α-spectrin and ankyrin-2, components of the endogenous spectrin cytoskeleton. The data suggest that high-affinity actin binding by SCA5 β-spectrin interferes with spectrin-actin cytoskeleton dynamics, leading to a loss of a cytoskeletal mechanism in distal dendrites required for dendrite stabilization and arbor outgrowth (Avery, 2017).

Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that causes gait and limb ataxia, slurred speech, and abnormal eye movements. SCA5 stems from autosomal dominant mutations in the SPTBN2 gene that encodes β-III-spectrin, a cytoskeletal protein predominantly expressed in the brain and enriched in cerebellar Purkinje cells. A necessary function of β-III-spectrin in Purkinje cells was demonstrated by β-III-spectrin-null mice, which show ataxic phenotypes and decreased Purkinje cell dendritic arborization. β-III-spectrin consists of an N-terminal actin-binding domain (ABD) followed by 17 spectrin-repeat domains and a C-terminal pleckstrin homology domain. SCA5 mutations that result in single amino acid substitutions or small in-frame deletions have been identified in the ABD and neighboring spectrin-repeat domains. In a SCA5 mouse model, expression in Purkinje cells of a β-III-spectrin transgene containing a spectrin-repeat domain mutation, E532_M544del, causes ataxic phenotypes and thinning of the cerebellar molecular layer that contains Purkinje cell dendrites. This suggests that the cellular mechanism underlying SCA5 pathogenesis is a Purkinje cell deficit linked to the loss of dendritic arborization (Avery, 2017).

The functional unit of β-III-spectrin is considered to be a heterotetrameric complex containing two β-spectrin subunits and two α-spectrin subunits. Through the β-spectrin subunits the spectrin heterotetramer binds and cross-links actin filaments. Multiple β-spectrin protein isoforms have been shown to form a spectrin-actin cytoskeletal structure that lines the plasma membrane of axons and dendrites. The spectrin-actin lattice is a highly conserved neuronal structure identified in the axons of a broad range of neuron types in mammals and in invertebrates, including Drosophila. A spectrin-actin lattice containing β-III-spectrin, or the homolog β-II-spectrin, was identified in the dendrites of hippocampal neurons. Recent studies suggest that the dendritic spectrin-actin cytoskeleton is a ubiquitous feature of neurons, prominent in both dendritic shafts and spines. The widespread localization of β-III-spectrin within the Purkinje cell dendritic arbor suggests that similar spectrin-actin interactions are important for Purkinje cell dendritic function (Avery, 2017).

The spectrin-actin cytoskeleton functions to organize integral membrane proteins through the spectrin adaptor ankyrin and provides mechanical stability to neuronal processes. A form of erythrocyte ankyrin, ankyrin-R, is expressed in Purkinje cells and appears to be required for Purkinje cell health and normal motor function. A hypomorphic ankyrin-R mutation, termed 'normoblastosis', causes Purkinje cell degeneration and ataxia in mice. The subcellular localization of ankyrin-R in the Purkinje cell soma and dendrites mirrors the distribution of β-III-spectrin, and recently β-III-spectrin was shown to physically interact with ankyrin-R. In β-III-spectrin-null mice, ankyrin-R is present in the soma but absent in Purkinje cell dendrites, suggesting that Purkinje cell degeneration and ataxic phenotypes observed in the absence of β-III-spectrin may be linked to a loss of ankyrin-R function in dendrites. A SCA5 mutation that results in a leucine 253-to-proline (L253P) substitution in the ABD of β-III-spectrin causes ectopically expressed β-III-spectrin and ankyrin-R to colocalize internally in HEK293T cells, in contrast to control cells where wild-type β-III-spectrin colocalizes with ankyrin-R at the plasma membrane. This previous study suggests that neurotoxicity caused by the L253P mutation may be connected to spectrin mislocalization and the concomitant mislocalization of ankyrin-R. However, it has not been established whether the L253P mutation affects the dendritic localization of β-spectrin or ankyrin proteins in any neuronal system (Avery, 2017).

This report extends an analysis of the β-III-spectrin L253P mutation, which was recently demonstrated to cause an ∼1,000-fold increase in the binding affinity of the β-III-spectrin ABD for actin filaments in vitro. The mutation is also destabilizing in vitro, causing the ABD to begin to unfold near physiological temperature. Given these results, a key question with important implications for the SCA5 disease mechanism is whether the previously described mislocalization of L253P β-III-spectrin in mammalian cells is driven by a loss of ABD-binding activity, as originally proposed, or instead is the consequence of increased ABD-binding activity. To address the mechanistic basis of β-III-spectrin dysfunction, this study has characterized the L235P mutant protein behavior in mammalian cells. In addition, a Drosophila SCA5 model was generated in which a Drosophila β-spectrin transgene containing the equivalent mutation is conditionally expressed in dendritic arborization sensory neurons. This study used the Drosophila model to analyze the impact of the mutation on dendritic morphology, an aspect of Purkinje cell dysfunction that potentially underlies SCA5 pathology. In living, fully intact larvae, the consequence were examined of the ABD mutation on dendritic arborization, β-spectrin subcellular localization, and the functional interaction of β-spectrin and ankyrin in dendrites (Avery, 2017).

The morphology of dendritic arbors dictates the connectivity of neuronal networks, integrating inputs and propagating signals. The question of how neurons modulate dendritic morphology is of keen interest in the study of neuronal function and neurodegeneration. For example, the molecular and cell biological mechanisms that control branch stability and remodeling within a dendritic field remain largely elusive. This report describes the consequence of a SCA5 mutation on the binding of β-III-spectrin to actin in mammalian cells and leveraged the Drosophila model system to reveal the impact of the SCA5 disease mutation on the neuronal spectrin-actin cytoskeleton and dendritic arborization. This work identifies an important cytoskeletal mechanism in distal dendrites required for formation of large, complex arbors, critical to the function of Purkinje cells targeted in hereditary ataxias (Avery, 2017).

The data suggest that high-affinity actin binding acts dominantly as a driver of L253P β-III-spectrin neurotoxicity by impacting the dynamics of the spectrin-actin network. Drosophila SCA5 β-spectrin containing the equivalent L253P mutation accumulates in the da neuron soma and is absent in distal dendritic regions, in contrast to wild-type β-spectrin that localizes throughout the arbor. In the axons of mammalian neurons the spectrin-actin lattice initially forms near the soma and propagates distally, suggesting that the loss of Drosophila SCA5 β-spectrin in distal dendrites reflects a defect in expansion of the spectrin-actin cytoskeleton from the soma into dendrites. Such an expansion defect may be a consequence of a slow dissociation rate that is typical of high-affinity molecular interactions. Specifically, high-affinity actin binding caused by the mutation may limit the pool of free β-spectrin molecules available to be recruited to an expanding cytoskeleton. Like the loss of Drosophila SCA5 β-spectrin in da neuron dendritic extensions, a reduction was observed of human L253P β-III-spectrin in HEK293T cell plasma membrane protrusions. The absence of L253P β-III-spectrin in filopodium-like and lamellipodium-like extensions, despite abundant localization elsewhere at the plasma membrane, suggests a partitioning between structurally dynamic and stable membrane regions. This partitioning supports the idea that high-affinity actin binding reduces the availability of β-III-spectrin to be recruited to newly formed membrane structures. The data predict that high-affinity binding of L253P β-III-spectrin to actin filaments within the neuronal spectrin-actin lattice negatively impacts Purkinje cell arborization and function by impeding the expansion of the spectrin-actin cytoskeleton in dynamically growing or remodeling dendritic branches and spines (Avery, 2017).

In addition to increasing actin-binding affinity, the L253P mutation destabilizes β-III-spectrin, causing the ABD to begin to unfold near physiological temperature in vitro. This denaturation raised the possibility that cellular phenotypes in mammalian cells are the consequence of ABD protein unfolding and loss of ABD-binding activity rather than elevated ABD-binding activity. Experiments do not support this interpretation, showing instead through co-IP assays that the mutant ABD retains high-affinity actin binding in cells. Indeed, in cultured mammalian cells, protein unfolding reflected in minor degradation products was detected only when the mutant ABD was highly overexpressed. Significantly, the high-affinity actin binding observed for the L253P mutation is mimicked by the alternative substitution, L253A, which, like the L253P mutation, is predicted to disrupt the normal hydrophobic contacts of leucine 253 in the β-III-spectrin ABD. In this case, no degradation of the L253A mutant is detected, and the increased protein stability of the L253A mutation was confirmed in vitro. In light of these results, the observation that the L253A and L253P mutations cause the same β-III-spectrin subcellular localization phenotypes in HEK293T cells supports the conclusion that the behavior of L253P β-III-spectrin is driven by increased ABD-binding activity. These results support the hypothesis that high-affinity actin binding contributes to the L253P β-III-spectrin neurotoxicity that underlies SCA5 pathology (Avery, 2017).

How do the L253P and L253A substitutions account for elevated actin-binding affinity? The location of the mutations in the CH2 domain is consistent with a suspected regulatory role for the domain in mediating actin binding. Biochemical studies of the isolated CH domains of β-spectrin or of the related α-actinin ABD previously documented actin-binding activity for the CH1 domain but not for the CH2 domain. Confinement of binding activity to the CH1 domain is further supported by a structural model for α-actinin ABD-actin complexes in which only a single CH domain is bound to actin filaments. Consistent with the idea that the L253P mutation disrupts a CH2 domain regulatory function, leucine 253 in the CH2 domain is predicted to interface with the CH1 domain and physically bridge the two domains through hydrophobic contacts. The decrease in hydrophobicity introduced by the L253P or L253A substitution is thus predicted to disrupt inter-CH domain contacts and relieve CH2 inhibition. Significantly, disease-causing mutations located in the CH2 domain of α-actinin or filamin also increase actin-binding affinity (Avery, 2017).

In addition to binding filaments of conventional actin, the β-III-spectrin ABD also interacts with ARP1, a component of the dynactin complex that facilitates transport mediated by microtubule motors. Consistent with an ARP1 interaction, a previous study reported that expression of SCA5 β-spectrin in Drosophila motoneurons impairs axonal transport. Given the ~75% similarity in actin and ARP1 primary structures, it is predicted that the L253P mutation will similarly enhance the binding of β-III-spectrin to ARP1. Current studies have not directly addressed this prediction. However, in a previous study, a bimolecular fluorescence complementation assay conducted in HEK293T cells overexpressing ARP1 concluded that the L253P mutation reduces the interaction of β-III-spectrin ABD with ARP1. Nonetheless, a direct test of how L253P β-III-spectrin impacts ARP1 binding is lacking. Indeed, ARP1-binding studies are not straightforward; the native ARP1 filament is difficult to purify, ARP1-specific antibodies are not available, and there is a strong propensity for ARP1 to form nonnative structures when overexpressed in cells. Further experiments are needed to fully understand the impact of the L253P mutation on ARP1 binding and intracellular vesicular transport (Avery, 2017).

In Drosophila, the lone homolog of β-III-spectrin, β-spectrin, localizes to both dendrites and axons, where the function of the spectrin cytoskeleton has been extensively studied. The impact of SCA5/L246P β-spectrin expression on synaptic organization at the neuromuscular junction (NMJ) has been reported. Spectrin RNAi also disrupts synaptic bouton organization but further leads to NMJ retraction, a phenotype not observed in SCA5 motoneurons. The current work shows that SCA5 β-spectrin is absent not only in distal dendrites but also at the axon terminus of da neurons. Thus, SCA5 β-spectrin may dominantly mislocalize the spectrin cytoskeleton not only in dendrites but also in distal axons. In light of these findings, the reduced size of the Drosophila NMJ reported in motor neurons expressing SCA5 β-spectrin may in part reflect a disruption of the spectrin cytoskeleton and associated ankyrin-2 (Avery, 2017).

This work points to a molecular mechanism in the somatodendritic compartment of neurons that enables the formation of large, complex dendritic arbors. As the dendritic arbor grows, dynamic actin assembly in the distal dendrites drives the formation of new terminal branches. It is suggested that during arbor growth expansion of the spectrin-actin cytoskeleton is required to stabilize terminal branches and allow for continued expansion of the arbor. To explain the mutant SCA5 arbor phenotypes, it is proposed that high-affinity binding of the mutant β-spectrin decreases spectrin-actin dynamics and consequently constrains expansion of the spectrin-actin cytoskeleton and stabilization of growing dendrites. The spectrin cytoskeleton has similarly been implicated in axonal growth and stabilization of synaptic junctions. In support of this cytoskeletal mechanism regulating dendritic arbor stability and potentially underlying SCA5 pathology, this study shows that the loss of β-spectrin, as well as ankyrin-2, in the distal dendrites of Drosophila da neurons correlates with a proximal shift in dendritic branching. Importantly, expression of mutant SCA5 β-spectrin and ankyrin-2 RNAi resulted in similar dendritic arborization defects, and SCA5 β-spectrin causes a loss of ankyrin-2 XL in distal dendrites. This study characterizes a progressive elimination of distal dendrites at segmental boundaries in SCA5 arbors. Moreover, lacking expansion of the spectrin-actin cytoskeleton in terminal dendrites, dynamic actin-based assembly drives complex terminal branching at the periphery of SCA5 arbors. However, the stability of the SCA5 terminal branching is compromised, and the outward growth of the arbor field is defective. One possibility is that impaired expansion of the spectrin-actin cytoskeleton and loss of ankyrin-2 in dendrites impacts localization of neuroglian, a cell-adhesion molecule required for arborization and which may mediate stability of dendritic branching in Drosophila da neurons. In Purkinje cells, it is predicted that L253P β-III-spectrin will similarly impair expansion of the spectrin-actin lattice, disrupting dendritic localization of critical membrane proteins ankyrin-R, EAAT4, and mGluR1α, and in consequence promoting defects in arborization and postsynaptic signaling that characterize SCA5 pathology (Avery, 2017).

Significantly, this model for the impact of a SCA5 mutation on cytoskeletal dynamics and distal arborization is similar to a disease model proposed for autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) in which a decrease in mitochondrial dynamics is suggested to disrupt distal Purkinje cell arborization. Like the mislocalization of SCA5 β-spectrin in da neurons, loss of function of the ARSACS disease protein sacsin, a mitochondrial protein, causes mitochondria to accumulate in the soma and proximal dendrites but fail to reach distal dendrites in mammalian neurons. Neurons such as Purkinje cells and da neurons that extend complex arbors appear to be especially vulnerable to disruptions to pathways in distal dendrites that support arborization, and this sensitivity possibly explains the cerebellar specificity of SCA5 pathology (Avery, 2017).

Fat body-derived Spz5 remotely facilitates tumor-suppressive cell competition through Toll-6-&alph-a;-Spectrin axis-mediated Hippo activation

Tumor-suppressive cell competition is an evolutionarily conserved process that selectively removes precancerous cells to maintain tissue homeostasis. Using the polarity-deficiency-induced cell competition model in Drosophila, this study identify Toll-6, a Toll-like receptor family member, as a driver of tension-mediated cell competition through α-Spectrin (α-Spec)-Yorkie (Yki) cascade. Toll-6 aggregates along the boundary between wild-type and polarity-deficient clones, where Toll-6 physically interacts with the cytoskeleton network protein α-Spec to increase mechanical tension, resulting in actomyosin-dependent Hippo pathway activation and the elimination of scrib mutant cells. Furthermore, this study show that Spz5 secreted from fat body, the key innate organ in fly, facilitates the elimination of scrib clones by binding to Toll-6. These findings uncover mechanisms by which fat bodies remotely regulate tumor-suppressive cell competition of polarity-deficient tumors through inter-organ crosstalk and identified the Toll-6-α-Spec axis as an essential guardian that prevents tumorigenesis via tension-mediated cell elimination (Kong, 2022).

Epithelial cells possess intrinsic mechanisms to outcompete and eliminate early precancerous cells to maintain homeostasis. For instance, in a mouse model of esophageal carcinogenesis, the majority of newly developed tumor clones are eliminated through cell competition by adjacent normal epithelium. Similarly, surveillance mechanisms also exist in Drosophila epithelium to actively remove oncogenic clones composed of polarity-deficient cells. Genetic studies in flies have uncovered numerous mechanisms that regulate tumor-suppressive cell competition, including c-Jun-N-terminal kinase (JNK) signaling activation-mediated cell elimination, direct cell-cell interaction, secreted factors from epithelial cells, and inter-organ crosstalk between insulin-producing cells and precancerous cell-bearing discs (Kong, 2022).

Initially identified in Drosophila, the Hippo pathway is an evolutionarily conserved signaling cascade that plays crucial roles in various physiological and pathological contexts, ranging from tumor progression and embryogenesis to stem cell renewal and immune surveillance. Apart from its well-established roles in controlling cell proliferation and cell death, numerous studies have proved that the Hippo pathway also functions as a key mechanotransducer to sense mechanical changes in the microenvironment. Despite the identification of multiple essential mechanosensitive signaling molecules including RAP2, MAP4K, Agrin, and Spectrin, it remains poorly understood how mechanical stimuli are transmitted from plasma membrane localized receptors to activate Hippo signaling cascade-mediated cellular responses, especially in intact tissues. This study, through a genetic screen in Drosophila, uncovered a regulatory mechanism whereby mechanical tension drives tumor-suppressive cell competition though the Hippo pathway. The genetic and biochemistry data uncovered Toll-6 as an essential regulator of Hippo signaling and further identified α-Spec as an essential downstream component that regulates cell competition via tension-mediated actomyosin activation. Moreover, this study further demonstrated that inter-organ communication is critical for the removal of precancerous cells at a systemic level and discovered fat body (FB)-derived Spz5 as a crucial ligand (Kong, 2022).

This study demonstrated that polarity-deficient oncogenic clones are eliminated through tension-dependent cell competition and has identified Toll-6 as a key membrane receptor that physically interacts and acts through α-Spec to activate the Hippo pathway. It has long been recognized that both extrinsic cues such as ligands and intrinsic factors such as stiffness and cell-cell contact-mediated mechanical cues can determine cell fate and affect cell proliferation, yet relatively little is known about how the cytoskeleton system contributes to the elimination of precancerous cells during cell competition in vivo. The data show that both &alpha-Spec and Rho1, two essential cytoskeleton regulators, accumulate and facilitate the elimination of scrib clones. In addition, α-Spec as a crucial linker that bridges Toll-6 activation-induced tensional changes to cytosolic Hippo pathway activation. Interestingly, studies in the mammalian system showed that RhoA (human Rho1 ortholog) is responsible for mechanical force-induced cell extrusion. Thus, a similar tension-mediated cell-elimination mechanism might exist in the mammalian system to actively remove unfitted precancerous cells (Kong, 2022).

TLRs play critical roles in the innate immune response. The Drosophila genome encodes nine TLRs, of which only Toll (Tl/Toll-1) has a clear function in innate immunity. Interestingly, a paradoxical role of Tl in regulating cell competition has been reported. Activation of Tl in polarity-deficient clones suppresses the elimination of losers, whereas in the Myc-induced cell competition model, increased Tl activity accelerates the elimination of losers. Apart from Tl, Toll-2, Toll-3, Toll-7, Toll-8, and Toll-9 have been implicated in regulating cell competition in different contexts, while Toll-4 and Toll-5 have little effect. It is noteworthy that none of above studies has investigated the role of Toll-6 in cell competition. The current data not only reveal Toll-6 as a crucial regulator of tumor-suppressive cell competition but also show how the mechanical tension-mediated Hippo cascade is initiated from the cell membrane through the Toll-6-&alpha-Spec axis. Notably, this study found that Toll-6 was not required for Myc-induced cell competition. Given that TLRs are highly conserved in vertebrates and the elimination of scrib-depleted cells also exists in the mammalian system, further experiments are necessary to determine whether analogous mechanisms exist in mammals and humans to regulate mechanical tension-induced, Hippo pathway-mediated tumor-suppressive cell competition (Kong, 2022).

Inter-organ communication is essential for proper development and homeostasis maintenance of multicellular organisms under both physiological and pathological conditions. The tumor progression process is also shaped by the interactions between tumor and other organs, including the immune system. Recent studies in Drosophila have provided insightful understanding of the complex crosstalk between organs during tumorigenesis. The FB is the major immune organ of Drosophila, and it has been shown that intestinal tumor progression or abdominal tumor transplantation promotes the wasting behavior of FBs. The current findings that the transcription of spz5 is increased in the FB from scrib clone bearing larvae to facilitate tumor-suppressive cell competition may provide an in vivo mechanistic understanding of the inter-organ communications between FBs and remotely colonized precancerous clones. Together, the ism) around the boundary between losers and winners, which recruits α-Spec and provokes Hippo pathway-dependent elimination of scrib-/- clones. Meanwhile, the presence of scrib-/- loser cells in the eye disc will trigger a systemic effect on the distal organs, including FBs, which results in the transcription upregulation and secretion of Spz5, in turn forming a feedforward loop to reinforce the tumor-suppressive cell competition by binding to Toll-6 (Kong, 2022).

Although biochemical and genetic data demonstrate that Toll-6 physically interacts with α-Spec and that α-Spec is required for the elimination of scrib clones, these experiments were unable to explain the molecular mechanisms by which Toll-6 recruits α-Spec and initiates the downstream signaling transduction. Another limitation is that because the substantial analysis relies heavily on genetics to infer mechanism, enough rigorous biochemistry data was not included to prove how the binding of Toll-6 with α-Spec triggers Hippo signaling activation. Additionally, this study found that FB-derived Spz5 is essential for the elimination of scrib clones through inter-organ communications, but it is not understood completely how the spz5 mRNA level is upregulated systematically in the FBs of larvae that bear scrib mutant clones. Future work will be required to dissect the transcriptome changes of FBs upon precancerous clone induction in distal organs. Finally, this study showed that Spz5 acts through Toll-6 to regulate cell competition, and it is known that Spz5 can bind to other TLRs to regulate both cell death and survival through a three-tier mechanism (Foldi, 2017), suggesting that Spz5 can trigger intracellular signal transduction through ligand receptor binding. Nonetheless, a question that remains unsolved is why a signaling network that relays cell mechanical properties (Toll-6-α-Spec axis) should be regulated by a chemical ligand/receptor interaction; it would be interesting to further explore the underlying mechanisms (Kong, 2022).


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: 12 September 2022 

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