The Spectrin cytoskeleton regulates the Hippo signalling pathway

The Spectrin cytoskeleton is known to be polarised in epithelial cells, yet its role remains poorly understood. Here, we show that the Spectrin cytoskeleton controls Hippo signalling. In the developing Drosophila wing and eye, loss of apical Spectrins (alpha/beta‐heavy dimers) produces tissue overgrowth and mis‐regulation of Hippo target genes, similar to loss of Crumbs (Crb) or the FERM‐domain protein Expanded (Ex). Apical beta‐heavy Spectrin binds to Ex and co‐localises with it at the apical membrane to antagonise Yki activity. Interestingly, in both the ovarian follicular epithelium and intestinal epithelium of Drosophila, apical Spectrins and Crb are dispensable for repression of Yki, while basolateral Spectrins (alpha/beta dimers) are essential. Finally, the Spectrin cytoskeleton is required to regulate the localisation of the Hippo pathway effector YAP in response to cell density human epithelial cells. Our findings identify both apical and basolateral Spectrins as regulators of Hippo signalling and suggest Spectrins as potential mechanosensors.     

Unexpected complexity in the mechanisms that target assembly of the spectrin cytoskeleton

The spectrin cytoskeleton assembles within discrete regions of the plasma membrane in a wide range of animal cell types. Although recent studies carried out in vertebrate systems indicate that spectrin assembly occurs indirectly through the adapter protein ankyrin, recent studies in Drosophila have established that spectrin can also assemble through a direct ankyrin-independent mechanism. Here we tested specific regions of the spectrin molecule for a role in polarized assembly and function. First, we tested mutant beta-spectrins lacking ankyrin binding activity and/or the COOH-terminal pleckstrin homology (PH) domain for their assembly competence in midgut, salivary gland, and larval brain. Remarkably, three different assembly mechanisms operate in these three cell types: 1) neither site was required for assembly in salivary gland; 2) only the PH domain was required in midgut copper cells; and 3) either one of the two sites was sufficient for spectrin assembly in larval brain. Further characterization of the PH domain revealed that it binds strongly to lipid mixtures containing phosphatidylinositol 4,5-bisphosphate (PIP(2)) but not phosphatidylinositol 3,4,5-trisphosphate. A K8Q mutation in the lipid binding region of the PH domain eliminated the PIP(2) interaction in vitro, yet the mutant protein retained full biological function in vivo. Reporter gene studies revealed that PIP(2) and the spectrin PH domain codistribute with one another in cells but not with authentic wild type alphabeta-spectrin. Thus, it appears that the PH domain imparts membrane targeting activity through a second mechanism that takes precedence over its PIP(2) binding activity.     

Posttranslational control of membrane-skeleton (ankyrin and alpha beta- spectrin) assembly in early myogenesis

Adult chicken skeletal muscle cells express polypeptides that are antigenically related to alpha-spectrin (Mr 240,000) and beta-spectrin (Mr 220,000-225,000), the major components of the erythrocyte membrane-skeleton, and to ankyrin (Mr 237,000; also termed goblin in chicken erythrocytes), which binds spectrin to the transmembrane anion transporter in erythrocytes. Comparative immunoblotting of SDS-solubilized extracts of presumptive myoblasts and fully differentiated myotubes cultured in vitro demonstrated that there is a dramatic accumulation of ankyrin and alpha- and beta-spectrin during myogenesis and a concomitant switch in the subunit composition of spectrin from alpha gamma to alpha beta. Analysis of early time points in myogenesis (12-96 h) revealed that these changes occur shortly after the main burst of cell fusion. To determine the temporal relationship between cell fusion and the accumulation of ankyrin and alpha- and beta-spectrin, we treated presumptive myoblasts with 2 mM EGTA, which resulted in the complete inhibition of cell fusion. The incorporation of [35S]methionine into total protein and, specifically, into alpha-, gamma-, and beta-spectrin remained the same in EGTA-treated and control cells. Analysis by immunoblotting of the amounts of ankyrin and alpha- and beta-spectrin in fusion-blocked cells revealed that there was no effect on accumulation for the first 19 h. However, there was then a dramatic cessation in their accumulation, and thereafter, the amount of each protein at steady state remained constant. Upon release from the EGTA block, the cells fused rapidly (less than 11 h), and the accumulation of ankyrin and alpha- and beta-spectrin was reinitiated after a lag period of 3-5 h at a rate similar to that in control cells. The inhibition in the accumulation of newly synthesized ankyrin, alpha-spectrin, and beta-spectrin in EGTA-treated myoblasts was not characteristic of all structural proteins, since the accumulation of the muscle-specific intermediate filament protein desmin was the same in control and fusion-blocked cells. These results show that in myogenesis, the synthesis of ankyrin and alpha- and beta-spectrin and their accumulation as a complex, although concurrent, are not coupled events. We hypothesize that the extent of assembly of these components of the membrane-skeleton in muscle cells is determined by a control mechanism(s) operative at the posttranslational level that is triggered near the time of cell fusion and the onset of terminal differentiation.     

Phosphorylation of ankyrin decreases its affinity for spectrin tetramer

The effects of phosphorylation on the interaction between spectrin and ankyrin were investigated. Spectrin and ankyrin were phosphorylated using purified human erythrocyte membrane and cytosolic (casein kinase A) kinases. These two kinases have similar properties as well as activities toward spectrin and ankyrin. Both kinases catalyzed the incorporation of about 2 mol of phosphate/mol of spectrin and about 7 mol of phosphate/mol of ankyrin. These phosphates were incorporated primarily into seryl and threonyl residues of the proteins. The phosphopeptide maps of ankyrin phosphorylated by the membrane kinase and casein kinase A were identical. Binding studies indicate that ankyrin exhibits different affinities for spectrin dimers (KD = 2.5 +/- 0.9 X 10(-6) M) and tetramers (KD = 2.7 +/- 0.8 X 10(-7) M). These dissociation constants were not appreciably affected by the phosphorylation of spectrin. On the other hand, phosphorylation of ankyrin was found to significantly reduce its affinity for either phosphorylated or unphosphorylated spectrin tetramers (KD = 1.2 +/- 0.1 X 10(-6) M) but not spectrin dimers (KD = 2.5 +/- 0.4 X 10(-6) M). The same results were obtained using either the membrane kinase or casein kinase A as the phosphorylating enzyme. The above observation suggests that ankyrin phosphorylation may provide an important mechanism for the regulation of the erythrocyte membrane cytoskeletal network.     

Expression and assembly of the erythroid membrane-skeletal proteins ankyrin (goblin) and spectrin in the morphogenesis of chicken neurons

The membrane-skeleton of adult chicken neurons in the cerebellum and optic system is composed of polypeptides structurally and functionally related to the erythroid proteins spectrin and ankyrin, respectively. Neuronal spectrin comprises two distinct complexes that share a common alpha subunit (Mr 240,000) but which have structurally distinct polymorphic subunits (beta' beta spectrin; Mr 220/225,000; gamma spectrin, Mr 235,000); the brain-specific form (alpha gamma spectrin or fodrin) and an erythrocyte-specific form (alpha beta' beta spectrin). Two structurally related isoforms of ankyrin have also been identified and are termed alpha (Mr 260,000) and beta (Mr 237,000) ankyrin. Immunofluorescence demonstrates that the variants of spectrin and ankyrin, respectively, have different distributions within neurons. On the one hand, alpha gamma spectrin and beta ankyrin are present throughout the neuron, in the perikaryon, dendrites, and axon, whereas alpha beta' spectrin and alpha ankyrin are localized exclusively in the perikaryon and dendrites where they are actively segregated from alpha gamma spectrin and other components of axonal transport. This asymmetric distribution of spectrin and ankyrin isoforms is established in distinct stages during neuronal morphogenesis. Early in cerebellar and retinal development, alpha gamma spectrin is expressed in mitotic cells. Subsequently beta ankyrin and alpha gamma spectrin are coexpressed in postmitotic cells and gradually accumulate on the plasma membrane in a uniform pattern throughout the neuron during the phase of cell growth. At the onset of synaptogenesis and the cessation of cell growth, their levels of synthesis decline sharply while the assembled proteins remained as stable membrane components. Concomitantly, there is a dramatic induction in the accumulation of alpha ankyrin and alpha beta' spectrin, whose assembly is limited to the plasma membrane of the perikarya and dendrites. These results demonstrate that two successive, developmentally regulated programs of ankyrin and spectrin expression and patterning on the plasma membrane are involved in the assembly of the spectrin-based asymmetry in the neuronal membrane-skeleton, and that their asymmetric distribution is actively maintained throughout the life of the neuron.     

Biogenesis of the avian erythroid membrane skeleton: receptor-mediated assembly and stabilization of ankyrin (goblin) and spectrin

Ankyrin is an extrinsic membrane protein in human erythrocytes that links the alpha beta-spectrin-based extrinsic membrane skeleton to the membrane by binding simultaneously to the beta-spectrin subunit and to the transmembrane anion transporter. To analyse the temporal and spatial regulation of assembly of this membrane skeleton, we investigated the kinetics of synthesis and assembly of ankyrin ( goblin ) with respect to those of spectrin in chicken embryo erythroid cells. Electrophoretic analysis of Triton X-100 soluble and cytoskeletal fractions show that at steady state both ankyrin and spectrin are detected exclusively in the cytoskeleton. In contrast, continuous labeling of erythroid cells with [35S]methionine, and immunoprecipitation of ankyrin and alpha- and beta-spectrin, reveals that newly synthesized ankyrin and spectrin are partitioned into both the cytoskeletal and Triton X-100 soluble fractions. The soluble pools of ankyrin and beta-spectrin reach a plateau of labeling within 1 h, whereas the soluble pool of alpha-spectrin is substantially larger and reaches a plateau more slowly, reflecting an approximately 3:1 ratio of synthesis of alpha- to beta-spectrin. Ankyrin and beta-spectrin enter the cytoskeletal fraction within 10 min of labeling, and the amount assembled into the cytoskeletal fraction exceeds the amount present in their respective soluble pools within 1 h of labeling. Although alpha-spectrin enters the cytoskeletal fraction with similar kinetics to beta-spectrin and ankyrin, and in amounts equimolar to beta-spectrin, the amount of cytoskeletal alpha-spectrin does not exceed the amount of soluble alpha-spectrin even after 3 h of labeling. Pulse-chase labeling experiments reveal that ankyrin and alpha- and beta-spectrin assembled into the cytoskeleton exhibit no detectable turnover, whereas the Triton X-100 soluble polypeptides are rapidly catabolized, suggesting that stable assembly of the three polypeptides is dependent upon their association with their respective membrane receptor(s). The existence in the detergent-soluble compartment of newly synthesized ankyrin and alpha- and beta-spectrin that are catabolized, rather than assembled, suggests that ankyrin and spectrin are synthesized in excess of available respective membrane binding sites, and that the assembly of these polypeptides, while rapid, is not tightly coupled to their synthesis. We hypothesize that the availability of the high affinity receptor(s) localized on the membrane mediates posttranslationally the extent of assembly of the three cytoskeletal proteins in the correct stoichiometry, their stability, and their spatial localization.     

Self-assembly of spectrin oligomers in vitro: a basis for a dynamic cytoskeleton

Purified human erythrocyte spectrin is able to form large oligomeric species without the collaboration of any other proteins. This reversible self-assembly process is both temperature and concentration dependent and seems to be mediated by the same kinds of low affinity noncovalent associations between spectrin monomers that promote tetramer formation. Low ionic strength extracts of erythrocyte membranes also contain these oligomeric species. These results support the idea that spectrin oligomers and the factors that regulate their formation may be responsible for both the stability and the versatility of the erythrocyte membrane cytoskeleton. It is postulated that the high concentrations of spectrin necessary for oligomerization are maintained in vivo by a high-affinity interaction with ankyrin. Such a coupling of high and low affinity interactions in multifunctional proteins may have significant implications for membrane structure and function.     

Spectrin oligomers: A structural feature of the erythrocyte cytoskeleton

Spectrin reversibly self-associates to high molecular weight oligomers through a concentration-driven process characterized by association constants of about 10⁵ mol^?1. This association is prominent under physiological conditions of pH, ionic strength, and temperature. It is disrupted by urea, but not Triton X-100. The process of spectrin association appears mathematically to resemble that for tropomyosin, although the mechanism is probably different. Spectrin association is weak compared to other prominent protein–protein associations in the red cell membrane skeleton. The linkage of these weak and strong associations suggests a process whereby the membrane skelton spontaneously assembles. Such affinity-modulated assembly involving weak associations is likely to be the focus of numerous membrane control mechanisms.     

Receptor clustering drives polarized assembly of ankyrin

Expression of the L1 family cell adhesion molecule neuroglian in Drosophila S2 cells leads to cell aggregation and polarized ankyrin accumulation at sites of cell-cell contact. Thus neuroglian adhesion generates a spatial cue for polarized assembly of ankyrin and the spectrin cytoskeleton. Here we characterized a chimera of the extracellular and transmembrane domains of rat CD2 fused to the cytoplasmic domain of neuroglian. The chimera was used to test the hypothesis that clustering of neuroglian at sites of adhesion generates the signal that activates ankyrin binding. Abundant expression of the chimera at the plasma membrane was not a sufficient cue to drive ankyrin assembly, since ankyrin remained diffusely distributed throughout the cytoplasm of CD2-neuroglian-expressing cells. However, ankyrin became highly enriched at sites of antibody-induced capping of CD2-neuroglian. Spectrin codistributed with ankyrin at capped sites. A green fluorescent protein-tagged ankyrin was used to monitor ankyrin distribution in living cells. Enhanced green fluorescent protein-ankyrin behaved identically to antibody-stained endogenous ankyrin, proving that the polarized accumulation of ankyrin was not an artifact of fixing and staining cells. We propose a model in which clustering of neuroglian induces a conformational change in the cytoplasmic domain that drives polarized assembly of the spectrin cytoskeleton.     

Phosphorylation of ankyrin down-regulates its cooperative interaction with spectrin and protein 3

Ankyrin mediates the primary attachment between beta spectrin and protein 3. Ankyrin and spectrin interact in a positively cooperative fashion such that ankyrin binding increases the extent of spectrin tetramer and oligomer formation (Giorgi and Morrow: submitted, 1988). This cooperative interaction is enhanced by the cytoplasmic domain of protein 3, which is prepared as a 45-41-kDa fragment generated by chymotryptic digestion of erythrocyte membranes. Using sensitive isotope-ratio methods and nondenaturing PAGE, we now demonstrate directly (1) the enhanced affinity of ankyrin for spectrin oligomers compared to spectrin dimers; (2) a selective stimulation of the affinity of ankyrin for spectrin oligomer by the 43-kDa cytoplasmic domain of protein 3; and (3) a selective reduction in the affinity of ankyrin for spectrin tetramer and oligomer after its phosphorylation by the erythrocyte cAMP-independent membrane kinase. The phosphorylation of ankyrin does not affect its binding to spectrin dimer. Ankyrin also enhances the rate of interconversion between dimer-tetramer-oligomer by 2-3-fold at 30 degrees C, and in the presence of the 43-kDa fragment, ankyrin stimulates the rate of oligomer interconversions by nearly 40-fold at this temperature. These results demonstrate a long-range cooperative interaction between an integral membrane protein and the peripheral cytoskeleton and indicate that this linkage may be regulated by covalent protein phosphorylation. Such interactions may be of general importance in nonerythroid cells.     

The actin cytoskeleton in presynaptic assembly

Dramatic morphogenetic processes underpin nearly every step of nervous system development, from initial neuronal migration and axon guidance to synaptogenesis. Underlying this morphogenesis are dynamic rearrangements of cytoskeletal architecture. Here we discuss the roles of the actin cytoskeleton in the development of presynaptic terminals, from the elaboration of terminal arbors to the recruitment of presynaptic vesicles and active zone components. The studies discussed here underscore the importance of actin regulation at every step in neuronal circuit assembly.     

Mapping the binding sites of human erythrocyte ankyrin for the anion exchanger and spectrin

This report describes initial characterization of the binding sites of ankyrin for spectrin and the anion exchanger using defined subfragments isolated from purified ankyrin domains. The spectrin-binding domain of ankyrin is comprised of two subdomains: an acidic, proline-rich region (pI = 4) involving the amino-terminal 80 residues from 828 to 908 and a basic region (pI = 8.8) that extends from 898 to 1386. The amino-terminal 70 amino acids of the spectrin-binding domain are critical for association with spectrin, since a subfragment missing this region is only 5% as active as the intact domain in displacing binding of spectrin to inside-out membrane vesicles, while deletion of the first 38 residues of the acidic domain results in a 10-fold reduction in activity. The anion exchanger-binding site is confined to an 89-kDa domain that was isolated and characterized as a globular molecule with approximately 30% alpha-helical configuration. A subfragment of the 89-kDa domain extending from residues 403 to 779 (or possibly 740) retains ability to associate with the anion exchanger. The 89-kDa domain is comprised of a series of tandem repeats of 33 amino acids that extend from residues 35 to 778 (Lux, S., John, K., and Bennett, V. (1990) Nature 344, 36-42). The activity of residues 403-779 demonstrates that the 33-amino acid repeats of the 89-kDa domain are responsible for association between ankyrin and the anion exchanger. The 33-amino acid repeating sequence of ankyrin represents an ancient motif also found in proteins of Drosophila, yeast, and Caenor habditis elegans. The finding that the 33-amino acid repeating sequence is involved in interaction with the anion exchanger implies that this motif may perform a role in molecular recognition in diverse proteins.     

Spectrin deficient inherited hemolytic anemias in the mouse: Characterization by spectrin synthesis and mRNA activity in reticulocytes

The 35 nucleotide spliced leader (SL) sequence is found on the 5' end of numerous trypanosome mRNAs, yet the tandemly organized reiteration units encoding this leader are not detectably linked to any of these structural genes. Here we report the presence of a class of discrete small SL RNA molecules that are derived from the genomic SL reiteration units of Trypanosoma brucei, Trypanosoma cruzi, and Leptomonas collosoma. These small SL RNAs are 135, 105, and 95 nucleotides, respectively, and contain a 5'-terminal SL or SL-like sequence. S1 nuclease analyses demonstrate that these small SL RNAs are transcribed from continuous sequence within the respective SL reiteration units. With the exception of the SL sequence and a concensus donor splice site immediately following it, these small RNAs are not well conserved. We suggest that the small SL RNAs may function as a donor of the SL sequence in an intermolecular process that places the SL at the 5' terminus of many trypanosomatid mRNAs.     

The notch ankyrin domain folds via a discrete, centralized pathway

The Notch ankyrin repeat domain contains seven ankyrin sequence repeats, six of which adopt very similar structures. To determine if folding proceeds along parallel pathways and the order in which repeats become structured during folding, we examined the effect of analogous destabilizing Ala-->Gly substitutions in each repeat on folding kinetics. We find that folding proceeds to an on-pathway kinetic intermediate through a transition state ensemble containing structure in repeats three through five. Repeats two, six, and seven remain largely unstructured in this intermediate, becoming structured in a second kinetic step that leads to the native state. These data suggest that the Notch ankyrin domain folds according to a discrete kinetic pathway despite structural redundancy in the native state and highlight the importance of sequence-specific interactions in controlling pathway selection. This centralized pathway roughly corresponds to a low energy channel through the experimentally determined energy landscape.