Transcriptional Regulation

The nuclear zinc-finger protein encoded by the hindsight (hnt) locus regulates several cellular processes in Drosophila epithelia, including the Jun N-terminal kinase (JNK) signaling pathway and actin polymerization. Defects in these molecular pathways may underlie the abnormal cellular interactions, loss of epithelial integrity, and apoptosis that occurs in hnt mutants, in turn causing failure of morphogenetic processes such as germ band retraction and dorsal closure in the embryo. To define the genetic pathways regulated by hnt, 124 deficiencies on the second and third chromosomes and 14 duplications on the second chromosome were assayed for dose-sensitive modification of a temperature-sensitive rough eye phenotype caused by the viable allele, hntpeb; 29 interacting regions were identified. Subsequently, 438 P-element-induced lethal mutations mapping to these regions and 12 candidate genes were tested for genetic interaction, leading to identification of 63 dominant modifier loci. A subset of the identified mutants also dominantly modify hnt308-induced embryonic lethality and thus represent general rather than tissue-specific interactors. General interactors include loci encoding transcription factors, actin-binding proteins, signal transduction proteins, and components of the extracellular matrix. Expression of several interactors was assessed in hnt mutant tissue. Five genes -- apontic (apt), Delta (Dl), decapentaplegic (dpp), karst (kst), and puckered (puc) -- regulate tissue autonomously and, thus, may be direct transcriptional targets of Hnt. Three of these genes -- apt, Dl, and dpp -- are also regulated nonautonomously in adjacent non-Hnt-expressing tissues. The expression of several additional interactors -- viking (vkg), Cg25, and laminin-alpha (LanA) -- is affected only in a nonautonomous manner (Wilk, 2004).

Protein Interactions

The native alpha;ßH polymer binds actin filaments with high affinity and has a typical spectrin morphology except that it is longer than most other spectrin isoforms and includes two knoblike structures that are attributed to a unique domain of the ßH subunit. The two knobs are often found connected to the intertwined strands near the center of the tetramer. The alpha;ßH molecules are often found in complexes of two or more tetramers associated with one another in the region of the knobs (Dubreuil, 1990).

To understand the role of the spectrin-based membrane skeleton in generating epithelial polarity, the distribution of membrane skeletal components was characterized in Drosophila ovarian follicle cells and in somatic clones of mutant cells that lack alpha-spectrin. Immunolocalization data reveal that wild-type follicle cells contain two populations of spectrin heterodimers: a network of alpha;ß heterodimers concentrated on the lateral plasma membrane and an alpha;ßHpopulation targeted to the apical surface. Induction of somatic clones lacking alpha-spectrin leads to follicle cell hyperplasia. Surprisingly, elimination of alpha-spectrin from follicle cells does not appear to prevent the assembly of conventional ß-spectrin and ankyrin at the lateral domain of the follicle cell plasma membrane. However, the alpha-subunit is essential for the correct localization of ßH-spectrin to the apical surface. As a consequence of disrupting the apical membrane skeleton, a distinct sub population of follicle cells undergoes unregulated proliferation, which leads to the loss of monolayer organization and disruption of the anterior-posterior axis of the oocyte. These results suggest that the spectrin-based membrane skeleton is required in a developmental pathway that controls follicle cell monolayer integrity and proliferation (Lee, 1997).

Two isoforms of spectrin have been described in Drosophila tissue culture cells: alpha;ß and alpha;ßH (Dubreuil, 1990). There is a single known alpha spectrin gene in Drosophila , and its product associates with either the ß or the ßH subunit (products of distinct genes [Dubreuil, 1990] to form spectrin heterotetramers. The specificity of polyclonal antibodies against the two ß spectrin isoforms was demonstrated in Western blots of total S2 cell proteins. The anti-ß spectrin antibody reacts with its 265-kDa antigen and the anti-ßH-spectrin antibody reacts with its 430-kDa antigen with no detectable cross-reactions. Despite its unusually large size, Drosophila ßH-spectrin is a bona fide ß subunit that forms spectrin tetramers resembling conventional spectrins (Dubreuil, 1996 and 1997).

Ankyrin and ß-spectrin have been used as markers of membrane skeleton assembly in neuroglian-expressing S2 cells (Dubreuil, 1996). Upon expression of neuroglian and formation of cell aggregates, ankyrin and ß-spectrin are selectively recruited to sites of cell-cell contact. The distribution of the alpha subunit of spectrin was compared with ankyrin using a monoclonal antibody. Unlike ankyrin, alpha-spectrin is constitutively associated with the plasma membrane of single S2 cells and neuroglian-expressing S2 cell aggregates. The lack of ankyrin colocalization with alpha-spectrin at nonadherent regions of the plasma membrane suggests that there is a population of spectrin in S2 cells that associates with the plasma membrane, independent of ankyrin (Dubreuil, 1997).

The isoform-specific ß-spectrin antibodies reveal two distinct spectrin distributions in control S2 cells and neuroglian-expressing S2 cell clusters. The sum of their staining patterns correspond to the broad distribution of alpha-spectrin. ß-spectrin is not detectably associated with the plasma membrane of control cells but it is recruited to the plasma membrane at sites of cell-cell contact in neuroglian-expressing cells. In contrast, the ßH-specific antibody stains the plasma membrane of all cells. ßH-spectrin appears to be uniformly distributed along the plasma membrane, although some variations in staining intensity are apparent (presumably because of the topology of the cell surface). Neuroglian-expressing S2 cell clusters exhibit the same uniform distribution of ßH-spectrin at the plasma membrane, with no apparent concentration of this isoform at sites of neuroglian-mediated cell-cell contact (Dubreuil, 1997).

Na,K-ATPase is known to interact directly with ankyrin: it colocalizes with spectrin and ankyrin at sites of E-cadherin-mediated cell-cell contact in mammalian cells. The distribution of the Na,K-ATPase was examined in neuroglian-expressing S2 cells using a monoclonal antibody against the alpha subunit of the chicken Na,K-ATPase, which reacts with the Drosophila Na,K-ATPase. Antibody staining reveals colocalization of the Na,K-ATPase with ankyrin at sites of cell-cell contact. Of the cell contacts that stained detectably with ankyrin antibody, 67% also stained with the Na,K-ATPase antibody. The Na,K-ATPase is weakly stained in a punctate pattern at the plasma membrane of nonadherent cells or at noncontact sites of adherent cells, presumably because of its low abundance in S2 cells relative to other Drosophila cell types that have been studied (e.g., salivary gland) (Dubreuil, 1997).

The distribution of alpha-, ß-, and ßH-spectrin, as well as ankyrin, neuroglian, and the Na,K-ATPase were all examined in vivo. The epithelial cells of the larval salivary gland abundantly expresses neuroglian at lateral regions of cell-cell contact. Ankyrin colocalizes with neuroglian at these sites, as observed in S2 cells. A similar staining pattern is detected with antibodies against alpha- and ß-spectrin. The Na,K-ATPase codistributes with ankyrin at lateral sites of cell-cell contact. Ankyrin and the Na,K-ATPase are also detected at the basal surface of cells, but neither protein is detected at the apical surface, facing the gland lumen. Whereas ßH-spectrin is uniformly distributed at the surface of cultured S2 cells, it is found almost exclusively at the apical domain of the salivary gland epithelium. In the same field, the Na,K-ATPase is concentrated at lateral sites of cell-cell contact. Thus, the two ß spectrin isoforms are segregated in a nonoverlapping distribution in the salivary gland epithelium, in contrast to their overlapping distribution in neuroglian-expressing S2 cells (Dubreuil, 1997).

Salivary glands from third instar larvae typically exhibit a thin epithelial cell layer surrounding a large gland lumen. The ßH-spectrin staining pattern in glands from first and second instar larvae and from embryos (Thomas, 1994) defines a relatively small lumen and a proportionately thicker epithelium. However, regardless of the stage examined, the distributions of alpha;ßH-spectrin at the apical surface and alpha;ß-spectrin/ankyrin at the lateral surface remain segregated (Dubreuil, 1997).

Lee (1997) has described the polarized distributions of alpha;ß and alpha;ßH-spectrins in the somatic follicle epithelium of the adult ovary. This paper examines the distribution of Neuroglian in relation to the follicle cell membrane skeleton. Neuroglian staining is concentrated along the lateral membranes of the epithelium along with ankyrin and ß spectrin. In contrast, ßH-spectrin is restricted to the apical surface facing the enclosed nurse cells and oocyte. Thus, in follicle cells as in the salivary gland, the two spectrin isoforms appear to respond independently to their respective positional cues at the cell surface (Dubreuil, 1997).

It is concluded Neuroglian has no apparent effect on the spectrin isoform alpha;ßH, which is constitutively associated with the plasma membrane in S2 cells. Another membrane marker, the Na,K-ATPase, codistributes with ankyrin and alpha;ß-spectrin at sites of neuroglian-mediated contact. The distributions of these markers in epithelial cells in vivo are consistent with the order of events observed in S2 cells. Neuroglian, ankyrin, alpha;ß-spectrin, and the Na,K-ATPase all colocalize at the lateral domain of salivary gland cells. In contrast, alpha;ßH-spectrin is sorted to the apical domain of salivary gland and somatic follicle cells. Thus, the two ß spectrin isoforms respond independently to positional cues at the cell surface: in one case the response is to an apically sorted receptor (Shotgun/E-cadherin) and in the other case to a locally activated cell-cell adhesion molecule (neuroglian). The results support a model in which the membrane skeleton behaves as a transducer of positional information within cells (Dubreuil, 1997).

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

To assess whether Lva interacts with other proteins, the native size of Lva was compared with other microfilament/microtubule-associated proteins (MMAPs). The S100 and the final protein (MMAP) fraction were passed separately over a gel filtration column, and fractions were assayed by immunoblot. alpha-Spectrin has been previously shown to copurify with ß- and ßH-Spectrin in two stable heterotetrameric complexes (alpha2ß2 and alpha2ßH2, respectively) and coimmunoprecipitates with ß- and ßH-Spectrin, but information on association of Lva with alpha-Spectrin only is presented for simplicity. Immunoblots show that in both the S100 and the MMAP fraction, Lva, CLIP190, alphaß-, and alphaßH-Spectrin coelute from the column with native molecular weights larger then their predicted molecular weights, indicating that each protein exists in large, stable complexes (Sisson, 2000).

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

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

The apical transmembrane protein Crumbs is necessary for both cell polarization and the assembly of the zonula adherens (ZA) in Drosophila epithelia. The apical spectrin-based membrane skeleton (SBMS) is a protein network that is essential for epithelial morphogenesis and ZA integrity, and exhibits close colocalization with Crumbs and the ZA in fly epithelia. These observations suggest that Crumbs may stabilize the ZA by recruiting the SBMS to the junctional region. Consistent with this hypothesis, it is reported that Crumbs is necessary for the organization of the apical SBMS in embryos and Schneider 2 cells, whereas the localization of Crumbs is not affected in karst mutants that eliminate the apical SBMS. The data indicate that specifically the 4.1 protein/ezrin/radixin/moesin (FERM) domain binding consensus, and in particular, an arginine at position 7 in the cytoplasmic tail of Crumbs is essential to efficiently recruit both the apical SBMS and the FERM domain protein, Moesin-like. Crumbs, ßHeavy-spectrin, and Moesin-like are all coimmunoprecipitated from embryos, confirming the existence of a multimolecular complex. It is proposed that Crumbs stabilizes the apical SBMS via Moesin-like and actin, leading to reinforcement of the ZA and effectively coupling epithelial morphogenesis and cell polarity (Médina, 2002).

The Crumbs-Stardust pathway is essential for polarity and has been shown to be a major apical signal for establishing the ZA at the apical-lateral boundary. The observation that mutations affecting ßH and Crumbs both cause a junctional phenotype, along with the close colocalization of both proteins in the marginal zone of epithelial cells, suggested a possible connection between the activities of these two proteins. Crumbs can indeed recruit apical ßH together with the FERM domain protein Moesin-like and actin. The data are in good agreement with the hypothesis that polarity cues are used to organize the SBMS, but this is the first time that this has been shown for an apical determinant (Médina, 2002).

Several lines of evidence indicate that Crumbs can recruit ßH into its complex: (a) ßH is mislocalized in embryos mutant for the truncation allele crumbs 8F105, in which the mutant Crumbs protein itself is mislocalized; (b) ßH mislocalization can be induced by overexpression of the Crumbs transmembrane and cytoplasmic domains in vivo; (c) ßH is recruited to Crumbs protein clusters in an S2 cell cocapping assay; (d) Crumbs can be coimmunoprecipitated with ßH; and (e) the protein-null allele crumbs11A22 acts as a dominant enhancer of hypomorphic karst alleles, strongly indicating that a reduction in the normal amount of Crumbs reduces the level of partially functional ßH at the membrane. Moreover, because the karst mutant alleles all produce COOH-terminally truncated proteins, these results further suggest that the Crumbs-ßH interaction site lies in the NH2-terminal portion of the latter. Finally, loss of Crumbs has been shown (Pellikka, 2002) to eliminate ßH from the stalk membrane of photoreceptors in the adult eye (Médina, 2002).

Current evidence indicates that ßH can be recruited to the membrane in several additional ways. First, it can associate with the specialized basal adherens junctions during cellularization in a Crumbs-independent manner. Second, it is found in the terminal web subtending brush borders in the midgut epithelium that does not express Crumbs. Finally, it has also been shown that ßH is only partially reduced in crumbs11A22 mutant follicle cell clones, indicating that in this Crumbs-expressing epithelium there are multiple mechanisms to recruit ßH. These data provide a compelling explanation for the modest nature of the karst-crumbs genetic interaction. By reducing Crumbs, only one of these pathways is affected. The observation that the karst1 allele produces readily detectable quantities of truncated product, most of which is not recruited to the membrane in any of these epithelia, suggests that there is a general and essential role of the COOH-terminal half of ßH in its stable membrane localization. Together, the above data are consistent with the multifunctional nature of spectrin membrane skeletons and with the idea that specific pathways recruit the SBMS to establish spatially distinct polarized membrane domains, whereas general COOH-terminal membrane association domains permit tight membrane association and network integration (Médina, 2002).

Partial rescue of crumbs mutants has been attained by the crumbsmyc-intra construct. This suggested that the transmembrane and cytoplasmic domains of Crumbs might be sufficient to concentrate ßH to some areas of the apical membrane. This result has been confirmed and extended, showing that the critical region for recruiting ßH is just 9 amino acids from position 6 through 14 of the cytoplasmic domain in the putative FERM domain binding site. Within this region, a conserved tyrosine residue at cytoplasmic domain position 10 (crucial for Crumbs function in vivo) and an arginine at position 7 are both required for this activity. It is worth noting that all Crumbs genes cloned so far contain a charged amino acid residue at position 7 in the cytoplasmic domain, suggesting that this is an evolutionarily conserved interaction site (Médina, 2002).

FERM domains are found in the protein 4.1 family of proteins that link the SBMS to cell-surface receptors as well as several other proteins which organize the cortical actin (ezrin/radixin/moesin). The founding member of this group, protein 4.1, was originally identified as a major component of the erythrocyte SBMS where it facilitates the interaction of spectrin with actin and the transmembrane protein Glycophorin C. Therefore, the presence of a conserved FERM binding domain in the Crumbs cytoplasmic domain suggests that Crumbs may bind to ßH via a FERM domain protein (Médina, 2002).

In Drosophila, the FERM domain family includes the proteins Coracle, Merlin, Moesin-like, and Expanded. Of these four proteins, Coracle is an unlikely candidate to bind to the Crumbs juxtamembrane domain since it is localized to the septate junctions basal to the ZA. However, the Merlin, Moesin-like, and Expanded proteins are localized in part or in whole at the ZA region in epithelia, and could thus be involved in the interaction between Crumbs and ßH. The fact that none of protein 4.1 family members known in Drosophila contains a spectrin-binding domain as defined by the archetypal protein 4.1 does not necessarily abrogate this hypothesis. ßH-spectrin is clearly recruited to the membrane by different mechanisms than its basolateral counterpart, and this specificity would likely be reflected in divergent interaction domains. In this work, it has been found that ßH and Moesin-like can both coimmunoprecipitate Crumbs. Furthermore, the capping assay and embryo expression evidence provide in vivo support for this result. Not only will Moesin-like cocap with the Crumbs cytoplasmic domain, but it is also dependent on exactly the same sequences that recruit ßH. These results, together with the existence of the consensus binding site for a FERM domain protein in Crumbs, strongly support the hypothesis that Moesin-like forms a bridge between Crumbs and the SBMS. A functional test of this relationship must wait until mutations in the Moesin-like locus become available. Thus, the current data, although highly suggestive, do not formally distinguish between the possibility of a Moesin-like bridge between Crumbs and the SBMS, and the existence of two separate complexes with direct interaction between Crumbs and ßH or Moesin-like in each. Significantly, actin does not cap consistently with Crumbs in S2 cells and is not present in immunoprecipitates. This suggests that other components present in epithelial cells are necessary for stabilization of the actin skeleton around the Crumbs complex. It also indicates that ßH is specifically recruited to the proposed complex and is not merely a passive arrival along with bulk actin (Médina, 2002).

These results indicate that Crumbs interacts with at least one protein network; a Moesin-like/Spectrin/actin-based network. However, it is unclear at present whether Moesin-like/ßH/actin coexists in the same complex with Crumbs. In the erythrocyte model, glycophorin C is linked to spectrin via a ternary complex containing protein 4.1 and the PDZ domain protein p55 bound to a topologically similar pair of binding sites to the two functional regions identified in the Crumbs cytoplasmic domain (Médina, 2002).

Because both the crumbs and karst phenotypes disrupt the ZA, it is hypothesized that Crumbs promotes the accumulation of ßH to the apicolateral region during gastrulation to orchestrate the fusion of spot adherens junctions and/or to stabilize the ZA. Moreover, the observation that karst mutants exhibit morphogenetic defects without any loss of epithelial polarity, whereas dlt mutants exhibit a strong polarity phenotype, suggests that the polarization and junction building functions of Crumbs are separate and parallel pathways. In support of this hypothesis, the FERM domain binding region of Crumbs is indeed required (Izaddoost, 2002) for correct organization of the ZA (Médina, 2002).

The loss of ßH function causes defects in cell shape change that are associated with apical contraction driven by an apically located actomyosin contractile ring. In this context the discovery that this spectrin isoform is complexed with Moesin-like is particularly provocative, since the activity of the latter is strongly correlated with modulation of cell shape and the actin cytoskeleton. Furthermore, the activity of moesin is modulated by phosphorylation in response to activation of Rho-associated kinase (ROK) in parallel with myosin II. Both Moesin and myosin light chain are activated by ROK phosphorylation and by ROK mediated inhibition of the myosin/moesin phosphatase. Therefore, it is speculated that ßH is part of the cytoskeletal network that facilitates such cell shape changes, and that in organizing spectrin at the membrane, Crumbs would appear to be acting as a molecular coordinator of polarity and morphogenesis. Furthermore, the finding that in human, mutations in CRB1 lead to pathologies such as retinitis pigmentosa (RP12) emphasizes the importance of deciphering the molecular networks associated with Crumbs in Drosophila. The human orthologue of ßH, ßV-spectrin, is strongly expressed in photoreceptor cells. This raises the exciting possibility that a similar interaction between CRB1 and ßV-spectrin exists in these cells. This will be examined in future work (Médina, 2002).

The C-terminal domain of Drosophila ßheavy-spectrin exhibits autonomous membrane association and modulates membrane area

Current models of cell polarity invoke asymmetric cues that reorganize the secretory apparatus to induce polarized protein delivery. An important step in this process is the stabilization of the protein composition in each polarized membrane domain. The spectrin-based membrane skeleton is thought to contribute to such stabilization by increasing the half-life of many proteins at the cell surface. Genetic evidence is consistent with a negative role for Drosophila ßHeavy-spectrin in endocytosis, but the inhibitory mechanism has not been elucidated. This study investigated the membrane binding properties of the C-terminal nonrepetitive domain of ßHeavy-spectrin through its in vivo expression in transgenic flies. This region was found to be a membrane-association domain that requires a pleckstrin homology domain for full activity, and robust membrane binding by such a C-terminal domain is shown to require additional contributions outside the pleckstrin homology. In addition, expression of the ßHeavy-spectrin C-terminal domain is shown to have a potent effect on epithelial morphogenesis. This effect is associated with its ability to induce an expansion in plasma membrane surface area. The membrane expansions adopt a very specific bi-membrane structure that sequesters both the C-terminal domain and the endocytic protein dynamin. These data provide supporting evidence for the inhibition of endocytosis by ßHeavy-spectrin, and suggest that the C-terminal domain mediates this effect through interaction with the endocytic machinery. Spectrin may be an active partner in the stabilization of polarized membrane domains (Williams, 2004).

Contemporary models of cell polarity invoke the cortical cytoskeleton as a stabilizing influence on asymmetric membrane domains. This study has investigated the membrane binding properties of the ßHeavy-spectrin (ßH) C-terminal segment 33. This domain autonomously localizes to the plasma membrane and, as expected from the analysis of vertebrate homologs, the pleckstrin homology (PH) domain is an important contributor to membrane binding. However, membrane binding conferred by the ßH PH domain is augmented by other interactions. In addition, expression of ßH segment 33 has a dominant effect on cell morphology and epithelial development. This dominant phenotype is associated with dramatic overgrowths of the plasma membrane that sequester the endocytic protein dynamin. These data support the proposed role of ßH as a downregulator of endocytosis and therefore a stabilizing factor for its associated membrane domains (Williams, 2004).

Previous data suggest that the C-terminal nonrepetitive domain of nonerythroid ß-spectrins is an important site of ankyrin-independent membrane association that functions by association with the phospholipid phosphatidylinositol-4,5-bisphosphate via a PH domain. Different isoforms of ß-spectrin have markedly divergent sequences surrounding this motif, which may also contribute to membrane attachment. ßH-spectrins do not associate with ankyrin, but they do contain a canonical spectrin PH domain, and such ankyrin-independent association may be an important contributor to membrane binding by the heavy isoforms (Williams, 2004).

The in vivo localization data presented in this study suggests that phospholipid binding is a necessary function of the ßH C-terminal membrane association domain because deletion of the PH domain disrupts membrane association. However, the PH domain plus its immediate C-terminal sequences did not exhibit robust membrane localization, indicating that neither phospholipid binding nor overlapping protein binding is sufficient for stable plasma membrane association. This suggests that phospholipid binding is part of a more complex anchoring mechanism, possibly serving a regulatory role (Williams, 2004).

ßH segment 33 does not exhibit conspicuous polarity, showing that it is unlikely to direct full-length ßH to its apical domain. ßH has been shown to be recruited to the membrane by the apical polarity determinant Crumbs, and null crumbs alleles dominantly enhance the phenotype arising from C-terminal truncations of ßH. These results suggested that Crumbs binds to the N-terminal half of ßH, or to alpha-spectrin. However, the avidity with which these truncated proteins bind to the membrane is low, as little if any is detectable at the membrane. This indicates that Crumbs-association is not sufficient for ßH-membrane binding and that more C-terminal parts of the protein must contribute through other interactions. The data presented here are consistent with a model in which Crumbs provides a polarized anchor for ßH, whereas the C-terminus contributes to its stable membrane association. However, the role of other sequences missing in the karst mutants must additionally be examined (Williams, 2004).

In this context, it is interesting to find that ßH is apparently recruited to the apical membrane of the salivary gland in the absence of alpha-spectrin. Other reports are beginning to indicate a significant degree of independence between the membrane binding of alpha- and ß- or ßH-spectrins. Together, such data raise the possibility that the requirement for an alpha/ß network per se among nonerythroid spectrin-based membrane skeletons SBMS may be a domain-specific feature, or secondary to membrane association (Williams, 2004).

Expression of ßH segment 33 causes a dominant apoptotic phenotype in susceptible tissues, whereas it disrupts epithelial morphogenesis and causes a membrane expansion phenotype elsewhere. These appear to represent two distinct functions, given that they are dependent on different sequences within the domain. The apoptotic phenotype requires the PH domain plus sequences to its C-terminal side and is specific to ßH; it is not seen on expression of the equivalent ß-spectrin fragment, and presumably reflects some aspect of the protein-lipid interactions defined by the ßHPH+3 construct. By contrast, the inhibition of morphogenesis and the induction of membrane expansion require additional protein interactions mediated by the sequences on the N-terminal side of the PH domain. This multiplicity of effects complicates the interpretation of genetic interaction experiments; however, current data are most consistent with ßH33 causing a gain-of-function phenotype. Thus, karst alleles do not dominantly enhance the ßH33 phenotype, whereas ßH overexpression is additive or synergistic, and ßH is not displaced by ßH33 expression. Clearly, there is much complexity within this region of the ßH molecule (Williams, 2004).

The C-terminal segment 33 of ßH causes a phenotype that is consistent with the potent inhibition of endocytosis, resulting in the sequestration of dynamin at the membrane. Given that these extensions have been observed in both the salivary gland and trachea, this is not a salivary gland specific effect, and thus reflects a more general function of ßH (Williams, 2004).

The SBMS has been suggested to facilitate membrane stability and cell polarity by limiting the lateral diffusion of proteins to sites of endocytosis and as a physical barrier to clathrin pit formation. In addition, a few reports of binding between SBMS proteins and endocytic components have also hinted at a more active role for the SBMS during endocytosis. the results support the idea that the SBMS stabilizes membrane domains by regulating membrane turnover, and extends these models by suggesting that the apical SBMS actively regulates protein turnover by nucleating a protein complex that modulates endocytosis. A primary role of this activity may be to down-regulate endocytosis in order to stabilize the protein composition of the associated membrane - a role that would fit well with models of cell polarity. Thus, the polarizing cues that recruit ßH may do so in part, to modulate endocytosis via segment 33 (Williams, 2004).

ßH collaborates with Crumbs, and an unidentified partner of Crumbs, to modulate the area of the photoreceptor apical stalk domains in the eye. It is not known if Crumbs acts to stimulate membrane delivery or to inhibit recovery, or both. On the basis of the data presented in this study, it is suggested that Crumbs recruits ßH to downregulate endocytosis. ßH could play a similar stabilizing role at adherens junctions, supporting the notion that the disruption of the ZA seen in karst mutants could arise from the inappropriate turnover of junctional components (Williams, 2004).

Crumbs activity and the modulation of apical surface area have been shown to be important factors in the morphogenesis of the salivary gland. Given that the turnover of junction proteins is required for epithelial morphogenesis, it is speculated that ßH segment 33 expression inhibits cell internalization because of its inhibitory role in endocytosis. Importantly, the membrane extensions cannot be a physical barrier to morphogenesis, since cell internalization ceases before their emergence. Whether there is a causal relationship between ßH segment 33 inhibition of endocytosis and cell internalization will be the subject of future investigations (Williams, 2004).

Crumbs also recruits the cortical actin organizer Moesin, and moesin mutations cause apical membrane overgrowth. However, loss-of-function moesin alleles do not eliminate ßH from the membrane in follicle cells, and so parallel modulation of membrane area by DMoesin and ßH seems probable (Williams, 2004).

The presence of dynamin, but not clathrin or amphiphysin in the ßH-induced membrane extensions, suggests that the endocytic process being inhibited is either clathrin-independent or is occurring in the later stages of clathrin-mediated endocytosis. The appearance of apparently normal mid-stage coated pits is consistent with both of these notions. Whichever pathway is inhibited, there must be a structural transition that releases dynamin from its tubular `pinching' assemblies into these more lamellar structures (Williams, 2004).

The abnormal bi-membrane phenotype is regarded as a chronic symptom of the inhibition of endocytosis by ßH segment 33, but their structure is nonetheless informative. The morphology of the membranes clearly indicates that ßH segment 33 is either capable of self-association leading to membrane adhesion, or that it is associated with a protein with this property. The dimensions of the bi-membrane are strongly reminiscent of membrane junctions produced by annexins. Furthermore, annexinVI has been associated with the proteolytic removal of spectrin to facilitate clathrin-mediated endocytosis in vertebrate cells. Therefore, it is speculated that a fly homologue of one of these annexin proteins is normally associated with the apical SBMS, and mediates the formation of these bi-membrane structures through a regulatory or structural association with ßH segment 33. Antibody and genetic reagents are currently being generated to test this hypothesis (Williams, 2004).

Spectrin tetramer formation is not required for viable development in Drosophila

The dominant paradigm for spectrin function is that (αβ)2-spectrin tetramers or higher order oligomers form membrane associated two-dimensional networks in association with F-actin to reinforce the plasma membrane. Tetramerization is an essential event in such structures. This study characterized the tetramerization interaction between α-spectrin and β-spectrins in Drosophila. Wild-type α-spectrin binds to both β- and βH-chains with high affinity, resembling other non-erythroid spectrins. However, α-specR22S, a tetramerization site mutant homologous to the pathological α-specR28S allele in humans, eliminates detectable binding to β-spectrin and reduces binding to βH-spectrin ~1000 fold. Even though spectrins are essential proteins, α-specR22S rescues α-spectrin mutants to adulthood with only minor phenotypes indicating that tetramerization, and thus conventional network formation, is not the essential function of non-erythroid spectrin. These data provide the first rigorous test for the general requirement for tetramer-based non-erythroid spectrin networks throughout an organism and find that they have very limited roles, in direct contrast to the current paradigm (Khanna, 2015).

karst: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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