alpha Spectrin


Myotube migration and the formation of muscle attachments are crucial events for the proper development of muscle patterning in the Drosophila embryo. T. Volk (1992) describes the identification in Drosophila of a new embryonic muscle-specific protein, MSP-300. This protein is initially expressed by muscle precursors at muscle-ectoderm and muscle-muscle attachment sites. As development continues, MSP-300 becomes associated with muscle myofibrillar network. Studies of the subcellular localization of this muscle-specific protein in primary embryonic cultured myotubes show that MSP-300 decorates actin filaments, and that it is specifically enriched in sites where actin microfilaments are linked to the plasma membrane. Migrating myotubes exhibit high levels of this protein at their leading edge, while in myotubes that have already developed sarcomeric architecture, the protein is localized mainly at the Z-discs. Sequence analysis reveals that the protein exhibits at least five spectrin-like repeats. Several properties are shared by MSP-300 and other members of the spectrin superfamily: these proteins associated with actin microfilaments, their sequences exhibit spectrin-like repeats and they are localized at sites where actin is linked to the plasma membrane. This protein could have a developmental role in the formation of muscle-ectoderm attachments and may be involved in myotube migration on the ectoderm (Volk, 1992). MSP-300 is distinct from alpha- and beta-spectrin and the beta-spectrin heavy chain coded for by karst.

Expression of vertebrate alpha spectrin isoforms

The spectrin-based membrane skeleton of skeletal muscle has been localized to "costameres," structures that lie on the cytoplasmic surface of the sarcolema over the Z lines and M lines of adjacent sarcomeres. Although their structure and function are still unclear, costameres are believed to link the contractile apparatus of superficial myofibrils to the sarcolemma and may also serve to stabilize the sarcolemma during the contractile cycle. Because the strength of contraction increases greatly after birth, both the expression and organization of spectrin and fodrin in the costameres of skeletal muscle may be developmentally regulated. Skeletal muscle contains spectrin (or spectrin I) and fodrin (or spectrin II) both members of the spectrin supergene family. Isoform-specific antibodies and cDNA probes were used to investigate the molecularforms, developmental expression, and subcellular localization of the spectrins in the skeletal muscle of the rat. Beta-spectrin (betaI) replaces beta-fodrin (betaII) at the sarcolemma as skeletal muscle fibers develop. As a result, adult muscle fibers contain only alpha-fodrin (alphaII) and the muscle isoform of beta-spectrin (betaISigma2). By contrast, other types of cells present in skeletal muscle tissue, including blood vessels and nerves, contain only alpha- and beta-fodrin. During late embryogenesis and early postnatal development, skeletal muscle fibers contain a previously unknown form of spectrin complex, consisting of alpha-fodrin, beta-fodrin, and the muscle isoform of beta-spectrin. These complexes associate with the sarcolemma to form linear membrane skeletal structures that otherwise resemble the structures found in the adult. These results suggest that the spectrin-based membrane skeleton of muscle fibers can exist in three distinct states during development (Zhou, 1998).

alpha-Spectrin mRNA is detectable at birth, in brain areas with perinatal neurogenesis, such as the cerebral cortex, hippocampus, thalamus,and olfactory bulb. alpha-Brain-spectrin mRNA increases gradually during the firstpostnatal days to reach a plateau between the second and the third week of life. In the young adult brain, the level of alpha-brain spectrin mRNA decreased globally. This spacio-temporal distribution argues for the involvement of the mRNA in the synthesis of both the erythroid and non-erythroid brain spectrin isoforms. In both the hippocampal formation and the cerebellum in situ hybridization signal variations are superimposable with neuronal maturation gradients. This pattern of variation, coupled with the known interaction of brain spectrins with other cytoskeletal proteins, agrees with the notion that brain spectrins may be involved in neuronal differentiation by way of the cytoskeletal lattice organization (Gelot, 1994).

The presence, localization, and beta spectrin associations in mouse brain have been determined for alpha erythroid spectrin, alpha SpI sigma, as well as alpha non-erythroid spectrin (alpha SpII sigma 1). Beta SpI sigma 2 is located in neuronal somata and dendrites in mouse cerebellum. beta SpII sigma 1 is located in the medullary layer, chiefly composed of axonal tracts. Antibodies specific for the alphasubunit of erythrocyte spectrin (alpha SpI sigma 1) localizes antigen to the somata and dendrites of cerebellar granule cell neurons, a pattern similar to that for the localization of the erythroid beta subunit (beta SpI sigma 2). In contrast thenon-erythroid alpha subunit (alpha SpII sigma 1) is localized to axons in thecerebellum corresponding to the pattern for the non-erythroid beta subunit (beta SpIIsigma 1). The distinct localization of antigens by antisera that recognize either thealpha subunit of red blood cell spectrin or the alpha subunit of non-erythroid brainspectrin, together with the correspondence of their localization with appropriate beta subunits, clearly indicate that brain contains at least two species of spectrin each with distinct alpha and beta subunits. There are minor populations of various hybrid tetramers in brain consisting of mixed erythroid and non-erythroid subunits (Clark, 1994).

Neuronal activity is an essential stimulus for induction of plasticity and normaldevelopment of the CNS. A novel immediate-early gene (IEG) cDNA has been identified that is rapidly induced in neurons by neural activity in experiments demonstrating adult and developmental neural plasticity. Both the mRNA and its encoded protein of this IEG are enriched in neuronal dendrites. Analysis of the deduced amino acid sequence indicates a region of homology with alpha-Spectrin, and the full-lengthprotein, prepared by in vitro transcription/translation, coprecipitates with F-actin.Confocal microscopy of the native protein in hippocampal neurons demonstrates thatthe IEG-encoded protein is enriched in the subplasmalemmal cortex of the cell bodyand dendrites and thus colocalizes with the actin cytoskeletal matrix. Accordingly, the gene and encoded protein has been called Arc (activity-regulated cytoskeleton-associated protein). These observations suggest that Arc may play a role in activity-dependent plasticity of dendrites (Lyford, 1995).

Beta subunit of spectrin

The complete primary structure of the general form of human beta-spectrin (beta G) has been deduced from cDNAs isolated from human brain. beta G-Spectrin is encoded by a gene located on human chromosome 2. beta G-Spectrin and erythrocyte beta-spectrin (beta R) share identical domain organization, with sequence identity of 60% and sequence similarity of 77%. beta-Spectrins have closely related N-terminal domains implicated in binding to actin, and 17 copies of a 106-residue repeat motif with consensus residues that are highly conserved between beta-spectrins as well as alpha-spectrins. C-terminal domains of beta G and the 270-kDa beta R-spectrins are candidate regions to associate with alpha-spectrin, and exhibit 75% similarity. beta G- and beta R-spectrins exhibit different patterns of expression in tissues and follow different developmental programs in those tissues where they are co-expressed. beta G-Spectrin is present in all tissues examined except for erythrocytes, while beta R-spectrin could be detected only in erythrocytes, brain, and heart. beta G- and beta R-Spectrins are both expressed in brain, but beta R appeared later in post-natal development and is highly enriched in cerebellum in contrast to the broad regional distribution of beta G-spectrin. beta-Spectrins are likely to perform related but distinct functions, with beta G in a general, constitutive role and beta R-spectrin involved in more specialized activities of differentiated cells (Hu, 1992).

The amino acid sequence of mouse brain beta spectrin (beta fodrin), deduced from the nucleotide sequence of complementary DNA clones, reveals that this non-erythroidbeta spectrin comprises 2363 residues, with a molecular weight of 274,449 Da. Brain beta spectrin contains three structural domains; the location of several functional domains are suggested, including f-actin, synapsin I, ankyrin and spectrin self association sites. There is striking homology and similar structural characteristics of brain beta spectrin repeats beta 11 and beta 12 to globins. In vitro analysis has demonstrated that heme is capable of specific attachment to brain spectrin, suggesting possible new functions in electron transfer, oxygen binding, nitric oxide binding or heme scavenging (Ma, 1993).

Isoforms of ankyrin (ankyrinR) are expressed in both the erythrocyte and the brain. Four cDNAs representing regulatory domains of ankyrinR expressed in the rat spleen and brain were cloned and sequenced. These different cDNAs were found to result from tissue-specific alternative mRNA processing of the ankyrinR regulatory domain. One of the isolated cDNAs was used to develop an antibody to brain isoforms of ankyrinR, and this antibody was used to study the localization of ankyrinR in the rat brain. The protein is found to be widely expressed in neurons of the metencephalon but limited to a discrete subset of neurons in the rat forebrain. In the thalamus and areas of the basal ganglia, these neurons are grouped in defined nuclei, whereas in the cortex, hippocampus, and caudate putamen they appear as isolated cells distributed randomly throughout these structures. A similar study using an antibody raised against erythrocyte spectrin (beta R) shows a comparable localization to that of ankyrinR. Both proteins were expressed late in the developing rat brain, as part of the maturation stage of neural development. These data suggest a specific role for these erythrocyte structural proteins in the postmitotic development of a subset of neurons in the rat brain (Lambert, 1993).

Voltage-gated sodium channels (VGSCs) are concentrated in the depths of the postsynaptic folds at mammalian neuromuscular junctions (NMJs) where they facilitate action potential generation during neuromuscular transmission. At the nodes of Ranvier and the axon hillocks of central neurons, VGSCs are associated with the cytoskeletal proteins, beta-spectrin and ankyrin, which may help to maintain the high local density of VGSCs. In skeletal muscle beta-spectrin is precisely colocalized with both VGSCs and ankyrinG, the nodal isoform of ankyrin. In end face views of rat NMJs, acetylcholine receptors (AChRs), and utrophin immunolabeling are organized in distinctive linear arrays corresponding to the crests of the postsynaptic folds. In contrast, beta-spectrin, VGSCs, and ankyrinG have a punctate distribution that extends laterally beyond the AChRs, consistent with a localization in the depths of the folds. Double antibody labeling shows that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG at the NMJ. Quantification of immunofluorescence in labeled transverse sections reveals that beta-spectrin is also concentrated in perijunctional regions, in parallel with an increase in labeling of VGSCs and ankyrinG, but not of dystrophin. These observations suggest that interactions with beta-spectrin and ankyrinG help to maintain the concentration of VGSCs at the NMJ and that a common mechanism exists throughout the nervous system for clustering VGSCs at a high density (Wood, 1998).

The expression of brain beta SpIIa and beta SpIb has been examined during mouse brain development. The 9 kb transcript that encodes beta SpIIa is present in fetal mouse brain tissue and increases to a maximal level in a 30-day-old mouse. There is a coordinate accumulation of the 7.8 kb alpha SpIIa mRNA (with beta SpIIa) during mouse brain development. The coordinate expression of alpha SpIIa and beta SpIIa at the mRNA and protein level allows formation of (alpha SpIIa/beta SpIIa) two fold tetramers early in premitotic neuronal development, and also avoids turnover of unassembled alpha and beta-subunits. An 11 kb transcript that encodes beta SpIb is not produced in embryonic tissue, and is first seen in a 6-day-old mouse. The protein translation products beta SpIIa and beta SpIb first appear in fetal mouse brain tissue at postnatal days 6 and 8, respectively. The expression of beta SpIb mRNA on postnatal days 6-8, and the appearance of these brain spectrin tetramers in postmitotic and postmigratory neurons of the cerebellum at the same time suggests that brain spectrin tetramers are involved in differentiated functions of the neuron (formation of cell-cell contacts, formation of dendritic processes and postsynaptic contacts). Thus, the data from the present study demonstrates that the expression of these two neuronal beta-spectrin isoforms is regulated at the level of mRNA expression (Zimmer, 1992).

ß-Spectrin is a major component of the membrane skeleton, a structure found at the plasma membrane of most animal cells. ß-Spectrin and the membrane skeleton have been proposed to stabilize cell membranes, generate cell polarity, or localize specific membrane proteins. The Caenorhabditis elegans homolog of ß-spectrin is encoded by the unc-70 gene. unc-70 null mutants develop slowly, and the adults are paralyzed and dumpy. However, the membrane integrity is not impaired in unc-70 animals, nor is cell polarity affected. Thus, ß-spectrin is not essential for general membrane integrity or for cell polarity. However, ß-spectrin is required for a subset of processes at cell membranes. In neurons, the loss of ß-spectrin leads to abnormal axon outgrowth. In muscles, a loss of ß-spectrin leads to disorganization of the myofilament lattice, discontinuities in the dense bodies, and a reduction or loss of the sarcoplasmic reticulum. These defects are consistent with ß-spectrin function in anchoring proteins at cell membranes (Hammarlund, 2000).

CeßS1 is predicted to contain 2,257 residues (~262 kD) and is 54.2% identical to human ß2sigma1 spectrin, which is the major nonerythrocyte ß-spectrin isoform in vertebrates. The C. elegans genome sequence is essentially complete, and BLAST searches fail to identify any other ß-spectrin homologs. Thus, unc-70 is likely to encode the only C. elegans ß-spectrin. Individual domains of CeßS1 have been compared to human ß2sigma1 and to Drosophila ß-spectrin to identify strongly conserved regions; four such regions have been found. The first includes the NH2-terminal actin-binding domain and the first two spectrin repeats that nucleate alphaß heterodimer formation and interact with adducin and other proteins. The second and third regions of conservation are centered on the 8th and 14th spectrin repeats, respectively; no specific functions have been previously localized to these repeats. Finally, the fourth region of conservation encompasses the last two spectrin repeats, which are essential for the formation of alpha2ß2 spectrin tetramers (Hammarlund, 2000).

Interaction of spectrin with Actin, Ankyrin, Protein 4.1, and Na+/K+ ATPase

The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells, homologs of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest functional linkages among ankyrin, fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, it has been shown that ankyrin binds to the ubiquitous (Na+/K+)ATPase, which has an asymmetrical distribution in polarized cells (Nelson, 1987).

In confluent MDCK cells and intact kidney proximal tubule cells, (Na+/K+)ATPase, fodrin, and analogs of human erythrocyte ankyrin are precisely colocalized in the basolateral domain at the ultrastructural level. This colocalization is only achieved in MDCK cells after confluence is attained. Erythrocyte ankyrin binds saturably to (Na+/K+)ATPase in a molar ratio of approximately 1 ankyrin to 4 (Na+/K+)ATPase's, with a kD of 2.6 microM. The binding of ankyrin to (Na+/K+)ATPase is inhibited by the 43-kD cytoplasmic domain of erythrocyte band 3. 125I-labeled ankyrin binds to the alpha subunit of (Na+/K+)ATPasee in vitro. There also appears to be a second minor membrane protein of approximately 240 kD that is associated with both erythrocyte and kidney membranes that binds 125I-labeled ankyrin avidly. The precise identity of this component is unknown. These results identify a molecular mechanism in the renal epithelial cell that may account for the polarized distribution of the fodrin-based cortical cytoskeleton (Morrow, 1989).

Protein 4.1's interaction with the erythroid skeletal proteins spectrin and actin and its essential role in regulating membrane strength are both attributable to expression of an alternatively spliced 63-nucleotide exon. The corresponding 21-amino acid (21-aa) cassette is within the previously identified spectrin-actin binding domain (10 kDa molecular mass) of erythroid protein 4.1. This cassette is absent, however, in several isoforms that are generated by tissue- and development-specific RNA splicing. Four isoforms of the 10-kDa domain were constructed for comparative assessment of functions particularly relevant to red cells. In vitro translated isoforms containing the 21-aa cassette, denoted 10k21 and 10k19,21, are able to bind spectrin, stabilize spectrin-actin complexes, and associate with red cell membrane. Isoforms replacing or lacking the 21-aa cassette, 10k19 and 10k0, do not function in these assays. A bacterially expressed fusion protein with glutathione-S-transferase, designated GST-10k21, congeals spectrin-actin into a network in vitro as found with purified protein 4.1. Additionally, incorporation of GST-10k21 into mechanically weak, 4.1-deficient membranes increases the mechanical strength of these membranes to normal. GST-10k19 did not function in these assays. These results show that the 21-aa sequence in protein 4.1 is critical to mechanical integrity of the red cell membrane. These results also allow the role of protein 4.1 in membrane mechanics to be interpreted primarily in terms of its spectrin-actin binding function. Alternatively expressed sequences within the 10-kDa domain of nonerythroid protein 4.1 are suggested to have different, yet to be defined functions (Discher, 1993).

A developmental alternative splicing switch, involving exon 16 of protein 4.1 pre-mRNA, occurs during mammalian erythropoiesis. By controlling expression of a 21-amino acid peptide required for high-affinity interaction of protein 4.1 with spectrin and actin, this switch helps to regulate erythrocyte membrane mechanical stability. Key aspects of protein 4.1 structure and function are conserved in nucleated erythroid cells of the amphibian Xenopus laevis. Analysis of protein 4.1 cDNA sequences cloned from Xenopus erythrocytes and oocytes show that tissue-specific alternative splicing of exon 16 also occurs in frogs. Importantly, functional studies with recombinant Xenopus erythroid 4.1 demonstrate specific binding to and mechanical stabilization of 4.1-deficient human erythrocyte membranes. Phylogenetic sequence comparison shows two evolutionarily conserved peptides that represent candidate spectrin-actin binding sites. Finally, in situ hybridization of early embryos shows high expression of 4.1 mRNA in ventral blood islands and in developing brain structures. These results demonstrate that regulated expression of structurally and functionally distinct protein 4.1 isoforms, mediated by tissue-specific alternative splicing, have been highly evolutionarily conserved. Moreover, both nucleated amphibian erythrocytes and their enucleated mammalian counterparts express 4.1 isoforms functionally competent for spectrin-actin binding (Winardi, 1995).

To study the functions of spectrin, Caco-2 intestinal epithelial cells were transfected with a plasmid conferring neomycin resistance and encoding either actin-binding or ankyrin-binding domains of beta G-spectrin fused with beta-galactosidase. These polypeptides, in principle, could interfere with the interaction of spectrin with actin or ankyrin, as well as block normal assembly of alpha- and beta-spectrin subunits. Cells expressing the fusion proteins represent only a small fraction of neomycin-resistant cells, but they can be detected based on expression of beta-galactosidase. Cells expressing spectrin domains exhibit a progressive decrease in amounts of endogenous beta G-spectrin, although alpha-Spectrin is still present. Beta G-spectrin-deficient cells lose epithelial cell morphology, become multinucleated, and eventually disappear after 10-14 d in culture. Spectrin-associated membrane proteins, ankyrin and adducin, as well as the (Na+/K+)ATPase, which binds to ankyrin, exhibit altered distributions in cells transfected with beta G-spectrin domains. E-cadherin and F-actin, in contrast to ankyrin, adducin, and the (Na+/K+)ATPase, are expressed, and they exhibit unaltered distribution in beta G-spectrin-deficient cells. These results establish that beta G-spectrin is essential for the normal morphology of epithelial cells, as well as for their maintenance in monolayer culture (Hu, 1995).

Miscellaneous spectrin interactions

A60 is a 60-kDa component of the axonal cortical cytoskeleton in CNS neurons. It appears to be neuron specific and is tightly bound to brain membranes. A60 in a partially purified soluble extract of brain membranes interacts selectively with brain but not erythrocyte spectrin. Because erythrocyte spectrin is more closely related to the dendritic form of spectrin than the axonal form, this raises the possibility that A60 localises in axons by interaction with the axonal form of spectrin only. A60 is not found in rat cerebellum before the day of birth. However, during postnatal development of the cerebellum (days 1-13) DR1 reactivity appears progressively. On postnatal day 1, a small population of cells in the mantle layer (presumptive Purkinje cells) is DR1 positive. There is no DR1 reactivity found in Purkinje cell axons during their initial phase of growth. By postnatal day 7, Purkinje cell bodies, initial dendritic segments, and the cerebellar white matter are all positive. This pattern of labelling is strengthened up until postnatal day 13. By contrast, in adult rat cerebellum, the location of A60 has changed so that it is most concentrated in axons, and dendritic staining is lost. These data indicate that A60 is a spectrin-binding component of the adult axonal membrane skeleton, the presence of which is only required in axons after the initial phase of growth (Hayes, 1994).

beta G spectrin subunit is responsible for high affinity membrane binding. Two regions of beta G spectrin were expressed in bacteria and demonstrated to contain fully functional binding site(s) for a subset of spectrin-binding sites in brain membranes depleted of peripheral proteins. One region, located near the NH2 terminus, is comprised of 106-residue repeats and requires repeats 2-7 for full activity. The other binding domain is located at the COOH terminus, which is the most variable between beta G and beta R spectrins; it is distinct from the 106-residue repeats, and contains a pleckstrin homology domain. NH2-terminal beta spectrin polypeptides interact with a membrane site(s) that recognizes both brain and erythrocyte isoforms of spectrin, is inhibited by calcium/calmodulin, and is not blocked by the COOH-terminal polypeptide. The COOH-terminal region associates with a membrane site(s) that is specific for brain spectrin, is not inhibited by calcium/calmodulin (See Drosophila Calmodulin), and is not blocked by the NH2-terminal polypeptide. These observations demonstrate membrane association of spectrin with at least two independent sites that differ with regard to regulation by calcium/calmodulin and in selectivity for spectrin isoforms (Davis, 1994).

NF2 is the most commonly mutated gene in benign tumours of the human nervous system. The NF2 protein, called schwannomin or merlin (see Drosophila Merlin), is absent in virtually all schwannomas, and many meningiomas and ependymomas. Using the yeast two-hybrid system, betaII-spectrin (also known as fodrin) has been identified as a schwannomin-binding protein. Interaction occurs between the carboxy-terminal domain of schwannomin isoform 2 and the ankyrin-binding region of betaII-spectrin. In contrast, isoform 1 of schwannomin interacts weakly with betaII-spectrin, presumably because of its strong self-interaction. Thus, alternative splicing of NF2 may regulate betaII-spectrin binding. Schwannomin co-immunoprecipitates with betaII-spectrin at physiological concentrations. The two proteins interact in vitro and co-localize in several target tissues and in STS26T cells. Three naturally occurring NF2 missense mutations show reduced, but not absent, betaII-spectrin binding, suggesting an explanation for the milder phenotypes seen in patients with missense mutations. STS26T cells treated with NF2 antisense oligonucleotides show alterations of the actin cytoskeleton. Schwannomin itself lacks the actin binding sites found in ezrin, radixin and moesin, suggesting that signaling to the actin cytoskeleton occurs via actin-binding sites on betaII-spectrin. Thus, schwannomin is a tumour suppressor directly involved in actin-cytoskeleton organization, which suggests that alterations in the cytoskeleton are an early event in the pathogenesis of some tumour types (Scoles, 1998).

AlphaII-spectrin is a major cortical cytoskeletal protein contributing to membrane organization and integrity. The Ca2+-activated binding of calmodulin to an unstructured insert in the 11th repeat unit of alphaII-spectrin enhances the susceptibility of spectrin to calpain cleavage but abolishes its sensitivity to several caspases and to at least one bacterially derived pathologic protease. Other regulatory inputs including phosphorylation by c-Src also modulate the proteolytic susceptibility of alphaII-spectrin. These pathways, acting through spectrin, appear to control membrane plasticity and integrity in several cell types. To provide a structural basis for understanding these crucial biological events, the crystal structure of a complex between bovine calmodulin and the calmodulin-binding domain of human alphaII-spectrin has been solved. The structure revealed that the entire calmodulin-spectrin-binding interface is hydrophobic in nature. The spectrin domain is also unique in folding into an amphiphilic helix once positioned within the calmodulin-binding groove. The structure of this complex provides insight into the mechanisms by which calmodulin, calpain, caspase, and tyrosine phosphorylation act on spectrin to regulate essential cellular processes (Simonovic, 2006).

Spectrin and the Golgi complex

Homologs of spectrin (a not-yet-cloned isoform, betaISigma* spectrin) and ankyrin (AnkG119 and an approximately 195-kDa ankyrin), two of the major components of the well-characterized erythrocyte plasma-membrane-skeleton, associate with the Golgi complex. ADP ribosylation factor (ARF) is a small G protein that controls the architecture and dynamics of the Golgi by mechanisms that remain incompletely understood. Activated ARF stimulates the in vitro association of betaISigma* spectrin with a Golgi fraction. The Golgi-associated betaISigma* spectrin contains epitopes characteristic of the betaISigma2 spectrin pleckstrin homology (PH) domain known to bind phosphatidylinositol 4,5-bisphosphate (PtdInsP2); ARF recruits betaISigma* spectrin by inducing increased PtdInsP2 levels in the Golgi. The stimulation of spectrin binding by ARF is independent of ARF's ability to stimulate phospholipase D or to recruit coat proteins (COP)-I and can be blocked by agents that sequester PtdInsP2. It is postulated that a PH domain within betaISigma* Golgi spectrin binds PtdInsP2 and acts as a regulated docking site for spectrin on the Golgi. Agents that block the binding of spectrin to the Golgi, either by blocking the PH domain interaction or a constitutive Golgi binding site within spectrin's membrane association domain I, inhibit the transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the medial compartment of the Golgi complex. Collectively, these results suggest that the Golgi-spectrin skeleton plays a central role in regulating the structure and function of this organelle (Godi, 1998).

Spectrin and axonal transport

Dynein-driven, dynactin-dependent vesicle transport was reconstituted using protein-free liposomes and soluble components from squid axoplasm. Dynein anddynactin, while necessary, are not the only essential cytosolic factors; axonal spectrin is also required. Spectrin is resident on axonal vesicles, and rebinds fromcytosol to liposomes or proteolysed vesicles, concomitant with their dynein-dynactin-dependent motility. Binding of purified axonal spectrin to liposomes requiresacidic phospholipids, as does motility. Using dominant negative spectrin polypeptides and a drug that releases PH domains from membranes, it has been shown that spectrin isrequired for linking dynactin, and thereby dynein, to acidic phospholipids in the membrane. This model is verified in the context of liposomes, isolated axonal vesicles, and whole axoplasm. It is concluded that spectrin has an essential role in retrograde axonal transport (Muresan, 2001).

It was initially quite surprising to discover that in squid axon cytosol, liposomes are driven exclusively toward microtubule minus ends. By contrast, most substrates such as silica or latex beads move in the opposite direction, driven by kinesin, which avidly adsorbs to practically any surface. Given the otherwise poor performance of cytoplasmic dynein in vitro, the extremely high levels of cytosol-induced minus end liposome motility and its dependence on acidic phospholipids prompted an examination of its physiological significance (Muresan, 2001).

Dynein-driven liposome motility in cytosol reflects a specific assembly process directed by acidic phospholipids. This process starts with the recruitment of spectrin, which in turn anchors dynactin and thereby dynein to the membrane. That protein-free liposomes recruit principal components of the retrograde transport machinery, much like bona fide organelles, is evident from the strikingly similar motility of liposomes in cytosol compared to that of native squid axon vesicles, and from the dependence of this motility on dynactin and spectrin (Muresan, 2001).

How could assembly of the dynein-dynactin machinery be directed to specific membranes if acidic phospholipids were involved in the linkage mechanism? Acidic phospholipids, such as phosphatidylserine, are primarily located on the cytoplasmic surface of organelles, including axonal vesicles. Also, lipid kinases may be activated at specific membranes where they catalyze the conversion of weakly acidic or neutral phospholipids (e.g., phosphatidylinositol or PC) to more acidic products (e.g., phosphoinositides or PA). It is proposed that recruitment of dynein to organelle membranes is fundamentally similar to other membrane-trafficking events such as vesicle budding, endocytosis, yeast vacuolar transport, and actin-based propulsion, in which assembly of a multicomponent complex at specific membranes involves binding to acidic phospholipids. To illustrate this analogy, the process of directed actin assembly at cell and organelle membranes is considered. First, N-WASP initiates localized actin assembly at specific membrane sites by binding to acidic phospholipids (i.e., phosphatidylinositol 4,5-bisphosphate), and second, to the actin-related protein complex, ARP2/3. A required small GTP binding protein, Cdc42, is thought to provide a basis for localized targeting. By analogy, squid axon spectrin also associates with acidic phospholipids, and also binds to an actin-related protein, ARP1, which in this case recruits dynactin. Identifying a GTP binding protein in this process would be an interesting prospect for the future (Muresan, 2001).

It is suspected that a PH domain in axonal spectrin may contribute to spectrin's interaction with acidic phospholipids and provide a degree of specificity to dynein recruitment. All known nonerythroid beta spectrins have PH domains, and the PH domains of many proteins, including betaII spectrin, bind specific acidic phospholipids. In addition, the binding of squid axon spectrin to both vesicles and protein-free liposomes is sensitive to neomycin, a drug that disrupts the interaction between PH domains and acidic phospholipids. By contrast, the synapsin III acidic phospholipid binding domain A, which is not a PH domain, fails to compete for membrane binding with axonal spectrin (Muresan, 2001).

The participation of other spectrin-membrane binding domains in binding to acidic phospholipids cannot be excluded. In addition to a PH domain, spectrins have two ankyrin-independent, direct membrane association domains that interact with purified brain membranes and are active in vivo. These studies do not address the role of interactions between integral membrane proteins and spectrin. Many such interactions exist, and their selective disruption can impair the trafficking of specific proteins in the secretory pathway. One way of reconciling these observations with the present findings is to envision that direct binding of spectrin to acidic phospholipids is sufficient to initiate a linkage to dynactin, but that more stable polyvalent interactions between the vesicle and spectrin occur in vivo (Muresan, 2001).

While the mechanism of dynein-dynactin recruitment to membranes via a spectrin meshwork bound to acidic phospholipids could explain the broad cargo selectivity of the dynein machinery, these findings do not exclude that dynein may attach to its many membranous cargoes in diverse ways. For example, in retinal photoreceptor cells, dynein appears to bind to membranes in the absence of dynactin via a direct interaction between one of its light chains (Tctex-1) and rhodopsin, a transmembrane protein involved in phototransduction (Muresan, 2001).

Previous reconstitution studies have established that the dynactin complex is an essential cofactor for dynein-driven vesicle transport. Those experiments, using cytosol and vesicles from chick embryonic tissues, show that dynactin and dynein together, but not dynein alone, reconstitute the motility of isolated KI-extracted vesicles. Since KI-extracted vesicles retain spectrin, an additional requirement for cytosolic spectrin may have been missed. In support of this possibility, it has been found that processive motility of KI-extracted squid axon vesicles is largely reconstituted using isolated dynein-dynactin (mean run length = 2.69 µm). However, it is noted that in the chick system, although dynactin alone increases dynein-driven motility of KI-extracted vesicles, it does not restore the high velocity of vesicle movement observed in cytosol. Full reconstitution requires a poorly characterized 9S protein complex, called Activator II, which, in combination with dynein, increases both the frequency and velocity of vesicle transport. Interestingly, spectrin is present in an Activator II fraction derived from squid axoplasm (Muresan, 2001).

In summary, by bridging the gap between biochemistry and motility, these experiments provide significant new evidence for a role of spectrin and acidic phospholipids in the recruitment of the dynein transport machinery to organelle membranes. Still needed in the future are biochemical studies using purified proteins as well as genetic approaches. In this respect, Drosophila and C. elegans, both of which show neuronal defects when spectrin is disrupted, may provide profitable systems for addressing further the role of spectrin in retrograde axonal transport (Muresan, 2001).

Mammalian alpha I-spectrin is a neofunctionalized polypeptide adapted to small highly deformable erythrocytes

Mammalian red blood cells, unlike those of other vertebrates, must withstand the rigors of circulation in the absence of new protein synthesis. Key to this is plasma membrane elasticity deriving from the protein spectrin, which forms a network on the cytoplasmic face. Spectrin is a tetramer (alphabeta)(2), made up of alphabeta dimers linked head to head. One component of erythrocyte spectrin, alphaI, is encoded by a gene unique to mammals. Phylogenetic analysis suggests that the other alpha-spectrin gene (alphaII) common to all vertebrates was duplicated after the emergence of amphibia, and that the resulting alphaI gene was preserved only in mammals. The activities of alphaI and alphaII spectrins differ in the context of the human red cell membrane. An alphaI-spectrin fragment containing the site of head-to-head interaction with the beta-chain binds more weakly than the corresponding alphaII fragment to this site. The latter competes so strongly with endogenous alphaI as to cause destabilization of membranes at 100-fold lower concentration than the alphaI fragment. The efficacies of alphaI/alphaII chimeras indicate that the partial structural repeat, which binds to the complementary beta-spectrin element, and the adjacent complete repeat together determine the strength of the dimer-dimer interaction on the membrane. Alignment of all available alpha-spectrin N-terminal sequences reveals three blocks of sequence unique to alphaI. Furthermore, human alphaII-spectrin is closer to fruitfly alpha-spectrin than to human alphaI-spectrin, consistent with adaptation of alphaI to new functions. It is concluded that alphaI-spectrin represents a neofunctionalized spectrin adapted to the rapid make and break of tetramers (Solomao, 2006; full text of article).

alphaII-spectrin is essential for assembly of the nodes of Ranvier in myelinated axons

Saltatory conduction in myelinated axons requires organization of the nodes of Ranvier, where voltage-gated sodium channels are prominently localized. Previous results indicate that alphaII-spectrin, a component of the cortical cytoskeleton, is enriched at the paranodes, which flank the node of Ranvier, but alphaII-spectrin's function has not been investigated. Starting with a genetic screen in zebrafish, a mutation was discovered in alphaII-spectrin (alphaII-spn) that disrupts nodal sodium-channel clusters in myelinated axons of the PNS and CNS. In alphaII-spn mutants, the nodal sodium-channel clusters are reduced in number and disrupted at early stages. Analysis of chimeric animals indicated that alphaII-spn functions autonomously in neurons. Ultrastructural studies show that myelin forms in the posterior lateral line nerve and in the ventral spinal cord in alphaII-spn mutants and that the node is abnormally long; these findings indicate that alphaII-spn is required for the assembly of a mature node of the correct length. alphaII-spectrin is enriched in nodes and paranodes at early stages and that the nodal expression diminishes as nodes mature. These results provide functional evidence that alphaII-spectrin in the axonal cytoskeleton is essential for stabilizing nascent sodium-channel clusters and assembling the mature node of Ranvier (Voss, 2007).

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

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