Essential control of an endothelial cell ISOC by the spectrin membrane skeleton.

Mechanism(s) underlying activation of store-operated Ca2+ entry currents, ISOC, remain incompletely understood. F-actin configuration is an important determinant of channel function, although the nature of interaction between the cytoskeleton and ISOC channels is unknown. We examined whether the spectrin membrane skeleton couples Ca2+ store depletion to Ca2+ entry. Thapsigargin activated an endothelial cell ISOC (-45 pA at -80 mV) that reversed at +40 mV, was inwardly rectifying when Ca2+ was the charge carrier, and was inhibited by La3+ (50 microM). Disruption of the spectrin-protein 4.1 interaction at residues A207-V445 of betaSpIISigma1 decreased the thapsigargin-induced global cytosolic Ca2+ response by 50% and selectively abolished the endothelial cell ISOC, without altering activation of a nonselective current through cyclic nucleotide-gated channels. In contrast, disruption of the spectrin-actin interaction at residues A47-K186 of betaSpIISigma1 did not decrease the thapsigargin-induced global cytosolic Ca2+ response or inhibit ISOC. Results indicate that the spectrin-protein 4.1 interaction selectively controls ISOC, indicating that physical coupling between calcium release and calcium entry is reliant upon the spectrin membrane skeleton.

Spectrin (betaSpIIsigma1) is an essential component of synaptic transmission.

The cellular mechanism that underlies the regulated release of synaptic vesicles during neurotransmission is not fully known. Our previous data has shown that brain spectrin (alphaSpIIsigma1/betaSpIIsigma1)2 is localized in axons and nerve terminals and we have shown that the beta subunit (betaSpIIsigma1) contains a synapsin-binding domain capable of interacting with synapsin and small synaptic vesicles in vitro and in vivo. These findings suggested a role for brain beta-spectrin in synaptic neurotransmission. To examine this possibility further, peptide-specific antibodies directed against epitopes within the synapsin-binding domain of brain beta-spectrin, or against flanking regions, were injected into the presynaptic neuron of synaptically paired rat hippocampal neurons in culture. Here, we show that the antibodies directed against the synapsin-binding domain specifically blocked synaptic neurotransmission.

Synthesis, assembly, and turnover of alpha and beta-erythroid and nonerythroid spectrins in rat hippocampal neurons.

The synthesis and turnover of alpha-erythroid, beta-erythroid, alpha-nonerythroid and beta-nonerythroid spectrins was investigated in cultured rat hippocampal neurons. [35S]methionine and subunit specific antibodies were used to label and immunoprecipitate newly synthesized spectrins in 12- to 14-day-old cultures. Synthesis experiments, performed under normal resting conditions, showed that the ratio of newly synthesized alpha-erythroid/beta-erythroid and alpha-nonerythroid/beta-nonerythroid spectrins is 1/1 (mol/mol) both in the soluble and insoluble fractions. Soluble and insoluble alpha and beta erythroid spectrin turn over rapidly (half-life=16-24 min). Soluble nonerythroid alpha-spectrin (half-life=80 min) and beta spectrin (half-life=53 min) turn over more slowly than their insoluble counterparts (30-34 min). The nonerythroid alpha spectrin turnover was significantly different (p<0.05) from the other measurements except for nonerythroid beta spectrin, indicating that these subunits are protected from rapid proteolytic degradation until they are assembled in the membrane skeleton.

The domain of brain beta-spectrin responsible for synaptic vesicle association is essential for synaptic transmission.

We have examined the interaction between synapsin I, the major phosphoprotein on the membrane of small synaptic vesicles, and brain spectrin. Using recombinant peptides we have localized the synapsin I attachment site upon the beta-spectrin isoform betaSpIISigmaI to a region of 25 amino acids, residues 211 through 235. This segment is adjacent to the actin binding domain and is within the region of the betaSpIISigmaI that we previously predicted as a candidate synapsin I binding domain based upon sequence homology. We used differential centrifugation techniques to quantitatively assess the interaction of spectrin with synaptic vesicles. Using this assay, high affinity saturable binding of recombinant betaSpIISigmaI proteins was observed with synaptic vesicles. Binding was only observed when the 25 amino acid synapsin I binding site was included on the recombinant peptides. Further, we demonstrate that antibodies directed against 15 amino acids of the synapsin I binding domain specifically blocked synaptic transmission in cultured hippocampal neurons. Thus, the synapsin I attachment site on betaSpIISigmaI spectrin comprises a approximately 25 amino acid segment of the molecule and interaction of these two proteins is an essential step for the process of neurotransmission.

Alpha-spectrins are major ubiquitinated proteins in rat hippocampal neurons and components of ubiquitinated inclusions in neurodegenerative disorders.

We have demonstrated that alpha-spectrins (alphaSpISigma* and alphaSpIISigma1) are major ubiquitinated proteins in terminally differentiated hippocampal neurons in culture. Western blotting experiments, using alphaSpISigma1, alphaSpIISigma1, and ubiquitin antibodies and lysates of 11-day-old cultured rat hippocampal neurons, have demonstrated that a single band comigrating with alphaSpISigma* and alphaSpIISigma1 in a 5% polyacrylamide sodium dodecyl sulfate gel is recognized by ubiquitin antibodies when (125)I-protein A is used for detection. Immunofluorescence staining of the 7- and 12 -day-old rat hippocampal neuron cultures using ubiquitin, alphaSpISigma1, and alphaSpIISigma1 antibodies demonstrated that all of these antibodies label neurons but not the astrocytes in the cultures. Immunoprecipitation of spectrin subunits in lysates of 12-day-old rat hippocampal neurons under stringent conditions (9.5 M urea) using alphaSpISigma1 and alphaSpIISigma1 antibodies followed by Western blot experiments of the immunoprecipitated spectrin subunits using alphaSpISigma1, alphaSpIISigma1 and ubiquitin antibodies confirmed that both alphaSpISigma* and alphaSpIISigma1 are ubiquitinated in rat hippocampal neurons. Furthermore, we demonstrated by immunohistochemistry that alpha-spectrins are components of the cytoplasmic ubiquitinated inclusions in hippocampal neurons in Alzheimer's and Parkinson's disease patients.

Measurement of the synthesis, turnover, and assembly of alpha- and beta-erythroid and nonerythroid spectrins in cultured rat hippocampal neurons.

We describe a method that has allowed us to measure the synthesis, turnover and assembly of alpha- and beta-erythroid and nonerythroid spectrins in cultured rat hippocampal neurons. For these studies, rat hippocampal cultures containing 74.5-83.0% neurons were established. B-27 (Gibco) supplement has been used to obtain an excellent long-term viability (up to 5 weeks) of hippocampal neurons in culture. For the synthesis, turnover, and assembly experiments the neurons were labeled with [35S]methionine, and chased with 10-fold excess of cold methionine for the turnover experiments. The cells were then lysed and immunoprecipitated with alpha, beta-erythroid, alpha, and beta-nonerythroid spectrin antibodies. Immunoprecipitated [35S]methionine-labeled spectrins of hippocampal neurons grown in vitro produced bands in 5% polyacrylamide minigels strong enough to be detected by the high sensitivity screens of a phosphorimager to generate graphs from which the synthesis or half-lives of alpha, beta-erythroid, alpha, and beta-nonerythroid spectrins were calculated. This method can be used to study the role of calpain, caspase-3, and the ubiquitin-proteasome system on the synthesis and turnover of erythroid and nonerythroid spectrins in resting and depolarized rat hippocampal neurons in culture.

Human alpha spectrin II and the FANCA, FANCC, and FANCG proteins bind to DNA containing psoralen interstrand cross-links.

Repair of DNA interstrand cross-links is a complex process critical to which is the identification of sites of damage by specific proteins. We have recently identified the structural protein nonerythroid alpha spectrin (alphaSpIISigma) as a component of a nuclear protein complex in normal human cells which is involved in the repair of DNA interstrand cross-links and have shown that it forms a complex with the Fanconi anemia proteins FANCA, FANCC, and FANCG. Using DNA affinity chromatography, we now show that alphaSpIISigma, present in HeLa cell nuclei, specifically binds to DNA containing psoralen interstrand cross-links and that the FANCA, FANCC, and FANCG proteins are bound to this damaged DNA as well. That spectrin binds directly to the cross-linked DNA has been shown using purified bovine brain spectrin (alphaSpIISigma1/betaSpIISigma1)2. Binding of the Fanconi anemia (FA) proteins to the damaged DNA may be either direct or indirect via their association with alphaSpIISigma. These results demonstrate a role for alpha spectrin in the nucleus as well as a new function for this protein in the cell, an involvement in DNA repair. alphaSpIISigma may bind to cross-linked DNA and act as a scaffold to help in the recruitment of repair proteins to the site of damage and aid in their alignment and interaction with each other, thus enhancing the efficiency of the repair process.

Spectrin ubiquitination and oxidative stress: potential roles in blood and neurological disorders.

This review covers the observations that erythrocyte spectrin has a E2 ubiquitin conjugating enzymatic activity that allows it to transfer ubiquitin to a target site in the alpha-spectrin repeats 20/21. The position of this ubiquitination site suggests that ubiquitination may regulate alpha beta spectrin heterodimer nucleation, spectrin-4.1-actin ternary complex formation, and adducin stimulated spectrin-actin attachment in the mature erythrocyte. In sickle cells, which contain altered redox status (high GSSG/GSH ratio), ubiquitin attachment to the E2 and target sites in alpha-spectrin is greatly diminished. We propose that this attenuated ubiquitination of spectrin may be due to glutathiolation of the E2 active site cysteine leading to diminished ubiquitin-spectrin adduct and conjugate formation. Furthermore we propose that lack of ubiquitin-spectrin complex formation leads to dysregulation of the membrane skeleton in mature SS erythrocytes and may diminish spectrin turnover in SS erythropoietic cells via the ubiquitin proteasome machinery. In hippocampal neurons, spectrin is the major ubiquitinated protein and a component of the cytoplasmic ubiquitinated inclusions observed in Alzheimer's and Parkinson's diseases. The two primary neuronal spectrin isoforms: alpha SpI Sigma*/beta SpI Sigma 2 and alpha SpII Sigma 1/beta SpII Sigma 1 are both ubiquitinated. Future work will resolve whether neuronal spectrins also contain E2-ubiquitin conjugating activity and the molecular basis for formation of ubiquitinated inclusions in neurological disorders.