Jon S. Morrow

Simultaneous degradation of alphaII-and betaII-spectrin by caspase 3 (CPP32) in apoptotic cells

The degradation of αII- and βII-spectrin during apoptosis in cultured human neuroblastoma SH-SY5Y cells was investigated. Immunofluorescent staining showed that the collapse of the cortical spectrin cytoskeleton is an early event following staurosporine challenge. This collapse correlated with the generation of a series of prominent spectrin breakdown products (BDPs) derived from both αII- and βII-subunits. Major C-terminal αII-spectrin BDPs were detected at ≈150, 145, and 120 kDa (αII-BDP150, αII-BDP145, and αII-BDP120, respectively); major C-terminal βII-spectrin BDPs were at ≈110 and 85 kDa (βII-BDP110 and βII-BDP85, respectively). N-terminal sequencing of the major fragments produced in vitro by caspase 3 revealed that αII-BDP150 and αII-BDP120 were generated by cleavages at DETD1185*S1186 and DSLD1478*S1479, respectively. For βII-spectrin, a major caspase site was detected at DEVD1457*S1458 , and both βII-BDP110 and βII-BDP85 shared a common N-terminal sequence starting with Ser1458. An additional cleavage site near the C terminus, at ETVD2146*S2147, was found to account for βII-BDP85. Studies using specific caspase or calpain inhibitors indicate that the pattern of spectrin breakdown during apoptosis differs from that during non-apoptotic cell death. We postulate that in concert with calpain, caspase rapidly targets critical sites in both αII- and βII-spectrin and thereby initiates a rapid dissolution of the spectrin-actin cortical cytoskeleton with apoptosis.

Rad9 Phosphorylation Sites Couple Rad53 to the Saccharomyces cerevisiae DNA Damage Checkpoint

Rad9 is required for the MEC1/TEL1-dependent activation of Saccharomyces cerevisiae DNA damage checkpoint pathways mediated by Rad53 and Chk1. DNA damage induces Rad9 phosphorylation, and Rad53 specifically associates with phosphorylated Rad9. We report here that multiple Mec1/Tel1 consensus [S/T]Q sites within Rad9 are phosphorylated in response to DNA damage. These Rad9 phosphorylation sites are selectively required for activation of the Rad53 branch of the checkpoint pathway. Consistent with the in vivo function in recruiting Rad53, Rad9 phosphopeptides are bound by Rad53 forkhead-associated (FHA) domains in vitro. These data suggest that functionally independent domains within Rad9 regulate Rad53 and Chk1, and support the model that FHA domain-mediated recognition of Rad9 phosphopeptides couples Rad53 to the DNA damage checkpoint pathway.

Dynactin-Dependent, Dynein-Driven Vesicle Transport in the Absence of Membrane Proteins: A Role for Spectrin and Acidic Phospholipids

We reconstituted dynein-driven, dynactin-dependent vesicle transport using protein-free liposomes and soluble components from squid axoplasm. Dynein and dynactin, while necessary, are not the only essential cytosolic factors; axonal spectrin is also required. Spectrin is resident on axonal vesicles, and rebinds from cytosol to liposomes or proteolysed vesicles, concomitant with their dynein-dynactin-dependent motility. Binding of purified axonal spectrin to liposomes requires acidic phospholipids, as does motility. Using dominant negative spectrin polypeptides and a drug that releases PH domains from membranes, we show that spectrin is required for linking dynactin, and thereby dynein, to acidic phospholipids in the membrane. We verify this model in the context of liposomes, isolated axonal vesicles, and whole axoplasm. We conclude that spectrin has an essential role in retrograde axonal transport.

The role of ankyrin and spectrin in membrane transport and domain formation

Recent discoveries reveal a Golgi-centric spectrin-ankyrin skeleton required for Golgi integrity and anterograde protein trafficking. Identification of specific functional domains in spectrin that mediate its association with motor proteins and the Golgi complex has allowed novel insights into the structure and function of the secretory pathway, and into how this process is controlled by ADP-ribosylation factor and phosphoinositides. Alternative models of Golgi spectrin function that have been recently proposed are reviewed.

Neural cell adhesion molecule (NCAM) association with PKCβ2 via βI spectrin is implicated in NCAM-mediated neurite outgrowth

In hippocampal neurons and transfected CHO cells, neural cell adhesion molecule (NCAM) 120, NCAM140, and NCAM180 form Triton X-100–insoluble complexes with βI spectrin. Heteromeric spectrin (αIβI) binds to the intracellular domain of NCAM180, and isolated spectrin subunits bind to both NCAM180 and NCAM140, as does the βI spectrin fragment encompassing second and third spectrin repeats (βI2–3). In NCAM120-transfected cells, βI spectrin is detectable predominantly in lipid rafts. Treatment of cells with methyl-β-cyclodextrin disrupts the NCAM120–spectrin complex, implicating lipid rafts as a platform linking NCAM120 and spectrin. NCAM140/NCAM180–βI spectrin complexes do not depend on raft integrity and are located both in rafts and raft-free membrane domains. PKCβ2 forms detergent-insoluble complexes with NCAM140/NCAM180 and spectrin. Activation of NCAM enhances the formation of NCAM140/NCAM180–spectrin–PKCβ2 complexes and results in their redistribution to lipid rafts. The complex is disrupted by the expression of dominant-negative βI2–3, which impairs binding of spectrin to NCAM, implicating spectrin as the bridge between PKCβ2 and NCAM140 or NCAM180. Redistribution of PKCβ2 to NCAM–spectrin complexes is also blocked by a specific fibroblast growth factor receptor inhibitor. Furthermore, transfection with βI2–3 inhibits NCAM-induced neurite outgrowth, showing that formation of the NCAM–spectrin–PKCβ2 complex is necessary for NCAM-mediated neurite outgrowth.

Disrupted synaptic development in the hypoxic newborn brain

Infants born prematurely risk significant life-long cognitive disability, representing a major pediatric health crisis. The neuropathology of this cohort is accurately modeled in mice subjected to sublethal postnatal hypoxia. Massively parallel transcriptome analysis using cDNA microchips (9,262 genes), combined with immunohistochemical and protein assays, reveals that sublethal hypoxia accentuates genes subserving presynaptic function, and it suppresses genes involved with synaptic maturation, postsynaptic function, and neurotransmission. Other significantly affected pathways include those involved with glial maturation, vasculogenesis, and components of the cortical and microtubular cytoskeleton. These patterns reveal a global dysynchrony in the maturation programs of the hypoxic developing brain, and offer insights into the vulnerabilities of processes that guide early postnatal cerebral maturation.

STRUCTURE OF THE ANKYRIN BINDING DOMAIN OF A ALPHA-Na,K-ATPase

The ankyrin 33-residue repeating motif, an L-shaped structure with protruding β-hairpin tips, mediates specific macromolecular interactions with cytoskeletal, membrane, and regulatory proteins. The association between ankyrin and α-Na,K-ATPase, a ubiquitous membrane protein critical to vectorial transport of ions and nutrients, is required to assemble and stabilize Na,K-ATPase at the plasma membrane. α-Na,K-ATPase binds both red cell ankyrin (AnkR, a product of the ANK1 gene) and Madin-Darby canine kidney cell ankyrin (AnkG, a product of the ANK3 gene) utilizing residues 142–166 (SYYQEAKSSKIMESFK NMVPQQALV) in its second cytoplasmic domain. Fusion peptides of glutathione S-transferase incorporating these 25 amino acids bind specifically to purified ankyrin (K d = 118 ± 50 nm). The three-dimensional structure (2.6 Å) of this minimal ankyrin-binding motif, crystallized as the fusion protein, reveals a 7-residue loop with one charged hydrophilic face capping a double β-strand. Comparison with ankyrin-binding sequences in p53, CD44, neurofascin/L1, and the inositol 1,4,5-trisphosphate receptor suggests that the valency and specificity of ankyrin binding is achieved by the interaction of 5–7-residue surface loops with the β-hairpin tips of multiple ankyrin repeat units.

Identification of a stomatin orthologue in vacuoles induced in human erythrocytes by malaria parasites. A role for microbial raft proteins in apicomplexan vacuole biogenesis.

When the human malaria parasite Plasmodium falciparum infects erythrocytes, proteins associated with host-derived detergent-resistant membrane (DRM) rafts are selectively recruited into the newly formed vacuole, but parasite proteins that contribute to raft-based vacuole development are unknown. In mammalian cells, DRM-associated integral membrane proteins such as caveolin-1 and flotillin-1 that form oligomers have been linked to the formation of DRM-based invaginations called caveolae. Here we show that the P. falciparum genome does not encode caveolins or flotillins but does contain an orthologue of human band 7 stomatin, a protein known to oligomerize, associate with non-caveolar DRMs and is distantly related to flotillins. Stomatins are members of a large protein family conserved in evolution and P. falciparum (Pf) stomatin appears to be a prokaryotic-like molecule. Evidence is presented that it associates with DRMs and may oligomerize, suggesting that these features are conserved in the stomatin family. Further, Pfstomatin is an integral membrane protein concentrated at the apical end of extracellular parasites, where it co-localizes with invasion-associated rhoptry organelles. A resident rhoptry protein, RhopH2 also resides in DRMs. This provides the first evidence that rhoptries of an apicomplexan parasite contain DRM rafts. Further, when the parasite invades erythrocytes, rhoptry Pfstomatin and RhopH2 are inserted into the newly formed vacuole. Thus, like caveolin-1 and flotillin-1, a stomatin may also associate with non-clathrin coated, DRM-enriched vacuoles. We propose a new model of invasion and vacuole formation involving DRM-based interactions of both host and parasite molecules.

Molecular and functional changes in spectrin from patients with hereditary pyropoikilocytosis.

The structural and functional properties of spectrin from normal and hereditary pyropoikilocytosis (HPP) donors from the two unrelated families were studied. The structural domains of the spectrin molecule were generated by mild tryptic digestion and analyzed by two-dimensional electrophoresis (isoelectric focusing; sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The alpha I-T80 peptide (Mr 80,000) is not detectable in two related HPP donors; instead, two new peptides (Mr 50,000 and 21,000) are generated and have been identified as fragments of the normal alpha I-T80. A third sibling has reduced levels of both the normal alpha I-T80 and the two new peptides. A similar analysis of spectrin from another HPP family indicates that their spectrins contain reduced amounts of the alpha I-T80 and the 50,000 and 21,000 fragments of the alpha I domain. The HPP donor also has other structural variations in the alpha I, alpha II, and alpha III domains. The alpha I-T80 domain of normal spectrin has been shown to be an important site for spectrin oligomerization (J. Morrow and V.T. Marchesi. 1981. J. Cell Biol. 88: 463-468), and in vitro assays indicate that HPP spectrin has an impaired ability to oligomerize. Ghost membranes from HPP donors are also more fragile than membranes from normal erythrocytes when measured by ektacytometry. In both the oligomerization and fragility assays, the degree of impairment is correlated with the amount of normal alpha I-T80 present in the spectrin molecule. We believe that a structural alteration in the alpha I-T80 domain perturbs normal in vivo oligomerization of spectrin, producing a marked decrease in erythrocyte stability.

Transforming growth factor beta induces caspase 3-independent cleavage of alphaII-spectrin (alpha-fodrin) coincident with apoptosis.

Transforming growth factor β (TGF-β) is a potent growth inhibitor and inducer of cell death in B-lymphocytes and is essential for immune regulation and maintenance of self-tolerance. In this report the mouse immature B cell line, WEHI 231, was used to examine the mechanisms involved in TGF-β-mediated apoptosis. Induction of apoptosis is detected as early as 8 h after TGF-β administration. Coincident with the onset of apoptosis, the cytoskeletal actin-binding protein, αII-spectrin (α-fodrin) is cleaved into 150-, 115-, and 110-kDa fragments. The broad spectrum caspase inhibitor (Boc-D-fmk (BD-fmk)) completely abolished TGF-β-induced apoptosis and αII-spectrin cleavage. Caspase 3, although present in WEH1 231 cells, was not activated by TGF-β, nor was its substrate, poly(ADP-ribose) polymerase. These results identify αII-spectrin as a novel substrate that is cleaved during TGF-β-induced apoptosis. Our data provide the first evidence of calpain and caspase 3-independent cleavage of αII-spectrin during apoptosis and suggests that TGF-β induces apoptosis and αII-spectrin cleavage via a potentially novel caspase. This report also provides the first direct evidence of caspase 3 activation in WEH1 231 cells and indicates that at least two distinct apoptotic pathways exist.