Irreversible deformation of the spectrin-actin lattice in irreversibly sickled cells

Irreversibly sickled cells (ISC's) are circulating erythrocytes in patients with sickle cell disease that retain a sickled shape even when oxygenated. Evidence points to a membrane defect that prevents the return of these cells to the normal biconcave shape. The erythrocyte membrane protein spectrin is believed to help control erythrocyte shape and deformability. Recent studies suggest that normally spectrin and an erythrocyte actin form a self-supporting, fibrillar, lattice-like network on the cytoplasmic membrane surface. When normal erythrocyte ghosts are extracted with Triton X-100 all the integral membrane proteins and most of the membrane lipids are removed, leaving a ghost-shaped residue composed principally of spectrin and actin. We concentrated ISC's from patients with sickle cell anemia and compared the morphology and protein composition of ghosts and Triton-extracted ghost residues prepared from these ISC's with similar preparations of reversibly sickable cells and normal cells. (a) Many ISC's formed ISC-shaped ghosts. (b) All ISC-shaped ghosts formed ISC-shaped Triton residues. (c) Spectrin, erythrocyte actin (Band 5), an unidentified Band 3 component, and Band 4.1 were the major protein components of the Triton residues. All membrane-associated sickle hemoglobin was removed by the Triton treatment. (d) No ISC-shaped ghosts or ISC-shaped Triton residues were formed when deoxygenated, sickled RSC's were lysed or Triton-extracted. ISC-shaped ghosts and Triton residues were never formed from normal cells. These observations suggest that a defect of the "spectrin-actin lattice" may be the primary abnormality of the ISC membrane. Since ISC's are rigid cells, the data support the postulate that spectrin is a major determinant of membrane deformability. Finally, they provide direct evidence that spectrin is important in determining erythrocyte shape.     

Identification of a candidate human spectrin Src homology 3 domain-binding protein suggests a general mechanism of association of tyrosine kinases with the spectrin-based membrane skeleton

Spectrin is a widely expressed protein with specific isoforms found in erythroid and nonerythroid cells. Spectrin contains an Src homology 3 (SH3) domain of unknown function. A cDNA encoding a candidate spectrin SH3 domain-binding protein was identified by interaction screening of a human brain expression library using the human erythroid spectrin (alphaI) SH3 domain as a bait. Five isoforms of the alphaI SH3 domain-binding protein mRNA were identified in human brain. Mapping of SH3 binding regions revealed the presence of two alphaI SH3 domain binding regions and one Abl-SH3 domain binding region. The gene encoding the candidate spectrin SH3 domain-binding protein has been located to human chromosome 10p11.2 --> p12. The gene belongs to a recently identified family of tyrosine kinase-binding proteins, and one of its isoforms is identical to e3B1, an eps8-binding protein (Biesova, Z., Piccoli, C., and Wong, W. T. (1997)Oncogene 14, 233-241). Overexpression of the green fluorescent protein fusion of the SH3 domain-binding protein in NIH3T3 cells resulted in cytoplasmic punctate fluorescence characteristic of the reticulovesicular system. This fluorescence pattern was similar to that obtained with the anti-human erythroid spectrin alphaI SigmaI/betaI SigmaI antibody in untransfected NIH3T3 cells; in addition, the anti-alphaI SigmaI/betaI SigmaI antibody also stained Golgi apparatus. Immunofluorescence obtained using antibodies against alphaI SigmaI/++betaI SigmaI spectrin and Abl tyrosine kinase but not against alphaII/betaII spectrin colocalized with the overexpressed green fluorescent protein-SH3-binding protein. Based on the conservation of the spectrin SH3 binding site within members of this protein family and published interactions, a general mechanism of interactions of tyrosine kinases with the spectrin-based membrane skeleton is proposed.     

Membrane dynamics of the water transport protein aquaporin-1 in intact human red cells

Aquaporin-1 (AQP1) is the prototype integral membrane protein water channel. Although the three-dimensional structure and water transport function of the molecule have been described, the physical interactions between AQP1 and other membrane components have not been characterized. Using fluorescein isothiocyanate-anti-Co3 (FITC-anti-Co3), a reagent specific for an extracellular epitope on AQP1, the fluorescence photobleaching recovery (FPR) and fluorescence imaged microdeformation (FIMD) techniques were performed on intact human red cells. By FPR, the fractional mobility of fluorescently labeled AQP1 (F-alphaAQP1) in the undeformed red cell membrane is 66 +/- 10% and the average lateral diffusion coefficient is (3.1 +/- 0.5) x 10(-11) cm2/s. F-alphaAQP1 fractional mobility is not significantly affected by antibody-induced immobilization of the major integral proteins band 3 or glycophorin A, indicating that AQP1 does not exist as a complex with these proteins. FIMD uses pipette aspiration of individual red cells to create a constant but reversible skeletal density gradient. F-alphaAQP1 distribution, like that of lipid-anchored proteins, is not at equilibrium after microdeformation. Over time, approximately 50% of the aspirated F-alphaAQP1 molecules migrate toward the membrane portion that had been maximally dilated, the aspirated cap. Based on the kinetics of migration, the F-alphaAQP1 lateral diffusion coefficient in the membrane projection is estimated to be 6 x 10(-10) cm2/s. These results suggest that AQP1 lateral mobility is regulated in the unperturbed membrane by passive steric hindrance imposed by the spectrin-based membrane skeleton and/or by skeleton-linked membrane components, and that release of these constraints by dilatation of the skeleton allows AQP1 to diffuse much more rapidly in the plane of the membrane.     

Functional link between phosphorylation state of membrane proteins and morphological changes of human erythrocytes

The Tyr-phosphorylation of the cytoplasmic domain of the major membrane-spanning band 3, rather than the Ser/Thr-phosphorylation of the membrane proteins (spectrin and band 3 itself), might be functionally related to certain morphological changes of human erythrocytes. This view is supported by the following lines of evidence: a) vanadate or its derivative pervanadate (vanadyl hydroperoxide), which markedly increase the Tyr-phosphorylation of band 3 (without practically affecting the Ser/Thr-phosphorylation of spectrin) promotes a crenation of human erythrocytes; b) okadaic acid, which selectively increases the Ser/Thr-phosphorylation of spectrin and other membrane proteins, does not promote any shape change, at least at a level detectable with scanning electron microscopy.     

Spectrin properties and the elasticity of the red blood cell membrane skeleton

Two models of spectrin elasticity are developed and compared to experimental measurements of the red blood cell (RBC) membrane shear modulus through the use of an elastic finite element model of the RBC membrane skeleton. The two molecular models of spectrin are: (i) An entropic spring model of spectrin as a flexible chain. This is a model proposed by several previous authors. (ii) An elastic model of a helical coiled-coil which expands by increasing helical pitch. In previous papers, we have computed the relationship between the stiffness of a single spectrin molecule (K) and the shear modulus of a network (mu), and have shown that this behavior is strongly dependent upon network topology. For realistic network models of the RBC membrane skeleton, we equate mu to micropipette measurements of RBCs and predict K for spectrin that is consistent with the coiled-coli molecular model. The value of spectrin stiffness derived from the entropic molecular model would need to be at least 30 times greater to match the experimental results. Thus, the conclusion of this study is that a helical coiled-coil model for spectrin is more realistic than a purely entropic model.     

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin-ankyrin G119 skeleton in Madin Darby canine kidney cells

Spectrin (betaISigma*) and ankyrin (AnkG119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of betaI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of alpha- and beta-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of beta-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as AnkG119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular-tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin-ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.     

Hereditary spherocytosis: diagnostic and anaemia-associated aberrations of ghost proteins

Various disorders of the red cell skeleton and membrane have been described in hereditary spherocytosis. To elucidate which aberrations could be used for identification of HS patients in a Danish population, we examined ghosts from 17 HS patients and 20 normals by use of SDS-gel scanning, native spectrin extraction, and limited tryptic digestion. Compared to normals, HS patients had significantly lowered alpha-spectrin (p < 0.004), protein 4.2 (p < 0.025), and actin (p < 0.05), and significantly increased anion-transporter (p < 3 x 10(-6)) and glyceraldehyde-3-phosphate dehydrogenase (G3PD, p < 0.04). Sixteen out of 17 HS patients could be identified by aberrations of the anion-transporter or protein 4.2 outside a 95% confidence interval for normals. Extraction of native spectrin and limited tryptic digest showed no difference between normals and HS patients. RBC separated into young and old fractions were used to examine the occurrence of protein aberrations associated with RBC age. Young RBC contained more G3PD (35%) and less protein 4.1 (6.5%) and actin (8.7%) than old. In male HS patients an increased G3PD content showed a linear correlation (p < 0.001) with a low concentration of blood haemoglobin. We conclude that aberrations of G3PD, and possibly protein 4.1, and actin, are associated with anaemia in HS. Increased anion-transporter or lowered protein 4.2 may be useful for diagnosis of HS, and were inherited in five out of six families where two generations were available.     

The Lyn-catalyzed Tyr phosphorylation of the transmembrane band-3 protein of human erythrocytes

Band-3 protein (approximately 95 kDa), the major and multifunctional transmembrane protein of human erythrocytes, has been shown to be phosphorylated by endogenous Tyr-protein kinases on different Tyr residues at its N and C cytoplasmic domains. Both the added p36syk (catalytic domain of p72syk) and Lyn kinases are able to phosphorylate the isolated cytoplasmic domain of band 3 (cdb3), yielded by chymotryptic digestion of band 3 in the isolated membranes (ghosts). However, the two Tyr-protein kinases exhibited different phosphorylation behaviours when added to the isolated erythrocyte membranes. More precisely, the added p36syk markedly Tyr phosphorylates the band-3 protein, whereas the added Lyn phosphorylates it very poorly. It is of interest that Lyn can associate with membranes and markedly phosphorylate band 3 when this latter protein has been previously phosphorylated by p36syk, i.e. the p36(syk)-catalyzed phosphorylation is proposed to be a prerequisite for the association of Lyn with the membrane (likely to band 3) and for the Lyn-catalyzed phosphorylation of different band-3 Tyr sites.     

Dynamic properties of ankyrin in T lymphocytes: colocalization with spectrin and protein kinase C beta

Ankyrin is a well characterized membrane skeletal protein which has been implicated in the anchorage of specific integral membrane proteins to the spectrin-based membrane skeleton in a number of systems. In this study, the organization of ankyrin was examined in lymphocytes in relation to T cell function. Light and electron microscope immunolocalization studies revealed extensive heterogeneity in the subcellular distribution of ankyrin in murine tissue-derived lymphocytes. While ankyrin can be localized at the lymphocyte plasma membrane, it can also be accumulated at some distance from the cell periphery, in small patches or in a single discrete, nonmembrane-bound structure. Double immunofluorescence studies demonstrated that ankyrin colocalizes with spectrin and with the signal transducing molecule protein kinase C beta (PKC beta) in tissue-derived lymphocytes, suggesting a functional association between these molecules in the lymphocyte cytoplasm. In addition, T lymphocyte activation-related signals and phorbol ester treatment, both of which lead to PKC activation, cause a rapid translocation of ankyrin, together with spectrin and PKC beta, to a single Triton X-100-insoluble aggregate in the cytoplasm. This finding suggests a mechanism for the reported appearance of PKC in the particulate fraction of cells after activation: activated lymphocyte PKC beta may interact with insoluble cytoskeletal elements like ankyrin and spectrin. Further evidence for a link between the subcellular organization of these proteins and PKC activity is provided by the observation that inhibitors of PKC activity cause their concomitant redistribution to the cell periphery. The dynamic nature of lymphocyte ankyrin and its ability to accumulate at sites distant from the plasma membrane are properties which may be unique to the lymphocyte form of the molecule. Its colocalization with PKC beta in the lymphocyte cytoplasm, together with its redistribution in response to physiological signals, suggests that structural protein(s) may play a role in signal transduction pathways in this cell type. Our data support the conclusion that ankyrin is not solely involved in anchorage of proteins at the plasma membrane in lymphoid cells.     

Molecular basis of spectrin deficiency in beta spectrin Durham. A deletion within beta spectrin adjacent to the ankyrin-binding site precludes spectrin attachment to the membrane in hereditary spherocytosis

We describe a spectrin variant characterized by a truncated beta chain and associated with hereditary spherocytosis. The clinical phenotype consists of a moderate hemolytic anemia with striking spherocytosis and mild spiculation of the red cells. We describe the biochemical characteristics of this truncated protein which constitutes only 10% of the total beta spectrin present on the membrane, resulting in spectrin deficiency. Analysis of reticulocyte cDNA revealed the deletion of exons 22 and 23. We show, using Southern blot analysis, that this truncation results from a 4.6-kb genomic deletion. To elucidate the basis for the decreased amount of the truncated protein on the membrane and the overall spectrin deficiency, we show that (a) the mutated gene is efficiently transcribed and its mRNA abundant in reticulocytes, (b) the mutant protein is normally synthesized in erythroid progenitor cells, (c) the stability of the mutant protein in the cytoplasm of erythroblasts parallels that of the normal beta spectrin, and (d) the abnormal protein is inefficiently incorporated into the membrane of erythroblasts. We conclude that the truncation within the beta spectrin leads to inefficient incorporation of the mutant protein into the skeleton despite its normal synthesis and stability. We postulate that this misincorporation results from conformational changes of the beta spectrin subunit affecting the binding of the abnormal heterodimer to ankyrin, and we provide evidence based on binding assays of recombinant synthetic peptides to inside-out-vesicles to support this model.     

Anion exchanger 1 (band 3) is required to prevent erythrocyte membrane surface loss but not to form the membrane skeleton

The red blood cell (RBC) membrane protein AE1 provides high affinity binding sites for the membrane skeleton, a structure critical to RBC integrity. AE1 biosynthesis is postulated to be required for terminal erythropoiesis and membrane skeleton assembly. We used targeted mutagenesis to assess AE1 function in vivo. RBCs lacking AE1 spontaneously shed membrane vesicles and tubules, leading to severe spherocytosis and hemolysis, but the levels of the major skeleton components, the synthesis of spectrin in mutant erythroblasts, and skeletal architecture are normal or nearly normal. The results indicate that AE1 does not regulate RBC membrane skeleton assembly in vivo but is essential for membrane stability. We postulate that stabilization is achieved through AE1-lipid interactions and that loss of these interactions is a key pathogenic event in hereditary spherocytosis.     

Erythrocyte membrane vesiculation: model for the molecular mechanism of protein sorting

Budding and vesiculation of erythrocyte membranes occurs by a process involving an uncoupling of the membrane skeleton from the lipid bilayer. Vesicle formation provides an important means whereby protein sorting and trafficking can occur. To understand the mechanism of sorting at the molecular level, we have developed a micropipette technique to quantify the redistribution of fluorescently labeled erythrocyte membrane components during mechanically induced membrane deformation and vesiculation. Our previous studies indicated that the spectrin-based membrane skeleton deforms elastically, producing a constant density gradient during deformation. Our current studies showed that during vesiculation the skeleton did not fragment but rather retracted to the cell body, resulting in a vesicle completely depleted of skeleton. These local changes in skeletal density regulated the sorting of nonskeletal membrane components. Highly mobile membrane components, phosphatidylethanolamine- and glycosylphosphatidylinositol-linked CD59 with no specific skeletal association were enriched in the vesicle. In contrast, two components with known specific skeletal association, band 3 and glycophorin A, were differentially depleted in vesicles. Increasing the skeletal association of glycophorin A by liganding its extrafacial domain reduced the fraction partitioning to the vesicle. We conclude that this technique of bilayer/skeleton uncoupling provides a means with which to study protein sorting driven by changes in local skeletal density. Moreover, it is the interaction of particular membrane components with the spectrin-based skeleton that determines molecular partitioning during protein sorting.     

ADP ribosylation factor regulates spectrin binding to the Golgi complex

Homologues of two major components of the well-characterized erythrocyte plasma-membrane-skeleton, spectrin (a not-yet-cloned isoform, betaI Sigma* spectrin) and ankyrin (AnkG119 and an approximately 195-kDa ankyrin), 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. We find that activated ARF stimulates the in vitro association of betaI Sigma* spectrin with a Golgi fraction, that the Golgi-associated betaI Sigma* spectrin contains epitopes characteristic of the betaI Sigma2 spectrin pleckstrin homology (PH) domain known to bind phosphatidylinositol 4,5-bisphosphate (PtdInsP2), and that ARF recruits betaI Sigma* spectrin by inducing increased PtdInsP2 levels in the Golgi. The stimulation of spectrin binding by ARF is independent of its ability to stimulate phospholipase D or to recruit coat proteins (COP)-I and can be blocked by agents that sequester PtdInsP2. We postulate that a PH domain within betaI Sigma* 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 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.     

Cytoplasmic regulation of the movement of E-cadherin on the free cell surface as studied by optical tweezers and single particle tracking: corralling and tethering by the membrane skeleton

The translational movement of E-cadherin, a calcium-dependent cell-cell adhesion molecule in the plasma membrane in epithelial cells, and the mechanism of its regulation were studied using single particle tracking (SPT) and optical tweezers (OT). The wild type (Wild) and three types of artificial cytoplasmic mutants of E-cadherin were expressed in L-cells, and their movements were compared. Two mutants were E-cadherins that had deletions in the COOH terminus and lost the catenin-binding site(s) in the COOH terminus, with remaining 116 and 21 amino acids in the cytoplasmic domain (versus 152 amino acids for Wild); these are called Catenin-minus and Short-tailed in this paper, respectively. The third mutant, called Fusion, is a fusion protein between E-cadherin without the catenin-binding site and alpha-catenin without its NH2-terminal half. These cadherins were labeled with 40-nm phi colloidal gold or 210-nm phi latex particles via a monoclonal antibody to the extracellular domain of E-cadherin for SPT or OT experiments, respectively. E-cadherin on the dorsal cell surface (outside the cell-cell contact region) was investigated. Catenin-minus and Short-tailed could be dragged an average of 1.1 and 1.8 micron by OT (trapping force of 0.8 pN), and exhibited average microscopic diffusion coefficients (Dmicro) of 1.2 x 10(-10) and 2.1 x 10(-10) cm2/s, respectively. Approximately 40% of Wild, Catenin-minus, and Short-tailed exhibited confined-type diffusion. The confinement area was 0.13 micron2 for Wild and Catenin-minus, while that for Short-tailed was greater by a factor of four. In contrast, Fusion could be dragged an average of only 140 nm by OT. Average Dmicro for Fusion measured by SPT was small (0.2 x 10(-10) cm2/s). These results suggest that Fusion was bound to the cytoskeleton. Wild consists of two populations; about half behaves like Catenin- minus, and the other half behaves like Fusion. It is concluded that the movements of the wild-type E-cadherin in the plasma membrane are regulated via the cytoplasmic domain by (a) tethering to actin filaments through catenin(s) (like Fusion) and (b) a corralling effect of the network of the membrane skeleton (like Catenin-minus). The effective spring constants of the membrane skeleton that contribute to the tethering and corralling effects as measured by the dragging experiments were 30 and 5 pN/micron, respectively, indicating a difference in the skeletal structures that produce these two effects.     

A new function for adducin. Calcium/calmodulin-regulated capping of the barbed ends of actin filaments

Adducin is a membrane skeleton protein originally described in human erythrocytes that promotes the binding of spectrin to actin and also binds directly to actin and bundles actin filaments. Adducin is associated with regions of cell-cell contact in nonerythroid cells, where it is believed to play a role in regulating the assembly of the spectrin-actin membrane skeleton. In this study we demonstrate a novel function for adducin; it completely blocks elongation and depolymerization at the barbed (fast growing) ends of actin filaments, thus functioning as a barbed end capping protein (Kcap approximately 100 nM). This barbed end capping activity requires the intact adducin molecule and is not provided by the NH2-terminal globular head domains alone nor by the COOH-terminal extended tail domains, which were previously shown to contain the spectrin-actin binding, calmodulin binding, and phosphorylation sites. A novel difference between adducin and other previously described capping proteins is that it is down-regulated by calmodulin in the presence of calcium. The association of stoichiometric amounts of adducin with the short erythrocyte actin filaments in the membrane skeleton indicates that adducin could be the functional barbed end capper in erythrocytes and play a role in restricting actin filament length. Our experiments also suggest novel possibilities for calcium regulation of actin filament assembly by adducin in erythrocytes and at cell-cell contact sites in nonerythroid cells.     

Transcriptional regulation of lysosomal acid lipase in differentiating monocytes is mediated by transcription factors Sp1 and AP-2

The spectrin membrane skeleton is a ubiquitous cytoskeletal structure with several cellular roles, including the maintenance of cell integrity, determination of cell shape and as a contributor to cell polarity. We have isolated mutations in the gene encoding &bgr ;Heavy-spectrin in Drosophila, and have named this essential locus karst. karst mutant individuals have a pleiotropic phenotype characterized by extensive larval lethality and, in adult escapers, rough eyes, bent wings, tracheal defects and infertility. Within karst mutant eyes, a significant number of ommatidia specifically lack photoreceptor R7 alongside more complex morphological defects. Immunolocalization of betaHeavy-spectrin in wild-type eye-antennal and wing imaginal discs reveals that betaHeavy-spectrin is present in a restricted subdomain of the membrane skeleton that colocalizes with DE-cadherin. We propose a model where normal levels of Sevenless signaling are dependent on tight cell-cell adhesion facilitated by the betaHeavy-spectrin membrane skeleton. Immunolocalization of betaHeavy-spectrin in the adult and larval midgut indicates that it is a terminal web protein, but we see no gross morphological defects in the adult apical brush border in karst mutant flies. Rhodamine phalloidin staining of karst mutant ovaries similarly reveals no conspicuous defect in the actin cytoskeleton or cellular morphology in egg chambers. This is in contrast to mutations in alpha-spectrin, the molecular partner of betaHeavy-spectrin, which affect cellular structure in both the larval gut and adult ovaries. Our results emphasize the fundamental contribution of the spectrin membrane skeleton to normal development and reveals a critical interplay between the integrity of a cell's membrane skeleton, the structure of cell-cell contacts and cell signaling.     

Temporal synthesis of band 3 oligomers during terminal maturation of mouse erythroblasts. Dimers and tetramers exist in the membrane as preformed stable species

Band 3, the anion transport protein of the erythrocyte membrane, exists in the membrane as a mixture of dimers (B3D) and tetramers (B3T). The dimers are not linked to the skeleton and constitute the free mobile band 3 fraction. The tetramers are linked to the skeleton by their interaction with ankyrin. In this report we have examined the temporal synthesis and assembly of band 3 oligomers into the plasma membrane during red cell maturation. The oligomeric state of newly synthesized band 3 in early and late erythroblasts was analyzed by size-exclusion high-pressure liquid chromatography of band 3 extracts derived by mild extraction of plasma membranes with the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether). This analysis revealed that at the early erythroblast stage, the newly synthesized band 3 is present predominantly as tetramers, whereas at the late stages of erythroid maturation, it is present exclusively as dimers. To examine whether the dimers and tetramers exist in the membrane as preformed stable species or whether they are interconvertible, the fate of band 3 species synthesized during erythroblast maturation was examined by pulse-chase analysis. We showed that the newly synthesized band 3 dimers and tetramers are stable and that there is no interconversion between these species in erythroblast membranes. Pulse-chase analysis followed by cellular fractionation showed that, in early erythroblasts, the newly synthesized band 3 tetramers are initially present in the microsomal fraction and later incorporated stably into the plasma membrane fraction. In contrast, in late erythroblasts the newly synthesized band 3 dimers move rapidly to the plasma membrane fraction but then recycle between the plasma membrane and microsomal fractions. Fluorescence photobleaching recovery studies showed that significant fractions of B3T and B3D are laterally mobile in early and late erythroblast plasma membranes, respectively, suggesting that many B3T-ankyrin complexes are unattached to the membrane skeleton in early erythroblasts and that the membrane skeleton has yet to become tightly organized in late erythroblasts. We postulate that in early erythroblasts, band 3 tetramers are transported through microsomes and stably incorporated into the plasma membrane. However, when ankyrin synthesis is downregulated in late erythroblasts, it appears that B3D are rapidly transported to the plasma membrane but then recycled between the plasma membrane and microsomal compartments. These observations may suggest novel roles for membrane skeletal proteins in stabilizing integral membrane protein oligomers at the plasma membrane and in regulating the endocytosis of such proteins.     

An in vivo method for studying afferent fibre activity from cervical paravertebral tissue during vertebral motion in anaesthetised cats

PURPOSE: The effect of ethanol and formate radicals on the major proteins of human erythrocyte membranes has been investigated. MATERIALS AND METHODS: Human erythrocyte ghosts and of erythrocyte ghosts stripped of peripheric proteins were irradiated in phosphate buffer with 100 mmol dm(-3) ethanol or 100 mmol dm(-3) formate under N2 or N2O. The alterations of the proteins were investigated by SDS-polyacrylamide gel electrophoresis and high-performance gel permeation chromatography. RESULTS: In contrast to previous results on ribonuclease and on serum albumin the ethanol radicals were found to have a higher efficiency to damage erythrocyte membrane proteins than the formate radicals. Spectrin (Bands 1 and 2) and capnophorin (Band 3) showed the highest radiation-induced loss of all membrane proteins. When cysteamine or dithiothreitol were added to the erythrocyte ghosts with a similar OH-scavenging capacity as ethanol or formate, no degradation or aggregation of the membrane proteins could be observed even after a dose as high as 1800 Gy. CONCLUSIONS: The results of this study confirm the high radiosensitivity of spectrin and capnophorin to primary radicals. Similarly to soluble proteins, membrane-associated proteins are more significantly damaged by ethanol radicals than by formate radicals.     

Cis-activation of L1-mediated ankyrin recruitment by TAG-1 homophilic cell adhesion

Neural cell adhesion molecules (CAMs) of the immunoglobulin (Ig) superfamily mediate not only cell aggregation but also growth cone guidance and neurite outgrowth. In this study we demonstrate that two neural CAMs, L1-CAM and TAG-1, induce the homophilic aggregation of Drosophila S2 cells but are unable to interact with each other when expressed on different cells (trans-interaction). However, immunoprecipitations from cells co-expressing L1-CAM and TAG-1 showed a strong cis-interaction between the two molecules in the plane of the plasma membrane. TAG-1 is linked to the membrane by a glycosylphosphatidylinositol (GPI) anchor and therefore is unable to directly interact with cytoplasmic proteins. In contrast, L1-CAM-mediated homophilic cell adhesion induces the selective recruitment of the membrane skeleton protein ankyrin to areas of cell contact. Immunolabeling experiments in which S2 cells expressing TAG-1 were mixed with cells co-expressing L1-CAM and TAG-1 demonstrated that the homophilic interaction between TAG-1 molecules results in the cis-activation of L1-CAM to bind ankyrin. This TAG-1-dependent recruitment of the membrane skeleton provides an example of how GPI-anchored CAMs are able to transduce signals to the cytoplasm. Furthermore, such interactions might ultimately result in the recruitment and the activation of other signaling molecules at sites of cell contacts.