Hereditary spherocytosis of man. Defective cytoskeletal interactions in the erythrocyte membrane.
ABSTRACT: Hereditary spherocytosis (HS) is an inherited abnormality of red cell shape and results from defective interactions amongst the components of the cytoskeleton. It is known that spectrin/actin dissociates in low ionic strength media from ghosts and cytoskeletons at a rate which is slower for HS than normal preparations. Hybridization experiments have established that this behaviour is not due to a defective spectrin or actin but resides in a spectrin-binding component of the membrane [Hill, Sawyer, Howlett & Wiley (1981) Biochem. J. 201, 259-266]. In the present study erythrocyte shells have been examined in low ionic strength media and a similar difference in the rate of solubilization has been revealed. Since band 4.1 (but not band 2.1) is a common component of cytoskeletons and shells it is possible that 4.1 may be abnormal in the HS condition. The interaction of band 4.1 with spectrin/actin was examined by low shear falling ball viscometry. The addition of a mixture of band 2.1 and 4.1 to a solution of actin and spectrin tetramer increased the viscosity due to cross-linking of the cytoskeletal elements by band 4.1. When band 2.1/4.1 mixtures were derived from five HS families the viscosity was increased to a greater extent than in the normal controls. This difference was not a result of alterations in the calcium dependence of the spectrin/actin-band 4.1 interaction. The results imply that band 4.1 may be defective in the HS condition.
Project description:Human erythrocytes possess a lattice work of extrinsic proteins on the inner face of the membrane (;cytoskeleton') that maintains the shape and deformability of the cell. The major proteins of the cytoskeleton are spectrin and actin, which are attached to the membrane by protein bands 2.1 (;ankyrin') and 4.1. The interactions of spectrin/actin with erythrocyte membranes from normal subjects and from patients with hereditary spherocytosis (HS) have been studied by using an air-driven ultracentrifuge, which can rapidly separate membranes from soluble proteins (150000g for 30s). The total amount of spectrin/actin in HS and normal ghosts is similar. However, the rate of dissociation of spectrin and actin from HS erythrocyte membranes at low ionic strength is significantly lower than that observed for normal membranes. Spectrin and actin isolated from either HS or normal membranes re-associated in a similar manner to spectrin/actin-depleted vesicles prepared from normal cells. Scatchard analysis showed an average binding capacity of 278mug/mg of membrane protein. However, spectrin/actin-depleted vesicles prepared from HS cells bound significantly less spectrin/actin prepared from either the normal or abnormal cells (average binding capacity 158mug/mg of membrane protein). The defect was defined further by studying the cytoskeleton obtained by Triton X-100 extraction of membranes. Under conditions of low ionic strength cytoskeletons prepared from HS membranes dissociated more slowly than those prepared from normal membranes, and only 80% of the protein from HS cytoskeletons could be solubilized after 180min compared with 100% for normal cytoskeletons. The difference between HS and normal membranes, which persists in isolated cytoskeletons, suggests that alterations in either the primary structure or the degree of phosphorylation of protein bands 2.1 or 4.1 may be central to the molecular basis of hereditary spherocytosis.
Project description:The erythrocyte membrane skeleton is the best understood cytoskeleton. Because its protein components have homologs in virtually all other cells, the membrane serves as a fundamental model of biologic membranes. Modern textbooks portray the membrane as a 2-dimensional spectrin-based membrane skeleton attached to a lipid bilayer through 2 linkages: band 3-ankyrin-beta-spectrin and glycophorin C-protein 4.1-beta-spectrin.(1-7) Although evidence supports an essential role for the first bridge in regulating membrane cohesion, rupture of the glycophorin C-protein 4.1 interaction has little effect on membrane stability.(8) We demonstrate the existence of a novel band 3-adducin-spectrin bridge that connects the spectrin/actin/protein 4.1 junctional complex to the bilayer. As rupture of this bridge leads to spontaneous membrane fragmentation, we conclude that the band 3-adducin-spectrin bridge is important to membrane stability. The required relocation of part of the band 3 population to the spectrin/actin junctional complex and its formation of a new bridge with adducin necessitates a significant revision of accepted models of the erythrocyte membrane.
Project description:Spectrin and protein 4.1 cross-link F-actin protofilaments into a network called the membrane skeleton. Actin and 4.1 bind to one end of beta-spectrin. The adjacent end of alpha-spectrin, called the EF-domain, is calmodulin-like, with calcium-dependent and calcium-independent EF-hands. It has no known function. However, the sph(1J)/sph(1J) mouse has very fragile red cells and lacks the last 13 amino acids in the EF-domain, suggesting the domain is critical for skeletal integrity. Using pulldown binding assays, we find the alpha-spectrin EF-domain either alone or incorporated into a mini-spectrin binds native and recombinant protein 4.2 at a previously identified region of 4.2 (G(3) peptide). Native 4.2 binds with an affinity comparable with other membrane skeletal interactions (K(d) = 0.30 microM). EF-domains bearing the sph(1J) mutation are inactive. Binding of protein 4.2 to band 3 (K(d) = 0.45 microM) does not interfere with the spectrin-4.2 interaction. Spectrin-4.2 binding is amplified by micromolar concentrations of Ca(2+) (but not Mg(2+)) by three to five times. Calmodulin also binds to the EF-domain (K(d) = 17 microM), and Ca(2+)-calmodulin blocks Ca(2+)-dependent binding of protein 4.2 but not Ca(2+)-independent binding. The data suggest that protein 4.2 is located near protein 4.1 at the spectrin-actin junctions. Because proteins 4.1 and 4.2 also bind to band 3, the erythrocyte anion channel, we suggest that one or both of these proteins cause a portion of band 3 to localize near the spectrin-actin junctions and provide another point of attachment between the membrane skeleton and the lipid bilayer.
Project description:Hereditary spherocytosis (HS) originates from defective anchoring of the cytoskeletal network to the transmembrane protein complexes of the red blood cell (RBC). Red cells in HS are characterized by membrane instability and reduced deformability and there is marked heterogeneity in disease severity among patients. To unravel this variability in disease severity, we analyzed blood samples from 21 HS patients with defects in ankyrin, band 3, ?-spectrin or ?-spectrin using red cell indices, eosin-5-maleimide binding, microscopy, the osmotic fragility test, Percoll density gradients, vesiculation and ektacytometry to assess cell membrane stability, cellular density and deformability. Reticulocyte counts, CD71 abundance, band 4.1 a:b ratio, and glycated hemoglobin were used as markers of RBC turnover. We observed that patients with moderate/severe spherocytosis have short-living erythrocytes of low density and abnormally high intercellular heterogeneity. These cells show a prominent decrease in membrane stability and deformability and, as a consequence, are quickly removed from the circulation by the spleen. In contrast, in mild spherocytosis less pronounced reduction in deformability results in prolonged RBC lifespan and, hence, cells are subject to progressive loss of membrane. RBC from patients with mild spherocytosis thus become denser before they are taken up by the spleen. Based on our findings, we conclude that RBC membrane loss, cellular heterogeneity and density are strong markers of clinical severity in spherocytosis.
Project description:Vertebrate 4.1 proteins have a spectrin-actin-binding (SAB) domain, which is lacking in all the invertebrate 4.1 proteins indentified so far, and it was therefore proposed that the SAB domain emerged with the advent of vertebrates during evolution. Here we demonstrated for the first time that amphioxus (an invertebrate chordate) protein 4.1, though lacking a recognizable SAB, was able to bind both spectrin and actin, with a binding capacity comparable to that of human protein 4.1. Detailed structure-activity analyses revealed that the unique domain U2/3 was a newly identified SAB-like domain capable of interacting with spectrin and actin, suggesting the presence of a "cryptic" SAB domain in amphioxus 4.1 protein. We also showed that amphioxus 4.1 protein gene was the common ancestor of vertebrate 4.1 protein genes, from which 4.1R, 4.1N, 4.1G, and 4.1B genes originated. This work will encourage further study on the structure-activity of invertebrate 4.1 protein and its interacting proteins.
Project description:Spectrin cytoskeletons are found in all metazoan cells, and their physical interactions between actin and ankyrins establish a meshwork that provides cellular structural integrity. With advanced super-resolution microscopy, the intricate spatial organization and associated functional properties of these cytoskeletons can now be analyzed with unprecedented clarity. Long neuronal processes like peripheral sensory and motor axons may be subject to intense mechanical forces including bending, stretching, and torsion. The spectrin-based cytoskeleton is essential to protect axons against these mechanical stresses. Additionally, spectrins are critical for the assembly and maintenance of axonal excitable domains including the axon initial segment and the nodes of Ranvier (NoR). These sites facilitate rapid and efficient action potential initiation and propagation in the nervous system. Recent studies revealed that pathogenic spectrin variants and diseases that protealyze and breakdown spectrins are associated with congenital neurological disorders and nervous system injury. Here, we review recent studies of spectrins in the nervous system and focus on their functions in axonal health and disease.
Project description:Various enteric bacterial pathogens target the host cell cytoskeletal machinery as a crucial event in their pathogenesis. Despite thorough studies detailing strategies microbes use to exploit these components of the host cell, the role of the spectrin-based cytoskeleton has been largely overlooked. Here we show that the spectrin cytoskeleton is a host system that is hijacked by adherent (Entropathogenic Escherichia coli [EPEC]), invasive triggering (Salmonella enterica serovar Typhimurium [S. Typhimurium]) and invasive zippering (Listeria monocytogenes) bacteria. We demonstrate that spectrin cytoskeletal proteins are recruited to EPEC pedestals, S. Typhimurium membrane ruffles and Salmonella containing vacuoles (SCVs), as well as sites of invasion and comet tail initiation by L. monocytogenes. Spectrin was often seen co-localizing with actin filaments at the cell periphery, however a disconnect between the actin and spectrin cytoskeletons was also observed. During infections with S. Typhimurium ?sipA, actin-rich membrane ruffles at characteristic sites of bacterial invasion often occurred in the absence of spectrin cytoskeletal proteins. Additionally, early in the formation of L. monocytogenes comet tails, spectrin cytoskeletal elements were recruited to the surface of the internalized bacteria independent of actin filaments. Further studies revealed the presence of the spectrin cytoskeleton during SCV and Listeria comet tail formation, highlighting novel cytoplasmic roles for the spectrin cytoskeleton. SiRNA targeted against spectrin and the spectrin-associated proteins severely diminished EPEC pedestal formation as well as S. Typhimurium and L. monocytogenes invasion. Ultimately, these findings identify the spectrin cytoskeleton as a ubiquitous target of enteric bacterial pathogens and indicate that this cytoskeletal system is critical for these infections to progress.
Project description:Protein 4.1 is an approximately 80-kD structural protein in the membrane skeleton which underlies and supports the erythrocyte plasma membrane. The preceding companion paper presents a biochemical study of two abnormal protein 4.1 species from individuals with the red blood cell disorder, hereditary elliptocytosis. These variants, "protein 4.1(68/65)" and "protein 4.1(95)," have altered molecular weights due to internal deletions and duplications apparently localized around the spectrin-actin binding domain. Here we use polymerase chain reaction (PCR) techniques to clone and sequence the corresponding mutant reticulocyte mRNAs, and correlate the deletion/duplication end points with exon boundaries of the gene. Protein 4.1(68/65) mRNA lacks sequences encoding the functionally important spectrin-actin binding domain due to a 240 nucleotide (nt) deletion spanning the codons for Lys407-Gly486. Protein 4.1(95) mRNA encodes a protein with two spectrin-actin binding domains by virtue of a 369 nt duplication of codons for Lys407-Gln529. These deletions and duplications correspond to gene rearrangements involving three exons encoding 21, 59, and 43 amino acids, respectively. The duplicated 21 amino acid exon in the 4.1(95) gene retains its proper tissue-specific expression pattern, being spliced into reticulocyte 4.1 mRNA and out of lymphocyte 4.1 mRNA.
Project description:Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including ?- and ?- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1?/? erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1?/? erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes.
Project description:Questions of if and when protein structures change within cells pervade biology and include questions of how the cytoskeleton sustains stresses on cells--particularly in mutant versus normal cells. Cysteine shotgun labeling with fluorophores is analyzed here with mass spectrometry of the spectrin-actin membrane skeleton in sheared red blood cell ghosts from normal and diseased mice. Sheared samples are compared to static samples at 37 °C in terms of cell membrane intensity in fluorescence microscopy, separated protein fluorescence, and tryptic peptide modification in liquid chromatography-tandem mass spectrometry (LC-MS/MS). Spectrin labeling proves to be the most sensitive to shear, whereas binding partners ankyrin and actin exhibit shear thresholds in labeling and both the ankyrin-binding membrane protein band 3 and the spectrin-actin stabilizer 4.1R show minimal differential labeling. Cells from 4.1R-null mice differ significantly from normal in the shear-dependent labeling of spectrin, ankyrin, and band 3: Decreased labeling of spectrin reveals less stress on the mutant network as spectrin dissociates from actin. Mapping the stress-dependent labeling kinetics of α- and β-spectrin by LC-MS/MS identifies Cys in these antiparallel chains that are either force-enhanced or force-independent in labeling, with structural analyses indicating the force-enhanced sites are sequestered either in spectrin's triple-helical domains or in interactions with actin or ankyrin. Shear-sensitive sites identified comprehensively here in both spectrin and ankyrin appear consistent with stress relief through forced unfolding followed by cytoskeletal disruption.