Simultaneous Loss of Mouse Dematin and beta-Adducin Results in Severe Erythrocyte Fragility and Hemolytic Anemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 809-809
Author(s):  
Anwar A. Khan ◽  
Huiqing Chen ◽  
Diana M. Gilligan ◽  
Luanne L. Peters ◽  
Joanne Messick ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Erythrocyte Membrane ◽  
Membrane Stability ◽  
Actin Binding ◽  
Junctional Complex ◽  
Filamentous Actin ◽  
Double Knockout ◽  
Erythrocyte Shape ◽  
Junctional Complexes ◽  
Simultaneous Loss

Abstract The mechanical strength and stability of the erythrocyte membrane are regulated by a network of proteins that participate in both horizontal and vertical interactions. The actin-containing junctional complexes, located at the tail ends of spectrin molecules, serve as the critical regulatory nodes for the maintenance of membrane stability. Dematin and beta-adducin, two actin-binding proteins of the junctional complex, are known to play essential roles in the regulation of erythrocyte shape and membrane stability, as revealed recently by the development of mouse knockout models. Here, we show that simultaneous loss of functional dematin (headpiece domain deletion) and beta-adducin results in severe fragility and abnormal shape of erythrocytes, despite the presence of major skeletal proteins. Adducin/Dematin Double Knockout (ADKO) mice are viable and can be distinguished at birth by their pallor with pronounced spleomegaly and regenerative hematopoiesis. Hematological evaluations show a reduction of erythrocytes, reduced hematocrit and hemoglobin, and a ~52% increase in the number of reticulocytes. The presence of a variety of misshapen and fragmented erythrocytes in the ADKO mice correlates with increased osmotic fragility and reduced erythrocyte life span in vivo. Despite an apparently normal composition of ghosts and skeletal proteins, the retention of spectrin in the ADKO erythrocyte plasma membrane is significantly compromised. Atomic force microscopy (AFM) revealed similar volume parameters in the four genotypes examined, but an increased grain size, and a decreased filament number in the ADKO erythrocyte membrane. In addition, highly aggregated, disassembled, and irregular features were visualized by AFM in the ADKO erythrocyte membrane. Staining of filamentous actin provided further evidence for the existence of large protein aggregates in the ADKO erythrocyte membrane. Together, these results demonstrate a crucial function of dematin and beta-adducin in the maintenance of erythrocyte shape and membrane stability, and more importantly, suggest the existence of an alternate mechanism for the linkage of junctional complexes to the plasma membrane.

Dematin and Adducin Provide a Critical Link between the Spectrin Cytoskeleton and the Erythrocyte Membrane Via Glucose Transporter-1.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1719-1719
Author(s):  
Anwar A. Khan ◽  
Toshihiko Hanada ◽  
Massimiliano Gaetani ◽  
Donghai Li ◽  
Brent C. Reed ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Erythrocyte Membrane ◽  
Glucose Transporter ◽  
Transmembrane Protein ◽  
Actin Binding ◽  
Junctional Complex ◽  
Head Region ◽  
Glucose Transporter 1 ◽  
Erythrocyte Shape ◽  
Spectrin Cytoskeleton

Abstract There is considerable interest in the elucidation of the mechanism that governs the linkage of elongated spectrin molecules to the erythrocyte plasma membrane. The mechanism by which the “head” region of the spectrin dimer, which participates in tetramer formation, binds to the membrane via ankyrin and band 3 has been reasonably well characterized. However, the mechanism by which the tail end of the spectrin dimer is anchored to the plasma membrane is not completely understood. Dematin and adducin are actin binding proteins located at the spectrin-actin junctions or “junctional complex” in the erythrocyte membrane. Individual suppression of their function in mice by the gene deletion exerts a modest effect on erythrocyte shape and membrane stability. In contrast, the combined deletion of dematin and adducin genes results in severe defects of erythrocyte shape, membrane instability, and hemolysis. Based on these findings, we proposed a model whereby dematin and adducin could function as a molecular bridge linking the junctional complex to the plasma membrane. Using a combination of cell surface labeling, immunoprecipitation, and vesicle proteomics, we have identified glucose transporter-1 as the receptor for dematin and adducin in the human erythrocyte membrane. This finding is the first description of a transmembrane protein that binds to dematin and adducin, thus providing a rationale for the attachment of the cytoskeletal junctional complex to the lipid bilayer via glucose transporter-1. Since homologues of dematin, adducin, and glucose transporter-1 exist in many non-erythroid cells, we propose that a conserved mechanism may exist that couples sugar and other related transporters to the actin cytoskeleton.

scholarly journals Tropomyosin modulates erythrocyte membrane stability

Blood ◽  
2006 ◽  
Vol 109 (3) ◽  
pp. 1284-1288 ◽  
Author(s):  
Xiuli An ◽  
Marcela Salomao ◽  
Xinhua Guo ◽  
Walter Gratzer ◽  
Narla Mohandas
Keyword(s):  
Erythrocyte Membrane ◽  
Ternary Complex ◽  
Mechanical Stability ◽  
Membrane Stability ◽  
Actin Binding ◽  
Erythrocyte Membranes ◽  
Junctional Complex ◽  
Protein 4.1 ◽  
Resealed Ghosts

Abstract The ternary complex of spectrin, actin, and 4.1R (human erythrocyte protein 4.1) defines the nodes of the erythrocyte membrane skeletal network and is inseparable from membrane stability under mechanical stress. These junctions also contain tropomyosin (TM) and the other actin-binding proteins, adducin, protein 4.9, tropomodulin, and a small proportion of capZ, the functions of which are poorly defined. Here, we have examined the consequences of selective elimination of TM from the membrane. We have shown that the mechanical stability of the membranes of resealed ghosts devoid of TM is grossly, but reversibly, impaired. That the decreased membrane stability of TM-depleted membranes is the result of destabilization of the ternary complex of the network junctions is demonstrated by the strongly facilitated entry into the junctions in situ of a β-spectrin peptide, containing the actin- and 4.1R-binding sites, after extraction of the TM. The stabilizing effect of TM is highly specific, in that it is only the endogenous isotype, and not the slightly longer muscle TM that can bind to the depleted membranes and restore their mechanical stability. These findings have enabled us identify a function for TM in elevating the mechanical stability of erythrocyte membranes by stabilizing the spectrin-actin-4.1R junctional complex.

Conditional Knockout-First Gene Disruption of Dematin Causes Precipitous Loss of Erythrocyte Membrane Stability and Severe Hemolytic Anemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 157-157
Author(s):  
Yunzhe Lu ◽  
Toshihiko Hanada ◽  
Athar H. Chishti
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Hemolytic Anemia ◽  
Erythrocyte Membrane ◽  
Half Life ◽  
Gene Disruption ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Wild Type

Abstract Dematin is an actin binding and bundling protein originally identified as a component of the erythrocyte membrane junctional complex. A widely expressed member of the villin-family of adaptor proteins, dematin regulates RhoA activity and cell shape in fibroblasts. Actin binding and bundling activity of dematin is regulated by phosphorylation of its headpiece domain by the cAMP-dependent protein kinase. Despite its extensive biochemical characterization, the physiological function of dematin in mature erythrocytes remains unknown. We used a conditional gene disruption strategy by generating a targeting construct that has the potential for full body gene knockout as well as tissue-specific deletion of dematin gene using the Cre-lox gene deletion system. Wild type, heterozygous, and homozygous progeny were obtained in a typical Mendelian ratio of 1:2:1. Dramatic splenomegaly in 7-week old full length dematin knockout (FLKO) mice was observed with the average spleen weight 10-fold higher than those of the wild type littermates. Flow cytometry showed a ~16-fold increase in reticulocytes (Fig.1A), which was also seen in the blood smear (Fig.1B,C). Severe hemolytic anemia is most likely the cause of relative pallor observed in FLKO mice at day 1 after birth. The adult FLKO mice continue to show relatively smaller body size as compared to wild type and heterozygous mice. These findings are consistent with severe anemia and compensatory erythropoiesis. FLKO mice exhibit typical signs of anisocytosis, microcytosis, macrocytosis, and polychromasia, which are indicative of tremendous variation in RBC cell size and the premature release of reticulocytes from the bone marrow. Moreover, additional RBC abnormalities, including poikilocytosis, acanthocytosis, fragmented RBC, and spherocytes, are consistent with severe hemolytic disease. By scanning EM, the FLKO erythrocytes showed dramatic variation in shape and size. The spherocytes, microcytic vesiculation, and the protruding structures are observed in FKLO mice, as well as extensive intravascular hemolysis (Fig. 1D,E). RBC half-life measurements in vivo by NHS-biotin labeling and flow cytometry showed mutant cells almost immediately cleared from the circulation in FLKO mice. A seven-week chase experiment showed that the half-life of RBCs was reduced from 22 days in wild type and heterozygous mice to less than 3 days in FLKO mice. The hematological phenotype of FLKO mice indicated reduced RBC count, hemoglobin, and hematocrit with increase in the RBC distribution width. Collectively, these findings indicate that the mechanical strength of RBC membrane strictly relies on the presence of full length dematin. We employed membrane fractionation, in vitro protein domain mapping, transmission/scanning electron microscopy, and dynamic deformability measurements to investigate the underlying mechanisms of extreme membrane fragility in FLKO erythrocytes. We also examined the protein profile of RBC ghosts. Surprisingly, the major cytoskeletal proteins remained unchanged in the FLKO ghosts; however, a marked reduction of spectrin, adducin, and actin was observed. When normalized against band 3, these proteins were reduced by 60%, 90%, and 90%, respectively. Since these membrane proteins are essential for RBC stability, our findings suggest a specific role of dematin in recruiting or maintaining a stable association of essential cytoskeletal proteins in the plasma membrane. These results raise the possibility that dematin may directly interact with adducin, and together anchor the spectrin molecules to the plasma membrane. Our findings provide the first in vivo evidence that dematin is essential for the maintenance of erythrocyte shape and membrane mechanical properties by regulating the integrity of the spectrin-actin junctions. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Function and interactions of the Ysc84/SH3yl1 family of actin- and lipid-binding proteins

2015 ◽  
Vol 43 (1) ◽  
pp. 111-116 ◽  
Author(s):  
Agnieszka N. Urbanek ◽  
Rebekah Chan ◽  
Kathryn R. Ayscough
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Regulatory Networks ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Morphological Changes ◽  
Lipid Binding ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Membrane Morphology

Understanding how actin filaments are nucleated, polymerized and disassembled in close proximity to cell membranes is an area of growing interest. Protrusion of the plasma membrane is required for cell motility, whereas inward curvature or invagination is required for endocytic events. These morphological changes in membrane are often associated with rearrangements of actin, but how the many actin-binding proteins of eukaryotes function in a co-ordinated way to generate the required responses is still not well understood. Identification and analysis of proteins that function at the interface between the plasma membrane and actin-regulatory networks is central to increasing our knowledge of the mechanisms required to transduce the force of actin polymerization to changes in membrane morphology. The Ysc84/SH3yl1 proteins have not been extensively studied, but work in both yeast and mammalian cells indicate that these proteins function at the hub of networks integrating regulation of filamentous actin (F-actin) with changes in membrane morphology.

scholarly journals Identification of adducin-binding residues on the cytoplasmic domain of erythrocyte membrane protein, band 3

2016 ◽  
Vol 473 (19) ◽  
pp. 3147-3158 ◽  
Author(s):  
Taina Franco ◽  
Haiyan Chu ◽  
Philip S. Low
Keyword(s):  
Erythrocyte Membrane ◽  
Membrane Integrity ◽  
Protein Band ◽  
Cytoplasmic Domain ◽  
Band 3 ◽  
Junctional Complex ◽  
Peripheral Protein ◽  
Protein Partners ◽  
Junctional Complexes ◽  
Ankyrin Protein

Two major complexes form structural bridges that connect the erythrocyte membrane to its underlying spectrin-based cytoskeleton. Although the band 3–ankyrin bridge may account for most of the membrane-to-cytoskeleton interactions, the linkage between the cytoplasmic domain of band 3 (cdb3) and adducin has also been shown to be critical to membrane integrity. In the present paper, we demonstrate that adducin, a major component of the spectrin–actin junctional complex, binds primarily to residues 246–264 of cdb3, and mutation of two exposed glutamic acid residues within this sequence completely abrogates both α- and β-adducin binding. Because these residues are located next to the ankyrin-binding site on cdb3, it seems unlikely that band 3 can bind ankyrin and adducin concurrently, reducing the chances of an association between the ankyrin and junctional complexes that would significantly compromise erythrocyte membrane integrity. We also demonstrate that adducin binds the kidney isoform of cdb3, a spliceoform that lacks the first 65 amino acids of erythrocyte cdb3, including the central strand of a large β-pleated sheet. Because kidney cdb3 is not known to bind any of the common peripheral protein partners of erythrocyte cdb3, including ankyrin, protein 4.1, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and phosphofructokinase, retention of this affinity for adducin was unexpected.

scholarly journals Actin Dependence of Polarized Receptor Recycling in Madin-Darby Canine Kidney Cell Endosomes

2002 ◽  
Vol 13 (1) ◽  
pp. 262-275 ◽  
Author(s):  
David R. Sheff ◽  
Ruth Kroschewski ◽  
Ira Mellman
Keyword(s):  
Plasma Membrane ◽  
Ethylenediaminetetraacetic Acid ◽  
Density Lipoprotein ◽  
Lateral Diffusion ◽  
Membrane Diffusion ◽  
Loss Of Function ◽  
Filamentous Actin ◽  
Early Endosomes ◽  
Basolateral Plasma Membrane ◽  
Junctional Complexes

Mammalian epithelial cell plasma membrane domains are separated by junctional complexes supported by actin. The extent to which actin acts elsewhere to maintain cell polarity remains poorly understood. Using latrunculin B (Lat B) to depolymerize actin filaments, several basolateral plasma membrane proteins were found to lose their polarized distribution. This loss of polarity did not reflect lateral diffusion through junctional complexes because a low-density lipoprotein receptor mutant lacking a functional endocytosis signal remained basolateral after Lat B treatment. Furthermore, Lat B treatment did not facilitate membrane diffusion across the tight junction as observed with ethylenediaminetetraacetic acid or dimethyl sulfoxide treatment. Detailed analysis of transferrin recycling confirmed Lat B depolarized recycling of transferrin from endosomes to the basolateral surface. Kinetic analysis suggested sorting was compromised at both basolateral early endosomes and perinuclear recycling endosomes. Despite loss of function, these two endosome populations remained distinct from each other and from early endosomes labeled by apically internalized ligand. Furthermore, apical and basolateral early endosomes were functionally distinct populations that directed traffic to a single common recycling endosomal compartment even after Lat B treatment. Thus, filamentous actin may help to guide receptor traffic from endosomes to the basolateral plasma membrane.

scholarly journals Adducin forms a bridge between the erythrocyte membrane and its cytoskeleton and regulates membrane cohesion

Blood ◽  
2009 ◽  
Vol 114 (9) ◽  
pp. 1904-1912 ◽  
Author(s):  
William A. Anong ◽  
Taina Franco ◽  
Haiyan Chu ◽  
Tahlia L. Weis ◽  
Emily E. Devlin ◽  
...  
Keyword(s):  
Erythrocyte Membrane ◽  
Membrane Skeleton ◽  
Band 3 ◽  
Membrane Stability ◽  
Junctional Complex ◽  
C Protein ◽  
Protein 4.1 ◽  
Protein Components ◽  
Actin Protein ◽  
Glycophorin C

Abstract 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–β-spectrin and glycophorin C–protein 4.1–β-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.

scholarly journals Sla2p Is Associated with the Yeast Cortical Actin Cytoskeleton via Redundant Localization Signals

1999 ◽  
Vol 10 (7) ◽  
pp. 2265-2283 ◽  
Author(s):  
Shirley Yang ◽  
M. Jamie T. V. Cope ◽  
David G. Drubin
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Coiled Coil ◽  
Intramolecular Interactions ◽  
The Other ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
C Terminus

Sla2p, also known as End4p and Mop2p, is the founding member of a widely conserved family of actin-binding proteins, a distinguishing feature of which is a C-terminal region homologous to the C terminus of talin. These proteins may function in actin cytoskeleton-mediated plasma membrane remodeling. A human homologue of Sla2p binds to huntingtin, the protein whose mutation results in Huntington’s disease. Here we establish by immunolocalization that Sla2p is a component of the yeast cortical actin cytoskeleton. Deletion analysis showed that Sla2p contains two separable regions, which can mediate association with the cortical actin cytoskeleton, and which can provide Sla2p function. One localization signal is actin based, whereas the other signal is independent of filamentous actin. Biochemical analysis showed that Sla2p exists as a dimer in vivo. Two-hybrid analysis revealed two intramolecular interactions mediated by coiled-coil domains. One of these interactions appears to underlie dimer formation. The other appears to contribute to the regulation of Sla2p distribution between the cytoplasm and plasma membrane. The data presented are used to develop a model for Sla2p regulation and interactions.

Dematin Is Essential for Calcium Mobilization in Platelets

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 365-365
Author(s):  
Adam Joseph Wieschhaus ◽  
Guy Le Breton ◽  
Athar H Chishti
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Platelet Activation ◽  
Western Blotting ◽  
Conflicts Of Interest ◽  
Calcium Mobilization ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Erythrocyte Membranes ◽  
Junctional Complex

Abstract Abstract 365 Dematin is an actin binding protein that was originally identified in the human erythrocyte membranes; however, dematin polypeptides are detectable in many non-erythroid cells. In erythrocytes, dematin is located at the spectrin-actin junctions thus linking the skeleton to the plasma membrane. This junctional complex serves to maintain both erythrocyte shape and membrane stability. Dematin is phosphorylated by multiple protein kinases, including the cAMP-dependent protein kinase and protein kinase C, and its actin bundling activity is regulated by phosphorylation. Dematin is also an excellent substrate of calpain-1, a calcium-dependent cysteine protease. Because of the functional similarities between the core components of the membrane skeleton in erythrocytes and platelets, we sought to investigate the physiological function of dematin in platelets. The remodeling of the actin cytoskeleton is known to regulate platelet activation and secretion of platelet granule contents. Western blotting demonstrated abundant expression of the ∼52 kDa polypeptide of dematin in both human and mouse platelets. To evaluate the functional role of dematin in platelets, we utilized the dematin headpiece knockout (HPKO) mouse model previously generated in our laboratory. Headpiece domain of dematin is an actin binding module sharing sequence similarity with the villin-family of cytoskeletal proteins. Importantly, the in vivo deletion of the headpiece domain of dematin resulted in substantial diminution of calcium mobilization in response to multiple agonists of platelet activation (Fig. 1). The reduced calcium mobilization in HPKO platelets was associated with significant inhibition of the platelet aggregation and granule secretion pathways. The HPKO platelets exhibit decreased activation of both the integrin αIIbβ3 receptor and RhoA upon platelet stimulation. Moreover, the HPKO platelets display aberrant morphology upon spreading on the fibrinogen and vWF-coated surfaces. Consistent with these findings, the HPKO mice show a significant clot retraction defect associated with a general tail bleeding phenotype. Mechanistically, the basal level of cAMP remained unaltered in the HPKO platelets suggesting the independence of the observed phenotype upon changes in the cAMP concentration. Immunofluorescence analysis indicated that dematin is associated with two platelet membrane compartments known to be involved in calcium fluxes, i.e., the dense tubular system (DTS) and the plasma membrane. Studies are currently in progress to identify the dematin-associated complex in platelets by Western blotting and proteomics approaches. Together, these results unveil dematin as a novel regulator of calcium homeostasis in platelets with functional implications for the development of new anti-platelet therapies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hereditary elliptocytosis with protein band 4.1 deficiency in the dog

Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 373-377 ◽  
Author(s):  
JE Smith ◽  
K Moore ◽  
M Arens ◽  
GA Rinderknecht ◽  
A Ledet
Keyword(s):  
Animal Model ◽  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Protein Band ◽  
Erythrocyte Deformability ◽  
Membrane Stability ◽  
Osmotic Sensitivity ◽  
Hereditary Elliptocytosis ◽  
Membrane Cytoskeleton ◽  
Glutathione Deficiency

Abstract A dog with persistent elliptocytosis was studied. The dog had membrane protein band 4.1 deficiency, microcytosis, shortened erythrocyte lifespan, increased osmotic sensitivity, and a mild glutathione deficiency. Erythrocyte deformability and membrane stability were adversely effected. The dog's parents had decreased band 4.1, decreased stability, and some elliptocytosis. This disorder in dogs closely resembles human patients with band 4.1 deficiency and should provide a valuable animal model to study the erythrocyte membrane cytoskeleton.

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