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.

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.

Erythrocyte Dematin Regulates Glucose Transport Via Akt Phosphorylation and 14-3-3zeta Association.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1990-1990
Author(s):  
Morvarid Mohseni ◽  
Anwar Khan ◽  
Athar H. Chishti
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Glucose Transport ◽  
Cell Biology ◽  
Glucose Transporter ◽  
Conflicts Of Interest ◽  
Adaptor Protein ◽  
Degradation Pathway ◽  
Actin Binding ◽  
Glucose Transporter 1

Abstract Abstract 1990 Poster Board I-1012 Erythrocyte dematin is a widely expressed actin-binding and bundling protein, and functions as a suppressor of RhoA signaling in fibroblasts (Mohseni and Chishti, Molecular Cell Biology 28: 4712-4718, 2008). Dematin is a substrate of multiple protein kinases, and its actin bundling activity is regulated by cAMP dependent protein kinase. Recently, we identified a novel interaction between dematin and glucose transporter-1 (GLUT1) that is critically important for erythrocyte shape and membrane mechanical properties (Khan et al., Journal of Biological Chemistry 283:14600-14609, 2008). Since homologues of dematin and GLUT1 exist in many non-erythroid cells, we proposed that a conserved mechanism might couple related sugar transporters, such as the insulin-responsive glucose transporter-4 (GLUT4), to the actin cytoskeleton via dematin. Immunocytochemistry established the presence of dematin in 3T3-L1 adipocytes, and a small pool of dematin and GLUT4-containing vesicles co-localized in 3T3-L1 cells under both basal and insulin-stimulated conditions. Plasma membrane sheet assays indicate that upon insulin stimulation, dematin translocates to the plasma membrane along with GLUT4, resulting in partial co-localization at the plasma membrane. Furthermore, dematin RNAi treated 3T3-L1 cells show reduced GLUT4 protein expression, suggesting that dematin may regulate a sub-population of GLUT4 via the lysosomal degradation pathway in adipocytes. Importantly, glucose transport was reduced by ∼28% in 3T3-L1 adipocytes depleted of dematin, and by ∼15% in the dematin headpiece knockout (HPKO) mouse primary adipocytes. Since a significant amount of dematin did not co-localize with GLUT4 in the cytosol and plasma membrane, biochemical interaction between dematin and GLUT4 could not be verified using immunoprecipitation and transfection assays. Although dematin does not bind directly to GLUT4 under these conditions, a possibility existed that this interaction may be transient and mediated through an adaptor protein. Interestingly, dematin contains seven 14-3-3 binding sites, and 14-3-3 adaptor has been shown to be functionally involved in GLUT4 trafficking. We demonstrate that phosphorylated dematin binds to 14-3-3 in 3T3-L1 adipocytes under both basal and insulin stimulated conditions. Mutagenesis studies identify serine-85 on dematin as the primary phospho-binding site for 14-3-3zeta. Furthermore, using pharmacological inhibitors, Akt is identified as the likely protein kinase that phosphorylates dematin to mediate the biochemical interactions between dematin and 14-3-3zeta. Together, our results identify erythrocyte dematin as a potential regulator of glucose transporter trafficking and degradation pathways in adipocytes with functional implications for glucose homeostasis, diabetes, and obesity. Disclosures: No relevant conflicts of interest to declare.

Reactive oxygen species downregulate glucose transport system in retinal endothelial cells

2011 ◽  
Vol 300 (4) ◽  
pp. C927-C936 ◽  
Author(s):  
Rosa Fernandes ◽  
Ken-ichi Hosoya ◽  
Paulo Pereira
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Plasma Membrane ◽  
Endothelial Cell ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Cell Damage ◽  
Proteasome Inhibition ◽  
Glucose Transporter 1 ◽  
Retinal Endothelial Cells

Retinal endothelial cells are believed to play an important role in the pathogenesis of diabetic retinopathy. In previous studies, we and others demonstrated that glucose transporter 1 (GLUT1) is downregulated in response to hyperglycemia. Increased oxidative stress is likely to be the event whereby hyperglycemia is transduced into endothelial cell damage. However, the effects of sustained oxidative stress on GLUT1 regulation are not clearly established. The objective of this study is to evaluate the effect of increased oxidative stress on glucose transport and on GLUT1 subcellular distribution in a retinal endothelial cell line and to elucidate the signaling pathways associated with such regulation. Conditionally immortalized rat retinal endothelial cells (TR-iBRB) were incubated with glucose oxidase, which increases the intracellular hydrogen peroxide levels, and GLUT1 regulation was investigated. The data showed that oxidative stress did not alter the total levels of GLUT1 protein, although the levels of mRNA were decreased, and there was a subcellular redistribution of GLUT1, decreasing its content at the plasma membrane. Consistently, the half-life of the protein at the plasma membrane markedly decreased under oxidative stress. The proteasome appears to be involved in GLUT1 regulation in response to oxidative stress, as revealed by an increase in stabilization of the protein present at the plasma membrane and normalization of glucose transport following proteasome inhibition. Indeed, levels of ubiquitinated GLUT1 increase as revealed by immunoprecipitation assays. Furthermore, data indicate that protein kinase B activation is involved in the stabilization of GLUT1 at the plasma membrane. Thus subcellular redistribution of GLUT1 under conditions of oxidative stress is likely to contribute to the disruption of glucose homeostasis in diabetes.

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 Increased glucose metabolism in Arid5b−/− skeletal muscle is associated with the down-regulation of TBC1 domain family member 1 (TBC1D1)

2020 ◽  
Vol 53 (1) ◽  
Author(s):  
Yuri Okazaki ◽  
Jennifer Murray ◽  
Ali Ehsani ◽  
Jessica Clark ◽  
Robert H. Whitson ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Energy Metabolism ◽  
Glucose Metabolism ◽  
Family Member ◽  
Glucose Transporter ◽  
Whole Body ◽  
Domain Family ◽  
Glucose Transporter 1

Abstract Background Skeletal muscle has an important role in regulating whole-body energy homeostasis, and energy production depends on the efficient function of mitochondria. We demonstrated previously that AT-rich interactive domain 5b (Arid5b) knockout (Arid5b−/−) mice were lean and resistant to high-fat diet (HFD)-induced obesity. While a potential role of Arid5b in energy metabolism has been suggested in adipocytes and hepatocytes, the role of Arid5b in skeletal muscle metabolism has not been studied. Therefore, we investigated whether energy metabolism is altered in Arid5b−/− skeletal muscle. Results Arid5b−/− skeletal muscles showed increased basal glucose uptake, glycogen content, glucose oxidation and ATP content. Additionally, glucose clearance and oxygen consumption were upregulated in Arid5b−/− mice. The expression of glucose transporter 1 (GLUT1) and 4 (GLUT4) in the gastrocnemius (GC) muscle remained unchanged. Intriguingly, the expression of TBC domain family member 1 (TBC1D1), which negatively regulates GLUT4 translocation to the plasma membrane, was suppressed in Arid5b−/− skeletal muscle. Coimmunofluorescence staining of the GC muscle sections for GLUT4 and dystrophin revealed increased GLUT4 localization at the plasma membrane in Arid5b−/− muscle. Conclusions The current study showed that the knockout of Arid5b enhanced glucose metabolism through the downregulation of TBC1D1 and increased GLUT4 membrane translocation in skeletal muscle.

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.

scholarly journals The phospholipid flippase ATP9A is required for the recycling pathway from the endosomes to the plasma membrane

2016 ◽  
Vol 27 (24) ◽  
pp. 3883-3893 ◽  
Author(s):  
Yoshiki Tanaka ◽  
Natsuki Ono ◽  
Takahiro Shima ◽  
Gaku Tanaka ◽  
Yohei Katoh ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Bilayers ◽  
Golgi Complex ◽  
Membrane Trafficking ◽  
Glucose Transporter ◽  
Glucose Transporter 1 ◽  
Recycling Endosomes ◽  
Type Iv ◽  
Late Endosomes ◽  
P Type

Type IV P-type ATPases (P4-ATPases) are phospholipid flippases that translocate phospholipids from the exoplasmic (or luminal) to the cytoplasmic leaflet of lipid bilayers. In Saccharomyces cerevisiae, P4-ATPases are localized to specific subcellular compartments and play roles in compartment-mediated membrane trafficking; however, roles of mammalian P4-ATPases in membrane trafficking are poorly understood. We previously reported that ATP9A, one of 14 human P4-ATPases, is localized to endosomal compartments and the Golgi complex. In this study, we found that ATP9A is localized to phosphatidylserine (PS)-positive early and recycling endosomes, but not late endosomes, in HeLa cells. Depletion of ATP9A delayed the recycling of transferrin from endosomes to the plasma membrane, although it did not affect the morphology of endosomal structures. Moreover, depletion of ATP9A caused accumulation of glucose transporter 1 in endosomes, probably by inhibiting their recycling. By contrast, depletion of ATP9A affected neither the early/late endosomal transport and degradation of epidermal growth factor (EGF) nor the transport of Shiga toxin B fragment from early/recycling endosomes to the Golgi complex. Therefore ATP9A plays a crucial role in recycling from endosomes to the plasma membrane.

scholarly journals DHHC9-mediated GLUT1 S-palmitoylation promotes glioblastoma glycolysis and tumorigenesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhenxing Zhang ◽  
Xin Li ◽  
Fan Yang ◽  
Chao Chen ◽  
Ping Liu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Plasma Membrane ◽  
Glucose Uptake ◽  
Posttranslational Modification ◽  
Poor Prognosis ◽  
Glucose Transporter ◽  
Transmembrane Protein ◽  
Glucose Supply ◽  
Glucose Transporter Glut1 ◽  
Transporter Glut1

AbstractGlucose transporter GLUT1 is a transmembrane protein responsible for the uptake of glucose into the cells of many tissues through facilitative diffusion. Plasma membrane (PM) localization is essential for glucose uptake by GLUT1. However, the mechanism underlying GLUT1 PM localization remains enigmatic. We find that GLUT1 is palmitoylated at Cys207, and S-palmitoylation is required for maintaining GLUT1 PM localization. Furthermore, we identify DHHC9 as the palmitoyl transferase responsible for this critical posttranslational modification. Knockout of DHHC9 or mutation of GLUT1 Cys207 to serine abrogates palmitoylation and PM distribution of GLUT1, and impairs glycolysis, cell proliferation, and glioblastoma (GBM) tumorigenesis. In addition, DHHC9 expression positively correlates with GLUT1 PM localization in GBM specimens and indicates a poor prognosis in GBM patients. These findings underscore that DHHC9-mediated GLUT1 S-palmitoylation is critical for glucose supply during GBM tumorigenesis.

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