scholarly journals Identification of a Novel Role for Dematin in Regulating Red Cell Membrane Function by Modulating Spectrin-Actin Interaction

2012 ◽  
Vol 287 (42) ◽  
pp. 35244-35250 ◽  
Author(s):  
Ichiro Koshino ◽  
Narla Mohandas ◽  
Yuichi Takakuwa
Keyword(s):  
Erythrocyte Membrane ◽  
Quartz Crystal ◽  
Mechanical Stability ◽  
Membrane Skeleton ◽  
Tail Region ◽  
Red Cell Membrane ◽  
Membrane Function ◽  
Pulldown Assay ◽  
Optimal Functioning ◽  
Protein 4.1R

The membrane skeleton plays a central role in maintaining the elasticity and stability of the erythrocyte membrane, two biophysical features critical for optimal functioning and survival of red cells. Many constituent proteins of the membrane skeleton are phosphorylated by various kinases, and phosphorylation of β-spectrin by casein kinase and of protein 4.1R by PKC has been documented to modulate erythrocyte membrane mechanical stability. In this study, we show that activation of endogenous PKA by cAMP decreases membrane mechanical stability and that this effect is mediated primarily by phosphorylation of dematin. Co-sedimentation assay showed that dematin facilitated interaction between spectrin and F-actin, and phosphorylation of dematin by PKA markedly diminished this activity. Quartz crystal microbalance measurement revealed that purified dematin specifically bound the tail region of the spectrin dimer in a saturable manner with a submicromolar affinity. Pulldown assay using recombinant spectrin fragments showed that dematin, but not phospho-dematin, bound to the tail region of the spectrin dimer. These findings imply that dematin contributes to the maintenance of erythrocyte membrane mechanical stability by facilitating spectrin-actin interaction and that phosphorylation of dematin by PKA can modulate these effects. In this study, we have uncovered a novel functional role for dematin in regulating erythrocyte membrane function.

scholarly journals Disorders of the erythrocyte membrane

2015 ◽  
Vol 9 (4) ◽  
pp. 323
Author(s):  
Sophia Delicou ◽  
Aikaterini Xydaki ◽  
Chryssanthi Kontaxi ◽  
Konstantinos Maragkos
Keyword(s):  
Surface Area ◽  
Erythrocyte Membrane ◽  
Structural Integrity ◽  
Mechanical Stability ◽  
Hereditary Spherocytosis ◽  
Membrane Surface ◽  
Membrane Skeleton ◽  
Inherited Disorders ◽  
Hereditary Elliptocytosis ◽  
Area Loss

Hemolytic anemia due to abnormalities of the erythrocyte membrane comprises an important group of inherited disorders. These include hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikilocytosis, and the hereditary stomatocytosis syndromes. The erythrocyte membrane skeleton composed of spectrin, actin, and several other proteins is essential for the maintenance of the erythrocyte shape, reversible deformability, and membrane structural integrity in addition to controlling the lateral mobility of integral membrane proteins. These disorders are characterized by clinical and laboratory heterogeneity and, as evidenced by recent molecular studies, by genetic heterogeneity. Defects in various proteins involved in linking the lipid bilayer to membrane skeleton result in loss in membrane cohesion leading to surface area loss and hereditary spherocytosis while defects in proteins involved in lateral interactions of the spectrin-based skeleton lead to decreased mechanical stability, membrane fragmentation and hereditary elliptocytosis. The disease severity is primarily dependent on the extent of membrane surface area loss. Treatment with splenectomy is curative in most patients.

scholarly journals Anatomy of the red cell membrane skeleton: unanswered questions

Blood ◽  
2016 ◽  
Vol 127 (2) ◽  
pp. 187-199 ◽  
Author(s):  
Samuel E. Lux
Keyword(s):  
Cell Membrane ◽  
Lipid Bilayer ◽  
Membrane Surface ◽  
Membrane Skeleton ◽  
Actin Binding ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Associated Proteins ◽  
Protein 4.1R ◽  
Cell Skeleton

Abstract The red cell membrane skeleton is a pseudohexagonal meshwork of spectrin, actin, protein 4.1R, ankyrin, and actin-associated proteins that laminates the inner membrane surface and attaches to the overlying lipid bilayer via band 3–containing multiprotein complexes at the ankyrin- and actin-binding ends of spectrin. The membrane skeleton strengthens the lipid bilayer and endows the membrane with the durability and flexibility to survive in the circulation. In the 36 years since the first primitive model of the red cell skeleton was proposed, many additional proteins have been discovered, and their structures and interactions have been defined. However, almost nothing is known of the skeleton’s physiology, and myriad questions about its structure remain, including questions concerning the structure of spectrin in situ, the way spectrin and other proteins bind to actin, how the membrane is assembled, the dynamics of the skeleton when the membrane is deformed or perturbed by parasites, the role lipids play, and variations in membrane structure in unique regions like lipid rafts. This knowledge is important because the red cell membrane skeleton is the model for spectrin-based membrane skeletons in all cells, and because defects in the red cell membrane skeleton underlie multiple hemolytic anemias.

scholarly journals Identification of a functional role for human erythrocyte sialoglycoproteins beta and gamma

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1068-1072
Author(s):  
ME Reid ◽  
JA Chasis ◽  
N Mohandas
Keyword(s):  
Erythrocyte Membrane ◽  
Material Properties ◽  
Functional Role ◽  
Mechanical Stability ◽  
Membrane Skeleton ◽  
Membrane Properties ◽  
Membrane Material ◽  
Skeletal Protein ◽  
Alpha Beta ◽  
Normal Erythrocyte

Four distinct erythrocyte membrane sialoglycoproteins (SGPs) denoted alpha, beta, gamma, and delta have been described, but their functions have not yet been defined. Recent evidence suggests that several of these SGPs associate with membrane skeletal proteins. Because the membrane skeletal protein network plays an important role in regulating the membrane material properties of deformability and mechanical stability, we wanted to determine whether the SGPs, through their interaction with the membrane skeleton, can modulate these membrane properties. We measured membrane mechanical stability and membrane deformability of erythrocytes that were deficient in either alpha, or delta or beta and gamma SGPs. Only erythrocytes deficient in beta and gamma SGP had altered membrane properties, as evidenced by marked decreases in both membrane mechanical stability (50% of normal) and membrane deformability (40% of normal). Erythrocytes deficient in either alpha or delta SGP had normal deformability and stability. Based on these data, we suggest that an interaction of beta and/or gamma SGP with the membrane skeleton plays a functionally important role in regulating normal erythrocyte membrane properties.

scholarly journals Cytoskeletal protein 4.1R negatively regulates T-cell activation by inhibiting the phosphorylation of LAT

Blood ◽  
2009 ◽  
Vol 113 (24) ◽  
pp. 6128-6137 ◽  
Author(s):  
Qiaozhen Kang ◽  
Yu Yu ◽  
Xinhong Pei ◽  
Richard Hughes ◽  
Susanne Heck ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mechanical Stability ◽  
Interleukin 2 ◽  
Humoral Response ◽  
Interferon Γ ◽  
Red Cell Membrane ◽  
Protein 4.1R

Abstract Protein 4.1R (4.1R) was first identified in red cells where it plays an important role in maintaining mechanical stability of red cell membrane. 4.1R has also been shown to be expressed in T cells, but its function has been unclear. In the present study, we use 4.1R-deficient mice to explore the role of 4.1R in T cells. We show that 4.1R is recruited to the immunologic synapse after T cell–antigen receptor (TCR) stimulation. We show further that CD4+ T cells of 4.1R−/− mice are hyperactivated and that they displayed hyperproliferation and increased production of interleukin-2 (IL-2) and interferon γ (IFNγ). The hyperactivation results from enhanced phosphorylation of LAT and its downstream signaling molecule ERK. The 4.1R exerts its effect by binding directly to LAT, and thereby inhibiting its phosphorylation by ZAP-70. Moreover, mice deficient in 4.1R display an elevated humoral response to immunization with T cell–dependent antigen. Thus, we have defined a hitherto unrecognized role for 4.1R in negatively regulating T-cell activation by modulating intracellular signal transduction.

scholarly journals Identification of a functional role for human erythrocyte sialoglycoproteins beta and gamma

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1068-1072 ◽  
Author(s):  
ME Reid ◽  
JA Chasis ◽  
N Mohandas
Keyword(s):  
Erythrocyte Membrane ◽  
Material Properties ◽  
Functional Role ◽  
Mechanical Stability ◽  
Membrane Skeleton ◽  
Membrane Properties ◽  
Membrane Material ◽  
Skeletal Protein ◽  
Alpha Beta ◽  
Normal Erythrocyte

Abstract Four distinct erythrocyte membrane sialoglycoproteins (SGPs) denoted alpha, beta, gamma, and delta have been described, but their functions have not yet been defined. Recent evidence suggests that several of these SGPs associate with membrane skeletal proteins. Because the membrane skeletal protein network plays an important role in regulating the membrane material properties of deformability and mechanical stability, we wanted to determine whether the SGPs, through their interaction with the membrane skeleton, can modulate these membrane properties. We measured membrane mechanical stability and membrane deformability of erythrocytes that were deficient in either alpha, or delta or beta and gamma SGPs. Only erythrocytes deficient in beta and gamma SGP had altered membrane properties, as evidenced by marked decreases in both membrane mechanical stability (50% of normal) and membrane deformability (40% of normal). Erythrocytes deficient in either alpha or delta SGP had normal deformability and stability. Based on these data, we suggest that an interaction of beta and/or gamma SGP with the membrane skeleton plays a functionally important role in regulating normal erythrocyte membrane properties.

scholarly journals Mapping of a spectrin-binding domain of human erythrocyte membrane protein 4.2

2002 ◽  
Vol 364 (3) ◽  
pp. 841-847 ◽  
Author(s):  
Debabrata MANDAL ◽  
Prasun K. MOITRA ◽  
Joyoti BASU
Keyword(s):  
Erythrocyte Membrane ◽  
Synthetic Peptide ◽  
Membrane Skeleton ◽  
Band 3 Protein ◽  
Fusion Peptides ◽  
Human Erythrocyte Membrane ◽  
Glutathione S Transferase ◽  
Protein 4.2 ◽  
N Terminus ◽  
Direct Binding

Protein 4.2 is a major component of the red blood cell membrane skeleton. Deficiency of protein 4.2 is linked with a variety of hereditary haemolytic anaemias. However, the interactions of protein 4.2 with other proteins of the erythrocyte membrane remain poorly understood. The major membrane-binding site for protein 4.2 resides on the cytoplasmic domain of band 3. Protein 4.2 interacts directly with spectrin in solution, suggesting that it stabilizes interactions between the membrane skeleton and the erythrocyte membrane. A 30kDa polypeptide, with its N-terminus corresponding to amino acid residue 269, derived by partial proteolysis of protein 4.2, was found to interact with biotinylated spectrin in gel renaturation assays. A series of overlapping glutathione S-transferase fusion peptides were constructed, and an α-helical domain encompassing residues 470–492 was found to be instrumental in mediating protein 4.2—spectrin interactions. Direct binding of a synthetic peptide, with the sequence corresponding to residues 470–492, to spectrin and the ability of the peptide to inhibit spectrin binding of protein 4.2 confirmed that these residues are crucial in mediating protein 4.2—spectrin interactions.

Plasma membrane-associate filament systems in cultured cells visualized by dry-cleaving

1983 ◽  
Vol 64 (1) ◽  
pp. 351-364
Author(s):  
D.A. Mesland ◽  
H. Spiele
Keyword(s):  
Cultured Cells ◽  
Hepatoma Cell ◽  
Cell Types ◽  
Membrane Skeleton ◽  
Adhesive Tape ◽  
Cell Organelles ◽  
Red Cell Membrane ◽  
Transmission Electron ◽  
General Network ◽  
Dry Cleaving

Substrate-attached critical-point-dried cells cleave along the level of the substrate-adherent membrane if removed by means of adhesive tape. The remaining membrane fragments on grids can be visualized three-dimensionally by means of stereo transmission electron microscopy. Attachment of cells may be achieved by active spreading of the cell, or artificially by poly-L-lysine adherence of prefixed cells. In 11 different cell types a filamentous network appears to remain associated with the cytoplasmic face of the membrane. In one hepatoma cell type virtually no filamentous network could be detected. Two general network morphologies are described: the hepatocytic network and the lymphoid network. Since no correspondence could be found between cytoplasmic structure and the structure of the membrane-associated network, and since cells generally cleave along the level of this network, excluding cell organelles, we conclude that it comprises a distinct structural system, analogous to the membrane skeleton of the red cell membrane.

scholarly journals Intrinsic material properties of the erythrocyte membrane indicated by mechanical analysis of deformation

Blood ◽  
1975 ◽  
Vol 45 (1) ◽  
pp. 29-43 ◽  
Author(s):  
EA Evans ◽  
PL La Celle
Keyword(s):  
Surface Area ◽  
Plastic Flow ◽  
Erythrocyte Membrane ◽  
Permanent Deformation ◽  
Volume Ratio ◽  
Total Surface Area ◽  
Water Soluble ◽  
Time Range ◽  
Extrinsic Factors ◽  
Red Cell Membrane

Abstract Deformation of the erythrocyte membrane by the micropipette technique permits analysis of intrinsic material characteristics of the membrane and provides a means to differentiate purely membrane factors from such extrinsic factors as surface area-to-volume ratio. Using small micropipettes (less than 0.5 microns radius) to deform cells, it is evident that the red cell membrane behaves like a solid for periods of time up to 5–10 min of sustained deformation; for long periods of strain, permanent deformations occur, indicative of the semi-solid structural character. In the time range in which the membrane behaves like a solid, the material is linearly elastic up to strains of 400%, implying a loose network structure in the membrane plane, and evaluation of the elastic parameter mu (mu for normal discocytes equals 7 x 10(-3) dynes/cm) suggests that the elements comprising the network may have a molecular weight of approximately that of the water-soluble membrane protein spectrin. Whether the network system is cross-linked or simply a polymer solution remains unanswered. Experimental data indicate that plastic flow of the membrane under conditions of protracted strain may lead to permanent deformation of the membrane, whereas uniform dilation of the membrane, requiring over 1000 times more energy than for plastic flow, results in membrane failure and lysis. Analyses of the data from larger micropipettes of limiting mean cylindrical diameter show their utility in evaluating extrinsic factors, e.g., surface area-to-volume relationships, which are related to the capability of the whole cell to form a new configuration with implicit resistance to total surface area change, as the cell enters narrow channels of the microcirculation. Thus, micropipettes with diameters in the 2.7–3.0-microns range can provide sensitive comparisons of cellular deformability of erythrocytes.

Phospholipid phosphorylation in erythrocyte of spontaneously hypertensive rats

1982 ◽  
Vol 243 (4) ◽  
pp. H590-H597 ◽  
Author(s):  
S. Koutouzov ◽  
P. Marche ◽  
J. F. Cloix ◽  
P. Meyer
Keyword(s):  
Erythrocyte Membrane ◽  
Spontaneously Hypertensive Rat ◽  
Intact Cells ◽  
Membrane Function ◽  
Spontaneously Hypertensive ◽  
Phosphatidylinositol Kinase ◽  
Inositol Lipids ◽  
Wistar Kyoto Rat ◽  
Series Of Experiments ◽  
Wistar Kyoto

The rapid turnover of phosphoinositides within membranes suggests that these lipids play an important role in membrane function. Since various abnormalities have been described in the erythrocyte membrane of the spontaneously hypertensive rat (SHR) we have studied the turnover of phosphoinositides in the erythrocyte of SHR and age-matched normotensive Wistar-Kyoto rat (WKY). This was achieved by measuring the incorporation of 32P into inositol lipids after incubation of 1) intact erythrocytes with [32P]orthophosphate and 2) isolated ghost membranes with [gamma-32P]ATP. In both series of experiments more than 99% of the radioactivity incorporated into lipids was into the polyphosphoinositides diphosphoinositide (DPI) and triphosphoinositide (TPI). In both intact erythrocytes and ghost membranes, the levels of 32P incorporated into DPI and TPI were significantly different in SHR than in WKY. Further analysis of factors known to influence the labeling of DPI and TPI indicated that this could be ascribed to decreased activities of phosphatidylinositol kinase and/or DPI kinase, with respect to ATP as substrate. Moreover comparison of data obtained in intact cells with those obtained with ghost membranes suggests that within the SHR erythrocyte, membrane-cytosol interactions may occur that could also be responsible for the alteration of phosphoinositide labeling observed in hypertensive animals. Since phosphoinositides have been reported to be involved in the Ca2+-gating system of membrane, our findings could be associated with the abnormal Ca2+ binding and transport recently described in SHR erythrocyte.

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