Reduction in flippase activity contributes to surface presentation of phosphatidylserine in human senescent erythrocytes.

Mature human erythrocytes circulate in blood for approximately 120 days, and senescent erythrocytes are removed by splenic macrophages. During this process, the cell membranes of senescent erythrocytes express phosphatidylserine, which is recognized as a signal for phagocytosis by macrophages. However, the mechanisms underlying phosphatidylserine exposure in senescent erythrocytes remain unclear. To clarify these mechanisms, we isolated senescent erythrocytes using density gradient centrifugation and applied fluorescence-labelled lipids to investigate the flippase and scramblase activities. Senescent erythrocytes showed a decrease in flippase activity but not scramblase activity. Intracellular ATP and K+ , the known influential factors on flippase activity, were altered in senescent erythrocytes. Furthermore, quantification by immunoblotting showed that the main flippase molecule in erythrocytes, ATP11C, was partially lost in the senescent cells. Collectively, these results suggest that multiple factors, including altered intracellular substances and reduced ATP11C levels, contribute to decreased flippase activity in senescent erythrocytes in turn to, present phosphatidylserine on their cell membrane. The present study may enable the identification of novel therapeutic approaches for anaemic states, such as those in inflammatory diseases, rheumatoid arthritis, or renal anaemia, resulting from the abnormally shortened lifespan of erythrocytes.

ATP11C T418N, a gene mutation causing congenital hemolytic anemia, reduces flippase activity due to improper membrane trafficking.

Distribution of phosphatidylserine (PS) in the erythrocyte membrane is essential for its activity. Flippase transports phospholipids from the outer to the inner leaflet of the lipid bilayer and maintains asymmetric distribution of phospholipids in the plasma membrane. ATP11C, a flippase, catalyzes PS flipping at the plasma membrane in association with cell cycle control protein 50A (CDC50A). ATP11C T418 N mutation causes 90% decrease in erythrocyte PS-flippase activity. However, the mechanism of the activity reduction remains unknown. To study the endogenous expression of ATP11C in erythrocytes, we produced a monoclonal antibody against human ATP11C. Immunoblotting analyses with this antibody revealed the absence of ATP11C in erythrocyte membranes derived from a patient with the T418 N mutation. Transiently expressed ATP11C wild-type in cultured cells localized in the cell membranes in the presence of CDC50A. Contrastingly, ATP11C T418 N mutants stacked at the endoplasmic reticulum (ER) even in the presence of CDC50A, suggesting improper intracellular trafficking. Expression of the T418 N mutant in cultured cells was lower than that in the wild-type. However, reduced expression of the T418 N mutant was partially restored by treatment with proteasome inhibitors, suggesting ER-associated degradation of the mutant protein. Cells expressing T418 N did not show flippase activity at the plasma membrane. These data show that the loss of PS-flippase activity in erythrocytes carrying ATP11C T418 N mutation is due to impaired enzymatic activity, improper membrane trafficking, and increased proteasome degradation.

Maintenance and regulation of asymmetric phospholipid distribution in human erythrocyte membranes: implications for erythrocyte functions.

PURPOSE OF REVIEW: The article summarizes new insights into the molecular mechanisms for the maintenance and regulation of the asymmetric distribution of phospholipids in human erythrocyte membranes. We focus on phosphatidylserine, which is primarily found in the inner leaflet of the membrane lipid bilayer under low Ca conditions (<1 µmol/l) and is exposed to the outer leaflet under elevated Ca concentrations (>1 µmol/l), when cells become senescent. Clarification of the molecular basis of phosphatidylserine flipping and scrambling is important for addressing long-standing questions regarding phosphatidylserine functions. RECENT FINDINGS: ATP11C, a P-IV ATPase, has been identified as a major flippase in analyses of patient erythrocytes with a 90% reduction in flippase activity. Phospholipid scramblase 1 (PLSCR1) has been defined as a Ca-activated scramblase that is completely suppressed by membrane cholesterol under low Ca concentrations. SUMMARY: For survival, phosphatidylserine surface exposure is prevented by cholesterol-mediated suppression of PLSCR1 under low Ca concentrations, irrespective of flipping by ATP11C. In senescent erythrocytes, PLSCR1 is activated by elevated Ca, resulting in phosphatidylserine exposure, allowing macrophage phagocytosis. These recent molecular findings establish the importance of the maintenance and regulation of phosphatidylserine distribution for both the survival and death of human erythrocytes.

An Unrecognized Function of Cholesterol: Regulating the Mechanism Controlling Membrane Phospholipid Asymmetry.

An asymmetric distribution of phospholipids in the membrane bilayer is inseparable from physiological functions, including shape preservation and survival of erythrocytes, and by implication other cells. Aminophospholipids, notably phosphatidylserine (PS), are confined to the inner leaflet of the erythrocyte membrane lipid bilayer by the ATP-dependent flippase enzyme, ATP11C, counteracting the activity of an ATP-independent scramblase. Phospholipid scramblase 1 (PLSCR1), a single-transmembrane protein, was previously reported to possess scrambling activity in erythrocytes. However, its function was cast in doubt by the retention of scramblase activity in erythrocytes of knockout mice lacking this protein. We show that in the human erythrocyte PLSCR1 is the predominant scramblase and by reconstitution into liposomes that its activity resides in the transmembrane domain. At or below physiological intracellular calcium concentrations, total suppression of flippase activity nevertheless leaves the membrane asymmetry undisturbed. When liposomes or erythrocytes are depleted of cholesterol (a reversible process in the case of erythrocytes), PS quickly appears at the outer surface, implying that cholesterol acts in the cell as a powerful scramblase inhibitor. Thus, our results bring to light a previously unsuspected function of cholesterol in regulating phospholipid scrambling.

A method for detailed analysis of the structure of mast cell secretory granules by negative contrast imaging.

Secretory granules (SGs) in mast cells contain various molecules that elicit allergy symptoms and are generally considered therapeutic targets. However, the biogenesis, maintenance, regulation, and recycling of these granules remain controversial, mainly due to the lack of suitable live-cell imaging methods. In this study, we applied negative contrast imaging with soluble green fluorescent protein (GFP) expressed in the cytoplasm as a method to validate structural information of mast cell SGs. We evaluated the accuracy of the method in detail, and we demonstrated that it can be used for quantitative analysis. Using this technique, secretory granules, the nucleus, mitochondria, and the cell body were visualized in individual RBL-2H3 mast cells without any influence. When combined with conventional multicolor fluorescence imaging, visualization of SG-associated proteins and SG-SG fusion was achieved. Moreover, 3D images were constructed based on this method, and detailed information on the number, size, and shape of individual SGs was obtained. We found that cell volume was correlated with SG number. In summary, the technique provides valuable and unique data, and will therefore advance SG research.

ATP11C is a major flippase in human erythrocytes and its defect causes congenital hemolytic anemia.

Phosphatidylserine is localized exclusively to the inner leaflet of the membrane lipid bilayer of most cells, including erythrocytes. This asymmetric distribution is critical for the survival of erythrocytes in circulation since externalized phosphatidylserine is a phagocytic signal for splenic macrophages. Flippases are P-IV ATPase family proteins that actively transport phosphatidylserine from the outer to inner leaflet. It has not yet been determined which of the 14 members of this family of proteins is the flippase in human erythrocytes. Herein, we report that ATP11C encodes a major flippase in human erythrocytes, and a genetic mutation identified in a male patient caused congenital hemolytic anemia inherited as an X-linked recessive trait. Phosphatidylserine internalization in erythrocytes with the mutant ATP11C was decreased 10-fold compared to that of the control, functionally establishing that ATP11C is a major flippase in human erythrocytes. Contrary to our expectations phosphatidylserine was retained in the inner leaflet of the majority of mature erythrocytes from both controls and the patient, suggesting that phosphatidylserine cannot be externalized as long as scramblase is inactive. Phosphatidylserine-exposing cells were found only in the densest senescent cells (0.1% of total) in which scramblase was activated by increased Ca(2+) concentration: the percentage of these phosphatidylserine-exposing cells was increased in the patient's senescent cells accounting for his mild anemia. Furthermore, the finding of similar extents of phosphatidylserine exposure by exogenous Ca(2+)-activated scrambling in both control erythrocytes and the patient's erythrocytes implies that suppressed scramblase activity rather than flippase activity contributes to the maintenance of phosphatidylserine in the inner leaflet of human erythrocytes.

Longitudinal PBL in Undergraduate Medical Education Develops Lifelong-Learning Habits and Clinical Competencies in Social Aspects.

Problem-based learning (PBL) is popular in medical education in Japan. We wished to understand the influence of PBL on the clinical competence of medical residents, using self-assessment and observer assessment. Tokyo Women's Medical University (TWMU) implemented PBL longitudinally (long-time) for four years, and on this basis we analyzed whether long-time PBL education is useful for clinical work. A self-assessment questionnaire was sent to junior and senior residents who were alumni of several schools, and an observation-based assessment questionnaire to senior doctors instructing them. Respondents were asked if they had used the PBL process in daily clinical tasks, and if so in what processes. Senior doctors were asked whether TWMU graduates perform differently from graduates of other schools. TWMU graduates answered "used a lot" and "used a little" with regard to PBL at significantly higher rates than other graduates. As useful points of PBL, they mentioned extracting clinical problems, solving clinical problems, self-directed leaning, positive attitude, collaboration with others, presentation, doctor-patient relations, self-assessment, and share the knowledge with doctors at lower levels and students. Observer assessments of TWMU graduates by senior doctors represented them as adaptive, good at presenting, good at listening to others' opinions, practical, selfish, and eager in their instructional practice. Longitudinal PBL can be a good educational method to develop lifelong-learning habits and clinical competencies especially in terms of the social aspect.

Phosphatidylinositol-4,5 bisphosphate (PIP(2)) inhibits apo-calmodulin binding to protein 4.1.

Membrane skeletal protein 4.1R(80) plays a key role in regulation of erythrocyte plasticity. Protein 4.1R(80) interactions with transmembrane proteins, such as glycophorin C (GPC), are regulated by Ca(2+)-saturated calmodulin (Ca(2+)/CaM) through simultaneous binding to a short peptide (pep11; A(264)KKLWKVCVEHHTFFRL) and a serine residue (Ser(185)), both located in the N-terminal 30 kDa FERM domain of 4.1R(80) (H·R30). We have previously demonstrated that CaM binding to H·R30 is Ca(2+)-independent and that CaM binding to H·R30 is responsible for the maintenance of H·R30 ß-sheet structure. However, the mechanisms responsible for the regulation of CaM binding to H·R30 are still unknown. To investigate this, we took advantage of similarities and differences in the structure of Coracle, the Drosophila sp. homologue of human 4.1R(80), i.e. conservation of the pep11 sequence but substitution of the Ser(185) residue with an alanine residue. We show that the H·R30 homologue domain of Coracle, Cor30, also binds to CaM in a Ca(2+)-independent manner and that the Ca(2+)/CaM complex does not affect Cor30 binding to the transmembrane protein GPC. We also document that both H·R30 and Cor30 bind to phosphatidylinositol-4,5 bisphosphate (PIP2) and other phospholipid species and that that PIP2 inhibits Ca(2+)-free CaM but not Ca(2+)-saturated CaM binding to Cor30. We conclude that PIP2 may play an important role as a modulator of apo-CaM binding to 4.1R(80) throughout evolution.

Unique structural changes in calcium-bound calmodulin upon interaction with protein 4.1R FERM domain: novel insights into the calcium-dependent regulation of 4.1R FERM domain binding to membrane proteins by calmodulin.

Calmodulin (CaM) binds to the FERM domain of 80 kDa erythrocyte protein 4.1R (R30) independently of Ca(2+) but, paradoxically, regulates R30 binding to transmembrane proteins in a Ca(2+)-dependent manner. We have previously mapped a Ca(2+)-independent CaM-binding site, pep11 (A(264)KKLWKVCVEHHTFFR), in 4.1R FERM domain and demonstrated that CaM, when saturated by Ca(2+) (Ca(2+)/CaM), interacts simultaneously with pep11 and with Ser(185) in A(181)KKLSMYGVDLHKAKD (pep9), the binding affinity of Ca(2+)/CaM for pep9 increasing dramatically in the presence of pep11. Based on these findings, we hypothesized that pep11 induced key conformational changes in the Ca(2+)/CaM complex. By differential scanning calorimetry analysis, we established that the C-lobe of CaM was more stable when bound to pep11 either in the presence or absence of Ca(2+). Using nuclear magnetic resonance spectroscopy, we identified 8 residues in the N-lobe and 14 residues in the C-lobe of pep11 involved in interaction with CaM in both of presence and absence of Ca(2+). Lastly, Kratky plots, generated by small-angle X-ray scattering analysis, indicated that the pep11/Ca(2+)/CaM complex adopted a relaxed globular shape. We propose that these unique properties may account in part for the previously described Ca(2+)/CaM-dependent regulation of R30 binding to membrane proteins.

Membrane peroxidation and methemoglobin formation are both necessary for band 3 clustering: mechanistic insights into human erythrocyte senescence.

Oxidative damage and clustering of band 3 in the membrane have been implicated in the removal of senescent human erythrocytes from the circulation at the end of their 120 day life span. However, the biochemical and mechanistic events leading to band 3 cluster formation have yet to be fully defined. Here we show that while neither membrane peroxidation nor methemoglobin (MetHb) formation on their own can induce band 3 clustering in the human erythrocytes, they can do so when acting in combination. We further show that binding of MetHb to the cytoplasmic domain of band 3 in peroxidized, but not in untreated, erythrocyte membranes induces cluster formation. Age-fractionated populations of erythrocytes from normal human blood, obtained by a density gradient procedure, have allowed us to examine a subpopulation, highly enriched in senescent cells. We have found that band 3 clustering is a feature of only this small fraction, amounting to ∼0.1% of total circulating erythrocytes. These senescent cells are characterized by an increased proportion of MetHb as a result of reduced nicotinamide adenine dinucleotide-dependent reductase activity and accumulated oxidative membrane damage. These findings have allowed us to establish that the combined effects of membrane peroxidation and MetHb formation are necessary for band 3 clustering, and this is a very late event in erythrocyte life. A plausible mechanism for the combined effects of membrane peroxidation and MetHb is proposed, involving high-affinity cooperative binding of MetHb to the cytoplasmic domain of oxidized band 3, probably because of its carbonylation, rather than other forms of oxidative damage. This modification leads to dissociation of ankyrin from band 3, allowing the tetrameric MetHb to cross-link the resulting freely diffusible band 3 dimers, with formation of clusters.

Novel mechanism of regulation of protein 4.1G binding properties through Ca2+/calmodulin-mediated structural changes.

Protein 4.1G (4.1G) is a widely expressed member of the protein 4.1 family of membrane skeletal proteins. We have previously reported that Ca(2+)-saturated calmodulin (Ca(2+)/CaM) modulates 4.1G interactions with transmembrane and membrane-associated proteins through binding to Four.one-ezrin-radixin-moesin (4.1G FERM) domain and N-terminal headpiece region (GHP). Here we identify a novel mechanism of Ca(2+)/CaM-mediated regulation of 4.1G interactions using a combination of small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy analyses. We document that GHP intrinsically disordered coiled structure switches to a stable compact structure upon binding of Ca(2+)/CaM. This dramatic conformational change of GHP inhibits in turn 4.1G FERM domain interactions due to steric hindrance. Based upon sequence homologies with the Ca(2+)/CaM-binding motif in protein 4.1R headpiece region, we establish that the 4.1G S(71)RGISRFIPPWLKKQKS peptide (pepG) mediates Ca(2+)/CaM binding. As observed for GHP, the random coiled structure of pepG changes to a relaxed globular shape upon complex formation with Ca(2+)/CaM. The resilient coiled structure of pepG, maintained even in the presence of trifluoroethanol, singles it out from any previously published CaM-binding peptide. Taken together, these results show that Ca(2+)/CaM binding to GHP, and more specifically to pepG, has profound effects on other functional domains of 4.1G.

Identification of a novel role for dematin in regulating red cell membrane function by modulating spectrin-actin interaction.

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 ß-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.

Dynamic secondary structural changes in Ca²âº-saturated calmodulin upon interaction with the antagonist, W-7.

Although the 3D structure of the Ca(2+)-bound CaM (Ca(2+)/CaM) complex with the antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), has been resolved, the dynamic changes in Ca(2+)/CaM structure upon interaction with W-7 are still unknown. We investigated time- and temperature-dependent dynamic changes in Ca(2+)/CaM interaction with W-7 in physiological conditions using one- and two-dimensional Fourier-transformed infrared spectroscopy (2D-IR). We observed changes in the α-helix secondary structure of Ca(2+)/CaM when complexed with W-7 at a molar ratio of 1:2, but not at higher molar ratios (between 1:2 and 1:5). Kinetic studies revealed that, during the initial 125s at 25°C, Ca(2+)/CaM underwent formation of secondary coil and turn structures upon binding to W-7. Variations in temperature that induced significant changes in the structure of the Ca(2+)/CaM complex failed to do so when Ca(2+)/CaM was complexed with W-7. We concluded that W-7 induced stepwise conformational changes in Ca(2+)/CaM that resulted in a rigidification of the complex and its inability to interact with target proteins and/or polypeptides.

Light scattering: a novel approach to analyze protein 4.1R FERM domain interaction with inside-out-vesicles in solution.

In this study, we describe a novel application for light scattering, a method widely used for separation of molecules in solution based on their size. We demonstrate that light scattering analysis can monitor the change in particle size of protein 4.1R prior to and after binding to red blood cell inside-out-vesicles in solution. Light scattering constitutes therefore a novel tool to analyze protein-binding association constants.

Characterization of cytoskeletal protein 4.1R interaction with NHE1 (Na(+)/H(+) exchanger isoform 1).

NHE1 (Na(+)/H(+) exchanger isoform 1) has been reported to be hyperactive in 4.1R-null erythrocytes [Rivera, De Franceschi, Peters, Gascard, Mohandas and Brugnara (2006) Am. J. Physiol. Cell Physiol. 291, C880-C886], supporting a functional interaction between NHE1 and 4.1R. In the present paper we demonstrate that 4.1R binds directly to the NHE1cd (cytoplasmic domain of NHE1) through the interaction of an EED motif in the 4.1R FERM (4.1/ezrin/radixin/moesin) domain with two clusters of basic amino acids in the NHE1cd, K(519)R and R(556)FNKKYVKK, previously shown to mediate PIP(2) (phosphatidylinositol 4,5-bisphosphate) binding [Aharonovitz, Zaun, Balla, York, Orlowski and Grinstein (2000) J. Cell. Biol. 150, 213-224]. The affinity of this interaction (K(d) = 100-200 nM) is reduced in hypertonic and acidic conditions, demonstrating that this interaction is of an electrostatic nature. The binding affinity is also reduced upon binding of Ca(2+)/CaM (Ca(2+)-saturated calmodulin) to the 4.1R FERM domain. We propose that 4.1R regulates NHE1 activity through a direct protein-protein interaction that can be modulated by intracellular pH and Na(+) and Ca(2+) concentrations.

Intracellular interactions between protein 4.1 and glycophorin C on transport vesicles, as determined by fluorescence correlation spectroscopy.

Interaction of protein 4.1 (4.1R) with the transmembrane protein glycophorin C (GPC) regulates the functions of erythrocyte membrane. Fluorescence correlation spectroscopy (FCS) was used to define the interaction of EGFP-4.1R with DsRed-GPC on transport vesicles (TVs) by measuring their fluctuation in living cells. DsRed-GPC expressed in HeLa cells was delivered to the plasma membrane through slow vesicle transport. EGFP-4.1R, which freely diffused in the cytosol when expressed alone, diffused slowly when co-expressed with DsRed-GPC, indicating association of EGFP-4.1R with TVs. Fluorescence cross-correlation spectroscopy (FCCS) showed direct interaction of EGFP-4.1R with DsRed-GPC on TVs. The present study demonstrates that 4.1R binds to GPC on TVs in living cells.

Enucleation of human erythroblasts involves non-muscle myosin IIB.

Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various cell-division inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34(+) cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts.