Microenvironmental stiffness mediates cytoskeleton re-organization in chondrocytes through laminin-FAK mechanotransduction / 国际口腔科学杂志·英文版

Microenvironmental biophysical factors play a fundamental role in controlling cell behaviors including cell morphology, proliferation, adhesion and differentiation, and even determining the cell fate. Cells are able to actively sense the surrounding mechanical microenvironment and change their cellular morphology to adapt to it. Although cell morphological changes have been considered to be the first and most important step in the interaction between cells and their mechanical microenvironment, their regulatory network is not completely clear. In the current study, we generated silicon-based elastomer polydimethylsiloxane (PDMS) substrates with stiff (15:1, PDMS elastomer vs. curing agent) and soft (45:1) stiffnesses, which showed the Young's moduli of ~450 kPa and 46 kPa, respectively, and elucidated a new path in cytoskeleton re-organization in chondrocytes in response to changed substrate stiffnesses by characterizing the axis shift from the secreted extracellular protein laminin β1, focal adhesion complex protein FAK to microfilament bundling. We first showed the cellular cytoskeleton changes in chondrocytes by characterizing the cell spreading area and cellular synapses. We then found the changes of secreted extracellular linkage protein, laminin β1, and focal adhesion complex protein, FAK, in chondrocytes in response to different substrate stiffnesses. These two proteins were shown to be directly interacted by Co-IP and colocalization. We next showed that impact of FAK on the cytoskeleton organization by showing the changes of microfilament bundles and found the potential intermediate regulators. Taking together, this modulation axis of laminin β1-FAK-microfilament could enlarge our understanding about the interdependence among mechanosensing, mechanotransduction, and cytoskeleton re-organization.

Relationship between PI3K/mTOR/RhoA pathway-regulated cytoskeletal rearrangements and phagocytic capacity of macrophages

The objective of this study was to investigate the relationship between PI3K/mTOR/RhoA signaling regulated cytoskeletal rearrangements and phagocytic capacity of macrophages. RAW264.7 macrophages were divided into four groups; blank control, negative control, PI3K-RNAi, and mTOR-RNAi. The cytoskeletal changes in the macrophages were observed. Furthermore, the phagocytic capacity of macrophages against Escherichia coli is reported as mean fluorescence intensity (MFI) and percent phagocytosis. Transfection yielded 82.1 and 81.5% gene-silencing efficiencies against PI3K and mTOR, respectively. The PI3K-RNAi group had lower mRNA and protein expression levels of PI3K, mTOR, and RhoA than the blank and negative control groups (Р<0.01). The mTOR-RNAi group had lower mRNA and protein levels of mTOR and RhoA than the blank and the negative control groups (Р<0.01). Macrophages in the PI3K-RNAi group exhibited stiff and inflexible morphology with short, disorganized filopodia and reduced number of stress fibers. Macrophages in the mTOR-RNAi group displayed pronounced cellular deformations with long, dense filopodia and an increased number of stress fibers. The PI3K-RNAi group exhibited lower MFI and percent phagocytosis than blank and negative control groups, whereas the mTOR-RNAi group displayed higher MFI and percent phagocytosis than the blank and negative controls (Р<0.01). Before and after transfection, the mRNA and protein levels of PI3K were both positively correlated with mTOR and RhoA (Р<0.05), but the mRNA and protein levels of mTOR were negatively correlated with those of RhoA (Р<0.05). Changes in the phagocytic capacity of macrophages were associated with cytoskeletal rearrangements and were regulated by the PI3K/mTOR/RhoA signaling pathway.

Dact1 involvement in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation by regulating cx43

Abstract Objective: To determine the role of the dishevelled binding antagonist of beta catenin 1 (DACT1) in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation (AF). Methods: The DACT1 expression and its associations with the degree of fibrosis and β-catenin in valvular disease patients were analyzed by immunohistochemistry and Masson's staining. DACT1 was overexpressed in the atrial myocyte cell line (HL-1) and the cardiac cell line (H9C2) by adenoviral vectors. Alterations in the fibrous actin (F-actin) content and organization and the expression of β-catenin were detected by flow cytometry, immunofluorescence, and Western blotting. Additionally, the association of DACT1 with gap junctions connexin 43 (Cx43) was detected by immunohistochemistry, immunofluorescence, and Western blotting. Results: Decreased cytoplasmic DACT1 expression in the myocardium was associated with AF (P=0.037) and a high degree of fibrosis (weak vs. strong, P=0.028; weak vs. very strong, P=0.029). A positive association was observed between DACT1 and β-catenin expression in clinical samples (P=0.028, Spearman's rho=0.408). Furthermore, overexpression of DACT1 in HL-1 and H9C2 cells induced an increase in β-catenin and subsequent partial colocalization of DACT1 and β-catenin. In addition, F-actin content and organization were enhanced. Interestingly, DACT1 was positively correlated with the Cx43 expression in clinical samples (P=0.048, Spearman's rho=0.370) and changed the Cx43 distribution in cardiac cell lines. Conclusion: DACT1 proved to be a novel AF-related gene by regulating Cx43 via cytoskeletal organization induced by β-catenin accumulation in cardiomyocytes. DACT1 could thus serve as a potential therapeutic marker for AF.

Interação parasita-célula hospedeira: modificação de proteínas de Tripanosoma cruzi durante adesão à matriz extracelular / Parasite-host cell interaction: modifications of Trypanosoma cruzi proteins during the adhesion to extracelular matrix

A doença de Chagas foi incialmente descrita em 1090 e após mais de 100 anos de investigações sobre essa doença, ainda pouco se sabe sobre os mecanismos ativados no parasita durante sua adesão e invasão à célula hospedeira. Glicoproteínas de massa molecular de 85kDa localizadas na membrana do parasita foram identificadas como principais elementos responsáveis pela interação com o hospedeiro. Essas proteínas também são capazes de se ligar a elementos da matriz extracelular (ECM) da célula hospedeira e esse evento parece ser crucial para modulação da adesão e invasão do parasita e consequente avanço da infecção. Embora diferentes elementos tenham sido identificados no hospedeiro como componentes da via de resposta a adesão ao parasita, as modificações induzidas pela sua ligação ao hospedeiro é ainda pouco conhecida. Modificações pós-traducionais de proteínas, incluindo a fosforilação, têm sido utilizadas por diferentes organismos na transdução de sinais extracelulares. Dessa forma, a identificação de proteínas diferencialmente fosforiladas durante a adesão de tripomastigotas de T. cruzi a ECM, fibronectina e laminina foi o objetivo dessa tese. Tripomastigotas foram incubados com ECM, fibronectina-, laminina- ou BSA- previamente aderidos em placas de cultura de células. Em seguida, os parasitas foram coletados e suas proteínas extraídas e separadas por 2D-PAGE. Os géis de eletroforese foram corados com Pro-Q Diamond (para identifiicação de proteínas fosforiladas) e posteriormente com coomassie colloidal (identificação de proteínas totais). Os spots com diferença significativa na coloração com Pro-Q Diamond (p< 0,05) foram identificados por LC-MS/MS. 54 spots foram diferencialmente fosforilados durante a adesão dos parasitas a ECM, dos quais 39 sofreram um aumento da intensidade de fosforilação e 15 uma redução. Já dos 43 spots diferencialmente fosforilados durante incubação com laminina, 16 aumentaram a fosforilação enquanto 27 sofreram redução da intensidade de fosforilação. Por fim, após incubação com fibronectina, dos 50 spots selecionados, 15 spots sofreram aumento da intensidade de fosforilação e 35 sofreram redução. Após identificação dos spots, as modificações por fosforilação/desfosforilação de proteínas de função desconhecida (hypothetical proteins), proteínas do citoesqueleto, proteínas do choque térmico (HSPs) e proteínas componentes do proteassomo do parasita foram as mais evidentes. A validação por immonoblotting de algumas proteínas identificadas indicou que a desfosforilação de proteínas do citoesqueleto junto com a fosforilação de proteínas do choque térmico são os principais eventos durante a resposta do parasita a adesão a ECM e a seus elementos. Além disso, a desfosforilação de ERK 1/2 observada indicou uma inativação dessa proteína em parasitas aderidos a fibronectina e laminina. Os resultados obtidos nessa tese sugerem uma provável relação entre modificações de proteínas do citoesqueleto e HSPs com a capacidade de internalização dos parasitas na célula hospedeira

The Chagas disease was firstly described in 1909. After more than 100 years of investigation about this sickness much less is known about the mechanism triggered in the parasite during the adhesion and invasion to the host cell. 85kDa glycoproteins were identified as the major element responsible for the attachment to the host. In addition, these proteins are able to binding to extracellular matrix elements and host cytoskeletal proteins and it event appears to be an essential step in host cell invasion by T. cruzi. Although downstream signal modifications have been studied in host cells upon parasite binding, the molecular changes induced on the parasite by ligand binding are largely unknown. Since post-translational modification of proteins by phosphorylation is one of the most important mechanisms employed by organisms to transduce external signals, identification of proteins modified upon adhesion of T. cruzi trypomastigotes to ECM, laminin and fibronectin of the host cell was pursued. Trypomastigotes (Y strain) were incubated with ECM, laminin-, fibronectin- or BSA-coated surfaces, followed by 2D-PAGE stained with Pro-Q Diamond (phosphorylated protein detection) followed by colloidal coomassie stain (total protein identification). Proteins with significant differences in Pro-Q Diamond stain (p<0.05) were identified by LC-MS/MS. 54 spots were differentially phosphorylated during parasite adhesion to ECM, in which 39 spots have increased their phosphorylation level and 15 have decreased their phosphorylation. From the 43 spots presenting modification to the phosphorylation on incubation with laminin, 16 corresponded to cases of increase of phosphorylation and 27 to cases of dephosphorylation. After incubation with fibronectin: from the 50 spots selected, 15 corresponded to increase of phosphorylation and 35 to dephosphorylation. The results show phosphorylation/dephosphorylation modifications of unknown proteins, parasite cytoskeletal proteins (alpha and beta tubulin and paraflagellar-rod proteins), heat shock proteins and proteasome proteins. The validation by immunoblotting of proteins and their phosphorylation intensities indicates that cytoskeletal protein dephosphorylation in addition to heat shock proteins phosphorylation are the most important event during the trypomastigotes adhesion to the ECM. Looking for downstream signaling, dephosphorylation of ERK1/2 was also shown in trypomastigotes adhered to fibronectin or laminin, suggesting its inactivation. Thereby, those results suggest a possible correlation between cytoskeletal proteins and HSPs modification and the ability of parasite to internalize into host cells

A likely role for a novel PH-domain containing protein, PEPP2, in connecting membrane and cytoskeleton

PH domains (pleckstrin homology) are well known to bind membrane phosphoinositides with different specificities and direct PH domain-containing proteins to discrete subcellular apartments with assistances of alternative binding partners. PH domain-containing proteins are found to be involved in a wide range of cellular events, including signalling, cytoskeleton rearrangement and vesicular trafficking. Here we showed that a novel PH domain-containing protein, PEPP2, displayed moderate phosphoinositide binding specificity. Full length PEPP2 associated with both plasma membrane and microtubules. The membrane-associated PEPP2 nucleated at cell-cell contacts and the leading edge of migrating cells. Overexpression of PEPP2 increased membrane microviscosity, indicating a potential role of PEPP2 in regulating function of membrane and microtubules.

The actin filament network associated to Sertoli cell ectoplasmic specializations

Junctional devices in Sertoli cells conform the blood-testis barrier and play a key role in maturation and differentiation of germ cells. The spacial distribution of ectoplasmic specializations of Sertoli cells was studied by beta-actin immunolabelling, using laser confocal and transmission electron microscopy. For confocal microscopy, beta-actin immunolabelling of ectoplasmic specializations was studied over the background of either prosaposin or glutaredoxin immunolabelling of the Sertoli cytoplasm. Labelling was found near the basal lamina, surrounding early spermatocytes (presumably in leptotene-zygotene) or at one of two levels in the seminiferous epithelium: (1) around deep infoldings of the Sertoli cell cytoplasm, in tubular stages before spermiation, and (2) in the superficial part of the seminiferous epithelium, in tubular stages after or during spermiation. For transmission electron microscopy, beta-actin immunolabelling of ectoplasmic specializations was also used. Ectoplasmic specializations were found at two different levels of the seminiferous epithelium. We also used freeze fracture to analyze the characteristics of tubulo-bulbar complexes, a known component of apical ectoplasmic specializations. Also, these different approaches allowed us to study the complex arrangement of the actin cytoskeleton of Sertoli cells branches, which surround germ cells in different stages of the spermatogenic cycle. Our results show a consistent labelling for beta-actin before, during and after the release of spermatozoa in the tubular lumen (spermiation) suggesting a significant role of the actin network in spermatic cell differentiation. In conclusion, significant interrelations among the beta-actin network, the junctional complexes of the blood-testis barrier and the ectoplasmic specializations were detected at different stages of the seminiferous cycle.

Histochemical Localization of Carbohydrates in Morphological Stages of Developing Human Minor Salivary Glands: A Comparative Study with Cytoskeletal Markers / Localización Histoquímica de Carbohidratos en el Desarrollo de Glándulas Salivales Menores Humanas: un Estudio Comparativo con los Marcadores del Citoesqueleto

Carbohydrates play a critical role in many cellular processes like disease, growth and development. In this work lectins, proteins that recognizes carbohydrate free or conjugated, were used as histochemical probes for carbohydrates localization in developing human minor salivary gland. Immunohistochemistry for traditional cytoskeleton markers (Cks 7, 8, 13, 14, 19, SMA and Vimentin) was performed and then compared whit lectin histochemistry for PNA, WGA, ConA and UEA-I, specifics for D-galactose, N-acetyl-glucosamine, glucose/mannose and L-fucose respectively. For this, specimens were obtained from tongues and lips of 15 human foetuses at 10-28 weeks of gestation. None of immune cytoskeleton markers were identified in the first stage of development differing from carbohydrate markers. UEA-I, WGA and PNA recognized their specific carbohydrate residues in all stages analyzed varying the staining intensity and cell types. Ck8 and N-acetyl-glucosamine were expressed in canalicular, branching and cytodifferentiation stages while SMA and glucose/mannose were observed in the cytodifferentiation stage one. ConA only recognized myoepithelial cells on cytodifferentiation stages because of this specificity ConA could be used as biomarker of myoepithelial cells on cytodifferentiation. Lectin histochemistry suggests that L-fucose, D-galactose e N-acetyl-glucosamine are intensily and previously expressed than traditional cytoskeletal markers in human minor salivary gland during development.

Los hidratos de carbono tienen un papel crítico en muchos procesos celulares, como la enfermedad, el crecimiento y el desarrollo. Fueron utilizadasas lectinas, proteínas que reconocen los hidratos de carbono libres o conjugados, como sondas de localización histoquímica de los carbohidratos en el desarrollo humano de la glándula salival menor. Se realizó inmunohistoquímica de los marcadores tradicionales del citoesqueleto (CKs 7, 8, 13, 14, 19, SMA y vimentina) y posterior comparación con la histoquímica de lectinas para PNA, WGA, ConA y la UEA-I, específicas para D-galactosa, N-acetil-glucosamina, glucosa/manosa y L-fucosa, respectivamente. Para ello, se obtuvieron muestras de la lengua y de los labios de 15 fetos humanos entre 10-28 semanas de gestación. Ninguno de los marcadores inmunológicos del citoesqueleto se identificaron en la primera etapa del desarrollo, diferente de los marcadores de hidratos de carbono. UEA-I, WGA y PNA reconocen sus residuos específicos de hidratos de carbono en todas las etapas analizadas variando la intensidad de la tinción y los tipos de células. CK8 y N-acetil-glucosamina se expresaron en etapas de canalización, ramificación y citodiferenciación mientras que SMA y la glucosa/manosa se observaron solamente en la etapa de citodiferenciación. ConA sólo se reconoció en las células mioepiteliales en etapas de citodiferenciación. Así, debido a esta especificidad, ConA podría utilizarse como marcador biológico de las células mioepiteliales en la citodiferenciación. La histoquímica de lectinas sugiere que L-fucosa, D-galactosa y N-acetil-glucosamina son intensamente expresadas durante el desarrollo como los marcadores tradicionales del citoesqueleto humanos en las glándulas salivales menores .

Biophysical aspects of cancer--electromagnetic mechanism.

Hypothesis of coherent vibration states in biological systems based on nonlinear interaction between longitudinal elastic and electric polarization fields with metabolic energy supply was formulated by Frohlich. Conditions for excitation of coherent states and generation of electromagnetic fields are satisfied in microtubules which form electrical polar structures. Numerical models are used for analysis of Frohlich's vibration states in cells. Reduction of activity and of energy production in mitochondria, and disintegration of cytoskeleton structures by phosphorylation on the pathway of cancer trasformation can diminish excitation of the Frohlich's vibration states and of the generated electromagnetic field, which results in disturbances of the interaction forces between cells. Interaction forces between cancer cells may be smaller than interaction forces between healthy cells and cancer cells as follows from numerical models. Mechanism of malignity, i.e. local invasion, detachment of cancer cells, and metastasis, is assumed to depend on the electromagnetic field.

Type II phosphatidylinositol 4-kinase(s) in cell signaling cascades.

Phosphorylated derivatives of phosphatidylinositol (PtdIns) are key components of many signaling cascades. Many isoforms of PtdIns kinases, PtdIns phosphate kinases and phosphatases use these lipids in amazing networks of signaling cascades that are yet to be understood fully. PtdIns 4-kinase(s) phosphorylates PtdIns at the 4th -OH position of inositol head group and are classified in to type II and III PtdIns 4-kinases. While type III PtdIns 4-kinases are implicated in vesicular trafficking, type II PtdIns 4-kinases are suggested to play a role in cell signaling, cytoskeletal rearrangements, cell motility and in microbial pathogenicity. This paper reviews the role of type II PtdIns 4-kinases in cell signaling cascades in health and disease.

Effect of estradiol on expression of cytoskeletal proteins during spermatogenesis in testis of sexually mature rats.

Earlier studies had shown that long term treatment with estradiol arrests spermatogenesis in adult male rats, at a dose of 0.1 mg/kg/day. The present study was therefore undertaken to ascertain the causes underlying the reduction in sperm counts by administering estradiol for a short term, at the dose of 0.1 mg/kg/day. Estradiol valerate was injected at a dose of 0.1 mg/kg/day, for a period of 10 days to one group of adult male rats, which were administered saline for 12 days prior to estradiol injection, and sacrificed after 22 days. The control group was administered saline for 22 days. The sera were analyzed for testosterone and FSH levels. One testis of each male was immersion fixed for histology, and for immunohistochemistry of two testicular cytoskeletal proteins, vimentin and vinculin. The contralateral testes were used for analysis of vimentin and vinculin gene expression by reverse transcriptase polymerase chain reaction (RTPCR) and western blotting. Another group exposed to estradiol for 10 days was injected with bromodeoxyuridine (BrdU), at a dose of 100 mg/kg/day, to ascertain the effect on germ cell proliferation, and sacrificed 12 days later, while estradiol treatment was continued till sacrifice. BrdU, at a dose of 100 mg/kg/day was injected i.p. to a group of control rats treated with saline for 10 days, and sacrificed 12 days later. The testes from both groups were immersion fixed for immunohistochemical detection of BrdU. Histology of estradiol treated testis showed predominance of tubules with round spermatids with accumulation of lipid droplets in Sertoli cell cytoplasm and decreased cell height, whereas controls showed elongating spermatids. BrdU immunolocalization in the testis, irrespective of treatment, indicated its incorporation in deoxyribonucleic acid (DNA) suggesting that estradiol sustained germ cell proliferation. Both vimentin and vinculin could be immunolocalized to the testis. The testicular levels of vimentin and vinculin, quantified after western blotting, were unaffected. The testicular expression of vimentin and vinculin seen by RTPCR was also unaffected. The study suggested that estradiol induced reduction in sperm counts was not due to adverse effects on proliferation. The observed predominance of seminiferous tubules showing round spermatids, accumulation of lipid droplets as compared to controls suggested that reduction in elongated spermatids occurred through reduced spermiation and phagocytosis. The study also suggested that reduction in Sertoli cell height after short-term estradiol treatment was not due to reduced expression of vimentin and vinculin, which could be maintained by estradiol. However, reduction in Sertoli cell height could have been due to suppression of FSH and testosterone, implicated in the polymerization of vimentin and organization of vinculin, two cytoskeletal proteins involved in inter-Sertoli or Sertoli-germ cell junctions. The study suggested that disorganization of Sertoli cell cytoskeleton and reduction in the volume of Sertoli cells could be an important factor for reduced efficiency of spermatogenesis after exposure to estrogenic molecules.

Inhibition of endocytotic functions in Dictyostelium discoideum treated with a carbamate pesticide.

Administration of a carbamate pesticide carbaryl (1-Naphthyl-N-methyl carbamate) at a concentration of 60 and 100 ppm greatly inhibits the endocytotic functions during growth of the cellular slime mold D. discoideum. The ingestion of fluorescien isothiocynate (FITC) labeled E. coli is reduced between 30 and 40% in the treated cells as compared to controls. Similarly, the uptake of FITC-labeled dextran, which has been used as fluid-phase marker for pinocytosis also show 40-50% inhibition in the treated cells. 3H-leucine uptake and incorporation are also inhibited in the treated cells. SDS-PAGE analysis of cytoskeletal proteins shows a higher actin association with the membrane of treated cells. The results demonstrate the detrimental effects of Carbamate on the soil microbe even at a very low concentration and the efficacy of the slime mold cells as a biosensor for the carbamate-induced cytotoxicity.

The cytoskeleton of the electric tissue of Electrophorus electricus, L

The electric eel Electrophorus electricus is a fresh water teleost showing an electrogenic tissue that produces electric discharges. This electrogenic tissue is distributed in three well-defined electric organs which may be found symmetrically along both sides of the eel. These electric organs develop from muscle and exhibit several biochemical properties and morphological features of the muscle sarcolema. This review examines the contribution of the cytoskeletal meshwork to the maintenance of the polarized organization of the electrocyte, the cell that contains all electric properties of each electric organ. The cytoskeletal filaments display an important role in the establishment and maintenance of the highly specialized membrane model system of the electrocyte. As a muscular tissue, these electric organs expresses actin and desmin. The studies that characterized these cytoskeletal proteins and their implications on the electrophysiology of the electric tissues are revisited.

Cross-linking of CD4 induces cytoskeletal association of CD4 and p56lck

A membrane glycoprotein CD4 functions as a co-receptor of a T lymphocyte. The co-receptor function has been attributed to a protein tyrosine kinase, p56lck, which is activated upon CD4 binding to MHC molecule. In this study, we present evidences that one of the pathways through which CD4 transmits its signal is cytoskeleton association of p56lck tyrosine kinase as well as CD4 itself. Cytoskeletal association of both proteins is inhibited by a tyrosine kinase inhibitor, genistein, indicating that tyrosine protein kinase activation is important for cytoskeletal association of CD4 and p56lck. Cytoskeletal association of these proteins by CD4 cross-linking is not affected by inhibitors of protein kinase C nor PI3-kinase. Taken together, these results suggest that CD4 cross-linking activates a tyrosine kinase which then induces the simultaneous association of CD4 and p56lck with cytoskeleton.

Parietal cells contain most of the myosin, filamin and actin present in rat gastric glands

The association of myosin and a filamin-like protein to the F-actin cytoskeleton of parietal cells was studied in the rat gastric mucosa. Myosin and the filamin-like protein were localized by indirect immunofluorescence microscopy while the distribution of actin was established by using FITC-phalloidin. These cytoskeletal proteins, concentrated in the parietal cells, changed their distribution in correlation with the hydrochloric acid (HCl) secretory state of the cells and the appearance of a developed intracellular canaliculus. Thus,in resting parietal cells, actin showed a patchy distribution, delimiting the poorly developed secretory canaliculi, while myosin and the filamin-like protein distributed diffusely over the cytoplasm. In secreting cells, F-actin was concentrated in the cytoplasmic projections filling the canalicular lumen, while myosin and the filamin-like protein were excluded from this region, concentrating in the adjoining cytoplasm. The present results show that myosin and the filamin-like protein change their association with the secretory membranes in relation to the development of the secretory canaliculus of parietal cells. In resting cells, both proteins associate with the endocellular secretory membranes. In secreting cells, the microvillar projections of the canalicular surface formed by these membranes bind F-actin, but exclude myosin and the filamin-like protein

Membrane-associated cytoskeleton and transbilayer phospholipid asymmetry.

Earlier studies have suggested that the membrane-associated cytoskeleton (membrane skeleton) in erythrocytes plays a major role in maintaining the transmembrane phospholipid asymmetry. But recently, it has been proposed that an ATP-dependent aminophospholipid pump is the sole determinant of this asymmetry in these cells. A critical analysis of the published data along with some unpublished results from the author's laboratory, however, indicate that both membrane skeleton and ATP-dependent aminophospholipid pump are required for maintaining the membrane phospholipid asymmetry in native erythrocytes.