Identification of new mutations in patients with hereditary spherocytosis by next-generation sequencing.

Hereditary spherocytosis (HS) is the most common inherited hemolytic anemia characterized by the presence of spherical-shaped erythrocytes on the peripheral blood smear, hemolysis, splenomegaly, jaundice, and gallstones. To date, mutations in at least five genes (ANK1, EPB42, SLC4A1, SPTA1, and SPTB) have been found to be associated with different subtypes of HS. Here, we aim to investigate the presence of novel as well as known mutations in 35 Chinese patients with clinically suspected HS. Whole-exome sequencing (WES) has identified 3 patients with SLC4A1, 16 patients with ANK1, and 16 patients with SPTB mutations, including 5 splicing, 12 nonsense, 9 frameshift, 7 missense, and 1 start-loss mutation, indicating that SPTB and ANK1 are the most frequently mutated genes in Chinese HS patients. Among 34 mutations identified, 21 were novel. Most of SPTB and ANK1 mutations were nonsense (8/16) and frameshift (6/16) mutations. By trio analysis of eight families we have confirmed six de novo mutations. In addition, genotype-phenotype analysis was also performed by comparing clinical manifestations among three groups of patients with SPTB, ANK1, and SLC4A1 mutations. It revealed that patients with ANK1 mutations had a significantly higher level of MCV and MCH but lower percentage of spherocytes compared with those carrying SPTB mutations. In conclusion, our results suggested that molecular diagnosis by next-generation sequencing (NGS) is a fast, economic, and accurate way to detect and identify pathogenic alterations of inherited diseases, highlighting the potential usage of NGS in clinical practice.

Association of Very Early Serum Levels of S100B, Glial Fibrillary Acidic Protein, Ubiquitin C-Terminal Hydrolase-L1, and Spectrin Breakdown Product with Outcome in ProTECT III.

Rapid risk-stratification of patients with acute traumatic brain injury (TBI) would inform management decisions and prognostication. The objective of this serum biomarker study (Biomarkers of Injury and Outcome [BIO]-Progesterone for Traumatic Brain Injury, Experimental Clinical Treatment [ProTECT]) was to test the hypothesis that serum biomarkers of structural brain injury, measured at a single, very early time-point, add value beyond relevant clinical covariates when predicting unfavorable outcome 6 months after moderate-to-severe acute TBI. BIO-ProTECT utilized prospectively collected samples obtained from subjects with moderate-to-severe TBI enrolled in the ProTECT III clinical trial of progesterone. Serum samples were obtained within 4 h after injury. Glial fibrillary acidic protein (GFAP), S100B, αII-spectrin breakdown product of molecular weight 150 (SBDP150), and ubiquitin C-terminal hydrolase-L1 (UCH-L1) were measured. The association between log-transformed biomarker levels and poor outcome, defined by a Glasgow Outcome Scale-Extended (GOS-E) score of 1-4 at 6 months post-injury, were estimated via logistic regression. Prognostic models and a biomarker risk score were developed using bootstrapping techniques. Of 882 ProTECT III subjects, samples were available for 566. Each biomarker was associated with 6-month GOS-E (p < 0.001). Compared with a model containing baseline patient variables/characteristics, inclusion of S100B and GFAP significantly improved prognostic capacity (p ≤ 0.05 both comparisons); conversely, UCH-L1 and SBDP did not. A final predictive model incorporating baseline patient variables/characteristics and biomarker data (S100B and GFAP) had the best prognostic capability (area under the curve [AUC] = 0.85, 95% confidence interval [CI]: CI 0.81-0.89). Very early measurements of brain-specific biomarkers are independently associated with 6-month outcome after moderate-to-severe TBI and enhance outcome prediction.

Clinical significance of detecting serum melatonin and SBDPs in brain injury in preterm infants.

BACKGROUND: To investigate the clinical values of serum melatonin and αII spectrin cleavage products (SBDPs) in assessing the severity of brain injury in preterm infants. METHODS: Sixty-four premature infants in total were selected and classified into the brain injury group (BI, n = 30) and the non-brain injury group (CON, n = 34) according to cranial imaging examination. The serum melatonin and SBDPs were detected by ELISA. All the preterm infants were received NBNA testing at 40 weeks of corrected gestational age. RESULTS: The levels of melatonin and SBDPs in the BI group were significantly higher than the CON group (p < 0.05) and the levels in the infants with severe brain injury were significantly higher than those with mild brain injury (p < 0.05), as well as exhibiting a negative correlation with the NBNA score at 40 weeks of corrected gestational age (p < 0.05). CONCLUSIONS: Detecting melatonin and SBDPs has clinical value in diagnosing and assessing the severity of brain injury in preterm infants.

Evaluation of alpha-II-spectrin breakdown products as potential biomarkers for early recognition and severity of aneurysmal subarachnoid hemorrhage.

This study assessed whether cytoskeletal protein alpha-II spectrin breakdown products (SBDP150, SBDP145, and SBDP120) would identify the presence of aSAH and be associated with severity (GCS score, WFNS grade and survival to hospital discharge). This prospective case-control study, conducted at a tertiary care Level I trauma center, enrolled adult patients with angiography confirmed aSAH who underwent ventriculostomy placement for cerebrospinal fluid (CSF) drainage. There were 40 patients enrolled in the study, 20 with aSAH and 20 control subjects. Patients with aSAH were a mean age of 54 (SD15) and 75% were female. There were significant differences in SBDP150, SBDP145, and SBDP120 CSF levels between patients with and without aSAH (p < 0.001), even in those presenting with a GCS Score of 15 and a WFNS Grade 1. The AUC for distinguishing aSAH from control subjects was 1.0 for SBDP150 and SBDP145, and 0.95 for SBDP120. SBDP150 and SBDP145 both yielded sensitivities and specificities of 100% and SBDP120 was 90% and 100% respectively. Moreover, there were significantly higher levels of SBDP150 and SBDP145 in the non-survivors than in the survivors (p < 0.001). This study demonstrates the potential that SBDP's have as biomarkers for recognition and severity of aSAH. A larger prospective study is warranted.

Temporal response profiles of serum ubiquitin C-terminal hydrolase-L1 and the 145-kDa alpha II-spectrin breakdown product after severe traumatic brain injury in children.

OBJECTIVE: Traumatic brain injury (TBI) is the leading cause of acquired disability among children. Brain injury biomarkers may serve as useful diagnostic and prognostic indicators for TBI. Levels of ubiquitin C-terminal hydrolase-L1 (UCH-L1) and the 145-kDa alpha II-spectrin breakdown product (SBDP-145) correlate with outcome in adults after severe TBI. The authors conducted a pilot study of these biomarkers in children after severe TBI to inform future research exploring their utility in this population. METHODS: The levels of UCH-L1 and SBDP-145 were measured in serum, and UCH-L1 in CSF from pediatric patients after severe TBI over 5 days after injury. Both biomarkers were also measured in age-matched control serum and CSF. RESULTS: Adequate numbers of samples were obtained in serum, but not CSF, to assess biomarker temporal response profiles. Using patients with samples from all time points, UCH-L1 levels increased rapidly and transiently, peaking at 12 hours after injury. SBDP-145 levels showed a more gradual and sustained response, peaking at 48 hours. The median serum UCH-L1 concentration was greater in patients with TBI than in controls (median [IQR] = 361 [187, 1330] vs 147 [50, 241] pg/ml, respectively; p < 0.001). Receiver operating characteristic (ROC) analysis revealed an AUC of 0.77. Similarly, serum SBDP-145 was greater in children with TBI than in controls (median [IQR] = 172 [124, 257] vs 69 [40, 99] pg/ml, respectively; p < 0.001), with an ROC AUC of 0.85. When only time points of peak levels were used for ROC analysis, the discriminability of each serum biomarker increased (AUC for UCH-L1 at 12 hours = 1.0 and for SBDP-145 at 48 hours = 0.91). Serum and CSF UCH-L1 levels correlated well in patients with TBI (r = 0.70, p < 0.001). CONCLUSIONS: Findings from this exploratory study reveal robust increases of UCH-L1 and SBDP-145 in serum and UCH-L1 in CSF obtained from children after severe TBI. In addition, important temporal profile differences were found between these biomarkers that can help guide optimal time point selection for future investigations of their potential to characterize injury or predict outcomes after pediatric TBI.

Prognostic utility of neuroinjury biomarkers in post out-of-hospital cardiac arrest (OHCA) patient management.

Despite aggressive intervention, patients who survive an out-of-hospital cardiac arrest (OHCA) generally have very poor prognoses, with nationwide survival rates of approximately 10-20%. Approximately 90% of survivors will have moderate to severe neurological injury ranging from moderate cognitive impairment to brain death. Currently, few early prognostic indicators are considered reliable enough to support patients' families and clinicians' in their decisions regarding medical futility. Blood biomarkers of neurological injury after OHCA may be of prognostic value in these cases. When most bodily tissues are oxygen-deprived, cellular metabolism switches from aerobic to anaerobic respiration. Neurons are a notable exception, however, being dependent solely upon aerobic respiration. Thus, after several minutes without circulating oxygen, neurons sustain irreversible damage, and certain measurable biomarkers are released into the circulation. Prior studies have demonstrated value in blood biomarkers in prediction of survival and neurologic impairment after OHCA. We hypothesize that understanding peptide biomarker kinetics in the early return of spontaneous circulation (ROSC) period, especially in the setting of refractory cardiac arrest, may assist clinicians in determining prognosis earlier in acute resuscitation. Specifically, during and after immediate resuscitation and return of ROSC, clinicians and families face a series of important questions regarding patient prognosis, futility of care and allocation of scarce resources such as the early initiation of extracorporeal cardiopulmonary resuscitation (ECPR). The ability to provide early prognostic information in this setting is highly valuable. Currently available, as well as potential biomarkers that could be good candidates in prognostication of neurological outcomes after OHCA or in the setting of refractory cardiac arrest will be reviewed and discussed.

Identification of aged bloodstains through mRNA profiling: Experiments results on selected markers of 30- and 50-year-old samples.

Remarkable progress has been made in recent years on the research of body fluid identification through messenger RNA(mRNA) profiling. In order to examine the viability of mRNA profiling as a method to identify aged bloodstains, this study tested two groups of bloodstain samples, dated 30 years and 50 years back respectively, on seven blood specific markers, i.e. HBB, HBA, GYPA, CD93, ALAS2, SPTB (91bp and 247bp primers), and PBGD. Test results indicate that HBA and HBB are the most stable markers in aged bloodstains, returning positive results in over 80% of the 50-year-old samples and over 90% of the 30-year-old samples. This finding proves mRNA profiling an effective way of identifying aged bloodstains.

Severe hemolysis and red cell fragmentation caused by the combination of a spectrin mutation with a thrombotic microangiopathy.

Two patients are described who presented with severe hemolysis and erythrocyte fragmentation. One patient had renal allograft rejection and disseminated intravascular coagulation, and the other had thrombotic thrombocytopenia purpura. The severity of hemolysis and the red cell abnormalities were considerably more profound than usually seen in patients with thrombotic microangiopathies. After evaluation of blood smears prepared before the onset of the disease and biochemical characterization of proteins of the red blood cell skeleton, a mutation of the skeletal protein spectrin, designated Sp alpha l/65, was identified. In the heterozygous form, this mutation manifests as mild, often asymptomatic, hereditary elliptocytosis. We conclude that in these two patients with thrombotic microangiopathy, the intrinsic red cell membrane instability resulting from the underlying skeletal defect aggravated the mechanical red cell fragmentation, producing morphological features similar to the severe hemolytic form of hereditary elliptocytosis or hereditary pyropoikilocytosis.

Quantification and characterization of regulin, a Mr-230,000 highly elongated protein of rabbit reticulocytes.

Procedures are described by which regulin in rabbit reticulocytes was quantified and isolated in relatively large amounts. In these cells the protein occurs at a ratio of about 1.1-1.6 regulin monomers/spectrin tetramer, corresponding to 80,000-100,000 molecules of Mr-230,000 regulin/cell. Erythrocytes contain less than 12% of the amount of regulin in reticulocytes and the protein has not been detected in non-erythroid cells. Regulin was found primarily in the cytosolic fraction of lysed reticulocytes. It appears to be unusually sensitive to proteolysis by Ca2+-activated thiol proteases. Isolation of Mr-230,000 undegraded regulin was accomplished by the use of protease inhibitors including N-ethylmaleimide. A striking characteristic of regulin is its tendency to aggregate in neutral solution of low ionic strength. Physical studies of the isolated protein indicate that it has a highly elongated form in solution. The protein has no known enzymatic activity but was shown previously to interact with and increase the enzymatic activity of a protein phosphatase. The properties of regulin suggest that it may have a structural function but it appears to be physically and immunologically distinct from known proteins. It is suggested that regulin may contribute to a gel matrix within the cytoplasm of reticulocytes.

Impaired echinocytic transformation of ankyrin- and spectrin-deficient erythrocytes in mice.

The membrane skeleton of the red blood cell plays an important role in the determination of cell deformability and cell shape. Under various in vitro conditions, red blood cells undergo an echinocytic or stomatocytic shape transformation. The mechanism of this fundamental process is not well understood. We have studied the red cell shape transformation in normoblastic anemia mice (nb/nb) and spherocytic anemia mice (sph/sph), which are deficient in ankyrin and spectrin, respectively. We found that both ankyrin-deficient cells (nb/nb) and spectrin-deficient cells (sph/sph) have a reduced capacity to undergo echinocytic transformation with various echinocytogenic treatments, that is, incubation with sodium salicylate (40 and 120 mM), calcium loading (50 microM A23187 + 2.2 mM Ca2+), or metabolic depletion (24 hr at 37 degrees C). These results suggest that the functional integrity of the membrane skeleton is essential for the maintenance and transformation of the red cell shape.

Cloning and nucleotide sequence of a mouse erythrocyte beta-spectrin cDNA.

A rabbit monospecific antibody for mouse beta-spectrin was used to screen a mouse anemic spleen cDNA expression library. A mouse beta-spectrin cDNA clone was isolated and identified by its ability to make mouse beta-spectrin-like antigens in Escherichia coli. This clone was used to probe total RNA from various mouse tissues. Anemic spleen RNA showed two strongly hybridizing RNA species of approximately 6 and 8 kb. Two very faintly hybridizing bands of about 6 kb and 10 kb could also be seen in total mouse brain RNA. All of these bands could be detected after hybridization under both stringent and nonstringent conditions. This suggests that erythroid beta-spectrin may also be expressed in the brain. No bands could be detected in kidney, liver, or spleen RNA. Southern blot analysis of mouse genomic DNA showed a single hybridizing band after digestion with several restriction endonucleases even under nonstringent conditions. Nucleotide sequencing of the cDNA insert revealed almost complete identity between the N-terminus of the deduced amino acid sequence of the cDNA clone and the C-terminal 15 amino acids of a peptide derived from the beta-8 repeat unit of human erythrocyte beta-spectrin. The deduced amino acid sequence contained most of the conserved amino acids characteristic of the 106 amino acid repeat unit first found in human alpha-spectrin and thus provides the first evidence for a complete 106 amino acid repeat unit structure in beta-spectrin.

Molecular determinants of clinical expression of hereditary elliptocytosis and pyropoikilocytosis.

The clinical severity of common hereditary elliptocytosis (HE) is highly variable, ranging from an asymptomatic carrier state to a severe hemolytic anemia. To elucidate the molecular basis of this variable clinical expression, we evaluated 56 subjects from 24 HE kindred, who carry alpha spectrin mutants characterized by a spectrin dimer (SpD) self-association defect related to a structural abnormality of the alpha I domain of spectrin. Twenty-nine subjects had common HE, 13 subjects have a closely related disorder, hereditary pyropoikilocytosis (HPP), and 14 are asymptomatic carriers. We compared the severity of hemolysis with the following biochemical parameters: (a) spectrin heterodimer self-association, as manifested by the percentage of SpD in the 4 degrees C low ionic strength spectrin extract; (b) spectrin structure, as examined by limited tryptic digestion of spectrin; and (c) spectrin content of the RBC membrane. Our analysis indicates that the severity of hemolysis may be correlated with quantitative differences in the percentage of SpD in the 4 degrees C spectrin extract, as well as the total spectrin content of the membrane. Thus, HPP subjects, who have the most severe hemolytic anemia, have the highest percentage of SpD as well as a decreased spectrin content. HE subjects and asymptomatic carriers, respectively, have a lower percentage of SpD and a normal spectrin content. Factors influencing these two determinants include functional differences between the individual spectrin mutants, the relative amounts of mutant spectrin present in the cells, the stability of mutant spectrin, and the possibility of a superimposed genetic defect involving spectrin synthesis.

Spectrin subtypes in mammalian brain: an immunoelectron microscopic study.

Spectrin is a major cytoskeletal component of the brain. At least 2 distinct spectrin subtypes are found in mammalian brain: brain spectrin(240/235) and brain spectrin(240/235E). In the present study spectrin subtypes were localized in the adult mouse brain by immunoelectron microscopy using antibodies that recognize each subtype. Brain spectrin(240/235E) was concentrated in neuronal cell bodies, dendrites, and postsynaptic terminals. It was also prominently associated with the plasma membrane, microtubules, filaments, mitochondria, endoplasmic reticulum, and nuclear envelope, and it appeared to interconnect structural elements within the cell. Brain spectrin(240/235E) also was localized to the plasma membrane, nuclear envelope, and cytoplasmic organelles of glial cell bodies. Brain spectrin(240/235) was detected in axons and presynaptic elements, where it was associated with the plasma membrane, microtubules, filaments, synaptic vesicles, and mitochondria. These results show that spectrin is distributed throughout the cytoplasm of neural cells, the location of spectrin is dependent on subtype, and the cytoplasmic surface of plasma membrane and organelles contains an extensive and intricate spectrin meshwork.

Diminished extractable spectrin in the erythrocytes of a patient with 'sporadic' hereditary spherocytosis.

A 51-year-old white man of Irish extraction was found to have apparent 'sporadic' hereditary spherocytosis with a reticulocyte count of 6%. Twelve of his 13 siblings were examined and found to be haematologically normal. The patient's erythrocytes were found to have a diminished amount of spectrin as compared to his siblings and to unrelated controls. It is suggested that the proband may represent either a new mutant or possibly double heterozygosity for two inherited biochemical variants of the red cell membrane skeleton which individually give no haematological abnormalities.

An actomyosin contractile mechanism for erythrocyte shape transformations.

The membrane skeleton of the human erythrocyte consists of many short actin filaments that are multiply cross-linked by long, flexible spectrin molecules into a continuous network in the plane of the membrane. The mechanical properties expected for this spectrin-actin network can account for the tensile strength of the erythrocyte membrane and for the remarkable deformability of the cells, yet not for their characteristic biconcave shape. Recently, an authentic vertebrate myosin as well as a non-muscle form of tropomyosin have been identified and purified from erythrocytes. The myosin is present with respect to the actin in an amount comparable to actin-myosin ratios in other non-muscle cells, and there is enough tropomyosin to almost completely coat all of the short actin filaments in the membrane skeleton. The implications of these unexpected discoveries for the molecular organization of the cytoskeleton are discussed, and a mechanism is proposed by which myosin could interact with the membrane-associated actin filaments to influence erythrocyte shape and membrane properties.

Spectrin degradation in intact red blood cells by phenylhydrazine.

The effects of phenylhydrazine on intact red cells and on red cell ghost membrane proteins were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In intact red cells 1 mM phenylhydrazine induced a marked decrease in intensity of the alpha- and beta-bands of spectrin without the formation of high molecular weight materials. Phenylhydrazine was also responsible for cross-linking of hemoglobin, which is apparent by the appearance of two new broad bands on the gel. Membrane glycoproteins were unaffected. Electrophoretic patterns of cytoskeletal proteins from phenylhydrazine-treated red cells obtained on two-dimensional SDS-polyacrylamide gels and stained with Coomassie blue or fluorescently labeled with monobromobimane indicated the presence of a new band between bands 4.2 and 5 at 60-65 kilodaltons (K). An immunoelectrophoretic blotting procedure utilizing polyclonal IgG antibodies for alpha- and beta-spectrin of the red cell cytoskeletal proteins revealed that the band observed at 60-65 K in the two-dimensional SDS-PAGE studies reacted with the antibodies. The presence or absence of glucose in the incubation medium and modification of oxyhemoglobin to met- or carboxyhemoglobin in the red cells did not protect the phenylhydrazine-mediated degradation of the major cytoskeletal proteins. Metal chelators and antioxidants had no effect on membrane protein changes. Ghost red cell proteins did not undergo changes at 1 mM phenylhydrazine in the presence or absence of hemoglobin, although at 5 mM phenylhydrazine the appearance of a faint high molecular weight band was observed. These results indicate that spectrin degradation without significant polymerization can be induced by phenylhydrazine.

A new abnormal variant of spectrin in black patients with hereditary elliptocytosis.

Seven black patients with mild hereditary elliptocytosis (HE) from five unrelated families were studied. The erythrocytes of these patients exhibited an abnormal thermal sensitivity (between 45 degrees C and 47 degrees C instead of 49 degrees C). An important defect of spectrin dimer self-association was detected in two ways: (1) the proportions of spectrin dimer (SpD) extracted from membranes at 4 degrees C under low ionic strength conditions were increased between 25% and 56% (normal value 15% +/- 2%); (2) the spectrin dimer----tetramer conversion in solution were defective with an association constant value between 0.4 and 2.4 X 10(5) M-1 for a normal value of 6 +/- 0.4 X 10(5) M-1. Spectrin (Sp) from HE patients and normal volunteers (32 black and 22 white subjects) was submitted to limited tryptic digestion, followed by one- or two-dimensional separation of the peptides. Peptide patterns of crude Sp from all seven HE patients exhibited a marked and reproducible decrease in 80,000-dalton peptide (previously identified as the dimer-dimer interaction domain of the alpha-chain) and a concomitant appearance of a novel 65,000-dalton peptide. A minor fragment at 28,000 daltons was also decreased. Tryptic digestion of HE spectrin dimer and tetramer (SpT), isolated after the SpD self-association procedure in solution, revealed modifications (decrease in the 80,000-dalton peptide and presence of a 65,000-dalton peptide) predominantly in HE SpD when peptide patterns of HE SpT were quite similar to control SpT patterns. Immunoblots with anti-alpha-chain antibodies revealed that the 65,000-dalton peptide derived from the alpha-chain. Kinetic studies of Sp digestion showed that the 65,000-dalton peptide did not result from further digestion of a 74,000 intermediate and was not a precursor of 46,000- to 50,000-dalton peptides. These results show a new structural defect of Sp-alpha-chain, associated with a defective Sp dimer self-association in HE.

The role of spectrin-dependent ATPase in erythrocyte shape maintenance.

The paper reports on the relationship between spectrin-dependent ATPase and erythrocyte shape. It can be seen from various experiments that different treatment of erythrocyte membrane which brings about alteration in activity of spectrin-dependent ATPase, also changes erythrocyte shape. We suggest that spectrin-dependent ATPase is a large actomyosin-like protein complex with some characteristics of contractile protein which, under suitable conditions, causes a physiological tension (contraction) of the membrane. Energetically rich state of spectrin-dependent ATPase seems to be responsible for this phenomenon.

[Analysis of erythrocytic spectrin in thalassemia and in Lepore hemoglobinosis]. / Analisi della spettrina eritrocitaria nella talassemia e nell'emoglobinosi lepore.

Total proteins and red cell membrane spectrin were determined in normal subjects, patients with hyperhaemolytic and anhaemolytic thalassaemia, and Hb Lepore from a single family. Electrophoresis on acrylamide gels was performed after solubilisation of the material in SDS using the whole membrane and spectrin. Amino acid composition was also determined after hot acid hydrolysis. Resin chromatography was employed to recognise acid, neutral and basic aminoo acids, glucosamine, and galactosamine. It was found that the significant changes in spectrin amino acid composition observed in thalassaemic subjects with peripheral hyperhaemolysis were not apparent in anhaemolytic patients, nor in the heterozygote, clinically asymptomatic carriers of Hb Lepore. These changes are certainly of importance on account of the structural alterations noted in the spectrin of the subjects concerned.

Lateral mobility of erythrocyte membrane proteins studied by the fluorescence photobleaching recovery technique.

Erythrocyte membrane peripheral and integral proteins have been isolated and purified, and the lateral diffusion of these proteins in a well-defined phospholipid bilayer matrix (dimyristoylphosphatidylcholine) has been studied by fluorescence photobleaching recovery measurements. Our own instrument for the recovery measurements is described and some data for lipid diffusions are compared with those previously reported by other investigators. The peripheral proteins (spectrin and band 4.1) diffuse rapidly on the lipid membrane in its fluid phase. The diffusion constant of approximately 5 x 10(-8) cm2 . s-1 (30 degrees C) was only a little smaller than that for lipid diffusion. The diffusion was greatly slowed down when the host lipid matrix became solid. The integral protein band 3 also diffuses rapidly in the fluid membrane. The diffusion constant of 1.6 x 10(-8) cm2 . s-1 (30 degrees C) was smaller than those for lipids and for the peripheral proteins. The lateral motion is compatible with diffusion of a cylinder with radius 3 nm in a two dimensional matrix with an inner viscosity of 2 poises and an inner thickness of 4 nm. The band 3 lateral motion was restricted by binding of the cytoskeletal component proteins (ankyrin, spectrin, actin, and band 4.1) to the reconstituted membranes. The diffusion constant decreased to half. The results provide a basis for the elucidation of transmembrane control mechanisms in more complex cellular systems.