Spectrin Filaments Research Articles

Coarse-Grained Modeling and Mechanical Behaviors of Actin–Spectrin–Microtubule Complex in Axonal Cytoskeleton

Axon is a long protrusion from the neuronal cell body whose interconnection forms the nervous network and allows biomechanical signals to be transmitted among neurons. From the mechanic’s point of view, the axon cytoskeleton consists of bundles of microtubules (MTs), cross-linked by microtubule-associated protein (MAP) tau, and supported by a periodic array of actin–spectrin rings. Yet, the fundamental question of how these actin–spectrin rings and microtubule bundles behave in synergy to provide the required mechanical strength to the whole axon is still poorly understood. Here, we developed a coarse-grained molecular dynamics model of axon to address this outstanding issue. We show that the dynamic response of spectrin filaments plays a vital role in the strain-softening of axon, which serves as a buffering mechanism for neurons to accommodate externally imposed deformation. Furthermore, the actin–spectrin structure is found to be essential for maintaining the mechanical stability of axon and allowing it to bear larger compressive forces. Our model and predictions not only explain recent experimental observations on axonal mechanics, but also provide insights on how to potentially modify the mechanical response of axonal cytoskeleton and therefore tune its capability in executing different biological duties.

Investigation of the role of Band 3 in red blood cell invasion by Francisella tularensis.

Francisella tularensis is a pathogenic gram-negative bacterium and the causative agent of the disease tularemia. As an intracellular pathogen, F. tularensis can infect and replicate within cells of the immune system, such as macrophages as well as non-immune cells including hepatocytes and epithelial cells. In all of the aforementioned cases, uptake is facilitated by the endocytic machinery and cytoskeletal rearrangements of the host cell. F. tularensis is also capable of invading erythrocytes - cells that are incapable of endocytosis or phagocytosis. Therefore, it quite likely that red blood cell invasion occurs through a distinct mechanism. Unlike the cytoskeleton of the nucleated host cells, erythrocytes are supported through a meshwork of spectrin filaments that are tethered by small actin bundles. We previously showed that actin rearrangement is not required for invasion. However, treatment of red blood cells with a snake venom that inhibits the activity of spectrin abrogates erythrocyte invasion. This venom acts through binding and inactivating Band 3, an anion transporter that complexes with spectrin. We therefore hypothesized that interaction with Band 3 is required for erythrocyte invasion. To test this, we generated Fab fragments from monoclonal and polyclonal antibodies specific to Band 3. These fragments will be used to treat erythrocytes during in vitro assays to determine the role of Band 3 during red blood cell invasion by F. tularensis. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and 1R15HL147135).

Statistical mechanics of asymmetric tethered membranes: Spiral and crumpled phases.

We develop the elastic theory for inversion-asymmetric tethered membranes and use it to identify and study their possible phases. Asymmetry in a tethered membrane causes spontaneous curvature, which in general depends on the local in-plane dilation of the tethered network. This in turn leads to long-range interactions between the local mean and Gaussian curvatures, which are not present in symmetric tethered membranes. This interplay between asymmetry and Gaussian curvature leads to a double-spiral phase not found in symmetric tethered membranes. At temperature T=0, tethered membranes of arbitrarily large size are always rolled up tightly into a conjoined pair of Archimedes' spirals. At finite T this spiral structure swells up significantly into algebraic spirals characterized by universal exponents, which we calculate. These spirals have long-range orientational order, and are the asymmetric analogs of statistically flat symmetric tethered membranes. We also find that sufficiently strong asymmetry can trigger a structural instability leading to crumpling of these membranes as well. This provides a mechanism for crumpling of asymmetric tethered membranes which is not present for symmetric membranes. We calculate the maximum linear extent L_{c} beyond which the membrane crumples, and calculate the universal dependence of L_{c} on the membrane parameters. By tuning the asymmetry parameter, L_{c} can be continuously varied, implying a scale-dependent crumpling. Our theory can be tested in controlled experiments on lipids with artificial deposits of spectrin filaments, in in vitro experiments on red blood cell membrane extracts, and on graphene coated on one side.

Modeling of the axon plasma membrane structure and its effects on protein diffusion.

The axon plasma membrane consists of the membrane skeleton, which comprises ring-like actin filaments connected to each other by spectrin tetramers, and the lipid bilayer, which is tethered to the skeleton via, at least, ankyrin. Currently it is unknown whether this unique axon plasma membrane skeleton (APMS) sets the diffusion rules of lipids and proteins in the axon. To answer this question, we developed a coarse-grain molecular dynamics model for the axon that includes the APMS, the phospholipid bilayer, transmembrane proteins (TMPs), and integral monotopic proteins (IMPs) in both the inner and outer lipid layers. We first showed that actin rings limit the longitudinal diffusion of TMPs and the IMPs of the inner leaflet but not of the IMPs of the outer leaflet. To reconcile the experimental observations, which show restricted diffusion of IMPs of the outer leaflet, with our simulations, we conjectured the existence of actin-anchored proteins that form a fence which restricts the longitudinal diffusion of IMPs of the outer leaflet. We also showed that spectrin filaments could modify transverse diffusion of TMPs and IMPs of the inner leaflet, depending on the strength of the association between lipids and spectrin. For instance, in areas where spectrin binds to the lipid bilayer, spectrin filaments would restrict diffusion of proteins within the skeleton corrals. In contrast, in areas where spectrin and lipids are not associated, spectrin modifies the diffusion of TMPs and IMPs of the inner leaflet from normal to confined-hop diffusion. Overall, we showed that diffusion of axon plasma membrane proteins is deeply anisotropic, as longitudinal diffusion is of different type than transverse diffusion. Finally, we investigated how accumulation of TMPs affects diffusion of TMPs and IMPs of both the inner and outer leaflets by changing the density of TMPs. We showed that the APMS structure acts as a fence that restricts the diffusion of TMPs and IMPs of the inner leaflet within the membrane skeleton corrals. Our findings provide insight into how the axon skeleton acts as diffusion barrier and maintains neuronal polarity.

A Minimal Coarse-Grained Molecular Dynamics Model of Axon Plasma Membrane with its Implication on the Diffusion Behavior of Axon Membrane Proteins

The axon initial segment (AIS) in the neuron has two crucial functions: maintaining the polarity of neurons and initiation of action potentials. One extraordinary feature of the AIS is the separation of somatodendritic and axonal domains which was confirmed by measuring the trajectory of beads attached to various axonal membrane proteins that there exists a diffusion barrier in the AIS restricting the motion of membrane proteins and lipids. According to the “fences and pickets” model, the periodic cytoskeleton and the highly concentrated transmembrane proteins in the AIS are capable of hindering the diffusion of other proteins and lipids through steric interaction and hydrodynamic friction effect. In this work, we established the first coarse-grained molecular dynamics model of axon plasma membrane which includes both axon membrane skeleton and axonal membranes to investigate the diffusion behavior of membrane proteins and lipids in the AIS from a mechanics aspect. First, we found that transmembrane proteins and proteins in the inner lipid leaflet are confined within the areas between the actin rings. We suspected and proved from the computational model that the size of actin associated protein junctions, which connected to the lipid bilayers may restrict the motion of proteins in the outer leaflet. Then we showed that the spectrin-lipid attraction level will affect the circumferential mobility of membrane proteins. Finally, the highly dense membrane proteins slowed down the motion of all membrane proteins even the lipids. We conclude that actin rings and spectrin filaments restrict the motion of membrane proteins along and around the axon, respectively. However, the main mechanism forming the diffusion barrier in the AIS is the accumulation of concentrated transmembrane proteins.

Quantitative mass spectrometry of human reticulocytes reveal proteome‐wide modifications during maturation

Erythropoiesis is marked by progressive changes in morphological, biochemical and mechanical properties of erythroid precursors to generate red blood cells (RBC). The earliest enucleated forms derived in this process, known as reticulocytes, are multi-lobular and spherical. As reticulocytes mature, they undergo a series of dynamic cytoskeletal re-arrangements and the expulsion of residual organelles, resulting in highly deformable biconcave RBCs (normocytes). To understand the significant, yet neglected proteome-wide changes associated with reticulocyte maturation, we undertook a quantitative proteomics approach. Immature reticulocytes (marked by the presence of surface transferrin receptor, CD71) and mature RBCs (devoid of CD71) were isolated from human cord blood using a magnetic separation procedure. After sub-fractionation into triton-extracted membrane proteins and luminal samples (isobaric tags for relative and absolute quantitation), quantitative mass spectrometry was conducted to identify more than 1800 proteins with good confidence and coverage. While most structural proteins (such as Spectrins, Ankyrin and Band 3) as well as surface glycoproteins were conserved, proteins associated with microtubule structures, such as Talin-1/2 and ß-Tubulin, were detected only in immature reticulocytes. Atomic force microscopy (AFM)-based imaging revealed an extended network of spectrin filaments in reticulocytes (with an average length of 48nm), which shortened during reticulocyte maturation (average spectrin length of 41nm in normocytes). The extended nature of cytoskeletal network may partly account for increased deformability and shape changes, as reticulocytes transform to normocytes.

Modeling of the axon membrane skeleton structure and implications for its mechanical properties.

Super-resolution microscopy recently revealed that, unlike the soma and dendrites, the axon membrane skeleton is structured as a series of actin rings connected by spectrin filaments that are held under tension. Currently, the structure-function relationship of the axonal structure is unclear. Here, we used atomic force microscopy (AFM) to show that the stiffness of the axon plasma membrane is significantly higher than the stiffnesses of dendrites and somata. To examine whether the structure of the axon plasma membrane determines its overall stiffness, we introduced a coarse-grain molecular dynamics model of the axon membrane skeleton that reproduces the structure identified by super-resolution microscopy. Our proposed computational model accurately simulates the median value of the Young’s modulus of the axon plasma membrane determined by atomic force microscopy. It also predicts that because the spectrin filaments are under entropic tension, the thermal random motion of the voltage-gated sodium channels (Nav), which are bound to ankyrin particles, a critical axonal protein, is reduced compared to the thermal motion when spectrin filaments are held at equilibrium. Lastly, our model predicts that because spectrin filaments are under tension, any axonal injuries that lacerate spectrin filaments will likely lead to a permanent disruption of the membrane skeleton due to the inability of spectrin filaments to spontaneously form their initial under-tension configuration.

Dielectric relaxations on erythrocyte membrane as revealed by spectrin denaturation

We studied the effect of spectrin denaturation at 49.5°C (TA) on the dielectric relaxations and related changes in the complex impedance, Z*, complex capacitance, C*, and dielectric loss curve of suspensions containing human erythrocytes, erythrocyte ghost membranes (EMs) and Triton-X-100 residues of EMs. The loss curve prior to, minus the loss curve after TA, resulted in a bell-shaped peak at 1.5MHz. The changes in the real and imaginary components of Z* and C* at TA, i.e., ΔZre, ΔZim, ΔCre and ΔCim, calculated in the same way, strongly varied with frequency. Between 1.0 and 12MHz the −ΔZim vs ΔZre, and ΔCim vs ΔCre plots depicted semicircles with critical frequencies, fcr, at 2.5MHz expressing recently reported relaxation of spectrin dipoles. Between 0.02 and 1.0MHz the −ΔZim vs ΔZre plot exhibited another relaxation whose fcr mirrored that of beta relaxation. This relaxation was absent on Triton-X-shells, while on erythrocytes and EMs it was inhibited by selective dissociation of either attachment sites between spectrin and bilayer. Considering above findings and inaccessibility of cytosole to outside field at such frequencies, the latter relaxation was assumed originating from a piezoelectric effect on the highly deformable spectrin filaments.

Role of band 3 in the erythrocyte membrane structural changes under thermal fluctuations -multi scale modeling considerations.

An attempt was made to discuss and connect various modeling approaches on various time and space scales which have been proposed in the literature in order to shed further light on the erythrocyte membrane rearrangement caused by the cortex-lipid bilayer coupling under thermal fluctuations. Roles of the main membrane constituents: (1) the actin-spectrin cortex, (2) the lipid bilayer, and (3) the trans membrane protein band 3 and their course-consequence relations were considered in the context of the cortex non linear stiffening and corresponding anomalous nature of energy dissipation. The fluctuations induce alternating expansion and compression of the membrane parts in order to ensure surface and volume conservation. The membrane structural changes were considered within two time regimes. The results indicate that the cortex non linear stiffening and corresponding anomalous nature of energy dissipation are related to the spectrin flexibility distribution and the rate of its changes. The spectrin flexibility varies from purely flexible to semi flexible. It is influenced by: (1) the number of band 3 molecules attached to single spectrin filaments, and (2) phosphorylation of the actin-junctions. The rate of spectrin flexibility changes depends on the band 3 molecules rearrangement.

Modeling of Vesiculation in Healthy and Defective Human Erythrocyte Membrane

We apply a two-component coarse-grained molecular dynamics (CGMD) red blood cell (RBC) membrane model which includes an explicit representation of the spectrin network, the lipid bilayer, and the band-3 proteins, to simulate vesiculation in the RBC membrane. We investigate vesiculation induced by the spontaneous curvature of the membrane domain and vesiculation induced by a stiffened cytoskeleton causing compression on the lipid bilayer. In addition, we model vesiculation in RBCs from patients suffering from the blood disorders of hereditary spherocytosis (HS) and elliptocytosis (HE). Our simulation results show that the spontaneous curvature of a membrane domain induces vesicles with a diameter less than 50 nm. We also found that compression on the membrane can cause the formation of vesicles having heterogeneous composition with a size similar to the size of the cytoskeleton corral. When both effects are taken into consideration, the compression on the membrane can facilitate the formation of vesicles originated from the membrane domain with the same spontaneous curvature. While the size of the vesicles induced by the compression in the normal RBC membrane is similar to the cytoskeleton corral size, the vesicle sizes become more diverse in HS RBCs because the constrain from the cytoskeleton on the lipid bilayer is reduced. When the vertical connectivity between the lipid bilayer and the cytoskeleton is elevated, multiple vesicles, with sizes similar to the cytoskeleton corral dimension, are generated from the compressed membrane. However, membrane with low vertical connectivity tends to produce larger vesicles under the same compression ratio as above. In HE RBCs, the reduced cytoskeleton connectivity facilitates the membrane vesiculation. It is noted that vesicles released from the HE RBCs could contain spectrin filaments while vesicles released from the HS RBCs are depleted of cytoskeleton components.

Erythrocyte Membrane Model with Explicit Description of the Lipid Bilayer and the Spectrin Network

The membrane of the red blood cell (RBC) consists of spectrin tetramers connected at actin junctional complexes, forming a two-dimensional (2D) sixfold triangular network anchored to the lipid bilayer. Better understanding of the erythrocyte mechanics in hereditary blood disorders such as spherocytosis, elliptocytosis, and especially, sickle cell disease requires the development of a detailed membrane model. In this study, we introduce a mesoscale implicit-solvent coarse-grained molecular dynamics (CGMD) model of the erythrocyte membrane that explicitly describes the phospholipid bilayer and the cytoskeleton, by extending a previously developed two-component RBC membrane model. We show that the proposed model represents RBC membrane with the appropriate bending stiffness and shear modulus. The timescale and self-consistency of the model are established by comparing our results with experimentally measured viscosity and thermal fluctuations of the RBC membrane. Furthermore, we measure the pressure exerted by the cytoskeleton on the lipid bilayer. We find that defects at the anchoring points of the cytoskeleton to the lipid bilayer (as in spherocytes) cause a reduction in the pressure compared with an intact membrane, whereas defects in the dimer-dimer association of a spectrin filament (as in elliptocytes) cause an even larger decrease in the pressure. We conjecture that this finding may explain why the experimentally measured diffusion coefficients of band-3 proteins are higher in elliptocytes than in spherocytes, and higher than in normal RBCs. Finally, we study the effects that possible attractive forces between the spectrin filaments and the lipid bilayer have on the pressure applied on the lipid bilayer by the filaments. We discover that the attractive forces cause an increase in the pressure as they diminish the effect of membrane protein defects. As this finding contradicts with experimental results, we conclude that the attractive forces are moderate and do not impose a complete attachment of the filaments to the lipid bilayer.

Hydrodynamic deformation reveals two coupled modes/time scales of red blood cell relaxation

We study the mechanical relaxation behavior of human red blood cells by observing the time evolution of shape change of cells flowing through microchannels with abrupt constrictions. We observe two types of relaxation processes. In the first fast process (τ1 ∼ 200 ms) the initially parachute shaped cells relax into cup-shaped cells (stomatocytes). These cells relax and reorient in a second relaxation process with a response time of τ1/2 ∼ 10 s into the equilibrium discoid shapes. The values for the relaxation times of single red blood cells in the population scatter significantly within the cell population between 0.11 s < τ1 < 0.52 s and 9 s < τ1/2 < 49 s, respectively. However, when plotting τ1/2 against τ1, we find a linear relationship between the two timescales and are able to relate both to the elastic properties of the spectrin cytoskeleton underlying the red cell's plasma membrane. Adenosine Triphosphate (ATP) enhances dissociation of spectrin filaments resulting in a reduced shear modulus. We modify the cytoskeleton connectivity by depletion and repletion of ATP and study the effect on relaxation. Both the linear relationship of timescales as well as the ATP dependence can be understood by theoretical models.

Native Ultrastructure of the Red Cell Cytoskeleton by Cryo-Electron Tomography

Erythrocytes possess a spectrin-based cytoskeleton that provides elasticity and mechanical stability necessary to survive the shear forces within the microvasculature. The architecture of this membrane skeleton and the nature of its intermolecular contacts determine the mechanical properties of the skeleton and confer the characteristic biconcave shape of red cells. We have used cryo-electron tomography to evaluate the three-dimensional topology in intact, unexpanded membrane skeletons from mouse erythrocytes frozen in physiological buffer. The tomograms reveal a complex network of spectrin filaments converging at actin-based nodes and a gradual decrease in both the density and the thickness of the network from the center to the edge of the cell. The average contour length of spectrin filaments connecting junctional complexes is 46 ± 15 nm, indicating that the spectrin heterotetramer in the native membrane skeleton is a fraction of its fully extended length (∼190 nm). Higher-order oligomers of spectrin were prevalent, with hexamers and octamers seen between virtually every junctional complex in the network. Based on comparisons with expanded skeletons, we propose that the oligomeric state of spectrin is in a dynamic equilibrium that facilitates remodeling of the network as the cell changes shape in response to shear stress.

Curling and Local Shape Changes of Red Blood Cell Membranes Driven by Cytoskeletal Reorganization

Human red blood cells (RBCs) lack the actin-myosin-microtubule cytoskeleton that is responsible for shape changes in other cells. Nevertheless, they can display highly dynamic local deformations in response to external perturbations, such as those that occur during the process of apical alignment preceding merozoite invasion in malaria. Moreover, after lysis in divalent cation-free media, the isolated membranes of ruptured ghosts show spontaneous inside-out curling motions at the free edges of the lytic hole, leading to inside-out vesiculation. The molecular mechanisms that drive these rapid shape changes are unknown. Here, we propose a molecular model in which the spectrin filaments of the RBC cortical cytoskeleton control the sign and dynamics of membrane curvature depending on whether the ends of the filaments are free or anchored to the bilayer. Computer simulations of the model reveal that curling, as experimentally observed, can be obtained either by an overall excess of weakly-bound filaments throughout the cell, or by the flux of such filaments toward the curling edges. Divalent cations have been shown to arrest the curling process, and Ca2+ ions have also been implicated in local membrane deformations during merozoite invasion. These effects can be replicated in our model by attributing the divalent cation effects to increased filament-membrane binding. This process converts the curl-inducing loose filaments into fully bound filaments that arrest curling. The same basic mechanism can be shown to account for Ca2+-induced local and dynamic membrane deformations in intact RBCs. The implications of these results in terms of RBC membrane dynamics under physiological, pathological, and experimental conditions is discussed.

Less is more: removing membrane attachments stiffens the RBC cytoskeleton

The polymerized network of the cytoskeleton of the red-blood cell (RBC) contains different protein components that maintain its overall integrity and attachment to the lipid bilayer. One of these key components is the band 3–ankyrin complex that attaches the spectrin filaments to the fluid bilayer. Defects in this particular component result in the shape transformation called spherocytosis, through the shedding of membrane nano-vesicles. We show here that this transition and membrane shedding can be explained through the increased stiffness of the network when the band 3–ankyrin complexes are removed. ATP-induced transient dissociations lead to network softening, which offsets the stiffening to some extent, and causes increased fragility of these mutant cells, as is observed.

Modulation of Lateral Diffusion in the Plasma Membrane by Protein Density

The rate of lateral diffusion of proteins over micron-scale distances in the plasma membrane (PM) of mammalian cells is much slower than in artificial membranes [1, 2]. Different models have been advanced to account for this discrepancy. They invoke either effects on the apparent viscosity of cell membranes through, for example, protein crowding [3, 4], or a role for cortical factors such as actin or spectrin filaments [1]. Here, we use photobleaching to test specific predictions of these models [5]. Neither loss of detectable cortical actin nor knockdown of spectrin expression has any effect on diffusion. Disruption of the PM by formation of ventral membrane sheets or permeabilization induces aggregation of membrane proteins, with a concomitant increase in rates of diffusion for the nonaggregated fraction. In addition, procedures that directly increase or decrease the total protein content of the PM in live cells cause reciprocal changes in lateral diffusion rates. Our data imply that slow diffusion over micron-scale distances is an intrinsic property of the membrane itself and that the density of proteins within the membrane is a significant parameter in determining rates of lateral diffusion.

Lateral Diffusion Anisotropy and Membrane Lipid/Skeleton Interaction in Outer Hair Cells

The organization of the plasma membrane of cells in lipid domains affects the way the membrane interacts with the underlying protein skeleton, which in turn affects the lateral mobility of lipid and protein molecules in the membrane. Membrane fluidity properties can be monitored by various approaches, the most versatile of which is fluorescence recovery after photobleaching (FRAP). We extended previous FRAP experiments on isolated cochlear outer hair cells (OHCs) by analyzing the two-dimensional pattern of lipid diffusion in the lateral membrane of these cells. We found that membrane lipid mobility in freshly isolated OHCs is orthotropic, diffusion being faster in the axial direction of the cell and slower in the circumferential direction. Increasing the cell’s turgor pressure by osmotic challenge reduced the axial diffusion constant, but had only a slight effect on circumferential diffusion. Our results suggest that lipid mobility in the OHC plasma membrane is affected by the presence of the cell’s orthotropic membrane skeleton. This effect could reflect interaction with spectrin filaments or with other membrane skeletal proteins. We also performed a number of FRAP measurements in temporal bone preparations preserving the structural integrity of the hearing organ. The diffusion rates measured for OHCs in this preparation were in good agreement with those obtained in isolated OHCs, and comparable to the mobility rates measured on the sensory inner hair cells. These observations support the idea that the plasma membranes of both types of hair cells share similar highly fluid phases in the intact organ. Lipid mobility was significantly slower in the membranes of supporting cells of the organ of Corti, which could reflect differences in lipid phase or stronger hindrance by the cytoskeleton in these membranes.

Dynamic simulations of membranes with cytoskeletal interactions

We describe a simulation algorithm for the dynamics of elastic membrane sheets over long length and time scales. Our model includes implicit hydrodynamic coupling between membrane and surrounding solvent and allows for arbitrary external forces acting on the membrane surface. In particular, the methodology is well suited to studying membranes in interaction with cytoskeletal filaments. We present results for the thermal undulations of a lipid bilayer attached to a regular network of spectrin filaments as a model for the red blood cell membrane. The dynamic fluctuations of the bilayer over the spectrin network are quantified and used to predict the macroscopic diffusion constant of band 3 on the surface of the red blood cell. We find that thermal undulations likely play a role in the mobility of band 3 in the plane of the erythrocyte membrane.

Red Blood Cell Membrane Fluctuations and Shape Controlled by ATP-Induced Cytoskeletal Defects

We show theoretically how adenosine 5′-triphosphate (ATP)-induced dynamic dissociations of spectrin filaments (from each other and from the membrane) in the cytoskeleton network of red blood cells (RBC) can explain in a unified manner both the measured fluctuation amplitude as well as the observed shape transformations as a function of intracellular ATP concentration. Static defects can be induced by external stresses such as those present when RBCs pass through small capillaries. We suggest that the partially freed actin at these defect sites may explain the activation of the CFTR membrane-bound protein and the subsequent release of ATP by RBCs subjected to deformations. Our theoretical predictions can be tested by experiments that measure the correlation between variations in the binding of actin to spectrin, the activity of CFTR, and the amount of ATP released.

Affinity purification of PSD-95-containing postsynaptic complexes.

A widely used method for the preparation of postsynaptic density (PSD) fractions consists of treatment of synaptosomal membranes with Triton X-100 and further purification by density gradient centrifugation. In the present study, the purity of this preparation was assessed by electron microscopic analysis. Thin-section and rotary shadow immuno-electron microscopy of the Triton X-100-derived PSD fraction shows many PSD-95-positive structures that resemble in situ PSDs in shape and size. However, the fraction also includes contaminants such as CaMKII clusters, spectrin filaments and neurofilaments. We used magnetic beads coated with an antibody against PSD-95 to further purify PSD-95-containing complexes from the Triton-derived PSD fraction. Biochemical analysis of the affinity-purified material shows a substantial reduction in the astrocytic marker glial fibrillary acidic protein and electron microscopic analysis shows mostly individual PSDs attached to magnetic beads. This preparation was used to assess the association of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-type glutamate receptors with the PSD-95-containing complex. AMPA receptors are demonstrated by immunoblotting to be present in the complex, although they do not co-purify exclusively with PSD-95, suggesting the existence of two pools of receptors, one associated with the PSD-95 scaffold and the other not. Of the AMPA receptor-anchoring proteins tested, SAP-97 is present in the affinity-purified preparation whereas GRIP is found only in trace amounts. These results imply that a subpopulation of AMPA receptors is anchored to the PSD-95-containing scaffold through interaction of GluR1 with SAP-97.