Association of deoxyribonuclease I with the pointed ends of actin filaments in human red blood cell membrane skeletons

We have characterized the interaction of bovine pancreatic deoxyribonuclease I (DNase I) with the filamentous (F-)actin of red cell membrane skeletons stabilized with phalloidin. The hydrolysis of [3H]DNA was used to assay DNase I. We found that DNase I bound to a homogenous class of approximately equal to 2.4 X 10(4) sites/skeleton with an association rate constant of approximately 1 X 10(6) M-1 S-1 and a KD of 1.9 X 10(-9) M at 20 degrees C. Phalloidin lowered the dissociation constant by approximately 1 order of magnitude. The DNase I which sedimented with the skeletons was catalytically inactive but could be reactivated by dissociation from the actin. Actin and DNA bound to DNase I in a mutually exclusive fashion without formation of a ternary complex. Phalloidin-treated red cell F-actin resembled rabbit muscle G-actin in all respects tested. Since the DNase I binding capacity of the skeletons corresponded to the number of actin protofilaments previously estimated by other methods, it seemed likely that the enzyme binding site was confined to one end of the filament. We confirmed this premise by showing that elongating the red cell filaments with rabbit muscle actin monomers did not appreciably add to their capacity to bind or inhibit DNase I. Saturation of skeletons with cytochalasin D or gelsolin, avid ligands for the barbed end of actin filaments, did not reduce their binding of DNase I. Furthermore, neither cytochalasin D nor DNase I alone blocked all of the sites for addition of monomeric pyrene-labeled rabbit muscle G-actin to phalloidin-treated skeletons; however, a combination of the two agents did so. In the presence of phalloidin, the polymerization of 300 nM pyrenyl actin on nuclei constructed from 5 nM gelsolin and 25 nM rabbit muscle G-actin was completely inhibited by 35 nM DNase I but not by 35 nM cytochalasin D. We conclude that DNase I associates uniquely with and caps the pointed (slow-growing or negative) end of F-actin. These results imply that the amino-terminal, DNase I-binding domain of the actin protomer is oriented toward the pointed end and is buried along the length of the actin filament.     

Depletion of membrane skeleton in red blood cell vesicles.

A possible physical interpretation of the partial detachment of the membrane skeleton in the budding region of the cell membrane and consequent depletion of the membrane skeleton in red blood cell vesicles is given. The red blood cell membrane is considered to consist of the bilayer part and the membrane skeleton. The skeleton is, under normal conditions, bound to the bilayer over its whole area. It is shown that, when in such conditions it is in the expanded state, some cell shape changes can induce its partial detachment. The partial detachment of the skeleton from the bilayer is energetically favorable if the consequent decrease of the skeleton expansion energy is larger than the corresponding increase of the bilayer-skeleton binding energy. The effect of shape on the skeleton detachment is analyzed theoretically for a series of the pear class shapes, having decreasing neck diameter and ending with a parent-daughter pair of spheres. The partial detachment of the skeleton is promoted by narrowing of the cell neck, by increasing the lateral tension in the skeleton and its area expansivity modulus, and by diminishing the attraction forces between the skeleton and the bilayer. If the radius of the daughter vesicle is sufficiently small relative to the radius of the parent cell, the daughter vesicle can exist either completely underlaid with the skeleton or completely depleted of the skeleton.     

Spectrin properties and the elasticity of the red blood cell membrane skeleton

Two models of spectrin elasticity are developed and compared to experimental measurements of the red blood cell (RBC) membrane shear modulus through the use of an elastic finite element model of the RBC membrane skeleton. The two molecular models of spectrin are: (i) An entropic spring model of spectrin as a flexible chain. This is a model proposed by several previous authors. (ii) An elastic model of a helical coiled-coil which expands by increasing helical pitch. In previous papers, we have computed the relationship between the stiffness of a single spectrin molecule (K) and the shear modulus of a network (μ), and have shown that this behavior is strongly dependent upon network topology. For realistic network models of the RBC membrane skeleton, we equate μ to micropipette measurements of RBCs and predict K for spectrin that is consistent with the coiled-coil molecular model. The value of spectrin stiffness derived from the entropic molecular model would need to be at least 30 times greater to match the experimental results. Thus, the conclusion of this study is that a helical coiled-coil model for spectrin is more realistic than a purely entropic model.     

A Highlights from MBoC Selection: Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane

Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e., do not exchange subunits with G-actin in the cytosol), this assumption has never been rigorously tested. Here we show that a subpopulation of human RBC actin filaments is indeed dynamic, based on rhodamine-actin incorporation into filaments in resealed ghosts and fluorescence recovery after photobleaching (FRAP) analysis of actin filament mobility in intact RBCs (∼25–30% of total filaments). Cytochalasin-D inhibition of barbed-end exchange reduces rhodamine-actin incorporation and partially attenuates FRAP recovery, indicating functional interaction between actin subunit turnover at the single-filament level and mobility at the membrane-skeleton level. Moreover, perturbation of RBC actin filament assembly/disassembly with latrunculin-A or jasplakinolide induces an approximately twofold increase or ∼60% decrease, respectively, in soluble actin, resulting in altered membrane deformability, as determined by alterations in RBC transit time in a microfluidic channel assay, as well as by abnormalities in spontaneous membrane oscillations (flickering). These experiments identify a heretofore-unrecognized but functionally important subpopulation of RBC actin filaments, whose properties and architecture directly control the biomechanical properties of the RBC membrane.     

Conformation and elasticity of the isolated red blood cell membrane skeleton.

We studied the structure and elasticity of membrane skeletons from human red blood cells (RBCs) during and after extraction of RBC ghosts with nonionic detergent. Optical tweezers were used to suspend individual cells inside a flow chamber, away from all surfaces; this procedure allowed complete exchange of medium while the low-contrast protein network of the skeleton was observed by high resolution, video-enhanced differential interference-contrast (DIC) microscopy. Immediately following extraction in a 5 mM salt buffer, skeletons assumed expanded, nearly spherical shapes that were uncorrelated with the shapes of their parent RBCs. Judging by the extent of thermal undulations and by their deformability in small flow fields, the bending rigidity of skeletons was markedly lower than that of either RBCs or ghosts. No further changes were apparent in skeletons maintained in this buffer for up to 40 min at low temperatures (T less than 10 degrees C), but skeletons shrank when the ionic strength of the buffer was increased. When the salt concentration was raised to 1.5 M, shrinkage remained reversible for approximately 1 min but thereafter became irreversible. When maintained in 1.5 M salt buffer for longer periods, skeletons continued to shrink, lost flexibility, and assumed irregular shapes: this rigidification was irreversible. At this stage, skeletons closely resembled those isolated in standard bulk preparations. We propose that the transformation to the rigid, irreversibly shrunken state is a consequence of spectrin dimer-dimer reconnections and that these structural rearrangements are thermally activated. We also measured the salt-dependent size of fresh and bulk extracted skeletons. Our measurements suggest that, in situ, the spectrin tethers are flexible, with a persistence length of approximately 10 nm at 150 mM salt.     

Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K(D(kin))=8.5 x 10(-8) M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K(D(kin))=3.8 x 10(-7) M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.     

Hereditary disorders of the red cell membrane skeleton

The hereditary hemolytic anemias include a heterogeneous class of disorders caused by defects in the proteins that constitute the membrane skeleton of the red blood cell. The combination of classical and molecular genetics together with clinical findings is rapidly improving our understanding of the basis of these defects.     

Model of red blood cell membrane skeleton: electrical and mechanical properties

A theoretical membrane skeleton model of erythrocyte has been developed and successfully applied to interpret electrical and mechanical properties of the red blood cell spectrin-actin network. The model is based on the structure of the membrane skeleton that is comprised of unit cells each containing an actin protofilament and shooting forth a few spectrin heterodimers. The loose ends of the heterodimers of adjacent cells can form bonds with each other giving rise to an integrated network. The number of bonds depends on the temperature. The bond length being excessive (2.6 times the distance between the centers of adjacent cells), the bonds are flexible, and can thus be regarded as entropy springs. The advanced model has been employed to calculate the shear modulus of the membrane skeleton as well as to establish its temperature dependence. In a wide range of temperatures mu(T) is a decreasing function well fitting the experimental data. The relationship between the membrane bilayer-free size of the skeleton and the ionic strength of the solution has been derived to appear in good agreement with the results obtained previously. Experimental data combined with the advanced theory yield the average number of heterodimers per unit cell, m0, as equal to ca. 5; the spectrin heterodimer charge has been estimated.     

Stress-free state of the red blood cell membrane and the deformation of its skeleton

The response of a red blood cell (RBC) to deformation depends on its membrane, a composite of a lipid bilayer and a skeleton, which is a closed, twodimensional network of spectrin tetramers as its bonds. The deformation of the skeleton and its lateral redistribution are studied in terms of the RBC resting state for a fixed geometry of the RBC, partially aspirated into a micropipette. The geometry of the RBC skeleton in its initial state is taken to be either two concentric circles, a references biconcave shape or a sphere. It is assumed that in its initial state the skeleton is distributed laterally in a homogeneous manner with its bonds either unstressed, presenting its stress-free state, or prestressed. The lateral distribution was calculated using a variational calculation. It was assumed that the spectrin tetramer bonds exhibit a linear elasticity. The results showed a significant effect of the initial skeleton geometry on its lateral distribution in the deformed state. The proposed model is used to analyze the measurements of skeleton extension ratios by the method of applying two modes of RBC micropipette aspiration.     

The cytoskeleton of the resting human blood platelet: structure of the membrane skeleton and its attachment to actin filaments

We used high-resolution EM and immunocytochemistry in combination with different specimen preparation techniques to resolve the ultrastructure of the resting platelet cytoskeleton. The periphery of the cytoskeleton, an electron-dense subplasmalemmal region in thin section electron micrographs, is a tightly woven planar sheet composed of a spectrin-rich network whose interstices contain GPIb/IX-actin-binding protein (ABP) complexes. This membrane skeleton connects to a system of curved actin filaments (F-actin) that emanate from a central oval core of F-actin cross-linked by ABP. The predominant interaction of the radial actin filaments with the membrane skeleton is along their sides, and the strongest connection between the membrane skeleton and F-actin is via ABP-GPIb ligands, although there is evidence for spectrin attaching to the ends of the radial actin filaments as well. Since a mechanical separation of the F-actin cores and radial F-actin-GPIb-ABP complexes from the underlying spectrin-rich skeleton leads to the latter's expansion, it follows that the spectrin-based skeleton of the resting cell may be held in a compressed form by interdigitating GPIb/IX complexes which are immobilized by radial F-actin-ABP anchors.     

Elasticity of the human red blood cell skeleton

We have measured by optical tweezers micromanipulations the area expansion and the shear moduli of spectrin skeletons freshly extracted from human red blood cells, in different controlled salinity conditions. At medium osmolarity (150 mOsm/kg), we measure KC=9.7+/-3.4 microN/m, muC=5.7+/-2.3 microN/m, KC/muC=2.1+/-0.7. When decreasing the osmolarity, both KC and muC decrease, while KC/muC is nearly constant and equal to about 2. This result is consistent with the predictions made when modeling the spectrin skeleton by a two-dimensional triangular lattice of springs. From the measured elastic moduli we estimate the persistence length of a spectrin filament: xi approximately 2.5 nm at 150 mOsm/kg.     

Influence of network topology on the elasticity of the red blood cell membrane skeleton.

A finite-element network model is used to investigate the influence of the topology of the red blood cell membrane skeleton on its macroscopic mechanical properties. Network topology is characterized by the number of spectrin oligomers per actin junction (phi a) and the number of spectrin dimers per self-association junction (phi s). If it is assumed that all associated spectrin is in tetrameric form, with six tetramers per actin junction (i.e., phi a = 6.0 and phi s = 2.0), then the topology of the skeleton may be modeled by a random Delaunay triangular network. Recent images of the RBC membrane skeleton suggest that the values for these topological parameters are in the range of 4.2 < phi a < 5.5 and 2.1 < phi s < 2.3. Model networks that simulate these realistic topologies exhibit values of the shear modulus that vary by more than an order of magnitude relative to triangular networks. This indicates that networks with relatively sparse nontriangular topologies may be needed to model the RBC membrane skeleton accurately. The model is also used to simulate skeletal alterations associated with hereditary spherocytosis and Southeast Asian ovalocytosis.     

Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K_D(kin) = 8.5 × 10^− 8 M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K_D(kin) = 3.8 × 10^− 7 M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.     

Interaction of the exported malaria protein Pf332 with the red blood cell membrane skeleton

Intra-erythrocytic Plasmodium falciparum malaria parasites synthesize and export numerous proteins into the red blood cell (RBC) cytosol, where some bind to the RBC membrane skeleton. These interactions are responsible for the altered antigenic, morphological and functional properties of parasite-infected red blood cells (IRBCs). Plasmodium falciparum protein 332 (Pf332) is a large parasite protein that associates with the membrane skeleton and who's function has recently been elucidated. Using recombinant fragments of Pf332 in in vitro interaction assays, we have localised the specific domain within Pf332 that binds to the RBC membrane skeleton to an 86 residue sequence proximal to the C-terminus of Pf332. We have shown that this region partakes in a specific and saturable interaction with actin (K_d = 0.60 µM) but has no detectable affinity for spectrin. The only exported malaria protein previously known to bind to actin is PfEMP3 but here we demonstrate that there is no competition for actin-binding between PfEMP3 and Pf332, suggesting that they bind to different target sequences in actin.     

Structural basis of membrane skeleton organization in red blood cells

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Influence of calmodulin on the human red cell membrane skeleton

The calcium receptor calmodulin interacts with components of the human red cell membrane skeleton as well as with the membrane. Under physiological salt conditions, calmodulin has a calcium-dependent affinity for spectrin, one of the major components of the membrane skeleton. It is apparent from our results that calmodulin inhibits the ability of erythrocyte spectrin (when preincubated with filamentous actin) to create nucleation centers and thereby to seed actin polymerization. The gelation of filamentous actin induced by spectrin tetramers is also inhibited by calmodulin. The inhibition is calcium dependent and decreases with increasing pH, similar to the binding of calmodulin to spectrin. Direct binding studies using aqueous two-phase partition indicate that calmodulin interferes with the binding of actin to spectrin. Even in the presence of protein 4.1, which is believed to stabilize the ternary complex, calmodulin has an inhibitory effect. Since calmodulin also inhibits the corresponding activities of brain spectrin (fodrin), it appears likely that calmodulin may modulate the organization of cytoskeletons containing actin and spectrin or spectrin analogues.     

An elastic network model based on the structure of the red blood cell membrane skeleton.

A finite element network model has been developed to predict the macroscopic elastic shear modulus and the area expansion modulus of the red blood cell (RBC) membrane skeleton on the basis of its microstructure. The topological organization of connections between spectrin molecules is represented by the edges of a random Delaunay triangulation, and the elasticity of an individual spectrin molecule is represented by the spring constant, K, for a linear spring element. The model network is subjected to deformations by prescribing nodal displacements on the boundary. The positions of internal nodes are computed by the finite element program. The average response of the network is used to compute the shear modulus (mu) and area expansion modulus (kappa) for the corresponding effective continuum. For networks with a moderate degree of randomness, this model predicts mu/K = 0.45 and kappa/K = 0.90 in small deformations. These results are consistent with previous computational models and experimental estimates of the ratio mu/kappa. This model also predicts that the elastic moduli vary by 20% or more in networks with varying degrees of randomness. In large deformations, mu increases as a cubic function of the extension ratio lambda 1, with mu/K = 0.62 when lambda 1 = 1.5.     

Intermolecular dynamics and function in actin filaments

Structural models of F-actin suggest that three segments in actin, the DNase I binding loop (residues 38-52), the hydrophobic plug (residues 262-274) and the C-terminus, contribute to the formation of an intermolecular interface between three monomers in F-actin. To test these predictions and also to assess the dynamic properties of intermolecular contacts in F-actin, Cys-374 pyrene-labeled skeletal alpha-actin and pyrene-labeled yeast actin mutants, with Gln-41 or Ser-265 replaced with cysteine, were used in fluorescence experiments. Large differences in Cys-374 pyrene fluorescence among copolymers of subtilisin-cleaved (between Met-47 and Gly-48) and uncleaved alpha-actin showed both intra- and intermolecular interactions between the C-terminus and loop 38-52 in F-actin. Excimer band formation due to intermolecular stacking of pyrene probes attached to Cys-41 and Cys-265, and Cys-41 and Cys-374, in mutant yeast F-actin confirmed the proximity of these residues on the paired sites (to within 18 A) in accordance with the models of F-actin structure. The dynamic properties of the intermolecular interface in F-actin formed by loop 38-52, plug 262-274 and the C-terminus may account for the observed cross-linking of these sites with reagents < 18 A. The functional importance of actin filament dynamics was demonstrated by the inhibition of the in vitro motility in the Gln-41-Cys-374 cross-linked actin filaments.     

Study of actin filament ends in the human red cell membrane

There is conflicting evidence concerning the state of the actin protofilaments in the membrane cytoskeleton of the human red cell. To resolve this uncertainty, we have analysed their characteristics with respect to nucleation of G-actin polymerization. The effects of cytochalasin E on the rate of elongation of the protofilaments have been measured in a medium containing 0.1 M-sodium chloride and 5 mM-magnesium chloride, using pyrene-labelled G-actin. At an initial monomer concentration far above the critical concentration for the negative ("pointed") end of F-actin, high concentrations of cytochalasin reduce the elongation rate of free F-actin by about 70%. The residual rate is presumed to correspond to the elongation rate at the negative ends. By contrast, the elongation rate on red cell ghosts or cytoskeletons falls to zero, allowing for the background of self-nucleated polymerization of the G-actin. The critical concentration of the actin in the red cell membrane has been measured after elongation of the filaments by added pyrenyl-G-actin in the same solvent. It was found to be 0.07 microM, compared with 0.11 microM under the same conditions for actin alone. This is consistent with prediction for the case of blocked negative ends on the red cell actin. The rate of elongation of actin filaments, free and in the red cell membrane cytoskeleton, has been measured as a function of the concentration of an added actin-capping protein, plasma gelsolin, with a high affinity for the positive ends. The elongation rate falls linearly with increasing gelsolin concentration until it approaches a minimum when the gelsolin has bound to all positive filament ends. The elongation rate at this point corresponds to the activity of the negative ends, and its ratio to the unperturbed polymerization rate (in the absence of capping proteins) is indistinguishable from zero in the case of ghosts, but about 1 : 4 in the case of F-actin. When ATP is replaced in the system by ADP, so that the critical concentrations at the two filament ends are equalized, the difference is equally well-marked: for F-actin, the rate at the equivalence point is about 40% of that in the absence of capping protein, whereas for ghosts the nucleated polymerization rate at the equivalence point is again zero, indicating that under these conditions the negative ends contribute little or not at all to the rate of elongation.(ABSTRACT TRUNCATED AT 400 WORDS)