Characterization of the actin filament capping state in human erythrocyte ghost and cytoskeletal preparations.

The narrow Gaussian-length-distribution of actin filaments forming the cytoskeleton of the human erythrocyte indicates the existence of strict mechanisms for length determination and maintenance. A similar regulation is achieved in striated muscle by the capping of both the ends of the thin filaments, which consequently prevents monomer exchange. However, the ability of erythroid cytoskeletal preparations to nucleate actin polymerization has led to the proliferation of the idea that at least the barbed ends of the actin filaments are uncapped. The mechanism by which the length of the filaments is thus maintained has been left open to debate. In an effort to resolve any doubt regarding length-maintenance in human erythrocytes we have characterized the capping state of the actin filaments in a number of different ghost and cytoskeletal preparations. Under conditions of sufficiently high bivalent-cation concentration the actin filaments retain functional caps at both the barbed and pointed ends. Hence filament capping at both ends prevents redistribution of the actin monomer in a similar manner to that proposed for the thin filaments of striated muscle. Actin filament uncapping is apparently caused by the centrifugal shearing stress imposed during ghost preparation. The uncapping is more pronounced when the bivalent-cation concentration is reduced or when the membrane is removed by detergents. The effects of bivalent cations seem to be mediated through the erythroid protein spectrin, consistent with the hypothesis of Wallis et al. [Wallis, Babitch and Wenegieme (1993) Biochemistry 32, 5045--5050] that the ability of spectrin to resist shearing stress is dependent on the degree of bound bivalent cations.

ATPase kinetics of the Dictyostelium discoideum myosin II motor domain.

Structural characterization of the mode of interaction of nucleotides bound to myosin has relied upon the crystal structure of the Dictyostelium discoideum myosin II motor domain. This fragment, denoted S1dC, lacks the regulatory domain and light chain subunits and may therefore fail to display the normal ATPase activity of the intact myosin molecule. Here we show that the elementary steps of the S1dC ATPase pathway and the effects of actin are similar to those of the complete myosin head fragment. This indicates that truncation at residue E759, with the removal of the light chain binding sites, is not crucial to catalytic activity. In particular, S1dC does not show the anomolous tight binding of ADP displayed by slightly shorter M754 construct reported elsewhere. We also show that the fluorescent analogue Cy3-EDA-ATP is a good substrate for S1dC and demonstrate the use of fluorescence correlation spectroscopy to determine the affinity of Cy3-EDA-ADP using microgram quantities of proteins.

Measurement of ATPase activities of myosin at the level of tracks and single molecules.

In order to determine the degree of mechanochemical coupling in actomyosin in vitro motility assays, it is desirable to measure the sliding velocity and the associated ATP turnover simultaneously at the single filament level. Actin sliding over tracks of immobilised heavy meromyosin (HMM) has been initiated by flash photolysis of caged ATP. Flash photolysis has also been used to displace fluorescent Cy3-EDA-nucleotides from HMM tracks to monitor the ATPase activity. These assays are now being combined using total internal reflectance fluorescence (TIRF) microscopy with a dual-view detection system for Cy3/Cy5 labels on ATP and actin respectively. In other experiments, we are exploring the use of the single molecule kinetic technique developed by Funatsu et al. (Nature 374, 555-559, 1995) to scale down ATPase assays of Dictyostelium myosin fragments and to elucidate the mechanism of regulation of the molluscan (scallop) myosin ATPase. Although fluctuations occur from the binding and release of Cy3-EDA-nucleotides during turnover and might provide a measure of the ATPase activity, other sources of fluctuations also need to be considered.

Purification and characterization of an alpha 1 beta 2 isoform of CapZ from human erythrocytes: cytosolic location and inability to bind to Mg2+ ghosts suggest that erythrocyte actin filaments are capped by adducin.

CapZ ("capping protein") is a heterodimeric actin capping protein that blocks actin filament assembly and disassembly at the fast growing (barbed) filament ends and is proposed to function in regulating actin filament dynamics as well as in stabilizing actin filament lengths in muscle and nonmuscle cells. We show here that erythrocytes contain a nonmuscle isoform of capZ (EcapZ) that is present exclusively in the cytosol and is not associated with the short actin filaments in the erythrocyte membrane skeleton. This is unlike other cell types where capZ is associated with cytoskeletal actin filaments and suggests that cytosolic EcapZ may be inactive, or alternatively, that the barbed ends are capped by adducin, a membrane skeleton protein that was shown recently to cap actin filament barbed ends in vitro [Kuhlman, P. A., Hughes, C. A., Bennett, V., & Fowler, V. M. (1996) J. Biol. Chem. 271, 7986]. To distinguish between these possibilities, we purified EcapZ from erythrocyte cytosol and characterized its biochemical and functional properties. Two-dimensional gel electrophoresis and western blotting reveals the EcapZ subunit composition to be alpha1beta2, as described for capZ from many other nonmuscle cells, with no evidence for posttranslational modifications. Purified EcapZ is fully functional in blocking actin elongation from barbed filament ends (Kcap approximately 1-5 nM) as well as in nucleating actin polymerization. Furthermore, cytosolic EcapZ binds to actin filament barbed ends, indicating that sequestering of EcapZ by a cytosolic inhibitory factor or insufficient amounts of EcapZ in cytosol also cannot account for its absence from the membrane skeleton. To test directly whether the barbed ends of the erythrocyte actin filaments were already capped, we measured binding of purified EcapZ to isolated membranes. Purified EcapZ does not cosediment with membranes prepared by hypotonic lysis in the presence of magnesium, suggesting that the barbed ends of the erythrocyte actin filaments are capped under these conditions but not by EcapZ. In contrast, purified EcapZ stoichiometrically reassociates with all the actin filament barbed ends in membranes prepared by hypotonic lysis in 5 mM sodium phosphate, pH 8.0 (5P8), conditions in which the barbed filament ends were previously reported to be uncapped. Comparison of the amounts of adducin associated with membranes prepared in the presence and absence of magnesium reveals that 60-80% of the adducin dissociates from the membrane during hemolysis and washing in 5P8 buffer, suggesting that the barbed ends become artifactually uncapped due to loss of adducin. The erythrocyte actin filaments may thus represent a specialized class of membrane-associated actin filaments that are capped by adducin instead of capZ.

In contrast to kindled seizures, the frequency of spontaneous epilepsy in the limbic status model correlates with greater aberrant fascia dentata excitatory and inhibitory axon sprouting, and increased staining for N-methyl-D-aspartate, AMPA and GABA(A) receptors.

This study determined whether there were differences in hippocampal neuron loss and synaptic plasticity by comparing rats with spontaneous epilepsy after limbic status epilepticus and animals with a similar frequency of kindled seizures. At the University of Virginia, Sprague-Dawley rats were implanted with bilateral ventral hippocampal electrodes and treated as follows; no stimulation (electrode controls; n=5): hippocampal stimulation without status (stimulation controls; n=5); and limbic status from continuous hippocampal stimulation (n=12). The limbic status group were electrographically monitored for a minimum of four weeks. Four rats had no recorded chronic seizures (status controls), and all three control groups showed no differences in hippocampal pathology and were therefore incorporated into a single group (controls). Eight limbic status animals eventually developed chronic epilepsy (spontaneous seizures) and an additional eight rats were kindled to a similar number and frequency of stage 5 seizures (kindled) as the spontaneous seizures group. At the University of California (UCLA) the hippocampi were processed for: (i) Niss1 stain for densitometric neuron counts; (ii) neo-Timm's histochemistry for mossy fiber sprouting; and (iii) immunocytochemical staining for glutamate decarboxylase, N-methyl-D-aspartate receptor subunit 2, AMPA receptor subunit 1 and the GABA(A) receptor. In the fascia dentata inner and outer molecular layers the neo-Timm's stain and immunoreactivity was quantified as gray values using computer image analysis techniques. Statistically significant results (P<0.05) showed the following. Compared to controls and kindled animals, rats with spontaneous seizures had: (i) lower neuron counts for the fascia dentata hilus, CA3 and CA1 stratum pyramidale; (ii) greater supragranular inner molecular layer mossy fiber staining; and (iii) greater glutamate decarboxylase immunoreactivity in both molecular layers. Greater supragranular excitatory mossy fiber and GABAergic axon sprouting correlated with: (i) increases in N-methyl-D-aspartate receptor subunit 2 inner molecular layer staining; (ii) more AMPA receptor subunit 1 immunoreactivity in both molecular layers; and (iii) greater outer than inner molecular layer GABA(A) immunoreactivity. Furthermore, in contrast to kindled animals, rats with spontaneous seizures showed that increasing seizure frequency per week and the total number of natural seizures positively correlated with greater Timm's and GABAergic axon sprouting, and with increases in N-methyl-D-aspartate receptor subunit 2 and AMPA receptor subunit 1 receptor staining. In this rat limbic status model these findings indicate that chronic seizures are associated with hippocampal neuron loss, reactive axon sprouting and increases in excitatory receptor plasticity that differ from rats with an equal frequency of kindled seizures and controls. The hippocampal pathological findings in the limbic status model are similar to those in humans with hippocampal sclerosis and mesial temporal lobe epilepsy, and support the hypothesis that synaptic reorganization of both excitatory and inhibitory systems in the fascia dentata is an important pathophysiological mechanism that probably contributes to or generates chronic limbic seizures.

Human fascia dentata anatomy and hippocampal neuron densities differ depending on the epileptic syndrome and age at first seizure.

This study determined fascia dentata anatomy and hippocampal neuron densities in patients with different epileptic syndromes. Based on presurgical data, patients were classified into: (a) pediatric patients (n=19); (b) temporal mass lesion cases (n=14); and (c) hippocampal sclerosis patients (n=31). Surgically removed hippocampi and autopsies (n=34) were studied for: (a) hippocampal neuron densities; (b) stratum granulosum (SG) widths and lengths; and (c) hilar areas. The number of granule cells and hilar neurons per tissue section were estimated from the neuron densities and fascia dentata area measurements. Results showed that compared with autopsies (p<0.05): (a) pediatric patients had similar SG and hilar areas; granule cell density was lower (but not hilar neuron density); and the estimated number of granule cells was lower (but not the number of hilar neurons); (b) the widths of SG and hilar areas were greater in mass lesion cases; the density of granule cells and hilar neurons was lower; and the total estimated numbers of granule cells and hilar neurons were similar to those of the autopsies; and (c) hippocampal sclerosis patients had wider, yet shorter SG; hilar areas were smaller; granule cell and hilar densities were lower; and the total estimated numbers of granule cells and hilar neurons were lower than those of the autopsy cases. The duration of the seizures did not correlate with lower fascia dentata neuron densities or estimates of total granule cell and hilar neurons. Furthermore, greater SG widths correlated with lower hilar and CA4 neuron densities, but not with age at first seizure or duration of epilepsy. These results indicate that the size of the fascia dentata SG and hilus along with hippocampal neuron densities differ between surgical patients with different epileptic syndromes, and a wider SG was associated with a lower density of end folium neurons. These findings support the hypothesis that hippocampal sclerosis and granule cell dispersion are not the consequence of repetitive seizures beginning at an early developmental age, but seem to differ depending on the type of epileptic syndrome.

X-ray crystal structure and solution fluorescence characterization of Mg.2'(3')-O-(N-methylanthraniloyl) nucleotides bound to the Dictyostelium discoideum myosin motor domain.

Mant (2'(3')-O-(N-methylanthraniloyl)) labeled nucleotides have proven to be useful tools in the study of the kinetic mechanism of the myosin ATPase by fluorescence spectroscopy. The sensitivity of the mant fluorophore to its local environment also makes it suitable to investigate the exposure of bound nucleotides to solvent from collisional quenching measurements. Here we present the crystal structure of mant-ADP and beryllium fluoride complexed with Dictyostelium discoideum myosin motor domain (S1dC) at 1.9 A resolution. We complement the structural approach with an investigation of the accessibility of the mant moiety to solvent using acrylamide quenching of fluorescence emission. In contrast to rabbit skeletal myosin subfragment 1, where the mant group is protected from acrylamide (Ksv=0.2 M-1), the fluorophore is relatively exposed when bound to Dictyostelium myosin motor domain (Ksv= 1.4 M-1). Differences between the Dictyostelium structure and that of vertebrate skeletal subfragment 1, in the region of the nucleotide binding pocket, are proposed as an explanation for the differences observed in the solvent accessibility of complexed mant-nucleotides. We conclude that protection of the mant group from acrylamide quenching does not report on overall closure of the nucleotide binding pocket but reflects more local structural changes.

The pathogenic and progressive features of chronic human hippocampal epilepsy.

To design useful experimental models of epilepsy, it is necessary to clearly understand the known clinical-pathologic features of the disease process. Studies of mesial temporal lobe epilepsy (MTLE) patients have identified several distinctive clinical and pathophysiologic characteristics and many of these can be analyzed in experimental models. For example, patients with typical MTLE have medical histories that often contain an initial precipitating injury (IPI), are likely to have hippocampal sclerosis in the surgical specimen, and have better seizure outcomes than patients with typical idiopathic temporal seizures (i.e. cryptogenic). Hippocampal from children as young as age 1 year with IPI histories also demonstrate neuron damage similar to adults with hippocampal sclerosis. Compared to IPI patients without seizures (i.e. trauma, hypoxia, etc.), IPI cases with severe seizures showed younger ages at the IPI, shorter latent periods, and longer durations of habitual MTLE. Hippocampal damage is often bilateral, however, the epileptogenic side shows hippocampal sclerosis and the opposite side usually shows only mild neuron losses. Moreover, MTLE patients show declines in hippocampal neuron densities with very long histories of habitual seizures (15 to 20 years), however, the additional neuron loss adds to the template of hippocampal sclerosis and occurs in limited subfields (granule cells, CA1 and prosubiculum). Hippocampal axon and synaptic reorganization is another pathologic feature of MTLE, and involves granule cell mossy fibers and axons immunoreactive for neuropeptide upsilon, somatostatin, and glutamate decarboxylase (which synthesizes GABA). Finally, MTLE patients with hippocampal sclerosis show increased granule cell mRNA levels for brain derived neurotropic factor, nerve growth factor, and neurotrophin-3 that correlate with mossy fiber sprouting or with declines in Ammon's horn neuron densities. Taken together, our data support the following concepts: (1) The pathogenesis of MTLE is associated with IPI histories that probably injure the hippocampus at some time prior to habitual seizure onsets, (2) most of the damage seems to occur with the IPI, (3) there can be additional neuron loss associated with long histories, (4) another pathologic feature of MTLE is axon reorganization of surviving fascia dentata and hippocampal neurons, and (5) reorganized axon circuits probably contribute to seizure or propagation.

Glutamate AMPA receptors in the fascia dentata of human and kainate rat hippocampal epilepsy.

The present study examined the relationship between the patterns and densities of glutamate AMPA receptor sub-units GluR1 and GluR2/3 in the molecular layer of the fascia dentata and aberrant mossy fiber neoinnervation in human and kainate rat hippocampal epilepsy. Because AMPA sub-units modulate the fast glutamate synaptic transmission, we hypothesized that the AMPA receptor densities would be related to the glutamate-secreting mossy fibers, which could then contribute to seizure generation. In human hippocampal epilepsy, we found that the immunocytochemical labeling of GluR1 and GluR2/3 dendrites was positively related to the densities and spatial locations of the densest, aberrant neo-Timm stained supragranular mossy fibers. We used quantitative densitometry for the mossy fibers. However, the relatively faint and punctate immunocytochemical staining of the receptors did not allow true quantitative densitometry of the dendritic trees because in human epilepsy granule cell densities were decreased on average 50% of normal. Nevertheless, visual observations did confirm spatial relations between dense fascia dentata inner molecular layer mossy fibers and dense AMPA receptor staining. In the outer molecular layer, the mossy fibers were present only in the lower portion, were not densely-stained, and the AMPA receptors were only faintly-labeled. Nevertheless, outer molecular layer AMPA receptor densities were usually present more distally than were the mossy fibers. Experiments were done using intrahippocampal kainate epileptic rats to test the time courses for the changes in mossy fibers and AMPA receptors. The upregulation of inner and outer molecular layer AMPA receptors occurred maximally within 5 days post-kainate injection, prior to any mossy fiber supragranular ingrowth. One hundred and eighty days after ipsilateral kainate the AMPA receptors were increased bilaterally in the inner and outer molecular layers despite the fact that the contralateral aberrant supragranular mossy fibers were minor in comparison to the dense ipsilateral mossy fiber hyperinnervation. These results suggest that in hippocampal epilepsy AMPA receptor numbers increase throughout the length of the molecular layer dendrites; however the AMPA receptor densities are greater in rough relation to the greatest aberrant mossy fiber presynaptic inputs. Interestingly, the receptor upregulation precedes the mossy fiber ingrowth and may play a role in initiating axonal sprouting or in maintaining the aberrant mossy fiber synapses.

Neuron loss, mossy fiber sprouting, and interictal spikes after intrahippocampal kainate in developing rats.

This study determined neuron losses, mossy fiber sprouting, and interictal spike frequencies in adult rats following intrahippocampal kainic acid (KA) injections during postnatal (PN) development. KA (0.4 micrograms/0.2 microliters; n = 64) was injected into one hippocampus and saline into the contralateral side between PN 7 to 30 days. Animals were sacrificed 28 to 256 days later, along with age-matched naive animals (controls; n = 20). Hippocampi were studied for: (1) Fascia dentata granule cell, hilar, and CA3c neuron counts; (2) neo-Timm's stained supragranular mossy fiber sprouting; and (3) hippocampal and intracerebral interictal spike densities (n = 13). Mossy fiber sprouting was quantified as the gray value differences between the inner and outer molecular layer. Statistically significant results (p < 0.05) showed the following: (1) Compared to controls, CA3c and hilar neuron counts were reduced in KA-hippocampi with injections at PN 7-10 and PN 12-14 respectively and counts decreased with older PN injections. Granule cell densities on the KA-side and saline injected hippocampi were not reduced compared to controls. (2) In adult rats, supragranular mossy fiber sprouting was observed in 2 of 7 PN 7 injected animals. Compared to controls, increased gray value differences, indicating mossy fiber sprouting, were found on the KA-side beginning with injuries at PN 12-14 and increasing with older PN injections. On the saline-side only PN 30 animals showed minimal sprouting. (3) Mossy fiber sprouting progressively increased on the KA-side with longer survivals in rats injured after PN 15. Sprouting correlated positively with later PN injections and longer post-injection survival intervals, and not with reduced hilar or CA3c neuron counts. (4) On the KA-side, mossy fiber gray value differences correlated positively with in vivo intrahippocampal interictal spike densities. These results indicate that during postnatal rat development intrahippocampal kainate excitotoxicity can occur as early as PN 7 and increases with older ages at injection. This rat model reproduces many of the pathologic, behavioral, and electrophysiologic features of human mesial temporal lobe epilepsy, and supports the hypothesis that hippocampal sclerosis can be the consequence of focal injury during early postnatal development that progressively evolves into a pathologic and epileptic focus.

A new function for adducin. Calcium/calmodulin-regulated capping of the barbed ends of actin filaments.

Adducin is a membrane skeleton protein originally described in human erythrocytes that promotes the binding of spectrin to actin and also binds directly to actin and bundles actin filaments. Adducin is associated with regions of cell-cell contact in nonerythroid cells, where it is believed to play a role in regulating the assembly of the spectrin-actin membrane skeleton. In this study we demonstrate a novel function for adducin; it completely blocks elongation and depolymerization at the barbed (fast growing) ends of actin filaments, thus functioning as a barbed end capping protein (Kcap approximately 100 nM). This barbed end capping activity requires the intact adducin molecule and is not provided by the NH2-terminal globular head domains alone nor by the COOH-terminal extended tail domains, which were previously shown to contain the spectrin-actin binding, calmodulin binding, and phosphorylation sites. A novel difference between adducin and other previously described capping proteins is that it is down-regulated by calmodulin in the presence of calcium. The association of stoichiometric amounts of adducin with the short erythrocyte actin filaments in the membrane skeleton indicates that adducin could be the functional barbed end capper in erythrocytes and play a role in restricting actin filament length. Our experiments also suggest novel possibilities for calcium regulation of actin filament assembly by adducin in erythrocytes and at cell-cell contact sites in nonerythroid cells.

Aberrant hippocampal mossy fiber sprouting correlates with greater NMDAR2 receptor staining.

This study determined in temporal lobe epilepsy patients and rats injected with intrahippocampal kainate (KA) whether fascia dentata molecular layer mossy fiber sprouting was associated with increases in NMDAR2 immunoreactivity (IR). Patients with hippocampal sclerosis (n = 11) were compared with those with temporal mass lesions (n = 7) and material obtained at autopsies (n = 4); and unilateral KA-injected rat hippocampi (n = 7) were compared with the contralateral saline-injected side and non-lesioned animals (n = 7; control). Hippocampi were studied for neo-Timm's stained mossy fiber sprouting and NMDAR2 IR. The staining was quantified as gray values (GV) using computer image analysis. Hippocampal sclerosis patients and KA-injected rats showed the greatest inner molecular layer (IML) mossy fiber sprouting and NMDAR2 staining. Compared with autopsies and patients with mass lesions, hippocampal sclerosis patients had greater IML neo-Timm's (p = 0.0018) and NMDAR2 staining (p = 0.0063). Similarly, compared with controls and saline-injected rats, KA-injected hippocampi showed greater IML mossy fiber sprouting and NMDAR2 IR (p = 0.0001). Furthermore, IML mossy fiber sprouting positively correlated with greater IML NMDAR2 staining in both human and experimental rat groups (p < 0.0099). These results support the hypothesis that in severely damaged hippocampi abnormal mossy fiber sprouting and concordant increases in IML NMDAR2 receptor staining may contribute or partially explain granule cell hyperexcitability and the pathophysiology of hippocampal epilepsy.

The kinetics of the interaction between the actin-binding domain of alpha-actinin and F-actin.

Measurement of the binding equilibrium for the interaction of alpha-actinin with F-actin is complicated by secondary reactions involving cross-linking and/or bundling of the actin filaments. To quantitate the initial binding event, we studied the interaction of the bacterially expressed actin-binding domain (ABD) of chick smooth muscle alpha-actinin with F-actin. Stopped-flow measurements revealed a quench in protein fluorescence and an enhancement in light scattering when ABD binds to F-actin yielding second order rate constants for association of 2 x 10(5), 1.8 x 10(6) and 4 x 10(6) M-1.s-1 at 5 degrees C, 15 degrees C and 25 degrees C, respectively. At the latter two temperatures the dissociation rate constants were 1.5 and 9.6s-1, giving equilibrium constants of 0.83 and 2.4 microM, respectively. Optical changes on mixing intact alpha-actinin with F-actin were dominated by secondary bundling events.

The identification and characterisation of an actin-binding site in alpha-actinin by mutagenesis.

We have shown previously that the N-terminal actin-binding domain of alpha-actinin retains activity when expressed in E. coli as a fusion protein with glutathione-S-transferase. In the present study we have made a series of N- and C-terminal deletions within this domain and show that an actin-binding site is contained within residues 120-134. Amino acid substitutions within this region indicate that several highly conserved hydrophobic residues are involved in binding to F-actin. The hypothesis that the interaction between alpha-actinin and F-actin is predominantly hydrophobic in nature is supported by the observation that binding is relatively independent of salt concentration.

Analysis of the actin-binding domain of alpha-actinin by mutagenesis and demonstration that dystrophin contains a functionally homologous domain.

To define the actin-binding site within the NH2-terminal domain (residues 1-245) of chick smooth muscle alpha-actinin, we expressed a series of alpha-actinin deletion mutants in monkey Cos cells. Mutant alpha-actinins in which residues 2-19, 217-242, and 196-242 were deleted still retained the ability to target to actin filaments and filament ends, suggesting that the actin-binding site is located within residues 20-195. When a truncated alpha-actinin (residues 1-290) was expressed in Cos cells, the protein localized exclusively to filament ends. This activity was retained by a deletion mutant lacking residues 196-242, confirming that these are not essential for actin binding. The actin-binding site in alpha-actinin was further defined by expressing both wild-type and mutant actin-binding domains as fusion proteins in E. coli. Analysis of the ability of such proteins to bind to F-actin in vitro showed that the binding site was located between residues 108 and 189. Using both in vivo and in vitro assays, we have also shown that the sequence KTFT, which is conserved in several members of the alpha-actinin family of actin-binding proteins (residues 36-39 in the chick smooth muscle protein) is not essential for actin binding. Finally, we have established that the NH2-terminal domain of dystrophin is functionally as well as structurally homologous to that in alpha-actinin. Thus, a chimeric protein containing the NH2-terminal region of dystrophin (residues 1-233) fused to alpha-actinin residues 244-888 localized to actin-containing structures when expressed in Cos cells. Furthermore, an E. coli-expressed fusion protein containing dystrophin residues 1-233 was able to bind to F-actin in vitro.