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Postsynaptic densities (PSDs) contain proteins that regulate synaptic transmission. We determined the positions of calcium/calmodulin-dependent protein kinase II (CaMKII) and PSD-95 within the three-dimensional structure of isolated PSDs using immunogold labeling, rotary shadowing, and electron microscopic tomography. The results show that all PSDs contain a central mesh immediately underlying the postsynaptic membrane. Label for PSD-95 is found on both the cytoplasmic and cleft sides of this mesh, averaging 12 nm from the cleft side. All PSDs label for PSD-95. The properties of CaMKII labeling are quite different. Label is virtually absent on the cleft sides of PSDs, but can be heavy on the cytoplasmic side at a mean distance of 25 nm from the cleft. In tomograms, CaMKII holoenzymes can be visualized directly, appearing as labeled, tower-like structures reflecting the 20 nm diameter of the holoenzyme. These towers protrude from the cytoplasmic side of the central mesh. There appears to be a local organization of CaMKII, as judged by fact that the nearest-neighbor distances are nearly invariant over a wide range of labeling density for CaMKII. The average density of CaMKII holoenzymes is highly variable, ranging from zero to values approaching a tightly packed state. This variability is significantly higher than that for PSD-95 and is consistent with an information storage role for CaMKII.

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 α-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.

We have previously reported the formation of calcium/calmodulin-dependent protein kinase II (CaMKII) clusters approximately 110 nm in diameter in hippocampal neurons in culture and in the intact adult brain, under conditions that simulate ischemic stress and increase [Ca(2+)](i) [Dosemeci et al. (2000) J. Neurosci. 20, 3076-3084; Tao-Cheng et al. (2001) Neuroscience 106, 69-78]. These observations suggest that ischemia-like conditions that prevail during the dissection of brain tissue for the preparation of hippocampal slices could lead to the formation of CaMKII clusters. We now show by pre-embedding immuno-electron microscopy that, indeed, CaMKII clusters are present in the CA1 pyramidal neurons in hippocampal slices from adult rats fixed immediately after dissection, and that the number of CaMKII clusters increases with the delay time between decapitation and fixation. Moreover, CaMKII clusters are typically localized near the endoplasmic reticulum. When acute slices are allowed to recover in oxygenated medium for 2 h, CaMKII clusters mostly disappear, indicating that clustering is reversible. Also, the postsynaptic density, another site for CaMKII accumulation under excitatory conditions, becomes thinner upon recovery. Treatment of recovered slices with high potassium for 90 s causes the re-appearance of CaMKII clusters in nearly all CA1 pyramidal cells examined. On the other hand, when dissociated hippocampal neurons in primary culture are exposed to the same depolarizing conditions, only approximately 25% of neurons exhibit CaMKII clusters, indicating a difference in the susceptibility of the neurons in culture and in acute slices to excitatory stimuli. Altogether these observations indicate that the effect of CaMKII clustering should be considered when interpreting experimental results obtained with hippocampal slices.

NMDA-induced modification of postsynaptic densities (PSDs) was studied by immunoelectron microscopy. Treatment of cultured hippocampal neurons with NMDA for 2 min promotes a 2.3 fold thickening of the PSD and a 4 fold increase in PSD-associated CaMKII immunolabel. These changes are reversed 5 min after the removal of NMDA and Ca2+ from the medium. In addition, following NMDA treatment, PSDs exhibit a 7.5 fold increase in labeling with an antibody specific to the (Thr286) phospho-form of CaMKII, indicating that CaMKII translocated to the PSD is phosphorylated. When the phosphatase inhibitors, calyculin A or okadaic acid, are included in the medium, the NMDA-induced thickening of the PSD as well as the increase in PSD-associated CaMKII immunolabeling are largely maintained (75% and 88% of the peak values respectively) at 5 min after removal of NMDA and Ca2+ from the medium. These results imply that NMDA receptors can mediate activity-induced changes in the PSD and that phosphatases of type 1 and/or 2A are involved in the reversal of these changes.

Tubulin carboxyterminal tail peptides are short (10-20 amino acids) acidic, unstructured peptides that vary significantly in sequence between tubulin isotypes and are exposed on the outer surface of microtubules. These peptides have, so far, eluded direct visualization. Electron microscopy tomography is used here to analyze stabilized microtubules embedded in a tungsten-based negative stain previously found to resist sustained electron beam irradiation. Sample stability is compatible with high electron doses, permitting collection of 142 images at various tilt angles with high signal-to-noise ratio. The resulting tomogram is reconstructed without the need of averaging multiple samples. After further optimizing collection of data and signal elaboration, images reveal the three dimensional structure of subunits as well as previously unresolved small stalks on the outer surface of the microtubule. Inspection of virtual sections demonstrates that the stalks are ∼ 2.5 nm long and ∼ 1 nm wide (at half length). Stalks protrude intermittently every ∼ 4 nm along the microtubule from the outer face of each monomer. These protrusions are well resolved from background in virtual sections perpendicular to the long axis of microtubule. These protrusions are likely the carboxyterminal peptides of tubulin. This hypothesis is supported by comparison with results obtained in the same way from microtubules whose C-terminals have been cleaved enzymatically. In summary, it is possible to resolve single peptides by amplitude contrast in a conventional electron microscope without the need of averaging data from multiple microtubules.

NMDA treatment of cultured hippocampal neurons causes recruitment of CYLD, as well as CaMKII, to the postsynaptic density (PSD), as shown by immunoelectron microscopy. Recruitment of CYLD, a deubiquitinase specific for K63-linked polyubiquitins, is blocked by pre-treatment with tatCN21, a CaMKII inhibitor, at a concentration that inhibits the translocation of CaMKII to the PSD. Furthermore, CaMKII co-immunoprecipitates with CYLD from solubilized PSD fractions, indicating an association between the proteins. Purified CaMKII phosphorylates CYLD on at least three residues (S-362, S-418, and S-772 on the human CYLD protein Q9NQC7-1) and promotes its deubiquitinase activity. Activation of CaMKII in isolated PSDs promotes phosphorylation of CYLD on the same residues and also enhances endogenous deubiquitinase activity specific for K63-linked polyubiquitins. Since K63-linked polyubiquitin conjugation to proteins inhibits their interaction with proteasomes, CaMKII-mediated recruitment and upregulation of CYLD is expected to remove K63-linked polyubiquitins and facilitate proteasomal degradation at the PSD.

Treatment of cultured hippocampal neurons with the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) in the absence of glucose mimics ischemic energy depletion and induces formation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) clusters, spherical structures with diameters of 75-175 nm [Dosemeci et al., J. Neurosci. 20 (2000) 3076-3084]. The demonstration that CaMKII clustering occurs in the intact, adult rat brain upon interruption of blood flow indicates that clustering is not confined to cell cultures. Application of N-methyl-D-aspartate (250 microM, 15 min) to hippocampal cultures also induces cluster formation, suggesting a role for Ca(2+). Indeed, intracellular Ca(2+) monitored with Fluo3-AM by confocal microscopy reaches a sustained high level within 5 min of CCCP treatment. The appearance of immunolabeled CaMKII clusters, detected by electron microscopy, follows the onset of the sustained increase in intracellular Ca(2+). Moreover, CaMKII does not cluster when the rise in intracellular Ca(2+) is prevented by the omission of extracellular Ca(2+) during CCCP treatment, confirming that clustering is Ca(2+)-dependent. A lag period of 1-2 min between the onset of high intracellular Ca(2+) levels and the formation of CaMKII clusters suggests that a sustained increase in Ca(2+) level is necessary for the clustering. CaMKII clusters disappear within 2 h of returning the cultures to normal incubation conditions, at which time no significant cell death is detected. These results indicate that pathological conditions that promote sustained episodes of Ca(2+) overload result in a transitory clustering of CaMKII into spherical structures. CaMKII clustering may represent a cellular defense mechanism to sequester a portion of the CaMKII pool, thereby preventing excessive protein phosphorylation.

Elevation of calcium during sustained synaptic activity may lead to the activation of the postsynaptic calcium-dependent protease calpain and thus could alter the integrity and localization of endogenous proteins. The distribution of anchoring proteins for neuroreceptors is an important determinant of the efficacy of neuronal transmission. Many of these anchoring proteins are concentrated within the postsynaptic density (PSD). In the present study, we examined the effects of calpain II on isolated PSDs using biochemical and electron microscopic techniques. Biochemical analysis reveals that PSD-95, a clustering molecule which anchors NMDA receptors by interaction with their NR2 subunits, as well as the NR2 subunits themselves, are cleaved by calpain. On the other hand, under conditions where all the PSD-95 protein is cleaved, actin and α-actinin—a protein thought to anchor NMDA receptors to actin filaments—remain intact. For analysis by electron microscopy, PSDs were adsorbed on glass, immunogold-labeled with an antibody to PSD-95, slam frozen, freeze dried, and rotary shadowed. Electron micrographs of replicas indicate that PSDs are disc-shaped and are composed of a lattice-like structure which labels with PSD-95 immunogold. After calpain treatment, PSDs adsorbed on glass become thinner overall and the lattice becomes fragmented. Altogether, these results suggest that calpain activity could produce changes in the organization of the PSD and, by cleaving PSD-95 associated with the PSD lattice, could modify the anchoring of NMDA receptors. Synapse 40:302–309, 2001. © 2001 Wiley-Liss, Inc.

Membrane fusion intermediates induced by the glycosylphosphatidylinositol-linked ectodomain of influenza hemagglutinin (GPI-HA) were investigated by rapid freeze, freeze-substitution, thin section electron microscopy, and with simultaneous recordings of whole-cell admittance and fluorescence. Upon triggering, the previously separated membranes developed numerous hourglass shaped points of membrane contact (approximately 10-130 nm waist) when viewed by electron microscopy. Stereo pairs showed close membrane contact at peaks of complementary protrusions, arising from each membrane. With HA, there were fewer contacts, but wide fusion pores. Physiological measurements showed fast lipid dye mixing between cells after acidification, and either fusion pore formation or the lack thereof (true hemifusion). For the earliest pores, a similar conductance distribution and frequency of flickering pores were detected for both HA and GPI-HA. For GPI-HA, lipid mixing was detected prior to, during, or after pore opening, whereas for HA, lipid mixing is seen only after pore opening. Our findings are consistent with a pathway wherein conformational changes in the ectodomain of HA pull membranes towards each other to form a contact site, then hemifusion and pore formation initiate in a small percentage of these contact sites. Finally, the transmembrane domain of HA is needed to complete membrane fusion for macromolecular content mixing.

Herpes simplex virus type I (HSV) typically enters peripheral nerve terminals and then travels back along the nerve to reach the neuronal cell body, where it replicates or enters latency. To monitor axoplasmic transport of HSV, we used the giant axon of the squid, Loligo pealei , a well known system for the study of axoplasmic transport. To deliver HSV into the axoplasm, viral particles stripped of their envelopes by detergent were injected into the giant axon, thereby bypassing the infective process. Labeling the viral tegument protein, VP16, with green fluorescent protein allowed viral particles moving inside the axon to be imaged by confocal microscopy. Viral particles moved 2.2 ± 0.26 μm/sec in the retrograde direction, a rate comparable to that of the transport of endogenous organelles and of virus in mammalian neurons in culture. Electron microscopy confirmed that 96% of motile (stripped) viral particles had lost their envelope but retained tegument, and Western blot analysis revealed that these particles had retained protein from capsid but not envelope. We conclude that ( i ) HSV recruits the squid retrograde transport machinery; ( ii ) viral tegument and capsid but not envelope are sufficient for this recruitment; and ( iii ) the giant axon of the squid provides a unique system to dissect the viral components required for transport and to identify the cellular transport mechanisms they recruit.

Cytoskeletal and postsynaptic density (PSD) fractions from forebrain contain discrete spherical structures that are immunopositive for Ca2+/calmodulin-dependent protein kinase II (CaMKII). Spherical structures viewed by rotary shadow electron microscopy have an average diameter of ∼100 nm and, in distinction to postsynaptic densities, do not immunolabel for PSD-95. These structures were purified to near homogeneity by extraction with the detergentN-lauryl sarcosinate. Biochemical analysis revealed that CaMKII accounts for virtually all of the protein in the purified preparation, suggesting that spherical structures are clusters of self-associated CaMKII. Exposure of cultured hippocampal neurons to a mitochondrial uncoupler in glucose-free medium promotes the formation of numerous CaMKII-immunopositive structures identical in size and shape to the CaMKII clusters observed in subcellular fractions. Clustering of CaMKII would reduce its kinase function by preventing its access to fixed substrates. On the other hand, clustering would not affect the ability of the large cellular pool of CaMKII to act as a calmodulin sink, as demonstrated by the Ca2+-dependent binding of gold-conjugated calmodulin to CaMKII clusters. We propose that the observed clustering of CaMKII into spherical structures is a protective mechanism preventing excessive protein phosphorylation upon loss of Ca2+homeostasis, without compromising calmodulin regulation.

Transient changes in the intracellular concentration of free calcium ([Ca2+])i) act as a trigger or modulator for a large number of important neuronal processes. Such transients can originate from voltage- or ligand-gated fluxes of Ca2+ into the cytoplasm from the extracellular space, or by ligand- or Ca2+(-)gated release from intracellular stores. Characterizing the sources and spatio-temporal patterns of [Ca2+]i transients is critical for understanding the role of different neuronal compartments in dendritic integration and synaptic plasticity. Optical imaging of fluorescent indicators sensitive to free Ca2+ is especially suited to studying such phenomena because this approach offers simultaneous monitoring of large regions of the dendritic tree in individual living central nervous system neurons. In contrast, energy-dispersive X-ray (EDX) microanalysis provides quantitative information on the amount and location of intracellular total, i.e., free plus bound, calcium (Ca) within specific subcellular dendritic compartments as a function of the activity state of the neuron. When optical measurements of [Ca2+]i transients and parallel EDX measurements of Ca content are used in tandem, and correlated simultaneously with electrophysiological measurements of neuronal activity, the combined information provides a relatively general picture of spatio-temporal neuronal total Ca fluctuations. To illustrate the kinds of information available with this approach, we review here results from our ongoing work aimed at evaluating the role of various Ca uptake, release, sequestration, and extrusion mechanisms in the generation and termination of [Ca2+]i transients in dendrites of pyramidal neurons in hippocampal slices during and after synaptic activity. Our observations support the long-standing speculation that the dendritic endoplasmic reticulum acts not only as an intracellular Ca2+ source that can be mobilized by a signal cascade originating at activated synapses, but also as a major intracellular Ca sink involved in active clearance mechanisms after voltage- and ligand-gated Ca2+ influx.

The application of immunoelectronmicroscopy to soluble proteins is limited because soluble proteins can redistribute during fixation. Fixation may also adversely affect the recognition of proteins associated with membranes. We show here how displacements of soluble proteins can be prevented and antigen sensitivity improved by freeze-substitution immunocytochemistry. The usefulness of this method for soluble cytoplasmic proteins is demonstrated for the twitchin protein in Aplysia muscle and the kinesin motor proteins in squid giant axons, in which the sizes of various cytoplasmic pools of kinesins are estimated. The utility for membrane proteins present in small numbers of copies is demonstrated by labeling a glutamate receptor subunit in mouse cerebellar cortex and the ZO-1 protein in tight junctions between MDCK cells. Thus, freeze-substitution immunocytochemistry can show the native distribution of both soluble and membrane proteins labeled with polyclonal antibodies and, at the same time, can reveal structural features comparable to those in chemically fixed or osmium freeze-substituted samples.

The squid giant axon provides an excellent model system for the study of actin-based organelle transport likely to be mediated by myosins, but the identification of these motors has proven to be difficult. Here the authors purified and obtained primary peptide sequence of squid muscle myosin as a first step in a strategy designed to identify myosins in the squid nervous system. Limited digestion yielded fourteen peptides derived from the muscle myosin which possess high amino acid sequence identities to myosin II from scallop (60-95%) and chick pectoralis muscle (31-83%). Antibodies generated to this purified muscle myosin were used to isolate a potential myosin from squid optic lobe which yielded 11 peptide fragments. Sequences from six of these fragments identified this protein as a myosin II. The other five sequences matched myosin II (50-60%, identities), and some also matched unconventional myosins (33-50%). A single band that has a molecular weight similar to the myosin purified from optic lobe copurifies with axoplasmic organelles, and, like the optic lobe myosin, this band is also recognized by the antibodies raised against squid muscle myosin II. Hence, this strategy provides an approach to the identification of a myosin associated with motile axoplasmic organelles.

Synaptic activity-dependent changes in the spatio-temporal distribution of calcium ions regulate important neuronal functions such as dendritic integration and synaptic plasticity, but the processes that terminate the free Ca2+transients associated with these changes remain unclear. We have characterized at the electron microscopic level the intracellular compartments involved in buffering free Ca2+transients in dendritic cytoplasm of CA3 neurons by measuring the larger changes in the concentrations of total Ca that persist for several minutes after neuronal activity. Quantitative energy-dispersive x-ray microanalysis of cryosections from hippocampal slice cultures rapidly frozen 3 min after afferent synaptic activity identified a subset of dendritic endoplasmic reticulum (ER) as a high-capacity Ca2+buffer. Calcium sequestration by cisterns of this subset of ER was graded, reversible, and dependent on a thapsigargin-sensitive Ca2+-ATPase. Sequestration was so robust that after repetitive high-frequency stimulation the Ca content of responsive ER cisterns increased as much as 20-fold. These results demonstrate that a subpopulation of ER is the major dendritic Ca sequestration compartment in the minutes after neuronal activity.

The organization of the cortical endoplasmic reticulum in the squid giant axon was investigated by rapid freeze and freeze-substitution electron microscopy, thereby eliminating the effects of fixatives on this potentially labile structure. Juvenile squid, which have thinner Schwann sheaths, were used in order to achieve freezing deep enough to include the entire axonal cortex. The smooth endoplasmic reticulum is composed of subaxolemmal and deeper cisternae, tubules, tethers and vesicles. The subaxolemmal cisternae make junctional contacts with the axolemma which are characterized by filamentous-granular bridging structures approximately 3 nm in diameter. The subaxolemmal junctions with the axolemma resemble the coupling junctions between the sarcoplasmic reticulum and the T-tubules in muscle. Reconstruction of short series of sections showed that a number of the elements of the endoplasmic reticulum were continuous but numerous separate vesicles were present as well. The morphology of endoplasmic reticulum as described here suggests that it is a highly dynamic entity as well as a Ca2+ sequestering organelle.

Homer is a postsynaptic density (PSD) scaffold protein that is involved in synaptic plasticity, calcium signaling and neurological disorders. Here, we use pre-embedding immunogold electron microscopy to illustrate the differential localization of three Homer gene products (Homer 1, 2, and 3) in different regions of the mouse brain. In cross-sectioned PSDs, Homer occupies a layer ∼30–100 nm from the postsynaptic membrane lying just beyond the dense material that defines the PSD core (∼30-nm-thick). Homer is evenly distributed within the PSD area along the lateral axis, but not at the peri-PSD locations within 60 nm from the edge of the PSD, where type I-metabotropic glutamate receptors (mGluR1 and 5) are concentrated. This distribution of Homer matches that of Shank, another major PSD scaffold protein, but differs from those of other two major binding partners of Homer, type I mGluR and IP3 receptors. Many PSD proteins rapidly redistribute upon acute (2 min) stimulation. To determine whether Homer distribution is affected by acute stimulation, we examined its distribution in dissociated hippocampal cultures under different conditions. Both the pattern and density of label for Homer 1, the isoform that is ubiquitous in hippocampus, remained unchanged under high K+ depolarization (90 mM for 2–5 min), N-methyl- d -asparic acid (NMDA) treatment (50 μM for 2 min), and calcium-free conditions (EGTA at 1 mM for 2 min). In contrast, Shank and calcium/calmodulin-dependent kinase II (CaMKII) accumulate at the PSD upon NMDA treatment, and CaMKII is excluded from the PSD complex under low calcium conditions.

The Salmonella typhimurium FliN protein has been proposed to form a mutually interacting complex with FliG and FliM, the switch complex, that is required for flagellar morphogenesis and function. We have used affinity chromatography for purification of extended flagellar basal bodies sufficient for quantitative analysis of their protein composition. The belled, extended structure is predominantly comprised of the switch complex proteins; with FliN present in the most copies (111 +/- 13). This explains why single, missense fliN, fliG or fliM mutations, found in many non-motile strains, can alter the belled morphology. Cell lysates from these strains contained the wild-type complement of FliG, FliM and FliN; but the basal bodies lacked the outer, cytoplasmic(C)-ring of the bell and were separated by sedimentation from FliM and FliN. The amount of FliG present in basal bodies from wild-type and one such mutant, FliN100LP, was comparable. These data show that: (1) the mutations define a FliG and FliMFliN multiple contact interface important for motility. (2) FliG is responsible for the increased size of the membrane-embedded MS-ring complex of belled relative to acid-treated basal bodies. (3) FliN, together with FliM, account for most of the C-ring. As a major component of the C-ring, FliN is distinct from the other proteins implicated in axial flagellar protein export. Inner, cytoplasmic rod basal substructure, seen by negative-stain and quick-freeze replica electron microscopy, may gate such export. Lack of connectivity between the cytoplasmic rod and ring substructures places contacts between FliG and FliMFliN at the periphery of the basal body, proximal to the flagellar intramembrane ring particles. This topology is consistent with models where torque results from interaction of circumferential arrays of the switch complex proteins with the ring particles.

Cryofixation followed by freeze substitution in osmium tetroxide was evaluated as a method for preparing biological specimens for immunoelectronmicroscopy. Samples were rapidly frozen by impact onto a sapphire block cooled with liquid nitrogen, substituted at −80°C in acetone containing osmium tetroxide, and embedded in epoxy resin. With this protocol, excellent ultrastructure can be combined with localization of antigens that otherwise would be inactivated by the osmium, but labeling may need to be enhanced by chemically etching the sections prior to staining. The effects of etching on various structures in the sections were investigated by examining the sections with atomic force microscopy, an approach that yields three-dimensional views of the surface of the section. A considerable part of the section was removed or collapsed by the etching, and these effects occurred differentially in several components of the tissue and with different etching protocols. Nevertheless, the results suggest that the partial removal of the plastic by etching of freeze-substituted tissue can be explored as a method for exposing fine biological structures for observation with atomic force microscopy. © 1996 Wiley-Liss, Inc.

We previously showed that axoplasmic organelles from the squid giant axon move toward the barbed ends of actin filaments and that KI-washed organelles separated from soluble proteins by sucrose density fractionation retain a 235-kDa putative myosin. Here, we examine the myosin-like activities of KI-washed organelles after sucrose density fractionation to address the question whether the myosin on these organelles is functional. By electron microscopy KI-washed organelles bound to actin filaments in the absence of ATP but not in its presence. Analysis of organelle-dependent ATPase activity over time and with varying amounts of organelles revealed a basal activity of 350 (range: 315–384) nmoles Pi/mg/min and an actin-activated activity of 774 (range: 560–988) nmoles/mg/min, a higher specific activity than for the other fractions. By video microscopy washed organelles moved in only one direction on actin filaments with a net velocity of 1.11 ± .03 μm/s and an instantaneous velocity of 1.63 ± 0.29 μm/s. By immunogold electronmicroscopy, 7% of KI-washed organelles were decorated with an anti-myosin antibody as compared to 0.5% with non-immune serum. Thus, some axoplasmic organelles have a tightly associated myosin-like activity.

Plus- and minus-end vesicle populations from squid axoplasm were isolated from each other by selective extraction of the minus-end vesicle motor followed by 5'-adenylyl imidodiphosphate (AMP-PNP)-induced microtubule affinity purification of the plus-end vesicles. In the presence of cytosol containing both plus- and minus-end motors, the isolated populations moved strictly in opposite directions along microtubules in vitro. Remarkably, when treated with trypsin before incubation with cytosol, purified plus-end vesicles moved exclusively to microtubule minus ends instead of moving in the normal plus-end direction. This reversal in the direction of movement of trypsinized plus-end vesicles, in light of further observation that cytosol promotes primarily minus-end movement of liposomes, suggests that the machinery for cytoplasmic dynein-driven, minus-end vesicle movement can establish a functional interaction with the lipid bilayers of both vesicle populations. The additional finding that kinesin overrides cytoplasmic dynein when both are bound to bead surfaces indicates that the direction of vesicle movement could be regulated simply by the presence or absence of a tightly bound, plus-end kinesin motor; being processive and tightly bound, the kinesin motor would override the activity of cytoplasmic dynein because the latter is weakly bound to vesicles and less processive. In support of this model, it was found that (a) only plus-end vesicles copurified with tightly bound kinesin motors; and (b) both plus- and minus-end vesicles bound cytoplasmic dynein from cytosol.

TatCN21 is a membrane permeable calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor derived from the inhibitor protein CaMKIIN. TatCN21 has been used to demonstrate the involvement of CaMKII in a variety of physiological and pathological phenomena, and it also limits excitotoxic damage in neurons. Here we use preembedding immunogold electron microscopy to examine the effect of tatCN21 on the redistribution of CaMKII in cultured hippocampal neurons. Incubation of cultures with tatCN21 (20 μM for 20 min) prior to exposure to N-methyl-d-asparic acid (NMDA) (50 μM for 2 min) inhibited both the accumulation of CaMKII at postsynaptic densities (PSDs) and CaMKII clustering in the dendrites. Under these conditions, CaMKII also formed morphologically distinct aggregates with polyribosomes near the PSD and in dendrites. Formation of these CaMKII-polyribosome aggregates requires the presence of both tatCN21 and calcium, and was augmented upon exposure to high K(+) or NMDA. CaMKII-polyribosome aggregates formed consistently with 20 μM tatCN21, but minimally or not at all with 5 μM. However, these aggregates are not induced by another CaMKII inhibitor, KN93. Formation of CaMKII-polyribosome aggregates was completely reversible within 1h after washout of tatCN21. Effects of tatCN21 were largely restricted to dendrites, with minimal effect in the soma. The effects of tatCN21 on CaMKII distribution can be used to dissect the mechanism of CaMKII involvement in cellular events.

Polyubiquitin chains on proteins flag them for distinct fates depending on the type of polyubiquitin linkage. While lysine48-linked polyubiquitination directs proteins to proteosomal degradation, lysine63-linked polyubiquitination promotes different protein trafficking and is involved in autophagy. Here we show that postsynaptic density (PSD) fractions from adult rat brain contain deubiquitinase activity that targets both lysine48 and lysine63-linked polyubiquitins. Comparison of PSD fractions with parent subcellular fractions by Western immunoblotting reveals that CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, is highly enriched in the PSD fraction. Electron microscopic examination of hippocampal neurons in culture under basal conditions shows immunogold label for CYLD at the PSD complex in approximately one in four synapses. Following depolarization by exposure to high K+, the proportion of CYLD-labeled PSDs as well as the labeling intensity of CYLD at the PSD increased by more than eighty percent, indicating that neuronal activity promotes accumulation of CYLD at the PSD. An increase in postsynaptic CYLD following activity, would promote removal of lysine63-polyubiquitins from PSD proteins and thus could regulate their trafficking and prevent their autophagic degradation.

The regulation of synaptic strength at γ-aminobutyric acid (GABA)-ergic synapses is dependent on the dynamic capture, retention, and modulation of GABA A-type receptors by cytoplasmic proteins at GABAergic postsynaptic sites. How these proteins are oriented and organized in the postsynaptic cytoplasm is not yet established. In order to better understand these structures and gain further insight into the mechanisms by which they regulate receptor populations at postsynaptic sites, we utilized electron tomography to examine GABAergic synapses in dissociated rat hippocampal cultures. GABAergic synapses were identified and selected for tomography using a set of criteria derived from the structure of immunogold-labeled GABAergic synapses. Tomography revealed a complex postsynaptic network composed of filaments that extend ∼100 nm into the cytoplasm from the postsynaptic membrane. The distribution of these postsynaptic filaments was strikingly similar to that of the immunogold label for gephyrin. Filaments were interconnected through uniform patterns of contact, forming complexes composed of 2-12 filaments each. Complexes did not link to form an integrated, continuous scaffold, suggesting that GABAergic postsynaptic specializations are less rigidly organized than glutamatergic postsynaptic densities.

Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) is a major component of postsynaptic densities (PSDs) involved in synaptic regulation. It has been previously shown that upon activity CaMKII from the spine reversibly aggregates at the cytoplasmic surfaces of PSDs, where it encounters various targets for phosphorylation. Targets for CaMKII are also present within the PSD, but there has been no reliable method to pinpoint whether, or where, CaMKII is located inside the PSD. Here we show that CaMKII can be mapped molecule-by-molecule within isolated PSDs using negative stain electron microscopy tomography. CaMKII molecules found in the core of the PSD may represent a pool distinct from the CaMKII residing at the cytoplasmic surface.

Electron tomography (ET) is a powerful technique for determining 3D ultrastructure at the supramolecular scale in sections of rapidly frozen, freeze-substituted, stained, embedded and sectioned cells and tissues. The application of conventional ET is limited to sections of thickness less than about 400 nm due to image blurring of multiply scattered electrons that are affected by the chromatic aberration of the objective lens. Many important structures such as pre- and post-synaptic nerve terminals in brain have larger dimensions so cannot be imaged in their entirety by conventional ET methods. We have therefore developed and applied tomography based on the scanning transmission electron microscope (STEM) operating at a beam energy of 300 keV. In STEM, the absence of imaging lenses after the specimen enables ET to be performed on plastic sections up to 1.5 micrometers in thickness without the deleterious effect of chromatic aberration on electrons that have undergone multiple inelastic scattering. Furthermore, it was found that the spatial resolution of 3D reconstructions obtained with an axial bright-field STEM detector was greatly improved relative to that obtained with a standard annular dark-field STEM detector, for regions in the lower half of micrometer thick sections. This advantage is attributable to the exclusion of electrons that have undergone multiple elastic scattering from the collection angles subtended by the axial bright-field detector. We have applied STEM tomography to image entire postsynaptic densities (PSDs) in dissociated cultures of rat hippocampus, in which specific proteins had been knocked down by RNAi. The technique allows multiple PSDs to be reconstructed from contiguous regions of neurons. The 3D ultrastructure reveals the role of key scaffolding proteins in the organization of PSDs.This work was supported by the Intramural Research Programs of NIBIB and NINDS, NIH.

Purkinje neurons in rat cerebellar slices injected with an oil drop saturated with 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC16(3) or DiI] to label the endoplasmic reticulum were observed by confocal microscopy. DiI spread throughout the cell body and dendrites and into the axon. DiI spreading is due to diffusion in a continuous bilayer and is not due to membrane trafficking because it also spreads in fixed neurons. DiI stained such features of the endoplasmic reticulum as densities at branch points, reticular networks in the cell body and dendrites, nuclear envelope, spines, and aggregates formed during anoxia nuclear envelope, spines, and aggregates formed during anoxia in low extracellular Ca2+. In cultured rat hippocampal neurons, where optical conditions provide more detail, DiI labeled a clearly delineated network of endoplasmic reticulum in the cell body. We conclude that there is a continuous compartment of endoplasmic reticulum extending from the cell body throughout the dendrites. This compartment may coordinate and integrate neuronal functions.

PSD-95, a membrane-associated guanylate kinase, is the major scaffolding protein in the excitatory postsynaptic density (PSD) and a potent regulator of synaptic strength. Here we show that PSD-95 is in an extended configuration and positioned into regular arrays of vertical filaments that contact both glutamate receptors and orthogonal horizontal elements layered deep inside the PSD in rat hippocampal spine synapses. RNA interference knockdown of PSD-95 leads to loss of entire patches of PSD material, and electron microscopy tomography shows that the patchy loss correlates with loss of PSD-95-containing vertical filaments, horizontal elements associated with the vertical filaments, and putative AMPA receptor-type, but not NMDA receptor-type, structures. These observations show that the orthogonal molecular scaffold constructed from PSD-95-containing vertical filaments and their associated horizontal elements is essential for sustaining the three-dimensional molecular organization of the PSD. Our findings provide a structural basis for understanding the functional role of PSD-95 at the PSD.

Synaptic vesicles contain a variety of proteins and lipids that mediate fusion with the pre-synaptic mem- brane. Although the structures of many synaptic vesicle proteins are known, an overall picture of how they are organized at the vesicle surface is lacking. In this paper, we describe a better method for the isolation of squid synaptic vesicles and characterize the results. For highly pure and intact synaptic vesicles from squid optic lobe, glycerol density gradient centrifugation was the key step. Different electron microscopic methods show that vesicle membrane surfaces are largely covered with structures corresponding to surface proteins. Each vesicle contains several stalked globular structures that extend from the vesicle surface and are consistent with the V-ATPase. BLAST search of a library of squid expressed sequence tags identifies 10 V- ATPase subunits, which are expressed in the squid stellate ganglia. Negative-stain tomography demonstrates directly that vesicles flatten during the drying step of negative stain- ing, and furthermore shows details of individual vesicles and other proteins at the vesicle surface.

The postsynaptic density (PSD) is a disc-shaped structure lining the cytoplasmic side of the postsynaptic membrane at glutamatergic excitatory synapses in the mammalian central nervous system. The PSD is a complex of regulatory proteins, membrane receptors and cell-adhesion molecules associated with a central scaffold (reviews: Sheng and Hoogenraad 2007, Feng and Zhang 2009). PSDs undergo changes in structure and composition upon stimulation (Geinisman et al., 1991, Hu et al., 1998, Dosemeci et al., 2001, Ehlers 2003), and these changes may impact synaptic strength. SynGAP, a Ras GTPase activating protein, is highly enriched in the PSD (Kim et al., 1998, Chen et al., 1998). Analysis of PSD fractions purified from adult rat forebrain shows that SynGAP is as abundant as PSD-95, and that the two are the most abundant proteins in the PSD fraction after CaMKII (Cheng et al., 2006). Yeast two-hybrid studies show that the α-isoform of SynGAP interacts with the PDZ domains of PSD-95 and SAP102, a similar MAGUK prominent in immature brain, via its C-terminal PDZ-binding domain (Kim et al., 1998). SynGAP and PSD-95 co-immunoprecipitate from solubilized PSD fractions (Chen et al., 1998). Knockout and overexpression implicate SynGAP in the inhibition of accumulation of AMPA receptors at the synapse and spine formation (Kim et al., 2003, Vazquez et al., 2004, Rumbaugh et al., 2006,). Heterozygous null mutation of SynGAP (SynGAP−/+) results in reduced induction of LTP in the CA1 region of the hippocampus (Komiyama et al., 2002). SynGAP−/+ and PSD-95−/− double mutants show enhanced induction of LTP, which is also observed in PSD-95−/− single mutants, suggesting that the role of SynGAP in the regulation of LTP is dependent on the presence of PSD-95 (Komiyama et al., 2002). The interaction of SynGAP with the PDZ domains of PSD-95 is well-characterized (Kim et al., 1998, Chen et al., 1998, Li et al., 2001). The PDZ-binding property of SynGAP, as well as its GTPase-activating property, has been shown to be necessary for the inhibitory action of SynGAP on accumulation of AMPA receptors at the synapse (Rumbaugh et al., 2006). The requirement for the PDZ-binding property of SynGAP suggests that direct association between SynGAP and PSD-95 is necessary for this inhibition. As the PDZ domains of PSD-95 lie within the PSD, close to the postsynaptic membrane (Chen et al., 2008), the association between SynGAP and PSD-95 would organize a laminar localization of SynGAP within the PSD. In the present study, we examine the distribution of SynGAP within the PSD under basal and excitatory conditions. The results add details to the picture of a dynamic PSD which reorganizes in response to synaptic activity.

Gap junctions in the epithelium and superficial fiber cells from young mice were examined in lenses prepared by rapid-freezing, and processed for freeze-substitution and freeze-fracture electron microscopy. There appeared to be three structural types of gap junction: one type between epithelial cells and two types between fiber cells. Epithelial gap junctions seen by freeze-sub stitution were ~20 nm thick and consistently associated with layers of dense material lying along both cytoplas mic surfaces. Fiber gap junctions, in contrast, were 1516 nm (type 1) or 17-18 nm thick (type 2), and had little associated cytoplasmic material. Type 1 fiber gap junctions were extensive in f lat expanses of cell membrane and had a thin, discontinuous central lamina, whereas type 2 fiber gap junctions were associated with the balland-socket domains and exhibited a dense, continuous central lamina. Both types of f iber gap junction had a diffuse arrangement of junctional intramembrane particles, whereas particles and pits of epithelial gap junc tions were in a tight, hexagonal configuration. The type 2 fiber gap junctions, however, had a larger particle size (~9 nm) than the type 1 (~7.5 nm). In addition, a large number of junctional particles typified the E-faces of both fiber types but not the epithelial type of gap junction. Gap junctions between fiber and epithelial cells had structural features of type 1 fiber gap junctions. These structural features suggest that the epithelial and the type 2 fiber gap junctions are of the common communicating type, whereas the type 1 fiber gap junctions may represent a new type of intercellular contact, not necessarily even derived from gap junction proteins, which serves an adhesive function in the lens.

Calcium calmodulin dependent kinase II (CaMKII) is sequestered in dendritic spines within seconds upon synaptic stimulation. The program Smoldyn was used to develop scenarios of single molecule CaMKII diffusion and binding in virtual dendritic spines. We first validated simulation of diffusion as a function of spine morphology. Additional cellular structures were then incorporated to simulate binding of CaMKII to the post-synaptic density (PSD); binding to cytoskeleton; or their self-aggregation. The distributions of GFP tagged native and mutant constructs in dissociated hippocampal neurons were measured to guide quantitative analysis. Intra-spine viscosity was estimated from fluorescence recovery after photo-bleach (FRAP) of red fluorescent protein. Intra-spine mobility of the GFP-CaMKII? constructs was measured, with hundred-millisecond or better time resolution, from FRAP of distal spine tips in conjunction with fluorescence loss (FLIP) from proximal regions. Different FRAP \ FLIP profiles were predicted from our Scenarios and provided a means to differentiate binding to the PSDs from self-aggregation. The predictions were validated by experiments. Simulated fits of the Scenarios provided estimates of binding and rate constants. We utilized these values to assess the role of self-aggregation during the initial response of native CaMKII holoenzymes to stimulation. The computations revealed that self-aggregation could provide a concentration-dependent switch to amplify CaMKII sequestration and regulate its activity depending on its occupancy of the actin cytoskeleton.

Electron microscopy has provided a structural basis for understanding chemical synapses. Here, we focus on a particular structure revealed by electron microscopy –the synaptic spinule (Tarrant and Routtenberg, 1977) – which consists of a double-membrane invagination inside synaptic terminals. The distribution and structural characteristics of spinules in brain have been described in detail (Sorra et al., 1998; Spacek and Harris, 2004) subsequent to their discovery (Westrum and Blackstad, 1962). Their presence in synapses is highly variable in different experimental systems and their functions remain undetermined. Some studies report spinule formation under pathological or excitatory conditions such as post-mortem effect (Tarrant and Routtenberg, 1977), or long-term potentiation (LTP, Schuster et al, 1990). Two studies report that synaptic spinules form quickly after stimulation. Synaptic spinules appear in bullfrog brain after 5–20 sec of high K+ depolarization (Van Harreveld and Trubatch, 1975). Another study combines electrical recording and simultaneous perfusion fixation to describe formation of spinules in rat hippocampal CA1 after 1 min of stimulation at 40 HZ (Applegate and Landfield, 1988). However, no quantitative morphometry was carried out, and most important, it was left unclear whether spinules are transient structures and if so, how quickly they disappear. Another major piece of missing information is whether spinules are present in synapses during basal activity. The closest approximation to a baseline for spinule formation derives from a study of bullfrog brains in which synaptic spinules were shown to be rare after cerebral cortex was simply dissected, allowed to recover, and then freeze-substituted for EM analysis (Van Harreveld and Trubatch, 1975). Most other studies on spinules have been carried out on rodent brains fixed by perfusion with aldehydes. However, perfusion fixation can result in unintended procedural delays, which in turn, can lead to synaptic activity and related structural changes (Tao-Cheng et al., 2007). Thus, in the present study, we turned to examining hippocampal slice cultures in order to have better control of the access of fixatives and the activity state of the synapses. We set out to explore the role of activity in the formation of synaptic spinules, and to find out whether basal levels of synaptic activity can produce spinules in slice cultures as well as in other experimental systems, such as acute brain slices, dissociated neuronal cultures and perfusion-fixed brains. This investigation required careful consideration of procedural factors such as tissue handling and fixation artifacts in the process of spinule formation. Finally, as a first step to elucidate the functional significance of synaptic spinules, we investigated the time course of spinule formation and disappearance during stimulation and after the cessation of excitatory conditions.

The phase behavior of synaptosomal membranes isolated from mammalian rat and marine invertebrate squid intact nerve endings was compared. Homogeneous liquid disordered (Lα) phase was observed at their body temperatures of 37 and ∼20°C for the rat and squid, respectively, using fluorescence microscopy and 1H-MAS NMR. Temperature decrease resulted in co-existence of an ordered phase with onset temperatures of 24°C for rat and 8°C for squid. Comprehensive analyses of the lipid composition by LC/MS explained the significant difference in the onset temperature: >2x higher content of 18:0 and 18:1 lipid chains in rat, which contributes to a higher transition temperature. The amount of ω-3 chains was ∼3x higher in squid than in rat due to a difference in the amounts of 22:6 and 20:5: squid membranes contained 2x more total 22:6 in major phospholipids (PC, PE, and PS) and cardiolipin (17:0,22:6,22:6,22:6-species). While prominent in rat, 20:4 was not found in squid. It is known for Lα phase PC that 16:0,22:6 is more ordered and thicker with a smaller area/lipid than 16:0,20:4. The high content of 22:6 in squid may contribute to order in the Lα phase at the body temperature (∼20°C) in contrast to the high content of 20:4 in rat at a higher body temperature (37°C). Fluorescence microscopy data showing the temperature-dependent phase behavior using giant unilamellar vesicles prepared with lipid compositions based on the synaptosomal measurements above will be discussed. Synaptosomal preparations of squid with body temperatures differing by ∼9°C will be compared.

Phosphorylation of synapsin I by CaMKII has been reported to mobilize synaptic vesicles from the reserve pool. In the present study, the distributions of alpha-CaMKII and of synapsin I were compared in synaptic boutons of unstimulated and stimulated hippocampal neurons in culture by immunogold electron microscopy. CaMKII and synapsin I are located in separate domains in presynaptic terminals of unstimulated neurons. Label for alpha -CaMKII typically surrounds synaptic vesicle clusters and is absent from the inside of the cluster in control synapses. In contrast, intense labeling for synapsin I is found within the vesicle clusters. Following 2 minutes of depolarization in high K(+), synaptic vesicles decluster and CaMKII label disperses and mingles with vesicles and synapsin I. These results indicate that, under resting conditions, CaMKII has limited access to the synapsin I in synaptic vesicle clusters. The peripheral distribution of CaMKII around vesicle clusters suggests that CaMKII-mediated declustering progresses from the periphery towards the center, with the depth of penetration into the synaptic vesicle cluster depending on the duration of CaMKII activation. Depolarization also promotes a significant increase in CaMKII immunolabel near the presynaptic active zone. Activity-induced redistribution of CaMKII leaves it in a position to facilitate phosphorylation of additional presynaptic proteins regulating neurotransmitter release.

The total molecular mass of individual postsynaptic densities (PSDs) isolated from rat forebrain was measured by scanning transmission EM. PSDs had a mean diameter of 360 nm and molecular mass of 1.10 ± 0.36 GDa. Because the mass represents the sum of the molecular masses of all of the molecules comprising a PSD, it becomes possible to derive the number of copies of each protein, once its relative mass contribution is known. Mass contributions of PSD-95, synapse-associated protein (SAP)97, and α-Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) were determined by quantitative gel electrophoresis of PSD fractions. The number of PSD-95 molecules per average PSD, contributing 2.3% of the mass of the PSD, was calculated to be 300, whereas the number of SAP97 molecules, contributing 0.9% of the mass of the PSD, was 90. The α-CaMKII holoenzymes, which contribute 6% of the mass when brains are homogenized within 2 min of interrupting blood flow, have 80 holoenzymes associated with a typical PSD. When blood flow is interrupted 15 min before homogenization, the average mass of PSDs increases by ≈40%. The additional α-CaMKII associated with PSDs accounts for up to 20% of this mass increase, representing the addition of 100–200 α-CaMKII holoenzymes.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

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.

Association of motor proteins with organelles is required for the motors to mediate transport. Because axoplasmic organelles move on actin filaments, they must have associated actin-based motors, most likely members of the myosin superfamily. To gain a better understanding of the roles of myosins in the axon we used the giant axon of the squid, a powerful model for studies of axonal physiology. First, a approximately 220 kDa protein was purified from squid optic lobe, using a biochemical protocol designed to isolate myosins. Peptide sequence analysis, followed by cloning and sequencing of the full-length cDNA, identified this approximately 220 kDa protein as a nonmuscle myosin II. This myosin is also present in axoplasm, as determined by two independent criteria. First, RT-PCR using sequence-specific primers detected the transcript in the stellate ganglion, which contains the cell bodies that give rise to the giant axon. Second, Western blot analysis using nonmuscle myosin II isotype-specific antibodies detected a single approximately 220 kDa band in axoplasm. Axoplasm was fractionated through a four-step sucrose gradient after 0.6 M KI treatment, which separates organelles from cytoskeletal components. Of the total nonmuscle myosin II in axoplasm, 43.2% copurified with organelles in the 15% sucrose fraction, while the remainder (56.8%) was soluble and found in the supernatant. This myosin decorates the cytoplasmic surface of 21% of the axoplasmic organelles, as demonstrated by immunogold electron-microscopy. Thus, nonmuscle myosin II is synthesized in the cell bodies of the giant axon, is present in the axon, and is associated with isolated axoplasmic organelles. Therefore, in addition to myosin V, this myosin is likely to be an axoplasmic organelle motor.

Depolarization of rat hippocampal neurons with a high concentration of external potassium induces a thickening of postsynaptic densities (PSDs) within 1.5–3 min. After high-potassium treatment, PSDs thicken 2.1-fold in cultured neurons and 1.4-fold in hippocampal slices compared with their respective controls. Thin-section immunoelectron microscopy of hippocampal cultures indicates that at least part of the observed thickening of PSDs can be accounted for by an accumulation of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) on their cytoplasmic faces. Indeed, PSD-associated gold label for CaMKII increases 5-fold after depolarization with potassium. The effects of high-potassium treatment on the composition and structure of the PSDs are mimicked by direct application of glutamate. In cultures, glutamate-induced thickening of PSDs and the accumulation of CaMKII on PSDs are reversed within 5 min of removal of glutamate and Ca 2+ from the extracellular medium. These results suggest that PSDs are dynamic structures whose thickness and composition are subject to rapid and transient changes during synaptic activity.

Electron micrographs of rotary shadowed replicas of α-Ca 2+ /calmodulin-dependent protein kinase II reveal a flower-shaped multimeric molecule with a central particle surrounded by 8–10 smaller peripheral particles. Peripheral particles are attached to the central particle by thin arms or “linkers.” Movement of peripheral particles to contact each other for autophosphorylation is likely to involve these linkers. It has generally been accepted that the segment 317–328 of the α-subunit constitutes the linker domain. In the present study we test this assumption by generating a mutant lacking the proposed sequence. The mutant has biochemical and morphological properties similar to those of the wild type, and a thin linker is occasionally observed in replicas from either type. The results indicate that the deleted sequence does not correspond to the linker domain. This conclusion, combined with observations from two recent studies which identify the C-terminal domain involved in oligomerization, narrows down the location of the linker domain within the sequence 330–354.

Neurotropic viruses, such as Herpes simplex virus type 1 (HSV1), first enter the axon terminal and are transported to the cell body where they replicate in the nucleus. Based on previous studies using whole animal assays (1), as well as assays of whole cells in vitro (2, 3), we know that HSV enters axons and travels as an unenveloped particle–capsid plus associated tegument proteins–in a retrograde direction toward the cell body. The rate of transport has been estimated to be 3–5 mm/hr (2). Although these earlier studies provided important insight into the delivery of virus to the nucleus, they were limited. What has been needed is an experimental system in which HSV can be applied to a particular region of a neuron at a known concentration and time, and the transport of the virus assayed quantitatively within the axon. With such an assay one could address questions about the real time motility of the viral particle and about the viral and cellular proteins that are essential for this behavior. The giant axon of the squid, Loligo pealei, serves as a powerful model for the molecular mechanisms of axoplasmic transport. The microtubule-based motor, kinesin, was discovered in squid (4); and microtubule-based transport in both anterograde and retrograde directions has been extensively characterized. Recently, actin-based transport of squid axoplasmic organelles has also been described (5, 6). Organelles move in either direction in the giant axon at 2–4 μm/s. Organelles isolated from squid axons move towards the barbed ends of actin filaments (6, 7) at 1.6 μm/s (8) and in either direction on microtubules at 2 μm/s (9). The axon contains long tracks of microtubule-actin filament bundles that appear to serve collectively as tracks for axonal transport (10). Thus, the squid axon contains all the requisite molecular machinery for transport. In this report, we describe the use of the squid giant axon as an alternative to previous approaches and present for the first time direct observation of the movement of HSV in living axons. To image the virus in the axon, we used a viral HSV strain in which a major tegument protein, viral protein (VP) 16, was generated as a fusion protein with GFP at the C terminal. This virus was grown in Vero cells, purified, and concentrated to a titer of about 1.0 × 109 pfu/ml. Different aliquots of the viral stock were treated in various ways to remove the viral envelope. This mimics the first step in viral infection in which the viral envelope fuses with the cell membrane, releasing the capsid together with its tegument into the cytosol. In vitro removal of the envelope was necessary for these experiments because injection of virus into the axon by-passes the normal membrane fusion step. Several treatments were found that produced motile, fluorescent particles in the axon after injection. The viral preparation was co-injected with non-fluorescent oil. The oil droplet was used to determine an appropriate focal plane and as a fixed reference marker, since it remains stationary after injection (11). The movement of viral particles was recorded on a laser scanning confocal microscope. Movement was sampled ~1 frame/3 s, and transport rates were determined by analyzing sequential frames. When we examined fields upstream from the oil droplet, i.e., closer to the cell body, we identified rapidly moving particles. One of these moved ~36 μm in 18 s for an overall rate of 1.9 μm/s in the retrograde direction (Fig. 1). At the end of this sequence, this particle went out of the plane of focus. Of 113 moving particles examined, all were found in the region of the axon between the oil droplet and the cell body, moving in the retrograde direction. Figure 1 Retrograde transport of a GFP-labeled viral particle in a living squid axon. The giant axon was dissected in Ca++-containing seawater and injected with >100pfu of GFP-VP16 labeled and extracted Herpes simplex virus. Still frames were taken from ... The effect on the virus of the pre-injection treatment was determined by electron microscopy of negatively stained preparations. Of the virus in those samples that produced motile particles when injected in the axon, ~90% had lost their surface envelope. Since the GFP-labeled protein, VP16, is a tegument protein, the moving particles we observed must include at least tegument, either as whole virus, tegument plus capsid, or simply as tegument aggregates. Since neither capsid nor membrane are GFP-labeled, we cannot know from these studies whether they also are capable of moving. We have demonstrated the rapid and preferential retrograde axonal transport of HSV particles in the living axon. The rate of movement of these viral particles in the squid giant axon is similar to the estimated rates of retrograde transport of this human virus in rat dorsal root ganglia in culture (2). Furthermore, this rate is consistent with the rates of retrograde transport of endogenous organelles in the squid axon and of isolated organelles on either microtubules or actin filaments. Both the direction and rate of movement suggest that a host cell motor molecule, such as the retrograde microtubule motor, dynein, may be co-opted by invading virus. Thus, by combining the power of the squid axon with a biochemical and genetic dissection of the virus, we expect to be able to identify the viral proteins required for transport, as well as the cellular transport machinery that they recruit.

A major issue in the slow transport of cytoskeletal proteins is the form in which they are transported. We have investigated the possibility that unpolymerized as well as polymerized cytoskeletal proteins can be actively transported in axons. We report the active transport of highly diffusible tubulin oligomers, as well as transport of the less diffusible neurofilament polymers. After injection into the squid giant axon, tubulin was transported in an anterograde direction at an average rate of 2.3 mm/day, whereas neurofilament was moved at 1.1 mm/day. Addition of the metabolic poisons cyanide or dinitrophenol reduced the active transport of both proteins to less than 10% of control values, whereas disruption of microtubules by treatment of the axon with cold in the presence of nocodazole reduced transport of both proteins to ≈20% of control levels. Passive diffusion of these proteins occurred in parallel with transport. The diffusion coefficient of the moving tubulin in axoplasm was 8.6 μm 2 /s compared with only 0.43 μm 2 /s for neurofilament. These results suggest that the tubulin was transported in the unpolymerized state and that the neurofilament was transported in the polymerized state by an energy-dependent nocodazole/cold-sensitive transport mechanism.

The postsynaptic density (PSD) is a macromolecular signaling assembly anchored in the postsynaptic membrane of nerve cells. It contains receptors, scaffold and signaling molecules, and cytoskeletal elements. Mass spectrometry of isolated PSDs reveals several hundred protein species [1]. The postsynaptic signal pathways have a major role in controlling synaptic strength, and ultimately may underlie the mechanisms involved in learning and memory. The fundamental molecular organization of the PSD in cultured hippocampal spines has recently been resolved by EM tomography. Three-dimensional reconstructions show an orthogonal assembly of horizontal scaffolding molecules associated with the vertically oriented filaments that in turn associate with major transmembrane structures [2]. Now that core individual proteins can be visualized with tomography, methods are needed to identify them. Structures can be matched with predicted sizes and shapes of proteins at specific cellular compartment, but a direct identifying label would be reassuring. Here, we label a key scaffolding molecule in the postsynaptic density, PSD-95 [3], by combining electron microscopic tomography with immunolabeling on cultured rat hippocampal neurons prepared by high pressure freezing, freeze substitution and cryoembedding. Dissociated cultured neurons grown on astrocytes in a 3 mm gold chamber for three weeks were initial fixed in 4% paraformaldehyde and incubated with primary antibody to PSD-95, followed by a Nanogold conjugated secondary antibody. After a short (6–8 min) silver enhancement, the cells underwent preparation by high pressure freezing, and Lowicryl HM-20 sections of thickness 100–200 nm were cut en face, photographed for dual axis tomography. We find that PSD-95 molecules can be individually labeled in the tomographic reconstructions. Our methods could have broad applications for identifying individual molecules in large protein complexes in neuronal cells.

The three-dimensional surface topology of rapid-frozen Salmonella typhimurium flagellar hook basal body complexes was studied by stereo-examination of thin-film metal replicas. The complexes contained the extended cytoplasmic structure, composed of the switch complex proteins; FliG, FliM, and FliN. Distinct nanometer-scale element arrays, separated by grooves, defined the outer surface of the cytoplasmic (C-) ring. The number of array elements was comparable to previously determined FliG and FliM copy numbers in the basal body. In addition to basal body complexes lacking C-rings, complexes containing incomplete C-rings were identified. The incomplete C-rings had lost segments of the proximal array. Basal bodies with the distal C-ring array alone were not found. These findings are compatible with the spatial organization of the flagellar switch suggested by previous biochemical data.

Organelles in the axoplasm from the squid giant axon move along exogenous actin filaments toward their barbed ends. An approximately 235-kDa protein, the only band recognized by a pan-myosin antibody in Western blots of isolated axoplasmic organelles, has been previously proposed to be a motor for these movements. Here, we purify this approximately 235-kDa protein (p235) from axoplasm and demonstrate that it is a myosin, because it is recognized by a pan-myosin antibody and has an actin-activated Mg-ATPase activity per mg of protein 40-fold higher than that of axoplasm. By low-angle rotary shadowing, p235 differs from myosin II and it does not form bipolar filaments in low salt. The amino acid sequence of a 17-kDa protein that copurifies with p235 shows that it is a squid optic lobe calcium-binding protein, which is more similar by amino acid sequence to calmodulin (69% identity) than to the light chains of myosin II (33% identity). A polyclonal antibody to this light chain was raised by using a synthetic peptide representing the calcium binding domain least similar to calmodulin. We then cloned this light chain by reverse transcriptase-PCR and showed that this antibody recognizes the bacterially expressed protein but not brain calmodulin. In Western blots of sucrose gradient fractions, the 17-kDa protein is found in the organelle fraction, suggesting that it is a light chain of the p235 myosin that is also associated with organelles.

Examination of directly frozen rough endoplasmic reticulum (ER) of retinal pigment epithelial cells by freeze-fracture and freeze-substitution revealed distinct paired transmembrane proteins associated with membrane ribosomes. Ribosomal subunits on intact ER membrane are directly visualized for the first time, providing a global view of the structure of the ribosome and the corresponding structures on the ER membrane. The ribosomal intersubunit cleft appears to be continuous with a cleft between paired transmembrane proteins that extends into the lumen of the ER. This continuous cleft may be the path taken by nascent polypeptides.

In order to explore how cytoskeletal proteins are moved by axonal transport, we injected fluorescent microtubules and actin filaments as well as exogenous particulates into squid giant axons and observed their movements by confocal microscopy. The squid giant axon is large enough to allow even cytoskeletal assemblies to be injected without damaging the axon or its transport mechanisms. Negatively charged, 10- to 500-nm beads and large dextrans moved down the axon, whereas small (70 kDa) dextrans diffused in all directions and 1000-nm beads did not move. Only particles with negative charge were transported. Microtubules and actin filaments, which have net negative charges, made saltatory movements down the axon, resulting in a net rate approximating that previously shown for slow transport of cytoskeletal elements. The present observations suggest that particle size and charge determine which materials are transported down the axon.