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Placozoa are millimeter-sized, flat, irregularly shaped ciliated animals that crawl on surfaces in warm oceans feeding on biofilms, which they digest externally. They stand out from other animals due to their simple body plans. They lack organs, body cavities, muscles and a nervous system and have only seven broadly defined morphological cell types, each with a unique distribution. Analyses of single cell transcriptomes of four species of placozoans revealed greater diversity of secretory cell types than evident from morphological studies, but the locations of many of these new cell types were unknown and it was unclear which morphological cell types they represent. Furthermore, there were contradictions between the conclusions of previous studies and the single cell RNAseq studies. To address these issues, we used mRNA probes for genes encoding secretory products expressed in different metacells in Trichoplax adhaerens to localize cells in whole mounts and in dissociated cell cultures, where their morphological features could be visualized and identified. The nature and functions of their secretory granules were further investigated with electron microscopic techniques and by imaging secretion in live animals during feeding episodes. We found that two cell types participate in disintegrating prey, one resembling a lytic cell type in mammals and another combining features of zymogen gland cells and enterocytes. We identified secretory epithelial cells expressing glycoproteins or short peptides implicated in defense. We located seven peptidergic cell types and two types of mucocytes. Our findings reveal mechanisms that placozoans use to feed and protect themselves from pathogens and clues about neuropeptidergic signaling. We compare placozoan secretory cell types with cell types in other animal phyla to gain insight about general evolutionary trends in cell type diversification, as well as pathways leading to the emergence of synapomorphies.

Importance: Natural language processing tools, such as ChatGPT (generative pretrained transformer, hereafter referred to as chatbot), have the potential to radically enhance the accessibility of medical information for health professionals and patients. Assessing the safety and efficacy of these tools in answering physician-generated questions is critical to determining their suitability in clinical settings, facilitating complex decision-making, and optimizing health care efficiency., Objective: To assess the accuracy and comprehensiveness of chatbot-generated responses to physician-developed medical queries, highlighting the reliability and limitations of artificial intelligence-generated medical information., Design, Setting, and Participants: Thirty-three physicians across 17 specialties generated 284 medical questions that they subjectively classified as easy, medium, or hard with either binary (yes or no) or descriptive answers. The physicians then graded the chatbot-generated answers to these questions for accuracy (6-point Likert scale with 1 being completely incorrect and 6 being completely correct) and completeness (3-point Likert scale, with 1 being incomplete and 3 being complete plus additional context). Scores were summarized with descriptive statistics and compared using the Mann-Whitney U test or the Kruskal-Wallis test. The study (including data analysis) was conducted from January to May 2023., Main Outcomes and Measures: Accuracy, completeness, and consistency over time and between 2 different versions (GPT-3.5 and GPT-4) of chatbot-generated medical responses., Results: Across all questions (n = 284) generated by 33 physicians (31 faculty members and 2 recent graduates from residency or fellowship programs) across 17 specialties, the median accuracy score was 5.5 (IQR, 4.0-6.0) (between almost completely and complete correct) with a mean (SD) score of 4.8 (1.6) (between mostly and almost completely correct). The median completeness score was 3.0 (IQR, 2.0-3.0) (complete and comprehensive) with a mean (SD) score of 2.5 (0.7). For questions rated easy, medium, and hard, the median accuracy scores were 6.0 (IQR, 5.0-6.0), 5.5 (IQR, 5.0-6.0), and 5.0 (IQR, 4.0-6.0), respectively (mean [SD] scores were 5.0 [1.5], 4.7 [1.7], and 4.6 [1.6], respectively; P = .05). Accuracy scores for binary and descriptive questions were similar (median score, 6.0 [IQR, 4.0-6.0] vs 5.0 [IQR, 3.4-6.0]; mean [SD] score, 4.9 [1.6] vs 4.7 [1.6]; P = .07). Of 36 questions with scores of 1.0 to 2.0, 34 were requeried or regraded 8 to 17 days later with substantial improvement (median score 2.0 [IQR, 1.0-3.0] vs 4.0 [IQR, 2.0-5.3]; P < .01). A subset of questions, regardless of initial scores (version 3.5), were regenerated and rescored using version 4 with improvement (mean accuracy [SD] score, 5.2 [1.5] vs 5.7 [0.8]; median score, 6.0 [IQR, 5.0-6.0] for original and 6.0 [IQR, 6.0-6.0] for rescored; P = .002)., Conclusions and Relevance: In this cross-sectional study, chatbot generated largely accurate information to diverse medical queries as judged by academic physician specialists with improvement over time, although it had important limitations. Further research and model development are needed to correct inaccuracies and for validation.

The synaptic cleft is the extracellular part of the synapse, bridging the pre- and postsynaptic membranes. The geometry and molecular organization of the cleft is gaining increased attention as an important determinant of synaptic efficacy. The present study by electron microscopy focuses on short-term morphological changes at the synaptic cleft under excitatory conditions. Depolarization of cultured hippocampal neurons with high K + results in an increased frequency of synaptic profiles with clefts widened at the periphery (open clefts), typically exhibiting patches of membranes lined by postsynaptic density, but lacking associated presynaptic membranes (18.0% open clefts in high K + compared to 1.8% in controls). Similarly, higher frequencies of open clefts were observed in adult brain upon a delay of perfusion fixation to promote excitatory/ischemic conditions. Inhibition of basal activity in cultured neurons through the application of TTX results in the disappearance of open clefts whereas application of NMDA increases their frequency (19.0% in NMDA vs. 5.3% in control and 2.6% in APV). Depletion of extracellular Ca 2+ with EGTA also promotes an increase in the frequency of open clefts (16.6% in EGTA vs. 4.0% in controls), comparable to that by depolarization or NMDA, implicating dissociation of Ca 2+ -dependent trans-synaptic bridges. Dissociation of transsynaptic bridges under excitatory conditions may allow perisynaptic mobile elements, such as AMPA receptors to enter the cleft. In addition, peripheral opening of the cleft would facilitate neurotransmitter clearance and thus may have a homeostatic and/or protective function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tao-Cheng, Moreira, Winters, Reese and Dosemeci.)

The chemical synapse is a complex machine separated into three parts: presynaptic, postsynaptic, and cleft. Super-resolution light microscopy has revealed alignment of presynaptic vesicle release machinery and postsynaptic neurotransmitter-receptors and scaffolding components in synapse spanning nanocolumns. Cryo-electron tomography confirmed that postsynaptic glutamate receptor-like structures align with presynaptic structures in proximity to synaptic vesicles into transsynaptic assemblies. In our electron tomographic renderings, nearly all transcleft structures visibly connect to intracellular structures through transmembrane structures to form transsynaptic assemblies, potentially providing a structural basis for transsynaptic alignment. Here, we describe the patterns of composition, distribution, and interactions of all assemblies spanning the synapse by producing three-dimensional renderings of all visibly connected structures in excitatory and inhibitory synapses in dissociated rat hippocampal neuronal cultures of both sexes prepared by high-pressure freezing and freeze-substitution. The majority of transcleft structures connect to material in both presynaptic and postsynaptic compartments. We found several instances of assemblies connecting to both synaptic vesicles and postsynaptic density scaffolding. Each excitatory synaptic vesicle within 30 nm of the active zone contacts one or more assembly. Further, intracellular structures were often shared between assemblies, entangling them to form larger complexes or association domains, often in small clusters of vesicles. Our findings suggest that transsynaptic assemblies physically connect the three compartments, allow for coordinated molecular organization, and may combine to form specialized functional association domains, resembling the light-level nanocolumns. SIGNIFICANCE STATEMENT A recent tomographic study uncovered that receptor-like cleft structures align across the synapse. These aligned structures were designated as transsynaptic assemblies and demonstrate the coordinated organization of synaptic transmission molecules between compartments. Our present tomographic study expands on the definition of transsynaptic assemblies by analyzing the three-dimensional distribution and connectivity of all cleft-spanning structures and their connected intracellular structures. While one-to-one component alignment occurs across the synapse, we find that many assemblies share components, leading to a complex entanglement of assemblies, typically around clusters of synaptic vesicles. Transsynaptic assemblies appear to form domains which may be the structural basis for alignment of molecular nanodomains into synapse spanning nanocolumns described by super-resolution light microscopy., (Copyright © 2023 the authors.)

Postsynaptic densities (PSDs) are large protein complexes associated with the postsynaptic membrane of excitatory synapses important for synaptic function including plasticity. Conventional electron microscopy (EM) typically depicts PSDs as compact disk-like structures of hundreds of nanometers in size. Biochemically isolated PSDs were also similar in dimension revealing a predominance of proteins with the ability to polymerize into an extensive scaffold; several EM studies noted their irregular contours with often small granular structures (<30 nm) and holes. Super-resolution light microscopy studies observed clusters of PSD elements and their activity-induced lateral movement. Furthermore, our recent EM study on PSD fractions after sonication observed PSD fragments (40-90 nm in size) separate from intact PSDs; however, such structures within PSDs remained unidentified. Here we examined isolated PSDs by cryo-EM tomography with our new approach of automatic segmentation that enables delineation of substructures and their quantitative analysis. The delineated substructures broadly varied in size, falling behind 30 nm or exceeding 100 nm and showed that a considerable portion of the substructures (>38%) in isolated PSDs was in the same size range as those fragments. Furthermore, substructures spanning the entire thickness of the PSD were found, large enough to contain both membrane-associated and cytoplasmic proteins of the PSD; interestingly, they were similar to nanodomains in frequency. The structures detected here appear to constitute the isolated PSD as modules of various compositions, and this modular nature may facilitate remodeling of the PSD for proper synaptic function and plasticity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jung, Chen and Reese.)

A-kinase anchoring protein 79-human/150-rodent (AKAP79/150) organizes signaling proteins to control synaptic plasticity. AKAP79/150 associates with the plasma membrane and endosomes through its N-terminal domain that contains three polybasic regions and two Cys residues that are reversibly palmitoylated. Mutations abolishing palmitoylation (AKAP79/150 CS) reduce its endosomal localization and association with the postsynaptic density (PSD). Here we combined advanced light and electron microscopy (EM) to characterize the effects of AKAP79/150 palmitoylation on its postsynaptic nanoscale organization, trafficking, and mobility in hippocampal neurons. Immunogold EM revealed prominent extrasynaptic membrane AKAP150 labeling with less labeling at the PSD. The label was at greater distances from the spine membrane for AKAP150 CS than WT in the PSD but not in extra-synaptic locations. Immunogold EM of GFP-tagged AKAP79 WT showed that AKAP79 adopts a vertical, extended conformation at the PSD with its N-terminus at the membrane, in contrast to extrasynaptic locations where it adopts a compact or open configurations of its N- and C-termini with parallel orientation to the membrane. In contrast, GFP-tagged AKAP79 CS was displaced from the PSD coincident with disruption of its vertical orientation, while proximity and orientation with respect to the extra-synaptic membrane was less impacted. Single-molecule localization microscopy (SMLM) revealed a heterogeneous distribution of AKAP150 with distinct high-density, nano-scale regions (HDRs) overlapping the PSD but more prominently located in the extrasynaptic membrane for WT and the CS mutant. Thick section scanning transmission electron microscopy (STEM) tomography revealed AKAP150 immunogold clusters similar in size to HDRs seen by SMLM and more AKAP150 labeled endosomes in spines for WT than for CS, consistent with the requirement for AKAP palmitoylation in endosomal trafficking. Hidden Markov modeling of single molecule tracking data revealed a bound/immobile fraction and two mobile fractions for AKAP79 in spines, with the CS mutant having shorter dwell times and faster transition rates between states than WT, suggesting that palmitoylation stabilizes individual AKAP molecules in various spine subpopulations. These data demonstrate that palmitoylation fine tunes the nanoscale localization, mobility, and trafficking of AKAP79/150 in dendritic spines, which might have profound effects on its regulation of synaptic plasticity., Competing Interests: AP was employed by University of Colorado when the research was conducted. She is currently employed by a company Recursion Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chen, Crosby, Feng, Purkey, Aronova, Winters, Crocker, Leapman, Reese and Dell’Acqua.)

TPMT and NUDT15 variants explain less than 25% of azathioprine-associated myelotoxicity. There are 25 additional genes in the thiopurine pathway that could also contribute to azathioprine myelotoxicity. We hypothesized that among TPMT and NUDT15 normal metabolizers, a score combining the genetically predicted expression of other proteins in the thiopurine pathway would be associated with a higher risk for azathioprine discontinuation due to myelotoxicity. We conducted a retrospective cohort study of new users of azathioprine who were normal TPMT and NUDT15 metabolizers. In 1201 White patients receiving azathioprine for an inflammatory disease, we used relaxed Least Absolute Shrinkage and Selection Operator (LASSO) regression to select genes that built a score for discontinuing azathioprine due to myelotoxicity. The score incorporated the predicted expression of AOX1 and NME1. Patients in the highest score tertile had a higher risk of discontinuing azathioprine compared to those in the lowest tertile (hazard ratio [HR] = 2.15, 95% confidence interval [CI] = 1.11-4.19, p = 0.024). Results remained significant after adjusting for a propensity score, including sex, tertile of calendar year at initial dose, initial dose, age at baseline, indication, prior TPMT testing, and the first 10 principal components of the genetic data (HR = 2.11, 95% CI = 1.08-4.13, p = 0.030). We validated the results in a cohort (N = 517 non-White patients and those receiving azathioprine to prevent transplant rejection) that included all other patients receiving azathioprine (HR = 2.00, (95% CI = 1.09-3.65, p = 0.024). In conclusion, among patients who were TPMT and NUDT15 normal metabolizers, a score combining the predicted expression of AOX1 and NME1 was associated with an increased risk for discontinuing azathioprine due to myelotoxicity., (© 2022 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Placozoa is a phylum of non-bilaterian marine animals. These small, flat organisms adhere to the substrate via their densely ciliated ventral epithelium, which mediates mucociliary locomotion and nutrient uptake. They have only six morphological cell types, including one, fiber cells, for which functional data is lacking. Fiber cells are non-epithelial cells with multiple processes. We used electron and light microscopic approaches to unravel the roles of fiber cells in Trichoplax adhaerens, a representative member of the phylum. Three-dimensional reconstructions of serial sections of Trichoplax showed that each fiber cell is in contact with several other cells. Examination of fiber cells in thin sections and observations of live dissociated fiber cells demonstrated that they phagocytose cell debris and bacteria. In situ hybridization confirmed that fiber cells express genes involved in phagocytic activity. Fiber cells also are involved in wound healing as evidenced from microsurgery experiments. Based on these observations we conclude that fiber cells are multi-purpose macrophage-like cells. Macrophage-like cells have been described in Porifera, Ctenophora, and Cnidaria and are widespread among Bilateria, but our study is the first to show that Placozoa possesses this cell type. The phylogenetic distribution of macrophage-like cells suggests that they appeared early in metazoan evolution., (© 2021. The Author(s).)

Trichoplax adhaerens is an enigmatic animal with an extraordinarily simple morphology and a cellular organization, which are the focus of current research. Protocols outlined here provide detailed descriptions of advanced techniques for light and electron microscopic studies of Trichoplax. Studies using these techniques have enhanced our understanding of cell type diversity and function in placozoans and have provided insight into the evolution, development, and physiology of this little understood group.

Postsynaptic density (PSD) fractions were isolated from rat forebrain and sonicated. Pellets from sonicated samples examined by electron microscopy revealed particles with an electron density similar to PSDs that appeared to be fragments of PSDs. Immuno-gold labeling confirmed that some of these contained PSD-95 and/or SynGAP. Biochemical analysis of supernatant and pellet fractions from sonicated samples showed almost complete recovery of several major PSD components (SynGAP, PSD-95, Shank3, Homer and Glutamate receptors) in the pellet, while the supernatant contained known contaminants of PSD fractions, such as glial acidic fibrillary protein and neurofilament protein, as well as actin and α-actinin, indicating susceptibility of these cytoskeletal elements to mechanical disruption. Size distributions of particulate material in control and sonicated samples were clearly different, with particles in the 40-90 nm range observed only in sonicated samples. Fragmentation of the PSD into subcomplexes containing major constituents suggests a patchwork structure consisting of weakly bound modules, that can be readily dissociated from each other through mechanical disruption. Modular organization and weak association between modules would endow the PSD with lateral structural flexibility.

The disk-shaped millimeter-sized marine animal, Trichoplax adhaerens , is notable because of its small number of cell types and primitive mode of feeding. It glides on substrates propelled by beating cilia on its lower surface and periodically pauses to feed on underlying microorganisms, which it digests externally. Here, a combination of advanced electron and light microscopic techniques are used to take a closer look at its secretory cell types and their roles in locomotion and feeding. We identify digestive enzymes in lipophils, a cell type implicated in external digestion and distributed uniformly throughout the ventral epithelium except for a narrow zone near its edge. We find three morphologically distinct types of gland cell. The most prevalent contains and secretes mucus, which is shown to be involved in adhesion and gliding. Half of the mucocytes are arrayed in a tight row around the edge of the ventral epithelium while the rest are scattered further inside, in the region containing lipophils. The secretory granules in mucocytes at the edge label with an antibody against a neuropeptide that was reported to arrest ciliary beating during feeding. A second type of gland cell is arrayed in a narrow row just inside the row of mucocytes while a third is located more centrally. Our maps of the positions of the structurally distinct secretory cell types provide a foundation for further characterization of the multiple peptidergic cell types in Trichoplax and the microscopic techniques we introduce provide tools for carrying out these studies., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Trichoplax adhaerens is a small, ciliated marine animal that glides on surfaces grazing upon algae, which it digests externally. It has no muscles or nervous system and only six cell types, all but two of which are embedded in its epithelium. The epithelial cells are joined by apical adherens junctions; neither tight junctions nor gap junctions are present. Monociliated epithelial cells on the lower surface propel gliding. The cilia beat regularly, but asynchronously, and transiently contact the substrate with each stroke. The animal moves in random directions in the absence of food. We show here that it exhibits chemotaxis, moving preferentially toward algae embedded in a disk of agar. We present a mathematical model to explain how coherent, directional movements could arise from the collective actions of a set of ciliated epithelial cells, each independently sensing and responding to a chemoattractant gradient. The model incorporates realistic values for viscoelastic properties of cells and produces coordinated movements and changes in body shape that resemble the actual movements of the animal. The model demonstrates that an animal can move coherently in search of food without any need for chemical signaling between cells and introduces a different approach to modeling behavior in primitive multicellular organisms., (Copyright © 2019 the Author(s). Published by PNAS.)

Analysis of affinity-purified PSD-95 complexes had previously identified a 'hypothetical protein', product of the gene FAM81A [1]. The present study examined the tissue and subcellular distribution of FAM81A protein and its expression levels during development. Comparison of different organs indicates selective expression of FAM81A protein in brain. FAM81A is expressed late in development, with a post-natal gradual increase in brain levels that parallels the expression of PSD-95. Comparison of subcellular fractions from adult brain shows that the distribution of FAM81A protein is similar to that of PSD-95, with a drastic enrichment in the postsynaptic density fraction. Immuno-electron microscopy of adult brain tissue reveals specific immunogold labeling for FAM81A protein at postsynaptic densities in the forebrain. The label for FAM81A protein is concentrated at the cytoplasmic edge of the electron-dense core of the postsynaptic density, with a mean distance of ∼33 nm from the postsynaptic membrane. These observations firmly establish FAM81A protein as a component of the postsynaptic density in the adult brain, suggesting a role in synaptic function., (Published by Elsevier B.V.)

Combining tomography with electron microscopy (EM) produces images at definition sufficient to visualize individual protein molecules or molecular complexes in intact neurons. When freeze-substituted hippocampal cultures in plastic sections are imaged by EM tomography, detailed structures emerging from 3D reconstructions reveal putative glutamate receptors and membrane-associated filaments containing scaffolding proteins such as postsynaptic density (PSD)-95 family proteins based on their size, shape, and known distributions. In limited instances, structures can be identified with enhanced immuno-Nanogold labeling after light fixation and subsequent freeze-substitution. Molecular identification of structure can be corroborated in their absence after acute protein knockdown or gene knockout. However, additional labeling methods linking EM level structure to molecules in tomograms are needed. A recent development for labeling structures for TEM employs expression of endogenous proteins carrying a green fluorescent tag, miniSOG, to photoconvert diaminobenzidine (DAB) into osmiophilic polymers. This approach requires initial mild chemical fixation but many of structural features in neurons can still be discerned in EM tomograms. The photoreaction product, which appears as electron-dense, fine precipitates decorating protein structures in neurons, may diffuse to fill cytoplasm of spines, thus obscuring specific localization of proteins tagged with miniSOG. Here we develop an approach to minimize molecular diffusion of the DAB photoreaction product in neurons, which allows miniSOG tagged molecule/complexes to be identified in tomograms. The examples reveal electron-dense clusters of reaction product labeling membrane-associated vertical filaments, corresponding to the site of miniSOG fused at the C -terminal end of PSD-95-miniSOG, allowing identification of PSD-95 vertical filaments at the PSD. This approach, which results in considerable improvement in the precision of labeling PSD-95 in tomograms without complications due to the presence of antibody complexes in immunogold labeling, may be applicable for identifying other synaptic proteins in intact neurons.

Densin is a scaffold protein known to associate with key elements of neuronal signaling. The present study examines the distribution of densin at the ultrastructural level in order to reveal potential sites that can support specific interactions of densin. Immunogold electron microscopy on hippocampal cultures shows intense labeling for densin at postsynaptic densities (PSDs), but also some labeling at extrasynaptic plasma membranes of soma and dendrites and endoplasmic reticulum. At the PSD, the median distance of label from the postsynaptic membrane was ~27 nm, with the majority of label (90%) confined within 40 nm from the postsynaptic membrane, indicating predominant localization of densin at the PSD core. Depolarization (90 mM K+ for 2 min) promoted a slight shift of densin label within the PSD complex resulting in 77% of label remaining within 40 nm from the postsynaptic membrane. Densin molecules firmly embedded within the PSD may target a minor pool of CaMKII to substrates at the PSD core., Competing Interests: The authors have declared that no competing interests exist.

Trichoplax adhaerens has only six cell types. The function as well as the structure of crystal cells, the least numerous cell type, presented an enigma. Crystal cells are arrayed around the perimeter of the animal and each contains a birefringent crystal. Crystal cells resemble lithocytes in other animals so we looked for evidence they are gravity sensors. Confocal microscopy showed that their cup-shaped nuclei are oriented toward the edge of the animal, and that the crystal shifts downward under the influence of gravity. Some animals spontaneously lack crystal cells and these animals behaved differently upon being tilted vertically than animals with a typical number of crystal cells. EM revealed crystal cell contacts with fiber cells and epithelial cells but these contacts lacked features of synapses. EM spectroscopic analyses showed that crystals consist of the aragonite form of calcium carbonate. We thus provide behavioral evidence that Trichoplax are able to sense gravity, and that crystal cells are likely to be their gravity receptors. Moreover, because placozoans are thought to have evolved during Ediacaran or Cryogenian eras associated with aragonite seas, and their crystals are made of aragonite, they may have acquired gravity sensors during this early era.

Trichoplax adhaerens is a flat, millimeter-sized marine animal that adheres to surfaces and grazes on algae. Trichoplax displays a repertoire of different feeding behaviors despite the apparent absence of a true nervous system with electrical or chemical synapses. It glides along surfaces to find food, propelled by beating cilia on cells at its ventral surface, and pauses during feeding by arresting ciliary beating. We found that when endomorphin-like peptides are applied to an animal, ciliary beating is arrested, mimicking natural feeding pauses. Antibodies against these neuropeptides label cells that express the neurosecretory proteins and voltage-gated calcium channels implicated in regulated secretion. These cells are embedded in the ventral epithelium, where they comprise only 4% of the total, and are concentrated around the edge of the animal. Each bears a cilium likely to be chemosensory and used to detect algae. Trichoplax pausing during feeding or spontaneously in the absence of food often induce their neighbors to pause as well, even neighbors not in direct contact. Pausing behavior propagates from animal to animal across distances much greater than the signal that diffuses from just one animal, so we presume that the peptides secreted from one animal elicit secretion from nearby animals. Signal amplification by peptide-induced peptide secretion explains how a small number of sensory secretory cells lacking processes and synapses can evoke a wave of peptide secretion across the entire animal to globally arrest ciliary beating and allow pausing during feeding., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Four-domain voltage-gated Ca 2+ (Ca v ) channels play fundamental roles in the nervous system, but little is known about when or how their unique properties and cellular roles evolved. Of the three types of metazoan Ca v channels, Ca v 1 (L-type), Ca v 2 (P/Q-, N- and R-type) and Ca v 3 (T-type), Ca v 3 channels are optimized for regulating cellular excitability because of their fast kinetics and low activation voltages. These same properties permit Ca v 3 channels to drive low-threshold exocytosis in select neurons and neurosecretory cells. Here, we characterize the single T-type calcium channel from Trichoplax adhaerens (TCa v 3), an early diverging animal that lacks muscle, neurons, and synapses. Co-immunolocalization using antibodies against TCa v 3 and neurosecretory cell marker complexin labeled gland cells, which are hypothesized to play roles in paracrine signaling. Cloning and in vitro expression of TCa v 3 reveals that, despite roughly 600 million years of divergence from other T-type channels, it bears the defining structural and biophysical features of the Ca v 3 family. We also characterize the channel's cation permeation properties and find that its pore is less selective for Ca 2+ over Na + compared with the human homologue Ca v 3.1, yet it exhibits a similar potent block of inward Na + current by low external Ca 2+ concentrations (i.e., the Ca 2+ block effect). A comparison of the permeability features of TCa v 3 with other cloned channels suggests that Ca 2+ block is a locus of evolutionary change in T-type channel cation permeation properties and that mammalian channels distinguish themselves from invertebrate ones by bearing both stronger Ca 2+ block and higher Ca 2+ selectivity. TCa v 3 is the most divergent metazoan T-type calcium channel and thus provides an evolutionary perspective on Ca v 3 channel structure-function properties, ion selectivity, and cellular physiology., (© 2017 Smith et al.)

Postsynaptic density protein 95 (PSD95) and synapse-associated protein 97 (SAP97) are homologous scaffold proteins with different N-terminal domains, possessing either a palmitoylation site (PSD95) or an L27 domain (SAP97). Here, we measured PSD95 and SAP97 conformation in vitro and in postsynaptic densities (PSDs) using FRET and EM, and examined how conformation regulated interactions with AMPA-type and NMDA-type glutamate receptors (AMPARs/NMDARs). Palmitoylation of PSD95 changed its conformation from a compact to an extended configuration. PSD95 associated with AMPARs (via transmembrane AMPAR regulatory protein subunits) or NMDARs [via glutamate ionotropic receptor NMDA-type subunit 2B (GluN2B) subunits] only in its palmitoylated and extended conformation. In contrast, in its extended conformation, SAP97 associates with NMDARs, but not with AMPARs. Within PSDs, PSD95 and SAP97 were largely in the extended conformation, but had different orientations. PSD95 oriented perpendicular to the PSD membrane, with its palmitoylated, N-terminal domain at the membrane. SAP97 oriented parallel to the PSD membrane, likely as a dimer through interactions of its N-terminal L27 domain. Changing PSD95 palmitoylation in PSDs altered PSD95 and AMPAR levels but did not affect NMDAR levels. These results indicate that in PSDs, PSD95 palmitoylation, conformation, and its interactions are dynamic when associated with AMPARs and more stable when associated with NMDARs. Altogether, our results are consistent with differential regulation of PSD95 palmitoylation in PSDs resulting from the clustering of palmitoylating and depalmitoylating enzymes into AMPAR nanodomains segregated away from NMDAR nanodomains., Competing Interests: The authors declare no conflict of interest.

Trichoplax adhaerens is the sole named member of Placozoa, an ancient metazoan phylum. This coin-shaped animal glides on ventral cilia to find and digest algae on the substrate. It has only six cell types, all but two of which are incorporated into the epithelium that encloses it. The upper epithelium is thin, composed of a pavement of relatively large polygonal disks, each bearing a cilium. The lower epithelium is thick and composed primarily of narrow ciliated cells that power locomotion. Interspersed among these cells are two different secretory cells: one containing large lipophilic granules that, when released, lyse algae under the animal; the other, less abundant, is replete with smaller secretory granules containing neuropeptides. All cells within both epithelia are joined by adherens junctions that are stabilized by apical actin networks. Cells are held in place during shape changes or under osmotic stress, but dissociate in low calcium. Neither tight, septate, nor gap junctions are evident, leaving only the adherens junction to control the permeability of the epithelium. Small (<4 kDa) fluorescent dextrans introduced into artificial seawater readily penetrate into the animal between the cells. Larger dextrans enter slowly, except in animals treated with reduced calcium, indicating that the adherens junctions form a circumferential belt around each cell that impedes diffusion into the animal. During feeding, the limited permeability of the adherens junctions helps to confine material released from lysed algae within the narrow space under the animal, where it is absorbed by endocytosis.

The postsynaptic density (PSD), apparent in electron micrographs as a dense lamina just beneath the postsynaptic membrane, includes a deeper layer, the "pallium", containing a scaffold of Shank and Homer proteins. Though poorly defined in traditionally prepared thin-section electron micrographs, the pallium becomes denser and more conspicuous during intense synaptic activity, due to the reversible addition of CaMKII and other proteins. In this Perspective article, we review the significance of CaMKII-mediated recruitment of proteins to the pallium with respect to both the trafficking of receptors and the remodeling of spine shape that follow synaptic stimulation. We suggest that the level and duration of CaMKII translocation and activation in the pallium will shape activity-induced changes in the spine.

Shank3 is a postsynaptic density (PSD) scaffold protein of the Shank family. Here we use pre-embedding immunogold electron microscopy to investigate factors influencing the distribution of Shank3 at the PSD. In dissociated rat hippocampal cultures under basal conditions, label for Shank3 was concentrated in a broad layer of the PSD, ~20-80 nm from the postsynaptic membrane. Upon depolarization with high K+ (90 mM, 2 min), or application of NMDA (50 μM, 2 min), both the labeling intensity at the PSD and the median distance of label from the postsynaptic membrane increased significantly, indicating that Shank3 molecules are preferentially recruited to the distal layer of the PSD. Incubation in medium supplemented with zinc (50 μM ZnCl2, 1 hr) also significantly increased labeling intensity for Shank3 at the PSD, but this addition of Shank3 was not preferential to the distal layer. When cells were incubated with zinc and then treated with NMDA, labeling intensity of Shank3 became higher than with either treatment alone and manifested a preference for the distal layer of the PSD. Without zinc supplementation, NMDA-induced accumulation of Shank3 at the PSD was transient, reversing within 30 min after return to control medium. However, when zinc was included in culture media throughout the experiment, the NMDA-induced accumulation of Shank3 was largely retained, including Shank3 molecules recruited to the distal layer of the PSD. These results demonstrate that activity induces accumulation of Shank3 at the PSD and that zinc stabilizes PSD-associated Shank3, possibly through strengthening of Shank-Shank association.

Synaptic transmission between neurons requires precise management of synaptic vesicles. While individual molecular components of the presynaptic terminal are well known, exactly how the molecules are organized into a molecular machine serving the storage and mobilization of synaptic vesicles to the active zone remains unclear. Here we report three filament types associated with synaptic vesicles in glutamatergic synapses revealed by electron microscope tomography in unstimulated, dissociated rat hippocampal neurons. One filament type, likely corresponding to the SNAREpin complex, extends from the active zone membrane and surrounds docked vesicles. A second filament type contacts all vesicles throughout the active zone and pairs vesicles together. On the third filament type, vesicles attach to side branches extending from the long filament core and form vesicle clusters that are distributed throughout the vesicle cloud and along the active zone membrane. Detailed analysis of presynaptic structure reveals how each of the three filament types interacts with synaptic vesicles, providing a means to traffic reserved and recycled vesicles from the cloud of vesicles into the docking position at the active zone., Significance Statement: The formation and release of synaptic vesicles has been extensively investigated. Explanations of the release of synaptic vesicles generally begin with the movement of vesicles from the cloud into the synaptic active zone. However, the presynaptic terminal is filled with filamentous material that would appear to limit vesicular diffusion. Here, we provide a systematic description of three filament types connecting synaptic vesicles. A picture emerges illustrating how the cooperative attachment and release of these three filament types facilitate the movement of vesicles to the active zone to become docked in preparation for release., (Copyright © 2016 the authors 0270-6474/16/363222-09$15.00/0.)

The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane-associated PSD-95-like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane-associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.

AIDA-1 is highly enriched in postsynaptic density (PSD) fractions and is considered a major component of the PSD complex. In the present study, immunogold electron microscopy was applied to determine localization as well as the activity-induced redistribution of AIDA-1 at the PSD using two antibodies that recognize two different epitopes. In cultured rat hippocampal neurons under basal conditions, immunogold label for AIDA-1 is mostly located within the dense core of the PSD, with a median distance of ~30 nm from the postsynaptic membrane. Under excitatory conditions, such as depolarization with high K+ (90 mM, 2 min) or application of NMDA (50 μM, 2 min), AIDA-1 label density at the PSD core is reduced to 40% of controls and the median distance of label from the postsynaptic membrane increases to ~55 nm. The effect of excitatory conditions on the postsynaptic distribution of AIDA-1 is reversed within 30 minutes after returning to control conditions. The reversible removal of AIDA-1 from the PSD core under excitatory conditions is similar to the redistribution of another abundant PSD protein, SynGAP. Both SynGAP-alpha1 and AIDA-1 are known to bind PSD-95. Activity-induced transient translocation of these abundant proteins from the PSD core could promote structural flexibility, vacate sites on PSD-95 for the insertion of other components and thus may create a window for synaptic modification.

Trichoplax is a small disk-shaped marine metazoan that adheres to substrates and locomotes by ciliary gliding. Despite having only six cell types and lacking synapses Trichoplax coordinates a complex sequence of behaviors culminating in external digestion of algae. We combine live cell imaging with electron microscopy to show how this is accomplished. When Trichoplax glides over a patch of algae, its cilia stop beating so it ceases moving. A subset of one of the cell types, lipophils, simultaneously secretes granules whose content rapidly lyses algae. This secretion is accurately targeted, as only lipophils located near algae release granules. The animal pauses while the algal content is ingested, and then resumes gliding. Global control of gliding is coordinated with precise local control of lipophil secretion suggesting the presence of mechanisms for cellular communication and integration.

The mechanisms controlling synapse growth and maintenance are of critical importance for learning and memory. The MAGUK family of synaptic scaffolding proteins is abundantly expressed at glutamatergic central synapses, but their importance in controlling the synaptic content of glutamate receptors is poorly understood. Here, we use a chained RNAi-mediated knockdown approach to simultaneously remove PSD-93, PSD-95, and SAP102, the MAGUKs previously shown to be responsible for synaptic localization of glutamate receptors. We find that MAGUKs are specifically responsible for creating functional synapses after initial spine formation by filling functionally silent spines with glutamate receptors. Removal of the MAGUKs causes a transient reduction in AMPA receptor quantal size followed by synaptic consolidation resulting in a normalization of quantal size at the few remaining functional synapses. Consolidation requires signaling through L-type calcium channels, CaM kinase kinase, and the GluA2 AMPA receptor subunit, akin to a homeostatic process., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Electron microscopy has revealed an abundance of material in the clefts of synapses in the mammalian brain, and the biochemical and functional characteristics of proteins occupying synaptic clefts are well documented. However, the detailed spatial organization of the proteins in the synaptic clefts remains unclear. Electron microscope tomography provides a way to delineate and map the proteins spanning the synaptic cleft because freeze substitution preserves molecular details with sufficient contrast to visualize individual cleft proteins. Segmentation and rendering of electron dense material connected across the cleft reveals discrete structural elements that are readily classified into five types at excitatory synapses and four types at inhibitory synapses. Some transcleft elements resemble shapes and sizes of known proteins and could represent single dimers traversing the cleft. Some of the types of cleft elements at inhibitory synapses roughly matched the structure and proportional frequency of cleft elements at excitatory synapses, but the patterns of deployments in the cleft are quite different. Transcleft elements at excitatory synapses were often evenly dispersed in clefts of uniform (18 nm) width but some types show preference for the center or edges of the cleft. Transcleft elements at inhibitory synapses typically were confined to a peripheral region of the cleft where it narrowed to only 6 nm wide. Transcleft elements in both excitatory and inhibitory synapses typically avoid places where synaptic vesicles attach to the presynaptic membrane. These results illustrate that elements spanning synaptic clefts at excitatory and inhibitory synapses consist of distinct structures arranged by type in a specific but different manner at excitatory and inhibitory synapses.

Shank and GKAP are scaffold proteins and binding partners at the postsynaptic density (PSD). The distribution and dynamics of Shank and GKAP were studied in dissociated hippocampal cultures by pre-embedding immunogold electron microscopy. Antibodies against epitopes containing their respective mutual binding sites were used to verify the expected juxtapositioning of Shank and GKAP. If all Shank and GKAP molecules at the PSD were bound to each other, the distribution of label for the two proteins should coincide. However, labels for the mutual binding sites showed significant differences in distribution, with a narrow distribution for GKAP located close to the postsynaptic membrane, and a wider distribution for Shank extending deeper into the cytoplasm. Upon depolarization with high K+, neither the intensity nor distribution of label for GKAP changed, but labeling intensity for Shank at the PSD increased to ~150% of controls while the median distance of label from postsynaptic membrane increased by 7.5 nm. These results indicate a preferential recruitment of Shank to more distal parts of the PSD complex. Conversely, upon incubation in Ca2+-free medium containing EGTA, the labeling intensity of Shank at the PSD decreased to ~70% of controls and the median distance of label from postsynaptic membrane decreased by 9 nm, indicating a preferential loss of Shank molecules in more distal parts of the PSD complex. These observations identify two pools of Shank at the PSD complex, one relatively stable pool, closer to the postsynaptic membrane that can bind to GKAP, and another more dynamic pool at a location too far away to bind to GKAP.

Syntaxins are a family of transmembrane proteins that participate in SNARE complexes to mediate membrane fusion events including exocytosis. Different syntaxins are thought to participate in exocytosis in different compartments of the nervous system such as the axon, the soma/dendrites or astrocytes. It is well known that exocytosis of synaptic vesicles at axonal presynaptic terminals involves syntaxin 1 but distributions of syntaxins on neuronal somal and dendritic, postsynaptic or astroglial plasma membranes are less well characterized. Here, we use pre-embedding immunogold labeling to compare the distribution of two plasma membrane-enriched syntaxins (1 and 4) in dissociated rat hippocampal cultures as well as in perfusion-fixed mouse brains. Comparison of Western blots of neuronal cultures, consisting of a mixture of hippocampal neurons and glia, with glial cultures, consisting of mostly astrocytes, shows that syntaxin 1 is enriched in neuronal cultures, whereas syntaxin 4 is enriched in glial cultures. Electron microscopy (EM)-immunogold labeling shows that syntaxin 1 is most abundant at the plasma membranes of axons and terminals, while syntaxin 4 is most abundant at astroglial plasma membranes. This differential distribution was evident even at close appositions of membranes at synapses, where syntaxin 1 was localized to the plasma membrane of the presynaptic terminal, including that at the active zone, while syntaxin 4 was localized to nearby peri-synaptic astroglial processes. These results show that syntaxin 4 is available to support exocytosis in astroglia., (Published by Elsevier Ltd.)

Background: Trichoplax adhaerens is the best-known member of the phylum Placozoa, one of the earliest-diverging metazoan phyla. It is a small disk-shaped animal that glides on surfaces in warm oceans to feed on algae. Prior anatomical studies of Trichoplax revealed that it has a simple three-layered organization with four somatic cell types., Results: We reinvestigate the cellular organization of Trichoplax using advanced freezing and microscopy techniques to identify localize and count cells. Six somatic cell types are deployed in stereotyped positions. A thick ventral plate, comprising the majority of the cells, includes ciliated epithelial cells, newly identified lipophil cells packed with large lipid granules, and gland cells. Lipophils project deep into the interior, where they alternate with regularly spaced fiber cells whose branches contact all other cell types, including cells of the dorsal and ventral epithelium. Crystal cells, each containing a birefringent crystal, are arrayed around the rim. Gland cells express several proteins typical of neurosecretory cells, and a subset of them, around the rim, also expresses an FMRFamide-like neuropeptide., Conclusions: Structural analysis of Trichoplax with significantly improved techniques provides an advance in understanding its cell types and their distributions. We find two previously undetected cell types, lipohil and crystal cells, and an organized body plan in which different cell types are arranged in distinct patterns. The composition of gland cells suggests that they are neurosecretory cells and could control locomotor and feeding behavior., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Shank is a specialized scaffold protein present in high abundance at the postsynaptic density (PSD). Using pre-embedding immunogold electron microscopy on cultured hippocampal neurons, we had previously demonstrated further accumulation of Shank at the PSD under excitatory conditions. Here, using the same experimental protocol, we demonstrate that a cell permeable CaMKII inhibitor, tatCN21, blocks NMDA-induced accumulation of Shank at the PSD. Furthermore we show that NMDA application changes the distribution pattern of Shank at the PSD, promoting a 7-10 nm shift in the median distance of Shank labels away from the postsynaptic membrane. Inhibition of CaMKII with tatCN21 also blocks this shift in the distribution of Shank. Altogether these results imply that upon activation of NMDA receptors, CaMKII mediates accumulation of Shank, preferentially at the distal regions of the PSD complex extending toward the cytoplasm., (Published by Elsevier Inc.)

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 ∼30-100nm 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 60nm 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 (2min) 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 (90mM for 2-5min), N-methyl-d-asparic acid (NMDA) treatment (50μM for 2min), and calcium-free conditions (EGTA at 1mM for 2min). 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., (Published by Elsevier Ltd.)

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.

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. 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 by 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., (Copyright © 2013 Wiley Periodicals, Inc.)

SynGAP, a protein abundant at the postsynaptic density (PSD) of glutamatergic neurons, is known to modulate synaptic strength by regulating the incorporation of AMPA receptors at the synapse. Two isoforms of SynGAP, α1 and α2, which differ in their C-termini, have opposing effects on synaptic strength. In the present study, antibodies specific for SynGAP-α1 and SynGAP-α2 are used to compare the distribution patterns of the two isoforms at the postsynaptic density (PSD) under basal and excitatory conditions. Western immunoblotting shows enrichment of both isoforms in PSD fractions isolated from adult rat brain. Immunogold electron microscopy of rat hippocampal neuronal cultures shows similar distribution of both isoforms at the PSD, with a high density of immunolabel within the PSD core under basal conditions. Application of NMDA promotes movement of SynGAP-α1 as well as SynGAP-α2 out of the PSD core. In isolated PSDs both isoforms of SynGAP can be phosphorylated upon activation of the endogenous CaMKII. Application of tatCN21, a cell-penetrating inhibitor of CaMKII, to hippocampal neuronal cultures blocks NMDA-induced redistribution of SynGAP-α1 and SynGAP-α2. Thus CaMKII activation promotes the removal of two distinct C-terminal SynGAP variants from the PSD.

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., (Published by Elsevier Ltd.)

Polyubiquitin chains on proteins flag them for distinct fates depending on the type of polyubiquitin linkage. While lysine48-linked polyubiquitination directs proteins to proteasomal 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., (Published by Elsevier Inc.)

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., (Copyright © 2012 Wiley Periodicals, Inc.)

The calcium calmodulin dependent kinase (CaMKII) is important for long-term potentiation at dendritic spines. Photo-activatable GFP (PaGFP) - CaMKII fusions were used to map CaMKII movements between and within spines in dissociated hippocampal neurons. Photo-activated PaGFP (GFP*) generated in the shaft spread uniformly, but was retained for about 1 s in spines. The differential localization of GFP*-CaMKII isoforms was visualized with hundred nanometer precision frame to frame using de-noising algorithms. GFP*-CaMKIIα localized to the tips of mushroom spines. The spatiotemporal profiles of native and kinase defective GFP*-CaMKIIβ, differed markedly from GFP*-CaMKIIα and mutant GFP*-CaMKIIβ lacking the association domain. CaMKIIβ bound to cortical actin in the dendrite and the stable actin network in spine bodies. Glutamate produced a transiently localized GFP*-CaMKIIα fraction and a soluble GFP*-CaMKIIβ fraction in spine bodies. Single molecule simulations of the interplay between diffusion and biochemistry of GFP* species were guided by the spatiotemporal maps and set limits on binding parameters. They highlighted the role of spine morphology in modulating bound CaMKII lifetimes. The long residence times of GFP*-CaMKIIβ relative to GFP*-CaMKIIα followed as consequence of more binding sites on the actin cytoskeleton than the post-synaptic density. These factors combined to retain CaMKII for tens of seconds, sufficient to outlast the calcium transients triggered by glutamate, without invoking complex biochemistry.

While negative staining can provide detailed, two-dimensional images of biological structures, the potential of combining tomography with negative staining to provide three-dimensional views has yet to be fully realized. Basic requirements of a negative stain for tomography are that the density and atomic number of the stain are optimal, and that the stain does not degrade or rearrange with the intensive electron dose (~10⁶ e/nm²) needed to collect a full set of tomographic images. A commercially available, tungsten-based stain appears to satisfy these prerequisites. Comparison of the surface structure of negatively stained influenza A virus with previous structural results served to evaluate this negative stain. The combination of many projections of the same structure yielded detailed images of single proteins on the viral surface. Corresponding surface renderings are a good fit to images of the viral surface derived from cryomicroscopy as well as to the shapes of crystallized surface proteins. Negative stain tomography with the appropriate stain yields detailed images of individual molecules in their normal setting on the surface of the influenza A virus.

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-CaMKIIα 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.

SynGAP is a Ras GTPase activating protein present at the postsynaptic density (PSD) in quantities matching those of the core scaffold protein PSD-95. SynGAP is reported to inhibit synaptic accumulation of AMPA receptors. Here, we characterize by immunogold electron microscopy the distribution of SynGAP at the PSD under basal and depolarizing conditions in rat hippocampal neuronal cultures. The PSD core, extending up to 40 nm from the postsynaptic membrane, typically shows label for SynGAP, while half of the synapses exhibit additional labeling in a zone 40-120 nm from the postsynaptic membrane. Upon depolarization with high K(+), labeling for SynGAP significantly decreases at the core of the PSD and concomitantly increases at the 40-120 nm zone. Under the same depolarization conditions, label for PSD-95, the presumed binding partner of SynGAP, does not change its localization at the PSD. Depolarization-induced redistribution of SynGAP is reversible and also occurs upon application of N-methyl-d-aspartic acid (NMDA). Activity-induced movement of SynGAP could vacate sites in the PSD core allowing other elements to bind to these sites, such as transmembrane AMPA receptor regulatory proteins (TARPs), and simultaneously facilitate access of SynGAP to CaMKII and Ras, elements of a regulatory cascade., (Published by Elsevier Ltd.)