Reese Thomas S.

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Reese
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Thomas S.
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  • Preprint
    Inhibition of phosphatase activity facilitates the formation and maintenance of NMDA-induced calcium/calmodulin-dependent protein kinase ii clusters in hippocampal neurons
    ( 2004-10-04) Tao-Cheng, Jung-Hwa ; Vinade, Lucia ; Winters, Christine A. ; Reese, Thomas S. ; Dosemeci, Ayse
    The majority of hippocampal neurons in dissociated cultures and in intact brain exhibit clustering of CaMKII into spherical structures with an average diameter of 110 nm when subjected to conditions that mimic ischemia and excitotoxicity (Tao-Cheng et al., 2001). Because clustering of CaMKII would reduce its effective concentration within the neuron, it may represent a cellular strategy to prevent excessive CaMKII-mediated phosphorylation during episodes of Ca2+ overload. Here we employ a relatively mild excitatory stimulus to promote sub-maximal clustering for the purpose of studying the conditions for the formation and disappearance of CaMKII clusters. Treatment with 30 µM NMDA for 2 min produced CaMKII clustering in ~15 percent of dissociated hippocampal neurons in culture, as observed by pre-embedding immunogold electron microscopy. These CaMKII clusters could be labeled with antibodies specific to the phospho form (Thr286) of CaMKII, suggesting that at least some of the CaMKII molecules in clusters are autophosphorylated. To test whether phosphorylation is involved in the formation and maintenance of CaMKII clusters, the phosphatase inhibitors calyculin A (5 nM) or okadaic acid (1 µM) were included in the incubation medium. With inhibitors more neurons exhibited CaMKII clusters in response to 2 min NMDA treatment. Furthermore, 5 min after the removal of NMDA and Ca2+, CaMKII clusters remained and could still be labeled with the phospho-specific antibody. In contrast, in the absence of phosphatase inhibitors, no clusters were detected 5 min after the removal of NMDA and Ca2+ from the medium. These results suggest that phosphatases type 1 and/or 2A regulate the formation and disappearance of CaMKII clusters.
  • Article
    Distribution of postsynaptic density (PSD)-95 and Ca2+/calmodulin-dependent protein kinase II at the PSD
    (Society for Neuroscience, 2003-12-03) Petersen, Jennifer D. ; Chen, Xiaobing ; Vinade, Lucia ; Dosemeci, Ayse ; Lisman, John E. ; Reese, Thomas S.
    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.
  • Article
    Persistent accumulation of calcium/calmodulin-dependent protein kinase II in dendritic spines after induction of NMDA receptor-dependent chemical long-term potentiation
    (Society for Neuroscience, 2004-10-20) Otmakhov, Nikolai ; Tao-Cheng, Jung-Hwa ; Carpenter, Stephen ; Asrican, Brent ; Dosemeci, Ayse ; Reese, Thomas S. ; Lisman, John E.
    Calcium/calmodulin-dependent protein kinase II (CaMKII) is a leading candidate for a synaptic memory molecule because it is persistently activated after long-term potentiation (LTP) induction and because mutations that block this persistent activity prevent LTP and learning. Previous work showed that synaptic stimulation causes a rapidly reversible translocation of CaMKII to the synaptic region. We have now measured green fluorescent protein (GFP)-CaMKIIα translocation into synaptic spines during NMDA receptor-dependent chemical LTP (cLTP) and find that under these conditions, translocation is persistent. Using red fluorescent protein as a cell morphology marker, we found that there are two components of the persistent accumulation. cLTP produces a persistent increase in spine volume, and some of the increase in GFP-CaMKIIα is secondary to this volume change. In addition, cLTP results in a dramatic increase in the bound fraction of GFP-CaMKIIα in spines. To further study the bound pool, immunogold electron microscopy was used to measure CaMKIIα in the postsynaptic density (PSD), an important regulator of synaptic function. cLTP produced a persistent increase in the PSD-associated pool of CaMKIIα. These results are consistent with the hypothesis that CaMKIIα accumulation at synapses is a memory trace of past synaptic activity.
  • Preprint
    Palmitoylation regulates glutamate receptor distributions in postsynaptic densities through control of PSD95 conformation and orientation
    ( 2016-08) Jeyifous, Okunola ; Lin, Eric I. ; Chen, Xiaobing ; Antinone, Sarah E. ; Mastro, Ryan ; Drisdel, Renaldo ; Reese, Thomas S. ; Green, William N.
    PSD95 and 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 electron microscopy, and examined how conformation regulated interactions with AMPA-type and NMDAtype glutamate receptors (AMPARs/NMDARs). Palmitoylation of PSD95 changed its conformation from a compact to an extended configuration. PSD95 associated with AMPARs (via TARP subunits) or NMDARs (via GluN2B subunits) only in its palmitoylated and extended conformation. In contrast, SAP97 in its extended conformation 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.
  • Article
    Envelope structure of Synechococcus sp. WH8113, a nonflagellated swimming cyanobacterium
    (BioMed Central, 2001-04-24) Samuel, Aravinthan D. T. ; Petersen, Jennifer D. ; Reese, Thomas S.
    Many bacteria swim by rotating helical flagellar filaments. Waterbury et al. discovered an exception, strains of the cyanobacterium Synechococcus that swim without flagella or visible changes in shape. Other species of cyanobacteria glide on surfaces. The hypothesis that Synechococcus might swim using traveling surface waves prompted this investigation. Results Using quick-freeze electron microscopy, we have identified a crystalline surface layer that encloses the outer membrane of the motile strain Synechococcus sp. WH8113, the components of which are arranged in a rhomboid lattice. Spicules emerge in profusion from the layer and extend up to 150 nm into the surrounding fluid. These spicules also send extensions inwards to the inner cell membrane where motility is powered by an ion-motive force. Conclusion The envelope structure of Synechococcus sp. WH8113 provides new constraints on its motile mechanism. The spicules are well positioned to transduce energy at the cell membrane into mechanical work at the cell surface. One model is that an unidentified motor embedded in the cell membrane utilizes the spicules as oars to generate a traveling wave external to the surface layer in the manner of ciliated eukaryotes.
  • Article
    Isolation and ultrastructural characterization of squid synaptic vesicles
    (Marine Biological Laboratory, 2011-04) Pekkurnaz, Gulcin ; Fera, Andrea ; Zimmerberg-Helms, Jessica ; DeGiorgis, Joseph A. ; Bezrukov, Ludmila ; Blank, Paul S. ; Mazar, Julia ; Reese, Thomas S. ; Zimmerberg, Joshua
    Synaptic vesicles contain a variety of proteins and lipids that mediate fusion with the pre-synaptic membrane. 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 staining, and furthermore shows details of individual vesicles and other proteins at the vesicle surface.