48 results on '"Kamasawa, Naomi"'
Search Results
2. Challenges of Sample Preparations for vEM-CLEM.
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Kamasawa, Naomi
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- 2024
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3. Presynaptic Rac1 controls synaptic strength through the regulation of synaptic vesicle priming.
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Keine, Christian, Al-Yaari, Mohammed, Radulovic, Tamara, Thomas, Connon I., Valino Ramos, Paula, Guerrero Given, Debbie, Ranjan, Mrinalini, Taschenberger, Holger, Kamasawa, Naomi, and Young Jr, Samuel M.
- Published
- 2022
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4. Selective activation of BK channels in small‐headed dendritic spines suppresses excitatory postsynaptic potentials.
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Tazerart, Sabrina, Blanchard, Maxime G., Miranda‐Rottmann, Soledad, Mitchell, Diana E., Navea Pina, Bruno, Thomas, Connon I., Kamasawa, Naomi, and Araya, Roberto
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DENDRITIC spines ,EXCITATORY postsynaptic potential ,CALCIUM-dependent potassium channels ,PYRAMIDAL neurons ,GLUTAMATE receptors ,VISUAL cortex - Abstract
Dendritic spines are the main receptacles of excitatory information in the brain. Their particular morphology, with a small head connected to the dendrite by a slender neck, has inspired theoretical and experimental work to understand how these structural features affect the processing, storage and integration of synaptic inputs in pyramidal neurons (PNs). The activation of glutamate receptors in spines triggers a large voltage change as well as calcium signals at the spine head. Thus, voltage‐gated and calcium‐activated potassium channels located in the spine head likely play a key role in synaptic transmission. Here we study the presence and function of large conductance calcium‐activated potassium (BK) channels in spines from layer 5 PNs. We found that BK channels are localized to dendrites and spines regardless of their size, but their activity can only be detected in spines with small head volumes (≤0.09 μm3), which reduces the amplitude of two‐photon uncaging excitatory postsynaptic potentials recorded at the soma. In addition, we found that calcium signals in spines with small head volumes are significantly larger than those observed in spines with larger head volumes. In accordance with our experimental data, numerical simulations predict that synaptic inputs impinging onto spines with small head volumes generate voltage responses and calcium signals within the spine head itself that are significantly larger than those observed in spines with larger head volumes, which are sufficient to activate spine BK channels. These results show that BK channels are selectively activated in small‐headed spines, suggesting a new level of dendritic spine‐mediated regulation of synaptic processing, integration and plasticity in cortical PNs. Key points: BK channels are expressed in the visual cortex and layer 5 pyramidal neuron somata, dendrites and spines regardless of their size.BK channels are selectively activated in small‐headed spines (≤0.09 μm3), which reduces the amplitude of two‐photon (2P) uncaging excitatory postsynaptic potentials (EPSPs) recorded at the soma.Two‐photon imaging revealed that intracellular calcium responses in the head of 2P‐activated spines are significantly larger in small‐headed spines (≤0.09 μm3) than in spines with larger head volumes.In accordance with our experimental data, numerical simulations showed that synaptic inputs impinging onto spines with small head volumes (≤0.09 μm3) generate voltage responses and calcium signals within the spine head itself that are significantly larger than those observed in spines with larger head volumes, sufficient to activate spine BK channels and suppress EPSPs. [ABSTRACT FROM AUTHOR]
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- 2022
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5. Gold In-and-Out: A Toolkit for Analyzing Subcellular Distribution of Immunogold-Labeled Membrane Proteins in Freeze-Fracture Replica Images.
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Guerrero-Given, Debbie, Goldin, Seth L., Thomas, Connon I., Anthony, Skylar A., Jerez, Diego, and Kamasawa, Naomi
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MEMBRANE proteins ,DISTRIBUTION (Probability theory) ,GRAPHICAL user interfaces ,IMMUNOGOLD labeling ,ION channels ,CELL receptors - Abstract
Integral membrane proteins such as ion channels, transporters, and receptors shape cell activity and mediate cell-to-cell communication in the brain. The distribution, quantity, and clustering arrangement of those proteins contribute to the physiological properties of the cell; therefore, precise quantification of their state can be used to gain insight into cellular function. Using a highly sensitive immunoelectron microscopy technique called sodium dodecyl sulfate-digested freeze-fracture replica immunogold labeling (SDS-FRL), multiple membrane proteins can be tagged with different sizes of immunogold particles at once and visualized two-dimensionally. For quantification, gold particles in the images must be annotated, and then different mathematical and statistical methods must be applied to characterize the distribution states of proteins of interest. To perform such analyses in a user-friendly manner, we developed a program with a simple graphical user interface called Gold In-and-Out (GIO), which integrates several classical and novel analysis methods for immunogold labeled replicas into one self-contained package. GIO takes an input of particle coordinates, then allows users to implement analysis methods such as nearest neighbor distance (NND) and particle clustering. The program not only performs the selected analysis but also automatically compares the results of the real distribution to a random distribution of the same number of particles on the membrane region of interest. In addition to classical approaches for analyzing protein distribution, GIO includes new tools to analyze the positional bias of a target protein relative to a morphological landmark such as dendritic spines, and can also be applied for synaptic protein analysis. Gold Rippler provides a normalized metric of particle density that is resistant to differences in labeling efficiency among samples, while Gold Star is useful for quantifying distances between a protein and landmark. This package aims to help standardize analysis methods for subcellular and synaptic protein localization with a user-friendly interface while increasing the efficiency of these time-consuming analyses. [ABSTRACT FROM AUTHOR]
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- 2022
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6. Adhesion GPCR Latrophilin 3 regulates synaptic function of cone photoreceptors in a transsynaptic manner.
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Yuchen Wang, Yan Cao, Hays, Cassandra L., Laboute, Thibaut, Ray, Thomas A., Guerrero-Given, Debbie, Ahuja, Abhimanyu S., Patil, Dipak, Rivero, Olga, Kamasawa, Naomi, Kay, Jeremy N., Thoreson, Wallace B., and Martemyanov, Kirill A.
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PHOTORECEPTORS ,CONES ,NEURAL transmission ,LABORATORY mice ,CALCIUM channels - Abstract
Cone photoreceptors mediate daylight vision in vertebrates. Changes in neurotransmitter release at cone synapses encode visual information and is subject to precise control by negative feedback from enigmatic horizontal cells. However, the mechanisms that orchestrate this modulation are poorly understood due to a virtually unknown landscape of molecular players. Here, we report a molecular player operating selectively at cone synapses that modulates effects of horizontal cells on synaptic release. Using an unbiased proteomic screen, we identified an adhesion GPCR Latrophilin3 (LPHN3) in horizontal cell dendrites that engages in transsynaptic control of cones. We detected and characterized a prominent splice isoform of LPHN3 that excludes a element with inhibitory influence on transsynaptic interactions. A gain-of-function mouse model specifically routing LPHN3 splicing to this isoform but not knockout of LPHN3 diminished CaV1.4 calcium channel activity profoundly disrupted synaptic release by cones and resulted in synaptic transmission deficits. These findings offer molecular insight into horizontal cell modulation on cone synaptic function and more broadly demonstrate the importance of alternative splicing in adhesion GPCRs for their physiological function. [ABSTRACT FROM AUTHOR]
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- 2021
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7. A deep learning approach to identifying immunogold particles in electron microscopy images.
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Jerez, Diego, Stuart, Eleanor, Schmitt, Kylie, Guerrero-Given, Debbie, Christie, Jason M., Hahn, William E., Kamasawa, Naomi, and Smirnov, Michael S.
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DEEP learning ,ELECTRON microscopy ,COLLOIDAL gold ,SOFTWARE development tools ,TISSUES ,IMMUNOHISTOCHEMISTRY ,GOLD nanoparticles - Abstract
Electron microscopy (EM) enables high-resolution visualization of protein distributions in biological tissues. For detection, gold nanoparticles are typically used as an electron-dense marker for immunohistochemically labeled proteins. Manual annotation of gold particle labels is laborious and time consuming, as gold particle counts can exceed 100,000 across hundreds of image segments to obtain conclusive data sets. To automate this process, we developed Gold Digger, a software tool that uses a modified pix2pix deep learning network capable of detecting and annotating colloidal gold particles in biological EM images obtained from both freeze-fracture replicas and plastic sections prepared with the post-embedding method. Gold Digger performs at near-human-level accuracy, can handle large images, and includes a user-friendly tool with a graphical interface for proof reading outputs by users. Manual error correction also helps for continued re-training of the network to improve annotation accuracy over time. Gold Digger thus enables rapid high-throughput analysis of immunogold-labeled EM data and is freely available to the research community. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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8. Cryo-Confocal Imaging for CLEM Mapping in Brain Tissues.
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Thomas, Connon I., Urban, Nicolai T., Sun, Ye, Colgan, Lesley A., Tu, Xun, Yasuda, Ryohei, and Kamasawa, Naomi
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BRAIN imaging ,FROZEN tissue sections ,CONFOCAL microscopy ,SCANNING electron microscopy ,SIGNAL-to-noise ratio - Abstract
In correlative light and electron microscopy (CLEM) workflows, identifying the same sub-cellular features in tissue by both light (LM) and electron microscopy (EM) remains a challenge. Furthermore, use of cryo-fixation for EM is desirable to capture rapid biological phenomena. Here, we describe a workflow that incorporates cryo-confocal laser scanning microscopy into the CLEM process, mapping cells in brain slices to re-image them with serial section scanning electron microscopy (ssSEM) array tomography. The addition of Airyscan detection increased the signal-to-noise ratio (SNR), allowing individual spines in thick frozen tissue to be visualized at a sufficient spatial resolution, providing a new tool for a CLEM approach to capture biological dynamics. [ABSTRACT FROM AUTHOR]
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- 2021
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9. A deep learning approach to identifying immunogold particles in electron microscopy images.
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Jerez, Diego, Stuart, Eleanor, Schmitt, Kylie, Guerrero-Given, Debbie, Christie, Jason M., Kamasawa, Naomi, and Smirnov, Michael S.
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ELECTRON microscopy ,DEEP learning ,IMMUNOGOLD labeling ,TISSUES ,GOLD nanoparticles - Abstract
Electron microscopy (EM) enables high-resolution visualization of protein distributions in biological tissues. For detection, gold nanoparticles are typically used as an electron-dense marker for immunohistochemically labeled proteins. Manual annotation of gold particle labels is laborious and time consuming, as gold particle counts can exceed 100,000 across hundreds of image segments to obtain conclusive data sets. To automate this process, we developed Gold Digger, a software tool that uses a modified pix2pix deep learning network capable of detecting and annotating colloidal gold particles in biological EM images obtained from both freeze-fracture replicas and plastic sections prepared with the post-embedding method. Gold Digger performs at near-human-level accuracy, can handle large images, and includes a user-friendly tool with a graphical interface for proof reading outputs by users. Manual error correction also helps for continued re-training of the network to improve annotation accuracy over time. Gold Digger thus enables rapid high-throughput analysis of immunogold-labeled EM data and is freely available to the research community. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
10. Cortical response selectivity derives from strength in numbers of synapses.
- Author
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Scholl, Benjamin, Thomas, Connon I., Ryan, Melissa A., Kamasawa, Naomi, and Fitzpatrick, David
- Abstract
Single neocortical neurons are driven by populations of excitatory inputs, which form the basis of neuronal selectivity to features of sensory input. Excitatory connections are thought to mature during development through activity-dependent Hebbian plasticity1, whereby similarity between presynaptic and postsynaptic activity selectively strengthens some synapses and weakens others2. Evidence in support of this process includes measurements of synaptic ultrastructure and in vitro and in vivo physiology and imaging studies3–8. These corroborating lines of evidence lead to the prediction that a small number of strong synaptic inputs drive neuronal selectivity, whereas weak synaptic inputs are less correlated with the somatic output and modulate activity overall6,7. Supporting evidence from cortical circuits, however, has been limited to measurements of neighbouring, connected cell pairs, raising the question of whether this prediction holds for a broad range of synapses converging onto cortical neurons. Here we measure the strengths of functionally characterized excitatory inputs contacting single pyramidal neurons in ferret primary visual cortex (V1) by combining in vivo two-photon synaptic imaging and post hoc electron microscopy. Using electron microscopy reconstruction of individual synapses as a metric of strength, we find no evidence that strong synapses have a predominant role in the selectivity of cortical neuron responses to visual stimuli. Instead, selectivity appears to arise from the total number of synapses activated by different stimuli. Moreover, spatial clustering of co-active inputs appears to be reserved for weaker synapses, enhancing the contribution of weak synapses to somatic responses. Our results challenge the role of Hebbian mechanisms in shaping neuronal selectivity in cortical circuits, and suggest that selectivity reflects the co-activation of large populations of presynaptic neurons with similar properties and a mixture of strengths.Live neuron imaging and electron microscopy reconstruction shows that the selectivity of cortical neuron responses to visual stimuli arises from the total number of synapses activated rather than being dominated by a small number of strong synaptic inputs. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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11. Targeting Functionally Characterized Synaptic Architecture Using Inherent Fiducials and 3D Correlative Microscopy.
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Thomas, Connon I., Ryan, Melissa A., Scholl, Benjamin, Guerrero-Given, Debbie, Fitzpatrick, David, and Kamasawa, Naomi
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- 2021
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12. ADAP1/Centaurin-α1 Negatively Regulates Dendritic Spine Function and Memory Formation in the Hippocampus.
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Szatmari, Erzsebet M., Moran, Corey, Cohen, Sarah, Jacob, Amanda, Parra-Bueno, Paula, Kamasawa, Naomi, Guerrero-Given, Debbie, Klein, Michael, Stackman Jr, Robert, and Yasuda, Ryohei
- Published
- 2021
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13. Interplay between cell-adhesion molecules governs synaptic wiring of cone photoreceptors.
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Yan Cao, Yuchen Wang, Dunn, Henry A., Orlandi, Cesare, Shultz, Nicole, Kamasawa, Naomi, Fitzpatrick, David, Wei Li, Zeitz, Christina, Hauswirth, William, and Martemyanov, Kirill A.
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PHOTORECEPTORS ,CONES ,NEUROTRANSMITTER receptors ,NERVOUS system ,NEURAL transmission - Abstract
Establishment of functional synaptic connections in a selective manner is essential for nervous system operation. In mammalian retinas, rod and cone photoreceptors form selective synaptic connections with different classes of bipolar cells (BCs) to propagate light signals. While there has been progress in elucidating rod wiring, molecular mechanisms used by cones to establish functional synapses with BCs have remained unknown. Using an unbiased proteomic strategy in cone-dominant species, we identified the cell-adhesion molecule ELFN2 to be pivotal for the functional wiring of cones with the ON type of BC. It is selectively expressed in cones and transsynaptically recruits the key neurotransmitter receptor mGluR6 in ON-BCs to enable synaptic transmission. Remarkably, ELFN2 in cone terminals functions in synergy with a related adhesion molecule, ELFN1, and their concerted interplay during development specifies selective wiring and transmission of cone signals. These findings identify a synaptic connectivity mechanism of cones and illustrate how interplay between adhesion molecules and postsynaptic transmitter receptors orchestrates functional synaptic specification in a neural circuit. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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14. Presynaptic development is controlled by the core active zone proteins CAST/ELKS.
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Radulovic, Tamara, Dong, Wei, Goral, R. Oliver, Thomas, Connon I., Veeraraghavan, Priyadharishini, Montesinos, Monica Suarez, Guerrero‐Given, Debbie, Goff, Kevin, Lübbert, Matthias, Kamasawa, Naomi, Ohtsuka, Toshihisa, and Young, Samuel M.
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NEURAL transmission ,NEUROPLASTICITY ,SYNAPTIC vesicles ,CALCIUM channels ,PROTEINS - Abstract
Key points: CAST/ELKS are positive regulators of presynaptic growth and are suppressors of active zone expansion at the developing mouse calyx of Held.CAST/ELKS regulate all three CaV2 subtype channel levels in the presynaptic terminal and not just CaV2.1.The half‐life of ELKS is on the timescale of days and not weeks.Synaptic transmission was not impacted by the loss of CAST/ELKS.CAST/ELKS are involved in pathways regulating morphological properties of presynaptic terminals during an early stage of circuit maturation. Many presynaptic active zone (AZ) proteins have multiple regulatory roles that vary during distinct stages of neuronal circuit development. The CAST/ELKS protein family are evolutionarily conserved presynaptic AZ molecules that regulate presynaptic calcium channels, synaptic transmission and plasticity in the mammalian CNS. However, how these proteins regulate synapse development and presynaptic function in a developing neuronal circuit in its native environment is unclear. To unravel the roles of CAST/ELKS in glutamatergic synapse development and in presynaptic function, we used CAST knockout (KO) and ELKS conditional KO (CKO) mice to examine how their loss during the early stages of circuit maturation impacted the calyx of Held presynaptic terminal development and function. Morphological analysis from confocal z‐stacks revealed that combined deletion of CAST/ELKS resulted in a reduction in the surface area and volume of the calyx. Analysis of AZ ultrastructure showed that AZ size was increased in the absence of CAST/ELKS. Patch clamp recordings demonstrated a reduction of all presynaptic CaV2 channel subtype currents that correlated with a loss in presynaptic CaV2 channel numbers. However, these changes did not impair synaptic transmission and plasticity and synaptic vesicle release kinetics. We conclude that CAST/ELKS proteins are positive regulators of presynaptic growth and are suppressors of AZ expansion and CaV2 subtype currents and levels during calyx of Held development. We propose that CAST/ELKS are involved in pathways regulating presynaptic morphological properties and CaV2 channel subtypes and suggest there is developmental compensation to preserve synaptic transmission during early stages of neuronal circuit maturation. Key points: CAST/ELKS are positive regulators of presynaptic growth and are suppressors of active zone expansion at the developing mouse calyx of Held.CAST/ELKS regulate all three CaV2 subtype channel levels in the presynaptic terminal and not just CaV2.1.The half‐life of ELKS is on the timescale of days and not weeks.Synaptic transmission was not impacted by the loss of CAST/ELKS.CAST/ELKS are involved in pathways regulating morphological properties of presynaptic terminals during an early stage of circuit maturation. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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15. Presynaptic Mitochondria Volume and Abundance Increase during Development of a High-Fidelity Synapse.
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Thomas, Connon I., Keine, Christian, Okayama, Satoko, Satterfield, Rachel, Musgrove, Morgan, Guerrero-Given, Debbie, Kamasawa, Naomi, and Young Jr., Samuel M.
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MITOCHONDRIA ,NEURAL transmission ,SYNAPSES ,SCANNING electron microscopy ,NEUROPLASTICITY - Abstract
The calyx of Held, a large glutamatergic presynaptic terminal in the auditory brainstem undergoes developmental changes to support the high action-potential firing rates required for auditory information encoding. In addition, calyx terminals are morphologically diverse, which impacts vesicle release properties and synaptic plasticity. Mitochondria influence synaptic plasticity through calcium buffering and are crucial for providing the energy required for synaptic transmission. Therefore, it has been postulated that mitochondrial levels increase during development and contribute to the morphological-functional diversity in the mature calyx. However, the developmental profile of mitochondrial volumes and subsynaptic distribution at the calyx of Held remains unclear. To provide insight on this, we developed a helper-dependent adenoviral vector that expresses the genetically encoded peroxidase marker for mitochondria, mito-APEX2, at the mouse calyx of Held. We developed protocols to detect labeled mitochondria for use with serial block face scanning electron microscopy to carry out semiautomated segmentation of mitochondria, high-throughput whole-terminal reconstruction, and presynaptic ultrastructure in mice of either sex. Subsequently, we measured mitochondrial volumes and subsynaptic distributions at the immature postnatal day (P)7 and the mature (P21) calyx. We found an increase of mitochondria volumes in terminals and axons from P7 to P21 but did not observe differences between stalk and swelling subcompartments in the mature calyx. Based on these findings, we propose that mitochondrial volumes and synaptic localization developmentally increase to support high firing rates required in the initial stages of auditory information processing. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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16. Screening Morphological Characteristics of Large Populations of Synaptic Vesicle Clouds and Active Zones from 3D EM Data.
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Thomas, Connon I, Anderson, Jordan R, Bolton, McLean, and Kamasawa, Naomi
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- 2023
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17. LRIT1 Modulates Adaptive Changes in Synaptic Communication of Cone Photoreceptors.
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Sarria, Ignacio, Yan Cao, Yuchen Wang, Ingram, Norianne T., Orlandi, Cesare, Kamasawa, Naomi, Kolesnikov, Alexander V., Pahlberg, Johan, Kefalov, Vladimir J., Sampath, Alapakkam P., and Martemyanov, Kirill A.
- Abstract
Cone photoreceptors scale dynamically the sensitivity of responses to maintain responsiveness across wide range of changes in luminance. Synaptic changes contribute to this adaptation, but how this process is coordinated at the molecular level is poorly understood. Here, we report that a cell adhesion-like molecule, LRIT1, is enriched selectively at cone photoreceptor synapses where it engages in a trans-synaptic interaction with mGluR6, the principal receptor in postsynaptic ON-bipolar cells. The levels of LRIT1 are regulated by the neurotransmitter release apparatus that controls photoreceptor output. Knockout of LRIT1 in mice increases the sensitivity of cone synaptic signaling while impairing its ability to adapt to background light without overtly influencing the morphology or molecular composition of photoreceptor synapses. Accordingly, mice lacking LRIT1 show visual deficits under conditions requiring temporally challenging discrimination of visual signals in steady background light. These observations reveal molecular mechanisms involved in scaling synaptic communication in the retina. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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18. Identification of a highly neurotoxic α-synuclein species inducing mitochondrial damage and mitophagy in Parkinson's disease.
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Grassi, Diego, Howard, Shannon, Minghai Zhou, Diaz-Perez, Natalia, Urban, Nicolai T., Guerrero-Given, Debbie, Kamasawa, Naomi, Volpicelli-Daley, Laura A., LoGrasso, Philip, and Lasmézas, Corinne Ida
- Subjects
ALPHA-synuclein ,NEUROTOXICOLOGY ,PARKINSON'S disease ,MITOCHONDRIA ,AUTOPHAGY - Abstract
Exposure of cultured primary neurons to preformed α-synuclein fibrils (PFFs) leads to the recruitment of endogenous α-synuclein and its templated conversion into fibrillar phosphorylated α-synuclein (pα-synF) aggregates resembling those involved in Parkinson's disease (PD) pathogenesis. Pα-synF was described previously as inclusions morphologically similar to Lewy bodies and Lewy neurites in PD patients. We discovered the existence of a conformationally distinct, nonfibrillar, phosphorylated α-syn species that we named "pα-syn*." We uniquely describe the existence of pα-syn* in PFF-seeded primary neurons, mice brains, and PD patients' brains. Through immunofluorescence and pharmacological manipulation we showed that pα-syn* results from incomplete autophagic degradation of pα-synF. Pα-synF was decorated with autophagic markers, but pα-syn* was not. Western blots revealed that pα-syn* was N- and C-terminally trimmed, resulting in a 12.5-kDa fragment and a SDS-resistant dimer. After lysosomal release, pα-syn* aggregates associated with mitochondria, inducing mitochondrial membrane depolarization, cytochrome C release, and mitochondrial fragmentation visualized by confocal and stimulated emission depletion nanoscopy. Pα-syn* recruited phosphorylated acetyl-CoA carboxylase 1 (ACC1) with which it remarkably colocalized. ACC1 phosphorylation indicates low ATP levels, AMPK activation, and oxidative stress and induces mitochondrial fragmentation via reduced lipoylation. Pα-syn* also colocalized with BiP, a master regulator of the unfolded protein response and a resident protein of mitochondria-associated endoplasmic reticulum membranes that are sites of mitochondrial fission and mitophagy. Pα-syn* aggregates were found in Parkin-positive mitophagic vacuoles and imaged by electron microscopy. Collectively, we showed that pα-syn* induces mitochondrial toxicity and fission, energetic stress, and mitophagy, implicating pα-syn* as a key neurotoxic α-syn species and a therapeutic target. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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19. The number and distribution of AMPA receptor channels containing fast kinetic GluA3 and GluA4 subunits at auditory nerve synapses depend on the target cells.
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Rubio, María, Matsui, Ko, Fukazawa, Yugo, Kamasawa, Naomi, Harada, Harumi, Itakura, Makoto, Molnár, Elek, Abe, Manabu, Sakimura, Kenji, and Shigemoto, Ryuichi
- Subjects
NEUROTRANSMITTER receptors ,AMPA receptors ,GLUTAMATE receptors ,NEURONS ,SYNAPSES - Abstract
The neurotransmitter receptor subtype, number, density, and distribution relative to the location of transmitter release sites are key determinants of signal transmission. AMPA-type ionotropic glutamate receptors (AMPARs) containing GluA3 and GluA4 subunits are prominently expressed in subsets of neurons capable of firing action potentials at high frequencies, such as auditory relay neurons. The auditory nerve (AN) forms glutamatergic synapses on two types of relay neurons, bushy cells (BCs) and fusiform cells (FCs) of the cochlear nucleus. AN-BC and AN-FC synapses have distinct kinetics; thus, we investigated whether the number, density, and localization of GluA3 and GluA4 subunits in these synapses are differentially organized using quantitative freeze-fracture replica immunogold labeling. We identify a positive correlation between the number of AMPARs and the size of AN-BC and AN-FC synapses. Both types of AN synapses have similar numbers of AMPARs; however, the AN-BC have a higher density of AMPARs than AN-FC synapses, because the AN-BC synapses are smaller. A higher number and density of GluA3 subunits are observed at AN-BC synapses, whereas a higher number and density of GluA4 subunits are observed at AN-FC synapses. The intrasynaptic distribution of immunogold labeling revealed that AMPAR subunits, particularly GluA3, are concentrated at the center of the AN-BC synapses. The central distribution of AMPARs is absent in GluA3-knockout mice, and gold particles are evenly distributed along the postsynaptic density. GluA4 gold labeling was homogenously distributed along both synapse types. Thus, GluA3 and GluA4 subunits are distributed at AN synapses in a target-cell-dependent manner. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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20. A novel region in the CaV2.1 α1 subunit C-terminus regulates fast synaptic vesicle fusion and vesicle docking at the mammalian presynaptic active zone.
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Lübbert, Matthias, Goral, R. Oliver, Satterfield, Rachel, Putzke, Travis, van den Maagdenberg, Arn M. J. M., Kamasawa, Naomi, and Young Jr, Samuel M.
- Published
- 2017
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21. Primary utricle structure of six Halimeda species and potential relevance for ocean acidification tolerance.
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Peach, Katherine E., Koch, Marguerite S., Blackwelder, Patricia L., Guerrero-Given, Debbie, and Kamasawa, Naomi
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OCEAN acidification ,UTRICLE & Saccule ,HALIMEDA ,SEDIMENTS ,CALCIFICATION - Abstract
Variations in utricle morphology may be responsible for different tolerances to ocean acidification (OA) within the macroalgal genus Halimeda, an important sediment producer on reefs. However, differences in species' utricle morphology and their relationship to calcification and crystal formation have not been well articulated. In the present study, we characterized the utricle morphologies of six Halimeda species. Primary utricle ultrastructure was quantitatively and qualitatively compared to tissue inorganic content and crystal microstructure. Morphologies differed across species and several morphometric relationships were revealed. Primary utricle size (r
2 =0.70) and diffusion pathway length (r2 =0.87) had inverse relationships with inorganic content based on regression analyses, and corresponded to crystal microstructure form. Species with large utricles and long diffusion pathways contained more narrow (~0.15 μm) aragonite needles and minimal micro-anhedral crystal formations. In contrast, species with small utricles and short diffusion pathways elucidated aggregates of micro-anhedral crystals and wider aragonite needles (~0.30 μm). Species' utricle characteristics generally corresponded to specific evolutionary lineages. Thus, characteristics of Halimeda utricle morphology may control long-term adaptive responses to OA, an idea articulated in the broader literature. [ABSTRACT FROM AUTHOR]- Published
- 2017
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22. Target- and input-dependent organization of AMPA and NMDA receptors in synaptic connections of the cochlear nucleus.
- Author
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Rubio, María E, Fukazawa, Yugo, Kamasawa, Naomi, Clarkson, Cheryl, Molnár, Elek, and Shigemoto, Ryuichi
- Abstract
ABSTRACT We examined the synaptic structure, quantity, and distribution of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)- and N-methyl-D-aspartate (NMDA)-type glutamate receptors (AMPARs and NMDARs, respectively) in rat cochlear nuclei by a highly sensitive freeze-fracture replica labeling technique. Four excitatory synapses formed by two distinct inputs, auditory nerve (AN) and parallel fibers (PF), on different cell types were analyzed. These excitatory synapse types included AN synapses on bushy cells (AN-BC synapses) and fusiform cells (AN-FC synapses) and PF synapses on FC (PF-FC synapses) and cartwheel cell spines (PF-CwC synapses). Immunogold labeling revealed differences in synaptic structure as well as AMPAR and NMDAR number and/or density in both AN and PF synapses, indicating a target-dependent organization. The immunogold receptor labeling also identified differences in the synaptic organization of FCs based on AN or PF connections, indicating an input-dependent organization in FCs. Among the four excitatory synapse types, the AN-BC synapses were the smallest and had the most densely packed intramembrane particles (IMPs), whereas the PF-CwC synapses were the largest and had sparsely packed IMPs. All four synapse types showed positive correlations between the IMP-cluster area and the AMPAR number, indicating a common intrasynapse-type relationship for glutamatergic synapses. Immunogold particles for AMPARs were distributed over the entire area of individual AN synapses; PF synapses often showed synaptic areas devoid of labeling. The gold-labeling for NMDARs occurred in a mosaic fashion, with less positive correlations between the IMP-cluster area and the NMDAR number. Our observations reveal target- and input-dependent features in the structure, number, and organization of AMPARs and NMDARs in AN and PF synapses. J. Comp. Neurol. 522:4023-4042, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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23. Retrograde Synaptic Signaling Mediated by K+ Efflux through Postsynaptic NMDA Receptors.
- Author
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Shih, Pei-Yu, Savtchenko, Leonid P., Kamasawa, Naomi, Dembitskaya, Yulia, McHugh, Thomas J., Rusakov, Dmitri A., Shigemoto, Ryuichi, and Semyanov, Alexey
- Abstract
Summary: Synaptic NMDA receptors (NMDARs) carry inward Ca
2+ current responsible for postsynaptic signaling and plasticity in dendritic spines. Whether the concurrent K+ efflux through the same receptors into the synaptic cleft has a physiological role is not known. Here, we report that NMDAR-dependent K+ efflux can provide a retrograde signal in the synapse. In hippocampal CA3-CA1 synapses, the bulk of astrocytic K+ current triggered by synaptic activity reflected K+ efflux through local postsynaptic NMDARs. The local extracellular K+ rise produced by activation of postsynaptic NMDARs boosted action potential-evoked presynaptic Ca2+ transients and neurotransmitter release from Schaffer collaterals. Our findings indicate that postsynaptic NMDAR-mediated K+ efflux contributes to use-dependent synaptic facilitation, thus revealing a fundamental form of retrograde synaptic signaling. [Copyright &y& Elsevier]- Published
- 2013
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24. Quantitative Localization of Cav2.1 (P/Q-Type) Voltage-Dependent Calcium Channels in Purkinje Cells: Somatodendritic Gradient and Distinct Somatic Coclustering with Calcium-Activated Potassium Channels.
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Indriati, Dwi Wahyu, Kamasawa, Naomi, Matsui, Ko, Meredith, Andrea L., Watanabe, Masahiko, and Shigemoto, Ryuichi
- Subjects
CALCIUM channels ,QUANTITATIVE research ,PURKINJE cells ,DENDRITIC cells ,POTASSIUM channels ,NEUROTRANSMITTERS ,GENE expression ,INTRACELLULAR calcium - Abstract
P/Q-type voltage-dependent calcium channels play key roles in transmitter release, integration of dendritic signals, generation of dendritic spikes, and gene expression. High intracellular calcium concentration transient produced by these channels is restricted to tens to hundreds of nanometers from the channels. Therefore, precise localization of these channels along the plasma membrane was long sought to decipher how each neuronal cell function is controlled. Here, we analyzed the distribution of Ca
v 2.1 subunit of the P/Q-type channel using highly sensitive SDS-digested freeze-fracture replica labeling in the rat cerebellar Purkinje cells. The labeling efficiency was such that the number of immunogold particles in each parallel fiber active zone was comparable to that of functional channels calculated from previous reports. Two distinct patterns of Cav 2.1 distribution, scattered and clustered, were found in Purkinje cells. The scattered Cav 2.1 had a somatodendritic gradient with the density of immunogold particles increasing 2.5-fold from soma to distal dendrites. The other population with 74-fold higher density than the scattered particles was found within clusters of intramembrane particles on the P-face of soma and primary dendrites. Both populations of Cav 2.1 were found as early as P3 and increased in the second postnatal week to a mature level. Using double immunogold labeling, we found that virtually all of the Cav 2.1 clusters were colocalized with two types of calcium-activated potassium channels, BK and SK2, with the nearest neighbor distance of ~40 nm. Calcium nanodomain created by the opening of Cav 2.1 channels likely activates the two channels that limit the extent of depolarization. [ABSTRACT FROM AUTHOR]- Published
- 2013
- Full Text
- View/download PDF
25. Evaluation of glutamate concentration transient in the synaptic cleft of the rat calyx of Held.
- Author
-
Budisantoso, Timotheus, Harada, Harumi, Kamasawa, Naomi, Fukazawa, Yugo, Shigemoto, Ryuichi, and Matsui, Ko
- Subjects
GLUTAMIC acid ,SPATIOTEMPORAL processes ,CLEFT palate ,SYNAPTIC vesicles ,NEUROTRANSMITTERS ,NEURONS - Abstract
Key points Spatiotemporal glutamate concentration profile in the cleft is a determinant of the EPSC amplitude and time course., Two key parameters required to describe this profile are the number of glutamate in a vesicle ( N
Glu ) and its diffusion coefficient ( DGlu ), both of which are unestablished., Using the rat calyx of Held synapse as a model, the distribution of AMPA receptors was mapped with SDS-digested freeze fracture replica labelling and their performance as glutamate sensors was evaluated with outside-out patch recordings., Based on these data, synaptic responses were simulated using various combinations of NGlu and DGlu , and an optimal range of the NGlu - DGlu combinations that could reproduce the recordings was determined., Using the estimated profile, we show that release from a single vesicle does not saturate the receptors, glutamate spillover does not affect the synaptic conductance amplitude, and synaptic response increases non-linearly with the number of multivesicular releases., Abstract Establishing the spatiotemporal concentration profile of neurotransmitter following synaptic vesicular release is essential for our understanding of inter-neuronal communication. Such profile is a determinant of synaptic strength, short-term plasticity and inter-synaptic crosstalk. Synaptically released glutamate has been suggested to reach a few millimolar in concentration and last for <1 ms. The synaptic cleft is often conceived as a single concentration compartment, whereas a huge gradient likely exists. Modelling studies have attempted to describe this gradient, but two key parameters, the number of glutamate in a vesicle ( NGlu ) and its diffusion coefficient ( DGlu ) in the extracellular space, remained unresolved. To determine this profile, the rat calyx of Held synapse at postnatal day 12-16 was studied where diffusion of glutamate occurs two-dimensionally and where quantification of AMPA receptor distribution on individual postsynaptic specialization on medial nucleus of the trapezoid body principal cells is possible using SDS-digested freeze-fracture replica labelling. To assess the performance of these receptors as glutamate sensors, a kinetic model of the receptors was constructed from outside-out patch recordings. From here, we simulated synaptic responses and compared them with the EPSC recordings. Combinations of NGlu and DGlu with an optimum of 7000 and 0.3 μm2 ms−1 reproduced the data, suggesting slow diffusion. Further simulations showed that a single vesicle does not saturate the synaptic receptors, and that glutamate spillover does not affect the conductance amplitude at this synapse. Using the estimated profile, we also evaluated how the number of multiple vesicle releases at individual active zones affects the amplitude of postsynaptic signals. [ABSTRACT FROM AUTHOR]- Published
- 2013
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26. Connexin composition in apposed gap junction hemiplaques revealed by matched double-replica freeze-fracture replica immunogold labeling.
- Author
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Rash JE, Kamasawa N, Davidson KG, Yasumura T, Pereda AE, Nagy JI, Rash, John E, Kamasawa, Naomi, Davidson, Kimberly G V, Yasumura, Thomas, Pereda, Alberto E, and Nagy, James I
- Abstract
Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identifying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing membranes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica immunogold labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques. However, freeze-fracture replica immunogold labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing. We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins. This approach allows unambiguous identification of connexins and determination of the membrane "sidedness" and the identities of connexin coupling partners in homotypic and heterotypic gap junctions of vertebrate neurons. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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27. Regulators of G protein signaling RGS7 and RGS11 determine the onset of the light response in ON bipolar neurons.
- Author
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Yan Cao, Pahlberg, Johan, Sarria, Ignacio, Kamasawa, Naomi, Sampath, Alapakkam P., and Martemyanov, Kirill A.
- Subjects
G proteins ,REGULATOR of G-protein-signaling proteins ,GUANOSINE triphosphatase ,PHOTORECEPTORS ,DENDRITES - Abstract
The time course of signaling via heterotrimeric G proteins is controlled through their activation by G-protein coupled receptors and deactivation through the action of GTPase accelerating proteins (GAPs). Here we identify RGS7 and RGS11 as the key GAPs in the mGluR6 pathway of retinal rod ON bipolar cells that set the sensitivity and time course of light-evoked responses. We showed using electroretinography and single cell recordings that the elimination of RGS7 did not influence dark-adapted light-evoked responses, but the concurrent elimination of RGS11 severely reduced their magnitude and dramatically slowed the onset of the response. In RGS7/RGS11 double-knockout mice, light-evoked responses in rod ON bipolar cells were only observed during persistent activation of rod photoreceptors that saturate rods. These observations are consistent with persistently high G-protein activity in rod ON bipolar cell dendrites caused by the absence of the dominant GAP, biasing TRPM1 channels to the closed state. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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28. Mechanisms Underlying Signal Filtering at a Multisynapse Contact.
- Author
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Budisantoso, Timotheus, Matsui, Ko, Kamasawa, Naomi, Fukazawa, Yugo, and Shigemoto, Ryuichi
- Subjects
CELLULAR signal transduction ,SYNAPSES ,ELECTROPHYSIOLOGY ,NEUROTRANSMITTERS ,AMPA receptors ,LABORATORY rats - Abstract
Visual information must be relayed through the lateral geniculate nucleus before it reaches the visual cortex. However, not all spikes created in the retina lead to postsynaptic spikes and properties of the retinogeniculate synapse contribute to this filtering. To understand the mechanisms underlying this filtering process, we conducted electrophysiology to assess the properties of signal transmission in the Long-Evans rat. We also performed SDS-digested freeze-fracture replica labeling to quantify the receptor and transporter distribution, as well as EM reconstruction to describe the 3D structure. To analyze the impact of transmitter diffusion on the activity of the receptors, simulations were integrated. We identified that a large contributor to the filtering is the marked paired-pulse depression at this synapse, which was intensified by the morphological characteristics of the contacts. The broad presynaptic and postsynaptic contact area restricts transmitter diffusion two dimensionally. Additionally, the presence of multiple closely arranged release sites invites intersynaptic spillover, which causes desensitization of AMPA receptors. The presence of AMPA receptors that slowly recover from desensitization along with the high presynaptic release probability and multivesicular release at each synapse also contribute to the depression. These features contrast with many other synapses where spatiotemporal spread of transmitter is limited by rapid transmitter clearance allowing synapses to operate more independently. We propose that the micrometer-order structure can ultimately affect the visual information processing. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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29. Pob1 Ensures Cylindrical Cell Shape by Coupling Two Distinct Rho Signaling Events During Secretory Vesicle Targeting.
- Author
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Nakano, Kentaro, Toya, Mika, Yoneda, Aki, Asami, Yukiko, Yamashita, Akira, Kamasawa, Naomi, Osumi, Masako, and Yamamoto, Masayuki
- Subjects
CELL morphology ,CELLULAR signal transduction ,EXOCYTOSIS ,CELL polarity ,MORPHOGENESIS ,SCHIZOSACCHAROMYCES pombe ,RHO GTPases ,PROTEINS - Published
- 2011
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30. Author Correction: A deep learning approach to identifying immunogold particles in electron microscopy images.
- Author
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Jerez, Diego, Stuart, Eleanor, Schmitt, Kylie, Guerrero-Given, Debbie, Christie, Jason M., Hahn, William E., Kamasawa, Naomi, and Smirnov, Michael S.
- Subjects
ELECTRON microscopy ,DEEP learning - Abstract
The Author Contributions section now reads: "Conceptualization: D.J., J.M.C., N.K., W.E.H., and M.S.; Investigation: D.J., E.S., K.S., and D.G-G.; Writing and Editing, D.J., E.S., D.G-G., J.M.C., N.K., W.E.H., and M.S.; Funding Acquisition: J.M.C.; Supervision: J.M.C, N.K., W.E.H., and M.S." In addition, Diego Jerez and Eleanor Stuart were incorrectly affiliated with 'Florida Atlantic University, 777 Glades Rd, Boca Raton, FL, 33431, USA'. The original article can be found online at https://doi.org/10.1038/s41598-021-87015-2. Correction to: I Scientific Reports i https://doi.org/10.1038/s41598-021-87015-2, published online 08 April 2021 William E. Hahn was omitted from the author list in the original version of this Article. [Extracted from the article]
- Published
- 2021
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31. Connexin45-Containing Neuronal Gap Junctions in Rodent Retina Also Contain Connexin36 in Both Apposing Hemiplaques, Forming Bihomotypic Gap Junctions, with Scaffolding Contributed by Zonula Occludens-1.
- Author
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Xinbo Li, Kamasawa, Naomi, Ciolofan, Cristina, Olson, Carl O., Shijun Lu, Davidson, Kimberly G. V., Yasumura, Thomas, Shigemoto, Ryuichi, Rash, John E., and Nagy, James I.
- Subjects
NEURAL circuitry ,TIGHT junctions ,RETINA ,NERVE tissue proteins ,GAP junctions (Cell biology) ,CONNEXINS - Abstract
Mammalian retinas contain abundant neuronal gap junctions, particularly in the inner plexiform layer (IPL), where the two principal neuronal connexin proteins are Cx36 and Cx45. Currently undetermined are coupling relationships between these connexins and whether both are expressed together or separately in a neuronal subtype-specific manner. Although Cx45-expressing neurons strongly couple with Cx36-expressing neurons, possibly via heterotypic gap junctions, Cx45 and Cx36 failed to form functional heterotypic channels in vitro. We now show that Cx36 and Cx45 coexpressed in HeLa cells were colocalized in immunofluorescent puncta between contacting cells, demonstrating targeting/scaffolding competence for both connexins in vitro. However, Cx36 and Cx45 expressed separately did not form immunofluorescent puncta containing both connexins, supporting lack of heterotypic coupling competence. In IPL, 87% of Cx45-immunofluorescent puncta were colocalized with Cx36, supporting either widespread heterotypic coupling or bihomotypic coupling. Ultrastructurally, Cx45 was detected in 9% of IPL gap junction hemiplaques, 90—100% of which also contained Cx36, demonstrating connexin coexpression and cotargeting in virtually all IPL neurons that express Cx45. Moreover, double replicas revealed both connexins in separate domains mirrored on both sides of matched hemiplaques. With previous evidence that Cx36 interacts with PDZ1 domain of zonula occludens-1 (ZO-1), we show that Cx45 interacts with PDZ2 domain of ZO-1, and that Cx36, Cx45, and ZO-1 coimmunoprecipitate, suggesting that ZO-1 provides for coscaffolding of Cx45 with Cx36. These data document that in Cx45-expressing neurons of IPL, Cx45 is almost always accompanied by Cx36, forming "bihomotypic" gap junctions, with Cx45 structurally coupling to Cx45 and Cx36 coupling to Cx36. [ABSTRACT FROM AUTHOR]
- Published
- 2008
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32. Gap junctions on hippocampal mossy fiber axons demonstrated by thin-section electron microscopy and freeze—fracture replica immunogold labeling.
- Author
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Hamzei-Sichani, Farid, Kamasawa, Naomi, Janssen, William G. M., Yasumura, Thomas, Davidson, Kimberly G. V., Hof, Patrick R., Wearne, Susan L., Stewart, Mark G., Young, Steven R., Whittington, Miles A., Rash, John E., and Traub, Roger D.
- Subjects
GAP junctions (Cell biology) ,CELL junctions ,CYTOPLASMIC granules ,ORGANELLES ,ELECTRON microscopy - Abstract
Gap junctions have been postulated to exist between the axons of excitatory cortical neurons based on electrophysiological. modeling, and dye-coupling data. Here, we provide ultrastructural evidence for axoaxonic gap junctions in dentate granule cells. Using combined confocal laser scanning microscopy, thin-section transmission electron microscopy, and grid-mapped freeze-fracture replica immunogold labeling, 10 close appositions revealing axoaxonic gap junctions (∼30–70 nm in diameter) were found between pairs of mossy fiber axons (∼100–200 nm in diameter) in the stratum lucidum of the CA3b field of the rat ventral hippocampus, and one axonal gap junction (∼100 connexons) was found on a mossy fiber axon in the CA3c field of the rat dorsal hippocampus. Immunogold labeling with two sizes of gold beads revealed that connexin36 was present in that axonal gap junction. These ultrastructural data support computer modeling and in vitro electrophysiological data suggesting that axoaxonic gap junctions play an important role in the generation of very fast (>`70 Hz) network oscillations and in the hypersynchronous electrical activity of epilepsy. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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33. Change in the Actin Cytoskeleton during Seismonastic Movement of Mimosa pudica.
- Author
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Kanzawa, Nobuyuki, Hoshino, Yoshinori, Chiba, Makiko, Hoshino, Daisuke, Kobayashi, Hidetaka, Kamasawa, Naomi, Kishi, Yoshiro, Osumi, Masako, Sameshima, Masazumi, and Tsuchiya, Takahide
- Subjects
ACTIN ,CYTOSKELETON ,SENSITIVE plant ,TURGOR ,IMMUNOFLUORESCENCE ,PHOSPHORYLATION - Abstract
The seismonastic movement of Mimosa pudica is triggered by a sudden loss of turgor pressure. In the present study, we compared the cell cytoskeleton by immunofluorescence analysis before and after movement, and the effects of actin- and microtubule-targeted drugs were examined by injecting them into the cut pulvinus. We found that fragmentation of actin filaments and microtubules occurs during bending, although the actin cytoskeleton, but not the microtubules, was involved in regulation of the movement. Transmission electron microscopy revealed that actin cables became loose after the bending. We injected phosphatase inhibitors into the severed pulvinus to examine the effects of such inhibitors on the actin cytoskeleton. We found that changes in actin isoforms, fragmentation of actin filaments and the bending movement were all inhibited after injection of a tyrosine phosphatase inhibitor. We thus propose that the phosphorylation status of actin at tyrosine residues affects the dynamic reorganization of actin filaments and causes seismonastic movement. [ABSTRACT FROM AUTHOR]
- Published
- 2006
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34. Neuronal connexin36 association with zonula occludens-1 protein (ZO-1) in mouse brain and interaction with the first PDZ domain of ZO-1.
- Author
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Li, Xinbo, Olson, Carl, Lu, Shijun, Kamasawa, Naomi, Yasumura, Thomas, Rash, John E., and Nagy, James I.
- Subjects
CONNEXINS ,MEMBRANE proteins ,NEURONS ,TIGHT junctions ,CELL junctions ,MICE - Abstract
Among the 20 members in the connexin family of gap junction proteins, only connexin 36 (Cx36) is firmly established to be expressed in neurons and to form electrical synapses at widely distributed interneuronal gap junctions in mammalian brain. Several connexins have recently been reported to interact with the PDZ domain-containing protein zonula occludens-1 (ZO-1), which was originally considered to be associated only with tight junctions, but has recently been reported to associate with other structures including gap junctions in various cell types. Based on the presence of sequence corresponding to a putative PDZ binding motif in Cx36, we investigated anatomical relationships and molecular association of Cx36 with ZO-1. By immunofluorescence, punctate Cx36/ZO-1 colocalization was observed throughout the central nervous system of wild-type mice, whereas labelling for Cx36 was absent in Cx36 knockout mice, confirming the specificity of the anti-Cx36 antibodies employed. By freeze-fracture replica immunogold labelling, Cx36 and ZO-1 in brain were found colocalized within individual ultrastructurally identified gap junction plaques, although some plaques contained only Cx36 whereas others contained only ZO-1. Cx36 from mouse brain and Cx36-transfected HeLa cells was found to coimmunoprecipitate with ZO-1. Unlike other connexins that bind the second of the three PDZ domains in ZO-1, glutathione S-transferase-PDZ pulldown and mutational analyses indicated Cx36 interaction with the first PDZ domain of ZO-1, which required at most the presence of the four c-terminus amino acids of Cx36. These results demonstrating a Cx36/ZO-1 association suggest a regulatory and/or scaffolding role of ZO-1 at gap junctions that form electrical synapses between neurons in mammalian brain. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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35. Effect of catalase-specific inhibitor 3-amino-1,2,4-triazole on yeast peroxisomal catalase in vivo
- Author
-
Ueda, Mitsuyoshi, Kinoshita, Hiroshi, Yoshida, Tomoko, Kamasawa, Naomi, Osumi, Masako, and Tanaka, Atsuo
- Subjects
CATALASE ,CANDIDA tropicalis - Abstract
3-Amino-1,2,4-triazole (3-AT) is known as an inhibitor of catalase to whose active center it specifically and covalently binds. Subcellular fractionation and immunoelectronmicroscopic observation of the yeast Candida tropicalis revealed that, in 3-AT-treated cells in which the 3-AT was added to the n-alkane medium from the beginning of cultivation, catalase transported into peroxisomes was inactivated and was present as insoluble aggregated forms in the organelle. The aggregation of catalase in peroxisomes occurred only in these 3-AT-treated cells and not in cells in which 3-AT was added at the late exponential growth phase. Furthermore, 3-AT did not affect the transportation of catalase into peroxisomes. The appearance of aggregation only in cells to which 3-AT was added from the beginning of cultivation suggests that, in the process of catalase transportation into yeast peroxisomes, some conformational change may take place and that correct folding may be inhibited by the binding of 3-AT to the active center of catalase. Accordingly, 3-AT will be an interesting compound for investigation of the transport machinery of the peroxisomal tetrameric catalase. [Copyright &y& Elsevier]
- Published
- 2003
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36. In situ localization of β-glucans in the cell wall of Schizosaccharomyces pombe.
- Author
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Humbel, Bruno M., Konomi, Mami, Takagi, Tomoko, Kamasawa, Naomi, Ishijima, Sanae A., and Osumi, Masako
- Published
- 2001
- Full Text
- View/download PDF
37. Aberrant chloroplasts in transgenic rice plants expressing a high level of maize NADP-dependent malic enzyme.
- Author
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Takeuchi, Yuu, Akagi, Hiromori, Kamasawa, Naomi, Osumi, Masako, and Honda, Hideo
- Subjects
CHLOROPLASTS ,MICROSCOPY ,ELECTRON microscopy ,TRANSGENIC rice ,TRANSGENIC organisms ,PLASTIDS - Abstract
NADP-dependent malic enzyme (NADP-ME) is a major decarboxylating enzyme in NADP-ME-type C
4 species such as maize and Flaveria. In this study, chloroplastic NADP-ME was transferred to rice (Oryza sativa L.) using a chimeric gene composed of maize NADP-ME cDNA under the control of rice light-harvesting chlorophyll-a/b-binding protein (Cab) promoter. There was a 20- to 70-fold increase in the NADP-ME activity in leaves of transgenic rice compared to that in wild-type rice plants. Immunocytochemical studies by electron microscopy showed that maize NADP-ME was mostly localized in chloroplasts in transgenic rice plants, and that the chloroplasts were agranal without thylakoid stacking. Chlorophyll content and photosystem II activity were inversely correlated with the level of NADP-ME activity. These results suggest that aberrant chloroplasts in transgenic plants may be caused by excessive NADP-ME activity. Based on these results and the known fact that only bundle sheath cells of NADP-ME species, among all three C4 subgroups, have agranal chloroplasts, we postulate that a high level of chloroplastic NADP-ME activity could strongly affect the development of chloroplasts. [ABSTRACT FROM AUTHOR]- Published
- 2000
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38. Molecular Breeding of Polysaccharide-Utilizing Yeast Cells by Cell Surface Engineering.
- Author
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UEDA, MITSUYOSHI, MURAI, TOSHIYUKI, SHIBASAKI, YUMI, KAMASAWA, NAOMI, OSUMI, MASAKO, and TANAKA, ATSUO
- Published
- 1998
- Full Text
- View/download PDF
39. Individual Expression of Candida tropicalis Peroxisomal and Mitochondrial Carnitine Acetyltransferase-Encoding Genes and Subcellular Localization of the Products in Saccharomyces cerevisiae.
- Author
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Kawachi, Hiroyuki, Atomi, Haruyuki, Ueda, Mitsuyoshi, Hashimoto, Noriko, Kobayashi, Keiko, Yoshida, Tomoko, Kamasawa, Naomi, Osumi, Masako, and Tanaka, Atsuo
- Subjects
CANDIDA tropicalis ,ACETYLTRANSFERASES ,MITOCHONDRIA ,SACCHAROMYCES cerevisiae ,PEROXISOMES - Abstract
In an n-alkane-assimilating yeast, Candida tropicalis, carnitine acetyltransferase (CAT; EC 2.3.1.7) was localized in both peroxisomes and mitochondria. Both CATs were encoded by one gene, CT-CAT, although the initiation sites of translation were suggested to be different. In the present study, the genes corresponding to the supposed C. tropicalis peroxisomal and mitochondrial CATs, which were truncated from the CT-CAT gene, were individually expressed in Saccharomyces cerevisiae, using the C. tropicalis isocitrate lyase promoter (UPR-ICL), which is inducible by oleic acid in concert with proliferation of peroxisomes in S. cerevisiae [Umemura, K., Atomi, H., Kanai, T., Teranishi, Y., Ueda, M., and Tanaka, A. (1995) Appl. Microbiol. Biotechnol. 43, 489–492]. The 71 kDa precursor of mitochondrial CAT, initiating at the first Met, was found to be processed to the mature size (66 kDa) in S. cerevisiae and immunoelectronmicroscopical observation revealed that this enzyme was localized in mitochondria. On the other hand, 68 kDa CAT, initiating at the second Met (residue No. 19), had no cleavable signal and was translocated into peroxisomes and cytosol, but not into mitochondria. The aniino-terniinal amino acid sequences of individually expressed CATs were identical to those of CATs isolated from alkane-grown C. tropicalls cells, respectively. These results demonstrated that only the 71 kDa protein yielded the 66 kDa protein and that peroxisomal and mitochondrial CATs arose from the difference in the initiation sites of translation. [ABSTRACT FROM AUTHOR]
- Published
- 1996
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- View/download PDF
40. Physiological Roles of Acetoacetyl-CoA Thiolase in n-Alkane-Utilizable Yeast, Candida tropicalis: Possible Contribution to Alkane Degradation and Sterol Biosynthesis.
- Author
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Kurihara, Tatsuo, Ueda, Mitsuyoshi, Kamasawa, Naomi, Osumi, Masako, and Tanaka, Atsuo
- Published
- 1992
41. βl-Oxidation of Butyrate, the Short-Chain-Length Fatty Acid, Occurs in Peroxisomes in the Yeast Candida tropicalis.
- Author
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Kurihara, Tatsuo, Ueda, Mitsuyoshi, Okada, Hirofumi, Kamasawa, Naomi, Naito, Nobuko, Osumi, Masako, and Tanaka, Atsuo
- Published
- 1992
42. Subcellular Destination of Mutant Peroxisomal Isocitrate Lyase Polypeptides of Candida tropicalis in Saccharomyces cerevisiae.
- Author
-
Kamasawa, Naomi, Yoshida, Tomoko, Kasahara, Mari, Kamada, Yasushi, Zou, Wen, Ueda, Mitsuyoshi, Tanaka, Atsuo, and Osumi, Masako
- Abstract
The peroxisomal isocitrate lyase (ICL) of an n-alkane-utilizable yeast, Candida tropicalis, could be transported into the peroxisomes of Saccharomyces cerevisiae MT8-1 cells grown in an oleic acid medium, even if strains were different. To identify the important regions to direct C. tropicalis ICL (CT-ICL) to its proper subcellular location in S. cerevisiae, we observed the subcellular localization of the expressed products of mutant CT-ICL genes in S. cerevisiae by immunoelectron microscopy. Under the control of the UPR-ICL promoter, which enhanced gene expression by proliferation of the peroxisomes, the following mutant CT-ICL genes were constructed and expressed: (i) truncated or mutant genes in the C-terminal region which apparently contained peroxisomal targeting signals, Δ550, Δ549–550, Δ548–550, Δ548–550 + SKL and Δ340–550; and (ii) four internal deletion mutant genes, Δ41–115, Δ41–239, Δ41–325 and Δ237–339. The results showed that three C-terminal amino acid residues were not essential for targeting the peroxisomes in this enzyme. In cells harboring the deleted internal regions of mutant ICL genes, the expressed polypeptides did not target the peroxisomes and the polypeptides, except for Δ41–115, were mistargeted to mitochondria. We observed an unknown structure which we called a “protein aggregate body (PAB),” in which various mutant CT-ICL polypeptides aggregated. [ABSTRACT FROM PUBLISHER]
- Published
- 1996
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43. Morphological Changes of Candida albicans Induced by BMY-28864, a Highly Water-soluble Pradimicin Derivative.
- Author
-
Numata, Keiichi, Ueki, Tomokazu, Naito, Nobuko, Yamada, Naoko, Kamasawa, Naomi, Oki, Toshikazu, and Osumi, Masako
- Abstract
The time course of BMY-28864-dependent morphological changes in A9540 was studied by electron microscopy. Scanning electron microscopy revealed that BMY-28864 often induced cell surface deformations such as abnormal swelling and bulging around budding sites and bud scars. The cell membrane damage was visualized by freeze-fracturing technique as deep pit-like invaginations. Transmission electron microscopy with thin-sectioned specimens also demonstrated that BMY-28864 induced cell membrane invaginations together with cell membrane detachment from the cell wall, nuclear membrane fragmentation and mitochondrial aberration. Statistical sequence analysis of the prominent BMY-28864-dependent morphological changes of the yeast cells led to the conclusion that BMY-28864 first attacked the cell membrane and then caused disintegration of other intracytoplasmic organelles, resulting in the lethal effect. [ABSTRACT FROM PUBLISHER]
- Published
- 1993
44. Light and Dark: Fluorescent and Electron Dense Labeling for Neuronal Cells Using a Novel Viral Vector.
- Author
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Thomas, Connon I., Kamasawa, Naomi, Okayama, Satoko, Keine, Christian, Satterfield, Rachel, Guerrero-Given, Debbie, and Young Jr, Samuel M.
- Published
- 2019
- Full Text
- View/download PDF
45. Author Correction: Cortical response selectivity derives from strength in numbers of synapses.
- Author
-
Scholl, Benjamin, Thomas, Connon I., Ryan, Melissa A., Kamasawa, Naomi, and Fitzpatrick, David
- Abstract
A Correction to this paper has been published: https://doi.org/10.1038/s41586-020-03158-8. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
46. Light and Dark: Fluorescent and Electron Dense Labeling for Neuronal Cells Using a Novel Viral Vector.
- Author
-
Thomas, Connon I., Kamasawa, Naomi, Okayama, Satoko, Keine, Christian, Satterfield, Rachel, Guerrero-Given, Debbie, and Young, Samuel M.
- Published
- 2018
- Full Text
- View/download PDF
47. High Throughput Correlation of Dendritic Spines: 2-photon in vivo live imaging to SEM Utilizing the Automated Tape-Collecting Ultramicrotome and Array Tomography.
- Author
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Thomas, Connon, Kamasawa, Naomi, Peter, Shani, Kuo-Sheng Lee, and Fitzpatrick, David
- Published
- 2017
- Full Text
- View/download PDF
48. CAST/ELKS Proteins Control Voltage-Gated Ca2+ Channel Density and Synaptic Release Probability at a Mammalian Central Synapse.
- Author
-
Dong, Wei, Radulovic, Tamara, Goral, R. Oliver, Thomas, Connon, Suarez Montesinos, Monica, Guerrero-Given, Debbie, Hagiwara, Akari, Putzke, Travis, Hida, Yamato, Abe, Manabu, Sakimura, Kenji, Kamasawa, Naomi, Ohtsuka, Toshihisa, and Jr.Young, Samuel M.
- Abstract
Summary In the presynaptic terminal, the magnitude and location of Ca 2+ entry through voltage-gated Ca 2+ channels (VGCCs) regulate the efficacy of neurotransmitter release. However, how presynaptic active zone proteins control mammalian VGCC levels and organization is unclear. To address this, we deleted the CAST/ELKS protein family at the calyx of Held, a Ca V 2.1 channel-exclusive presynaptic terminal. We found that loss of CAST/ELKS reduces the Ca V 2.1 current density with concomitant reductions in Ca V 2.1 channel numbers and clusters. Surprisingly, deletion of CAST/ELKS increases release probability while decreasing the readily releasable pool, with no change in active zone ultrastructure. In addition, Ca 2+ channel coupling is unchanged, but spontaneous release rates are elevated. Thus, our data identify distinct roles for CAST/ELKS as positive regulators of Ca V 2.1 channel density and suggest that they regulate release probability through a post-priming step that controls synaptic vesicle fusogenicity. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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