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Once upon a time the statistics of quantal release were fashionable: "n" available vesicles (fusion sites), each with probability "p" of releasing a quantum. The story was not so simple, a nice paradigm to be abandoned. Biophysicists, experimenting with "black films," explained the astonishing rapidity of spike- induced release: calcium can trigger the fusion of lipidic vesicles with a lipid bilayer, by masking the negative charges of the membranes. The idea passed away, buried by the discovery of NSF, SNAPs, SNARE proteins and synaptotagmin, Munc, RIM, complexin. Electrophysiology used to be a field for few adepts. Then came patch clamp, and multielectrode arrays and everybody became electrophysiologists. Now, optogenetics have blossomed, and the whole field has changed again. Nice surprise for me, when Alvarez de Toledo demonstrated that release of transmitters could occur through the transient opening of a pore between the vesicle and the plasma-membrane, no collapse of the vesicle in the membrane needed: my mentor Bruno Ceccarelli had cherished this idea ("kiss and run") and tried to prove it for 20 years. The most impressive developments have probably regarded IT, computers and all their applications; machine learning, AI, and the truly spectacular innovations in brain imaging, especially functional ones, have transformed cognitive neurosciences into a new extraordinarily prolific field, and certainly let us imagine that we may finally understand what is going on in our brains. Cellular neuroscience, on the other hand, though the large public has been much less aware of the incredible amount of information the scientific community has acquired on the cellular aspects of neuronal function, may indeed help us to eventually understand the mechanistic detail of how the brain work. But this is no more in the past, this is the future. [ABSTRACT FROM AUTHOR]

Synaptic dysfunction is an early feature in Alzheimer's disease (AD) pathogenesis and a major morphological correlate of memory deficits. Given the main synaptic location of N-methyl-D-aspartate receptors (NMDARs), their dysregulation has been implicated in these pathological effects. Here, to detect possible alterations in the expression and synaptic localisation of the GluN1 subunit in the brain of amyloidogenic APP/PS1 mice, we employed histoblot and SDS-digested freeze-fracture replica labelling (SDS-FRL) techniques. Histoblots showed that GluN1 expression was significantly reduced in the hippocampus in a layer-dependent manner, in the cortex and the caudate putamen of APP/PS1 transgenic mice at 12 months of age but was unaltered at 1 and 6 months. Using quantitative SDS-FRL, we unravelled the molecular organisation of GluN1 in seven excitatory synapse populations at a high spatial resolution in the CA1 and CA3 fields and the DG of the hippocampus in 12-month-old APP/PS1 mice. In the CA1 field, the labelling density for GluN1 in the excitatory synapses established on spines and interneurons, was significantly reduced in APP/PS1 mice compared to age-matched wild-type mice in the stratum lacunosum-moleculare but unaltered in the stratum radiatum. In the CA3 field, synaptic GluN1 was reduced in mossy fibre-CA3 pyramidal cell synapses but unaltered in the A/C-CA3 pyramidal cell synapses. In the DG, the density of GluN1 in granule cell-perforant pathway synapses was reduced in APP/PS1 mice. Altogether, our findings provide evidence of specific alterations of synaptic GluN1 in the trisynaptic circuit of the hippocampus in Aβ pathology. This differential vulnerability in the disruption of NMDARs may be involved in the mechanisms causing abnormal network activity of the hippocampal circuit and cognitive impairment characteristic of APP/PS1 mice. [ABSTRACT FROM AUTHOR]

Abstract Synaptic dysfunction is an early feature in Alzheimer’s disease (AD) pathogenesis and a major morphological correlate of memory deficits. Given the main synaptic location of N-methyl-D-aspartate receptors (NMDARs), their dysregulation has been implicated in these pathological effects. Here, to detect possible alterations in the expression and synaptic localisation of the GluN1 subunit in the brain of amyloidogenic APP/PS1 mice, we employed histoblot and SDS-digested freeze-fracture replica labelling (SDS-FRL) techniques. Histoblots showed that GluN1 expression was significantly reduced in the hippocampus in a layer-dependent manner, in the cortex and the caudate putamen of APP/PS1 transgenic mice at 12 months of age but was unaltered at 1 and 6 months. Using quantitative SDS-FRL, we unravelled the molecular organisation of GluN1 in seven excitatory synapse populations at a high spatial resolution in the CA1 and CA3 fields and the DG of the hippocampus in 12-month-old APP/PS1 mice. In the CA1 field, the labelling density for GluN1 in the excitatory synapses established on spines and interneurons, was significantly reduced in APP/PS1 mice compared to age-matched wild-type mice in the stratum lacunosum-moleculare but unaltered in the stratum radiatum. In the CA3 field, synaptic GluN1 was reduced in mossy fibre-CA3 pyramidal cell synapses but unaltered in the A/C-CA3 pyramidal cell synapses. In the DG, the density of GluN1 in granule cell-perforant pathway synapses was reduced in APP/PS1 mice. Altogether, our findings provide evidence of specific alterations of synaptic GluN1 in the trisynaptic circuit of the hippocampus in Aβ pathology. This differential vulnerability in the disruption of NMDARs may be involved in the mechanisms causing abnormal network activity of the hippocampal circuit and cognitive impairment characteristic of APP/PS1 mice.

Voltage‐gated CaV2.1 (P/Q‐type) Ca2+ channels play a crucial role in regulating neurotransmitter release, thus contributing to synaptic plasticity and to processes such as learning and memory. Despite their recognized importance in neural function, there is limited information on their potential involvement in neurodegenerative conditions such as Alzheimer's disease (AD). Here, we aimed to explore the impact of AD pathology on the density and nanoscale compartmentalization of CaV2.1 channels in the hippocampus in association with GABAB receptors. Histoblotting experiments showed that the density of CaV2.1 channel was significantly reduced in the hippocampus of APP/PS1 mice in a laminar‐dependent manner. CaV2.1 channel was enriched in the active zone of the axon terminals and was present at a very low density over the surface of dendritic tree of the CA1 pyramidal cells, as shown by quantitative SDS‐digested freeze‐fracture replica labelling (SDS‐FRL). In APP/PS1 mice, the density of CaV2.1 channel in the active zone was significantly reduced in the strata radiatum and lacunosum‐moleculare, while it remained unaltered in the stratum oriens. The decline in Cav2.1 channel density was found to be associated with a corresponding impairment in the GABAergic synaptic function, as evidenced by electrophysiological experiments carried out in the hippocampus of APP/PS1 mice. Remarkably, double SDS‐FRL showed a co‐clustering of CaV2.1 channel and GABAB1 receptor in nanodomains (~40–50 nm) in wild type mice, while in APP/PS1 mice this nanoarchitecture was absent. Together, these findings suggest that the AD pathology‐induced reduction in CaV2.1 channel density and CaV2.1‐GABAB1 de‐clustering may play a role in the synaptic transmission alterations shown in the AD hippocampus. Therefore, uncovering these layer‐dependent changes in P/Q calcium currents associated with AD pathology can benefit the development of future strategies for AD management. [ABSTRACT FROM AUTHOR]

Once upon a time the statistics of quantal release were fashionable: “n” available vesicles (fusion sites), each with probability “p” of releasing a quantum. The story was not so simple, a nice paradigm to be abandoned. Biophysicists, experimenting with “black films,” explained the astonishing rapidity of spike-induced release: calcium can trigger the fusion of lipidic vesicles with a lipid bilayer, by masking the negative charges of the membranes. The idea passed away, buried by the discovery of NSF, SNAPs, SNARE proteins and synaptotagmin, Munc, RIM, complexin. Electrophysiology used to be a field for few adepts. Then came patch clamp, and multielectrode arrays and everybody became electrophysiologists. Now, optogenetics have blossomed, and the whole field has changed again. Nice surprise for me, when Alvarez de Toledo demonstrated that release of transmitters could occur through the transient opening of a pore between the vesicle and the plasma-membrane, no collapse of the vesicle in the membrane needed: my mentor Bruno Ceccarelli had cherished this idea (“kiss and run”) and tried to prove it for 20 years. The most impressive developments have probably regarded IT, computers and all their applications; machine learning, AI, and the truly spectacular innovations in brain imaging, especially functional ones, have transformed cognitive neurosciences into a new extraordinarily prolific field, and certainly let us imagine that we may finally understand what is going on in our brains. Cellular neuroscience, on the other hand, though the large public has been much less aware of the incredible amount of information the scientific community has acquired on the cellular aspects of neuronal function, may indeed help us to eventually understand the mechanistic detail of how the brain work. But this is no more in the past, this is the future.

N‐methyl‐d‐aspartate receptors (NMDARs) are pivotal players in the synaptic transmission and synaptic plasticity underlying learning and memory. Accordingly, dysfunction of NMDARs has been implicated in the pathophysiology of Alzheimer disease (AD). Here, we used histoblot and sodium dodecylsulphate‐digested freeze‐fracture replica labelling (SDS‐FRL) techniques to investigate the expression and subcellular localisation of GluN1, the obligatory subunit of NMDARs, in the hippocampus of P301S mice. Histoblots showed that GluN1 expression was significantly reduced in the hippocampus of P301S mice in a laminar‐specific manner at 10 months of age but was unaltered at 3 months. Using the SDS‐FRL technique, excitatory synapses and extrasynaptic sites on spines of pyramidal cells and interneuron dendrites were analysed throughout all dendritic layers in the CA1 field. Our ultrastructural approach revealed a high density of GluN1 in synaptic sites and a substantially lower density at extrasynaptic sites. Labelling density for GluN1 in excitatory synapses established on spines was significantly reduced in P301S mice, compared with age‐matched wild‐type mice, in the stratum oriens (so), stratum radiatum (sr) and stratum lacunosum‐moleculare (slm). Density for synaptic GluN1 on interneuron dendrites was significantly reduced in P301S mice in the so and sr but unaltered in the slm. Labelling density for GluN1 at extrasynaptic sites showed no significant differences in pyramidal cells, and only increased density in the interneuron dendrites of the sr. This differential alteration of synaptic versus extrasynaptic NMDARs supports the notion that the progressive accumulation of phospho‐tau is associated with changes in NMDARs, in the absence of amyloid‐β pathology, and may be involved in the mechanisms causing abnormal network activity of the hippocampal circuit. [ABSTRACT FROM AUTHOR]

Tau pathology is a hallmark of Alzheimer's disease (AD) and other tauopathies, but how pathological tau accumulation alters the glutamate receptor dynamics driving synaptic dysfunction is unclear. Here, we determined the impact of tau pathology on AMPAR expression, density, and subcellular distribution in the hippocampus of P301S mice using immunoblot, histoblot, and quantitative SDS-digested freeze-fracture replica labeling (SDS-FRL). Histoblot and immunoblot showed differential regulation of GluA1 and GluA2 in the hippocampus of P301S mice. The GluA2 subunit was downregulated in the hippocampus at 3 months while both GluA1 and GluA2 subunits were downregulated at 10 months. However, the total amount of GluA1-4 was similar in P301S mice and in age-matched wild-type mice. Using quantitative SDS-FRL, we unraveled the molecular organization of GluA1-4 in various synaptic connections at a high spatial resolution on pyramidal cell spines and interneuron dendrites in the CA1 field of the hippocampus in 10-month-old P301S mice. The labeling density for GluA1-4 in the excitatory synapses established on spines was significantly reduced in P301S mice, compared to age-matched wild-type mice, in the strata radiatum and lacunosum-moleculare but unaltered in the stratum oriens. The density of synaptic GluA1-4 established on interneuron dendrites was significantly reduced in P301S mice in the three strata. The labeling density for GluA1-4 at extrasynaptic sites was significantly reduced in several postsynaptic compartments of CA1 pyramidal cells and interneurons in the three dendritic layers in P301S mice. Our data demonstrate that the progressive accumulation of phospho-tau is associated with alteration of AMPARs on the surface of different neuron types, including synaptic and extrasynaptic membranes, leading to a decline in the trafficking and synaptic transmission, thereby likely contributing to the pathological events taking place in AD. [ABSTRACT FROM AUTHOR]

Tau pathology is a hallmark of Alzheimer’s disease (AD) and other tauopathies, but how pathological tau accumulation alters the glutamate receptor dynamics driving synaptic dysfunction is unclear. Here, we determined the impact of tau pathology on AMPAR expression, density, and subcellular distribution in the hippocampus of P301S mice using immunoblot, histoblot, and quantitative SDS-digested freeze-fracture replica labeling (SDS-FRL). Histoblot and immunoblot showed differential regulation of GluA1 and GluA2 in the hippocampus of P301S mice. The GluA2 subunit was downregulated in the hippocampus at 3 months while both GluA1 and GluA2 subunits were downregulated at 10 months. However, the total amount of GluA1-4 was similar in P301S mice and in age-matched wild-type mice. Using quantitative SDS-FRL, we unraveled the molecular organization of GluA1-4 in various synaptic connections at a high spatial resolution on pyramidal cell spines and interneuron dendrites in the CA1 field of the hippocampus in 10-month-old P301S mice. The labeling density for GluA1-4 in the excitatory synapses established on spines was significantly reduced in P301S mice, compared to age-matched wild-type mice, in the strata radiatum and lacunosum-moleculare but unaltered in the stratum oriens. The density of synaptic GluA1-4 established on interneuron dendrites was significantly reduced in P301S mice in the three strata. The labeling density for GluA1-4 at extrasynaptic sites was significantly reduced in several postsynaptic compartments of CA1 pyramidal cells and interneurons in the three dendritic layers in P301S mice. Our data demonstrate that the progressive accumulation of phospho-tau is associated with alteration of AMPARs on the surface of different neuron types, including synaptic and extrasynaptic membranes, leading to a decline in the trafficking and synaptic transmission, thereby likely contributing to the pathological events taking place in AD.

The amygdala plays a crucial role in attaching emotional significance to environmental cues. Its intercalated cell masses (ITC) are tight clusters of GABAergic neurons, which are distributed around the basolateral amygdala complex. Distinct ITC clusters are involved in the acquisition and extinction of conditioned fear responses. Previously, we have shown that fear memory retrieval reduces the AMPA/NMDA ratio at thalamic afferents to ITC neurons within the dorsal medio-paracapsular cluster. Here, we investigate the molecular mechanisms underlying the fear-mediated reduction in the AMPA/NMDA ratio at these synapses and, in particular, whether specific changes in the synaptic density of AMPA receptors underlie the observed change. To this aim, we used a detergent-digested freeze-fracture replica immunolabeling technique (FRIL) approach that enables to visualize the spatial distribution of intrasynaptic AMPA receptors at high resolution. AMPA receptors were detected using an antibody raised against an epitope common to all AMPA subunits. To visualize thalamic inputs, we virally transduced the posterior thalamic complex with Channelrhodopsin 2-YFP, which is anterogradely transported along axons. Using face-matched replica, we confirmed that the postsynaptic elements were ITC neurons due to their prominent expression of μ-opioid receptors. With this approach, we show that, following auditory fear conditioning in mice, the formation and retrieval of fear memory is linked to a significant reduction in the density of AMPA receptors, particularly at spine synapses formed by inputs of the posterior intralaminar thalamic and medial geniculate nuclei onto identified ITC neurons. Our study is one of the few that has directly linked the regulation of AMPA receptor trafficking to memory processes in identified neuronal networks, by showing that fear-memory induced reduction in AMPA/NMDA ratio at thalamic-ITC synapses is associated with a reduced postsynaptic AMPA receptor density.

Macroautophagy (hereafter autophagy) is an intracellular degradative pathway in budding yeast cells. Certain lipid types play essential roles in autophagy; yet the precise mechanisms regulating lipid composition during autophagy remain unknown. Here, we explored the role of the Osh family proteins in the modulating lipid composition during autophagy in budding yeast. Our results showed that osh1 - osh7 ∆ deletions lead to autophagic dysfunction, with impaired GFP-Atg8 processing and the absence of autophagosomes and autophagic bodies in the cytosol and vacuole, respectively. Freeze-fracture electron microscopy (EM) revealed elevated phosphatidylinositol 4-phosphate (PtdIns(4)P) levels in cytoplasmic and luminal leaflets of autophagic bodies and vacuolar membranes in all deletion mutants. Phosphatidylserine (PtdSer) levels were significantly decreased in the autophagic bodies and vacuolar membranes in osh4 ∆ and osh5 ∆ mutants, whereas no significant changes were observed in other osh deletion mutants. Furthermore, we identified defects in autophagic processes in the osh4 ∆ and osh5 ∆ mutants, including rare autophagosome formation in the osh5 ∆ mutant and accumulation of autophagic bodies in the vacuole in the osh4 ∆ mutant, even in the absence of the proteinase inhibitor PMSF. These findings suggest that Osh4p and Osh5p play crucial roles in the transport of PtdSer to autophagic bodies and autophagosome membranes, respectively. The precise control of lipid composition in the membranes of autophagosomes and autophagic bodies by Osh4p and Osh5p represents an important regulatory mechanism in autophagy. [Display omitted] • Defects in autophagy in yeast osh1 - osh7 ∆: osh4 ts mutant • Increase in PtdIns(4)P in the membrane of yeast osh1 - osh7 ∆: osh4 ts mutant • Decrease in PtdSer in the membrane of yeast osh1 - osh7 ∆: osh4 ts mutant • Osh5p is essential for autophagosome formation • Osh4p is essential for degradation of autophagic bodies in the vacuole • Decrease in PtdSer in the membranes of yeast osh4 ∆ and osh5 ∆ mutants [ABSTRACT FROM AUTHOR]

Membrane microdomains or rafts, sterol- and sphingolipid-rich microdomains in the plasma membrane have been studied extensively in mammalian cells. Recently, rafts were found to mediate virulence in a variety of parasites, including Toxoplasma gondii. However, it has been difficult to examine a two-dimensional distribution of lipid molecules at a nanometer scale. We tried to determine the distribution of glycosphingolipids GM1 and GM3, putative raft components in the T. gondii cell membrane in this study, using a rapid-frozen and freeze-fractured immuno-electron microscopy method. This method physically stabilized molecules in situ, to minimize the probability of artefactual disruption. Labeling of GM3, but not GM1, was observed in the exoplasmic (or luminal), but not the cytoplasmic, leaflet of the inner membrane complex (IMC) in T. gondii infected in human foreskin fibroblast-1 (HFF-1). No labeling was detected in any leaflet of the T. gondii plasma membrane. In contrast to HFF-1, T. gondii infected in mouse fibroblast (MF), labelings of both GM1 and GM3 were detected in the IMC luminal leaflet, although GM1′s gold labeling density was very low. The same freeze-fracture EM method showed that both GM1 and GM3 were expressed in the exoplasmic leaflet of the MF plasma membrane. However, labeling of only GM3, but not GM1, was detected in the exoplasmic leaflet of the HFF-1 plasma membrane. These results suggest that GM1 or GM3, localized in the IMC, is obtained from the plasma membranes of infected host mammalian cells. Furthermore, the localization of microdomains or rafts in the luminal leaflets of the intracellular confined space IMC organelle of T. gondii suggests a novel characteristic of rafts.

The hippocampus plays key roles in learning and memory and is a main target of Alzheimer's disease (AD), which causes progressive memory impairments. Despite numerous investigations about the processes required for the normal hippocampal functions, the neurotransmitter receptors involved in the synaptic deficits by which AD disables the hippocampus are not yet characterized. By combining histoblots, western blots, immunohistochemistry and high‐resolution immunoelectron microscopic methods for GABAB receptors, this study provides a quantitative description of the expression and the subcellular localization of GABAB1 in the hippocampus in a mouse model of AD at 1, 6 and 12 months of age. Western blots and histoblots showed that the total amount of protein and the laminar expression pattern of GABAB1 were similar in APP/PS1 mice and in age‐matched wild‐type mice. In contrast, immunoelectron microscopic techniques showed that the subcellular localization of GABAB1 subunit did not change significantly in APP/PS1 mice at 1 month of age, was significantly reduced in the stratum lacunosum‐moleculare of CA1 pyramidal cells at 6 months of age and significantly reduced at the membrane surface of CA1 pyramidal cells at 12 months of age. This reduction of plasma membrane GABAB1 was paralleled by a significant increase of the subunit at the intracellular sites. We further observed a decrease of membrane‐targeted GABAB receptors in axon terminals contacting CA1 pyramidal cells. Our data demonstrate compartment‐ and age‐dependent reduction of plasma membrane‐targeted GABAB receptors in the CA1 region of the hippocampus, suggesting that this decrease might be enough to alter the GABAB‐mediated synaptic transmission taking place in AD. [ABSTRACT FROM AUTHOR]

Metabotropic glutamate receptor subtype 5 (mGlu5) is implicated in the pathophysiology of Alzheimer’s disease (AD). However, its alteration at the subcellular level in neurons is still unexplored. Here, we provide a quantitative description on the expression and localisation patterns of mGlu5 in the APP/PS1 model of AD at 12 months of age, combining immunoblots, histoblots and high-resolution immunoelectron microscopic approaches. Immunoblots revealed that the total amount of mGlu5 protein in the hippocampus, in addition to downstream molecules, i.e., Gq/11 and PLCβ1, was similar in both APP/PS1 mice and age-matched wild type mice. Histoblots revealed that mGlu5 expression in the brain and its laminar expression in the hippocampus was also unaltered. However, the ultrastructural techniques of SDS-FRL and pre-embedding immunogold demonstrated that the subcellular localisation of mGlu5 was significantly reduced along the neuronal surface of hippocampal principal cells, including CA1 pyramidal cells and DG granule cells, in APP/PS1 mice at 12 months of age. The decrease in the surface localisation of mGlu5 was accompanied by an increase in its frequency at intracellular sites in the two neuronal populations. Together, these data demonstrate, for the first time, a loss of mGlu5 at the plasma membrane and accumulation at intracellular sites in different principal cells of the hippocampus in APP/PS1 mice, suggesting an alteration of the excitability and synaptic transmission that could contribute to the cognitive dysfunctions in this AD animal model. Further studies are required to elucidate the specificity of mGlu5-associated molecules and downstream signalling pathways in the progression of the pathology.

Cryo-planing SEM provides a powerful 3D view of internal and external tissue and cell features. However, specimen preparation is tedious because it requires custom apparatus and difficult cryogenic manipulations. We present an easy method using mPrep/s capsules that provide a "handle" to hold and orient specimens throughout cryo-preparation and imaging. Cryo-facing is done with a cryo-ultramicrotome, requiring no custom equipment while providing accurate control of the image plane. We show this with fresh Christmas cactus leaves and aldehyde-fixed kidney tissue imaged by room temperature SEM after freeze-drying, demonstrating the method operating without an expensive cryo-SEM. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 4‐phophate (PtdIns(4)P) is an essential signaling molecule in the Golgi body, endosomal system, and plasma membrane and functions in the regulation of membrane trafficking, cytoskeletal organization, lipid metabolism and signal transduction pathways, all mediated by direct interaction with PtdIns(4)P‐binding proteins. PtdIns(4)P was recently reported to have functional roles in autophagosome biogenesis. LC3 and GABARAP subfamilies and a small GTP‐binding protein, Rab7, are localized on autophagosomal membranes and participate at each stage of autophagosome formation and maturation. To better understand autophagosome biogenesis, it is essential to determine the localization of PtdIns(4)P and to examine its relationship with LC3 and GABARAP subfamilies and Rab7. To analyze PtdIns(4)P distribution, we used an electron microscopy technique that labels PtdIns(4)P on the freeze‐fracture replica of intracellular biological membranes, which minimizes the possibility of artificial perturbation because molecules in the membrane are physically immobilized in situ. Using this technique, we found that PtdIns(4)P is localized on the cytoplasmic, but not the luminal (exoplasmic), leaflet of the inner and outer membranes of autophagosomes. Double labeling revealed that PtdIns(4)P mostly colocalizes with Rab7, but not with LC3B, GABARAP, GABARAPL1 and GABARAPL2. Rab7 plays essential roles in autophagosome maturation and in autophagosome‐lysosome fusion events. We suggest that PtdIns(4)P is localized to the cytoplasmic leaflet of the autophagosome at later stages, which may illuminate the importance of PtdIns(4)P at the later stages of autophagosome formation. Quick‐freezing and freeze‐fracture replica labeling method revealed that phosphatidylinositol 4‐phosphate (PtdIns(4)P) was localized on the cytoplasmic leaflet in the inner and outer membranes of the autophagosome in the Torin1‐treated autophagy‐induced Huh7 cells. PtdIns(4)P was colocalized with Rab7, but not LC3B, GABARAP, GABARAPL1 or GABARAPL2 on the autophagosomal membranes. Rab7 was reported to play essential roles for the maturation of autophagosome formation, suggesting that PtdIns(4)P appears with Rab7 at the later and mature stages of autophagosome formation. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 4-phosphate (PtdIns(4)P) is the immediate precursor of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), which is located on the cytoplasmic leaflet of the plasma membrane and has been reported to possess multiple cellular functions. Although PtdIns(4)P and PtdIns(4,5)P 2 have been reported to localize to multiple intracellular compartments and to each function as regulatory molecules, their generation, regulation and functions in most intracellular compartments are not well-defined. To analyze PtdIns(4)P and PtdIns(4,5)P 2 distributions, at a nanoscale, we employed an electron microscopy technique that specifically labels PtdIns(4)P and PtdIns(4,5)P 2 on the freeze-fracture replica of intracellular biological membranes. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilized in situ . Using this technique, we found that PtdIns(4)P was localized to the cytoplasmic leaflet of Golgi apparatus and vesicular-shaped structures. The PtdIns(4,5)P 2 labeling was observed in the cytoplasmic leaflet of the mitochondrial inner membrane and vesicular-shaped structures. Double labeling of PtdIns(4)P and PtdIns(4,5)P 2 with endosome markers illustrated that PtdIns(4)P and PtdIns(4,5)P 2 were mainly localized to the late endosome/lysosome and early endosome, respectively. PtdIns(4)P and PtdIns(4,5)P 2 were colocalized in some endosomes, with the two phospholipids separated into distinct microdomains on the same endosomes. This is the first report showing, at a nanoscale, segregation of PtdIns(4)P- and PtdIns(4,5)P 2 -enriched microdomains in the endosome, of likely importance for endosome functionality. [ABSTRACT FROM AUTHOR]

We developed a new method to observe distribution of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] using electron microscopy. In freeze-fracture replicas of quick-frozen samples, PtdIns(3,5)P2 was labeled specifically using recombinant ATG18 tagged with glutathione S-transferase and 4×FLAG, which was mixed with an excess of recombinant PX domain to suppress binding of ATG18 to phosphatidylinositol 3-phosphate. Using this method, PtdIns(3,5)P2 was found to be enriched in limited domains in the yeast vacuole and mammalian endosomes. In the yeast vacuole exposed to hyperosmolar stress, PtdIns(3,5)P2 was distributed at a significantly higher density in the intramembrane particle (IMP)-deficient liquid-ordered domains than in the surrounding IMP-rich domains. In mammalian cells, PtdIns(3,5)P2 was observed in endosomes of tubulo-vesicular morphology labeled for RAB5 or RAB7. Notably, distribution density of PtdIns(3,5)P2 in the endosome was significantly higher in the vesicular portion than in the tubular portion. The nano-scale distribution of PtdIns(3,5)P2 revealed in the present study is important to understand its functional roles in the vacuole and endosomes.

Early intermediate structures of liposome-liposome fusion events were captured by freeze-fracture electron microscopic (EM) technique. The images show the morphology of the fusion interface at several different stages of the fusion event. One of the intermediates was captured at a serendipitous stage of two vesicles’ membranes (both leaflets) merging and their contents starting to intermix clearly showing the fusion interface with a previously unseen fusion rim. From the morphological information a hypothetical sequence of the fusion event and corresponding lipid structural arrangements are described.

Early intermediate structures of liposome-liposome fusion events were captured by freeze-fracture electron microscopic (EM) technique. The images show the morphology of the fusion interface at several different stages of the fusion event. One of the intermediates was captured at a serendipitous stage of two vesicles’ membranes (both leaflets) merging and their contents starting to intermix clearly showing the fusion interface with a previously unseen fusion rim. From the morphological information a hypothetical sequence of the fusion event and corresponding lipid structural arrangements are described.

Phosphatidylinositol 4-phosphate [PtdIns(4)P] is the immediate precursor of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ], which is localized to the cytoplasmic leaflet of the plasma membrane and has been reported to possess multiple cell biological functions. Direct evidence showing the distribution of PtdIns(4)P pools at a nanoscale when the plasma membrane PtdIns(4,5)P 2 is hydrolyzed by agonist stimulation is lacking. To analyze the distribution of PtdIns(4)P at a nanoscale, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the plasma membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilized in situ . Using this technique, we observed no PtdIns(4)P in the caveolae of normal cultured human fibroblasts, although PtdIns(4,5)P 2 has been shown to be highly concentrated in them in our previous report. When cells were stimulated with angiotensin II, the level of PtdIns(4)P in the undifferentiated membrane transiently decreased to 64.3% at 10 s, began to increase at 30 s and largely increased to 341.9% at 40 s, and then returned to the initial level at 130 s after the stimulation. Interestingly, PtdIns(4)P localized at the caveolae at 70 and 130 s after the stimulation. These results suggest that the level of the PtdIns(4)P pool in the plasma membrane is sensitive and the distribution of PtdIns(4)P dramatically changes by agonist stimulation, and there are active sites of production or replenishment of PtdIns(4)P at undifferentiated membrane and caveolar areas. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 3,5-bisphosphate ( PtdIns(3,5) P2) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5) P2 using freeze-fracture electron microscopy. GST-ATG18-4× FLAG was used to label PtdIns(3,5) P2 and its binding to phosphatidylinositol 3-phosphate ( PtdIns(3)P) was blocked by an excess of the p40phox PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5) P2 was concentrated in intramembrane particle ( IMP)-deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP-deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid-disordered domain marker. In yeast lacking either PtdIns(3,5) P2 or its effector, Atg18p, the IMP-deficient, PtdIns(3)P-rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5) P2 metabolism is defective. In HeLa cells, PtdIns(3,5) P2 was significantly enriched in the vesicular domain of RAB5- and RAB7-positive endosome/lysosomes of the tubulo-vesicular morphology. This biased distribution of PtdIns(3,5) P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5) P2-rich and -deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality. [ABSTRACT FROM AUTHOR]

In this study we describe the early changes of the myelin sheath following surgical nerve crush. We used the freeze-fracture technique to better evaluate myelin alterations during an early stage of Wallerian degeneration. Rat sural nerves were experimentally crushed and animals were sacrificed by transcardiac perfusion 30 h after surgery. Segments of the nerves were processed for routine transmission electron microscopy and freeze-fracture techniques. Our results show that 30 h after the lesion there was asynchrony in the pattern of Wallerian degeneration, with different nerve fibers exhibiting variable degrees of axon disruption. This was observed by both techniques. Careful examination of several replicas revealed early changes in myelin membranes represented by vacuolization and splitting of consecutive lamellae, rearrangement of intramembranous particles and disappearance of paranodal transverse bands associated or not with retraction of paranodal myelin terminal loops from the axolemma. These alterations are compatible with a direct injury to the myelin sheath following nerve crush. The results are discussed in terms of a similar mechanism underlying both axon and myelin breakdown.

Background and Aims Cryopreservation is the only long-term conservation strategy available for germplasm of recalcitrant-seeded species. Efforts to cryopreserve this form of germplasm are hampered by potentially lethal intracellular freezing events; thus, it is important to understand the relationships among cryo-exposure techniques, water content, structure and survival. Methods Undried embryonic axes of Acer saccharinum and those rapidly dried to two different water contents were cooled at three rates and re-warmed at two rates. Ultrastructural observations were carried out on radicle and shoot tips prepared by freeze-fracture and freeze-substitution to assess immediate (i.e. pre-thaw) responses to cooling treatments. Survival of axes was assessed in vitro. Key Results Intracellular ice formation was not necessarily lethal. Embryo cells survived when crystal diameter was between 0·2 and 0·4 µm and fewer than 20 crystals were distributed per μm2 in the cytoplasm. Ice was not uniformly distributed within the cells. In fully hydrated axes cooled at an intermediate rate, the interiors of many organelles were apparently ice-free; this may have prevented the disruption of vital intracellular machinery. Intracytoplasmic ice formation did not apparently impact the integrity of the plasmalemma. The maximum number of ice crystals was far greater in shoot apices, which were more sensitive than radicles to cryo-exposure. Conclusions The findings challenge the accepted paradigm that intracellular ice formation is always lethal, as the results show that cells can survive intracellular ice if crystals are small and localized in the cytoplasm. Further understanding of the interactions among water content, cooling rate, cell structure and ice structure is required to optimize cryopreservation treatments without undue reliance on empirical approaches. [ABSTRACT FROM PUBLISHER]

Scanning electron microscopy operated at cryogenic temperature (cryo-SEM) is a powerful tool for investigating surface and cross-sectional nanostructures of water-containing samples. Typically, cryo-SEM samples are frozen just before observation in specific metal carriers. However, pre-frozen samples are also of interest, such as frozen food and freeze-stored animal samples. In such cases, sample mounting with a defined orientation is required, but there has been a lack of ideal conductive adhesives that can be used without increasing the sample temperature. Here, we developed a mixture of graphite oxide and 1,3-butanediol as an adhesive, capable of gluing samples at dry-ice temperature and is frozen below that temperature. Dispersion of graphite oxide increased the conductivity and reduced the charge-up contrast. Acquisition of energy-dispersive X-ray spectrum, cross-sectional ion milling, and high-resolution imaging were successfully achieved using the adhesive. We tested and confirmed the usefulness of this new adhesive by applying it to cryo-SEM surface imaging of diatomite, freeze-fractured cross-sectional imaging of chicken liver, and ion milling cross-sectional imaging of a deep-sea snail. The new adhesive is not only useful for food science and field-preserved biological samples but also potentially applicable to wider fields such as archaeological and biological samples preserved under permafrost.

The freeze-fracture technique was used to analyse the organization of the plasma membrane, as well as membranes of cytoplasmic organelles, of the pathogenic protozoan Trichomonas vaginalis. Rosettes formed by 4 to 14 intramembranous particles were seen on the fracture faces of the membrane lining the anterior flagella as well as in fracture faces of the plasma membrane enclosing the anterior region of the protozoan and in cytoplasmic organelles. Special organization of the membrane particles were also seen in the region of association of the recurrent flagellum to the cell body.

Abstract: Sindbis Virus (SV), the prototype alphavirus in the family togaviridae, infects both mammalian and insect cells. The ability of SV to infect cells possessing significantly different biochemical environments suggests that there may be a common mode of entry into each cell type. Previous studies show that up to 4h post infection cells are permeable to small ions and alpha sarcin suggesting that the plasma membrane is compromised as infection takes place. Thin-section electron microscopy has also shown SV to bind to the plasma membrane and lose its electron dense core through a pore like structure developed upon interaction of the virus with the cell surface. Using freeze-fracture replicas, thin-sections and antibody labeling the data presented herein show virus associated with intramembrane particles on mosquito cells. These data suggest that the intramembrane particles associated with SV may be part of the pore structure consisting of virus proteins and cell receptor. [Copyright &y& Elsevier]

The freezefracture technique provides large and detailed ultrastructural views of cellular membranes in the transmission electron microscope. The major goal of freezefracture cytochemistry is the identification and localization of membrane components such as proteins or lipids for studying their distribution, dynamics or their relationship to specialized membrane domains. The most versatile and effective technique in freezefracture cytochemistry is termed freezefracture replica immunolabeling (FRIL). Using this technique chemically unfixed cellular samples are rapidly cryofixed in their natural habitat. They are fractured, replicated and immobilized by platinumcarbon evaporation followed by a careful sodium dodecyl sulfate (SDS) treatment. The detergent SDS removes most of the cellular material except those molecules that are in direct contact to the platinum/carbon replica. Immunogold labeling of these replicabound biomolecules visualizes their spatial organization in high resolution on planar views of cellular membranes. [ABSTRACT FROM AUTHOR]

Spermatogenesis requires connexin 43 (Cx43).This study examines normal gene transcription, translation, and phosphorylation of Cx43 to define its role on germ cell growth and Sertoli cell's differentiation, and identifies abnormalities arising from spontaneous autoimmune orchitis (AIO) in mink, a seasonal breeder and a natural model for autoimmunity. Northern blot analysis detected 2.8- and a 3.7-kb Cx43 mRNA bands in seminiferous tubule-enriched fractions. Cx43 mRNA increased in seminiferous tubule-enriched fractions throughout development and then seasonally with the completion of spermatogenesis. Cx43 protein levels increased transiently during the colonization of the tubules by the early-stage spermatocytes. Cx43 phosphorylated (PCx43) and nonphosphorylated (NPCx43) in Ser368 decreased during the periods of completion of meiosis and Sertoli cell differentiation, while Cx43 mRNA remained elevated throughout. PCx43 labeled chiefly the plasma membrane except by stage VII when vesicles were also labeled in Sertoli cells. Vesicles and lysosomes in Sertoli cells and the Golgi apparatus in the round spermatids were NPCx43 positive. A decrease in Cx43 gene expression was matched by a Cx43 protein increase in the early, not the late, phase of AIO. Total Cx43 and PCx43 decreased with the advance of orchitis. The study makes a novel finding of gap junctions connecting germ cells. The data indicate that Cx43 protein expression and phosphorylation in Ser368 are stage-specific events that may locally influence the acquisition of meiotic competence and the Sertoli cell differentiation in normal testis. AIO modifies Cx43 levels, suggesting changes in Cx43-mediated intercommunication and spermatogenic activity in response to cytokines imbalances in Sertoli cells. [ABSTRACT FROM AUTHOR]

Abstract: Viral factories are complex structures in the infected cell where viruses compartmentalize their life cycle. Rubella virus (RUBV) assembles factories by recruitment of rough endoplasmic reticulum (RER), mitochondria and Golgi around modified lysosomes known as cytopathic vacuoles or CPVs. These organelles contain active replication complexes that transfer replicated RNA to assembly sites in Golgi membranes. We have studied the structure of RUBV factory in three dimensions by electron tomography and freeze-fracture. CPVs contain stacked membranes, rigid sheets, small vesicles and large vacuoles. These membranes are interconnected and in communication with the endocytic pathway since they incorporate endocytosed BSA-gold. RER and CPVs are coupled through protein bridges and closely apposed membranes. Golgi vesicles attach to the CPVs but no tight contacts with mitochondria were detected. Immunogold labelling confirmed that the mitochondrial protein p32 is an abundant component around and inside CPVs where it could play important roles in factory activities. [ABSTRACT FROM AUTHOR]

Abstract: The structure of water-in-paraffin oil emulsions stabilized by both cationic surfactant (CTAB) and silicon oxide nanoparticles is investigated. A transition in the geometry of the droplets, which from spherical turns polymorphous, is discussed on the basis of scanning electron microscopy observations. The freeze-fracture technique is employed in addition to a sublimation procedure helping the visualization of the inner structure of the droplets. Their deformation is shown to be a consequence of the interplay between nanoparticles, CTAB and interfacial properties. For polymorphous droplets the existence of a network of self assembled particles bridging them from part to part is evidenced. [Copyright &y& Elsevier]

Abstract: The effects of sulfadiazine (SD), one of the generally used antibiotics was studied on bacteria-membrane mimetic model systems consisting of pure dipalmitoylphosphatidylethanolamine (DPPE) and DPPE/dipalmitoylphosphatidylglycerol (DPPG) at 95/5, 80/20 and 50/50 DPPE/DPPG ratios by using differential scanning calorimetry (DSC), simultaneous small- and wide-angle X-ray scattering (SWAXS) and freeze-fracture technique. In the presence of SD, varied between 10-3 and 1 SD/lipid molar ratios, the 95/5 DPPE/DPPG system shows tendentious destruction in the layer arrangement which is accompanied by minor perturbations in the thermotropic behaviour. Moreover, at this lipid composition the addition of SD results in the formation of stacks of extremely extended flat bilayers. Systems having a higher DPPG molar ratio exhibit complex and diffuse morphologies. At 50/50 DPPE/DPPG ratio DPPG and SD act together and form large spherical vesicles. The uniform morphology is not accompanied by a regular lamellar arrangement. The range of the SD/lipid ratio, where the SD molecules are embedded into the lipid bilayers, extends to about 10-1. Over this limit the separation of SD molecules can be observed at all investigated DPPE/DPPG ratios. [Copyright &y& Elsevier]

Abstract: The effects of three aminoglycosides (AGs), tobramycin, streptomycin and spectinomycin on 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE)–1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-1-glycerol] (DPPG)/water systems with 0.05 and 0.2 DPPG/DPPE+DPPG molar fractions were examined by using differential scanning calorimetry (DSC), simultaneous small- and wide-angle X-ray scattering (SWAXS), and freeze-fracture transmission electronmicroscopy (FF-TE) at 10−1 AG/lipid molar ratio in the doped systems. The effects induced by the AGs strongly depend on DPPG/DPPE+DPPG molar fraction. In the presence of the AGs regular lyotropic structures form as opposed to the complex and badly correlated layer structures of the pure 0.2 DPPG/DPPE+DPPG molar fraction system. The investigated AGs possess different structural and thermotropic effects: tobramycin and streptomycin which are true aminoglycosides decrease the main transition enthalpy in both model systems and induce the formation of correlated layer arrangements, while the occurrence of spectinomycin, a non-aminoglycoside aminocyclitol still ranked as an AG, results in a hexagonal phase as it can be deduced from the SAXS patterns and visually observed in the electronmicrographs. [Copyright &y& Elsevier]

Abstract: p-Boronophenylalanine (BPA) is a clinically approved boron neutron capture therapy (BNCT) agent currently being used in clinical trials of glioblastoma multiforme, melanoma and liver metastases. Secondary ion mass spectrometry (SIMS) observations from the Cornell SIMS Laboratory provided support for using a 6h infusion of BPA, instead of a 2h infusion, for achieving higher levels of boron in brain tumor cells. These observations were clinically implemented in Phase II experimental trials of glioblastoma multiforme in Sweden. However, the mechanisms for higher BPA accumulation with longer infusions have remained unknown. In this work, by using 13C15N-labeled phenylalanine and T98G human glioblastoma cells, comparisons between the 10B-delivery of BPA and the accumulation of labeled phenylalanine after 2 and 6h treatments were made with a Cameca IMS-3f SIMS ion microscope at 500nm spatial resolution in fast frozen, freeze-fractured, freeze-dried cells. Due to the presence of the Na–K-ATPase in the plasma membrane of most mammalian cells, the cells maintain an approximately 10/1 ratio of K/Na in the intracellular milieu. Therefore, the quantitative imaging of these highly diffusible species in the identical cell in which the boron or labeled amino acid was imaged provides a rule-of-thumb criterion for validation of SIMS observations and the reliability of the cryogenic sampling. The labeled phenylalanine was detected at mass 28, as the 28(13C15N)− molecular ion. Correlative analysis with optical and confocal laser scanning microscopy revealed that fractured freeze-dried glioblastoma cells contained well-preserved ultrastructural details with three discernible subcellular regions: a nucleus or multiple nuclei, a mitochondria-rich perinuclear cytoplasmic region and the remaining cytoplasm. SIMS analysis revealed that the overall cellular signals of both 10B from BPA and 28CN− from labeled phenylalanine increased approximately 1.6-fold between the 2 and 6h exposures. However, the subcellular distribution of 10B was different than the 28CN in the mitochondria-rich perinuclear cytoplasmic region: 10B was reduced in this region, but 28CN was not. These observations indicate that: (i) a comparable higher accumulation of BPA and phenylalanine at 6h versus 2h plausibly represents a similar time-dependent entry mechanism through the plasma membrane in response to cellular requirements for the amino acid in glioblastoma cells and (ii) intracellular processes, especially those implicated with mitochondria, can plausibly recognize BPA as a different molecule than phenylalanine and may significantly differ in its sequestration and metabolism. For further understanding cell cycle influence on BPA accumulation, DNA-synthesizing S-phase cells were compared with non-S-phase cells. SIMS observations revealed that after 1h exposure to BPA, S-phase cells contained elevated levels of 10B in their nucleus in comparison to the nucleus of non-S-phase cells. Consequently, one reason that longer BPA exposures would increase its accumulation in most tumor cells will be the movement of the cell cycle through the S-phase. These observations suggest further cell cycle research in BPA-mediated BNCT and may have special significance for brain tumors since tumor cells are primarily the only cells in the brain with active proliferation characteristics. This study also shows the need for cryogenic sampling for subcellular measurements in BNCT, as even a brief thaw of frozen samples can result in gross redistribution of boron in subcellular compartments. [Copyright &y& Elsevier]

We have defined regions of the skeletal muscle ryanodine receptor (RyR1) essential for bidirectional signaling with dihydropyridine receptors (DHPRs) and for the organization of DHPR into tetrad arrays by expressing RyR1-RyR3 chimerae in dyspedic myotubes. RyR1-RyR3 constructs bearing RyR1 residues 1-1681 restored wild-type DHPR tetrad arrays and, in part, skeletal-type excitation-contraction (EC) coupling (orthograde signaling) but failed to enhance DHPR Ca2+ currents (retrograde signaling) to WI RyR1 levels. Within this region, the D2 domain (amino acids 1272-1455), although ineffective on its own, dramatically enhanced the formation of tetrads and EC coupling rescue by constructs that otherwise are only partially effective. These findings suggest that the orthograde signal and DHPR tetrad formation require the contributions of numerous RyR regions. Surprisingly, we found that RyR3, although incapable of supporting EC coupling or tetrad formation, restored a significant level of Ca2+ current, revealing a functional interaction with the skeletal muscle DHPR. Thus, our data support the hypotheses that (i) the structural/functional link between RyR1 and the skeletal muscle DHPR requires multiple interacting regions, (ii) the D2 domain of RyR1 plays a key role in stabilizing this interaction, and (iii) a form of retrograde signaling from RyR3 to the DHPR occurs in the absence of direct protein- protein interactions. [ABSTRACT FROM AUTHOR]

This study primarily measured and established a morphometric spectrum of cross-sectional areas for foot and ankle ligaments with the use of a new freeze-fracture technique that was independent of ligament cross-sectional shape. Ligament morphometry measurements were made on a total of 121 bone-ligament-bone preparations that were harvested from 26 fresh (unembalmed) male cadaver feet. We used the traditional digital caliper method to measure length and the freeze-fracture technique to measure cross-sectional area. Cross-sectional area values ranged from 21.36 to 170.48 mm² and length values from 5.01 to 37.45 mm. In addition, area-to-length ratios ranged from 0.60 to 27.02 mm. Compared with the freeze-fracture technique, the digital caliper method, which assumed that ligament crosssectional shape was rectangular, resulted in an approximately 35% difference in cross-sectional area. [ABSTRACT FROM AUTHOR]

In the light of the recently published structure of GlpF and AQP1, we have analysed the nature of the residues which could be involved in the formation of the selectivity filter of aquaporins, glycerol facilitators and aquaglyceroporins. We demonstrate that the functional specificity for major intrinsic protein (MIP) channels can be explained on one side by analysing the polar environment of the residues that form the selective filter. On the other side, we show that the channel selectivity could be associated with the oligomeric state of the membrane protein. We conclude that a non-polar environment in the vicinity of the top of helix 5 could allow aquaglyceroporins and GlpF to exist as monomers within the hydrophobic environment of the membrane. [Copyright &y& Elsevier]

Low density Triton X-100-insoluble plasma membrane microdomains can be isolated from different mammalian cell types and are proposed to be involved in membrane trafficking, cell morphogenesis and signal transduction. Heterotrimeric G-proteins and their receptors are often associated with such domains, suggesting that these structures are involved in G-protein-coupled signaling. Here we report that detergent-insoluble plasma membrane microdomains also exist in higher plants and contain about 15% of membrane-bound heterotrimeric G-protein beta-subunit (Gβ). Plasma membrane microdomains were isolated from tobacco leaves. They have low buoyant density relative to the surrounding plasma membrane, and are insoluble in Triton X-100 at 4 °C. Detergent-insoluble vesicles were examined by freeze-fracture electron microscopy. They have sizes in the range 100–400 nm, and often contain aggregated protein complexes. The majority of plasma membrane proteins cannot be detected in the Triton X-100-insoluble fraction, while few polypeptides are highly enriched. We identified six proteins with molecular masses of 22, 28, 35, 60, 67 and 94 kDa in detergent-insoluble fractions that are glycosylphosphatidylinositol (GPI)-anchored. [ABSTRACT FROM AUTHOR]

Metabotropic glutamate receptor subtype 5 (mGlu5) is implicated in the pathophysiology of Alzheimer's disease (AD). However, its alteration at the subcellular level in neurons is still unexplored. Here, we provide a quantitative description on the expression and localisation patterns of mGlu5 in the APP/PS1 model of AD at 12 months of age, combining immunoblots, histoblots and high-resolution immunoelectron microscopic approaches. Immunoblots revealed that the total amount of mGlu5 protein in the hippocampus, in addition to downstream molecules, i.e., Gq/11 and PLCβ1, was similar in both APP/PS1 mice and age-matched wild type mice. Histoblots revealed that mGlu5 expression in the brain and its laminar expression in the hippocampus was also unaltered. However, the ultrastructural techniques of SDS-FRL and pre-embedding immunogold demonstrated that the subcellular localisation of mGlu5 was significantly reduced along the neuronal surface of hippocampal principal cells, including CA1 pyramidal cells and DG granule cells, in APP/PS1 mice at 12 months of age. The decrease in the surface localisation of mGlu5 was accompanied by an increase in its frequency at intracellular sites in the two neuronal populations. Together, these data demonstrate, for the first time, a loss of mGlu5 at the plasma membrane and accumulation at intracellular sites in different principal cells of the hippocampus in APP/PS1 mice, suggesting an alteration of the excitability and synaptic transmission that could contribute to the cognitive dysfunctions in this AD animal model. Further studies are required to elucidate the specificity of mGlu5-associated molecules and downstream signalling pathways in the progression of the pathology. [ABSTRACT FROM AUTHOR]

The ultrastructure and supramolecular organization of photosynthetic membranes in four species of marine red algae (Porphyra yezoensis, Porphyra leucosticta, Erythrocladia subintegra, Pterothamnion crispum) were studied both in replicas of rapidly frozen cells and in ultrathin sections. Because the thylakoids in red algal chloroplasts are not stacked, freeze-fractured thylakoid membranes exhibit only two types of fracture faces (EF and PF). In all four species the PF reveals numerous, tightly packed, but randomly distributed particles (density range from 3530 to 4970 particles μm-2 for Porphyra yezoensis). The particle size distribution is uniform in the two types of fracture faces with an average diameter of about 11.3-11.6 nm in all four algae. The phycobilisomes of Porphyra yezoensis and Erythrocladia subintegra have the approximate shape of a compressed rotation ellipsoid with the following dimensions; Porphyra yezoensis, length 52.8 ± 1.2 nm, thickness 24.4 ± 0.6 nm, height 34.6 ± 1 nm; Erythrocladia subintegra, length 45.1 ± 0.7 nm, thickness 23.1 ± 0.5 nm, height 29.4 ± 0.9 nm. In Porphyra yezoensis, Porphyra leucosticta and Erythrocladia subintegra both the phycobilisomes and EF particles are organized into rows with a spacing of 70-80 nm in Porphyra yezoensis, 65-75 nm in Porphyra leucosticta and 53.8-55 nm in Erythrocladia subintegra. EF particle complexes with four subunits were found in Porphyra yezoensis. In Pterothamnion crispum the shape of the phycobilisomes is hemiellipsoidal. The phycobilisomes seem to be organized into rows; their centre to centre periodicity is c. 60 nm. Porphyra yezoensis cells possess about 770 ± 58 phycobilisomes μm-2 of thylakoid surface, whereas the number of EF particles is about 1270 ± 203 μm-2 Thus, the ratio of phycobilisomes to EF particles is c. 0.6. In Erythrocladia subintegra the ratio of phycobilisomes to EF particles is about 0.7. Of specia; interest is the occurence of a crystalline matrix in pyrenoids of Prophyra leucosticta and the absence of phycobilisomes in the intrapyrenoid thylakoids of Porphyra yezoensis and Erythrocladia subintegra. [ABSTRACT FROM AUTHOR]

Vascular bundles of petioles below wilted leaves of Nymphoides peltata (S.G. Gmel. O. Kuntze) were frozen intact and freeze-fractured for electron microscopy. Cell walls in them appeared drawn in against the helical thickenings of xylem vessels. By contrast, walls round vessels which had been frozen in vascular bundles below turgid leaves, and walls round vessels which had been fixed, embedded and sectioned, were straight or bulged outwards slightly. Walls bulged outwards slightly also from cut vessels filled with sucrose solution before freezing. Movement of vessel walls could produce the clicks audible when water cavitates in vessels, and might explain a variable resistance to the flow of water through plants. [ABSTRACT FROM AUTHOR]