Intramembranous Particles Research Articles

Neural networks with functional inputs for multi-class supervised classification of replicated point patterns

A spatial point pattern is a collection of points observed in a bounded region of the Euclidean plane or space. With the dynamic development of modern imaging methods, large datasets of point patterns are available representing for example sub-cellular location patterns for human proteins or large forest populations. The main goal of this paper is to show the possibility of solving the supervised multi-class classification task for this particular type of complex data via functional neural networks. To predict the class membership for a newly observed point pattern, we compute an empirical estimate of a selected functional characteristic. Then, we consider such estimated function to be a functional variable entering the network. In a simulation study, we show that the neural network approach outperforms the kernel regression classifier that we consider a benchmark method in the point pattern setting. We also analyse a real dataset of point patterns of intramembranous particles and illustrate the practical applicability of the proposed method.

A Microtubule-Associated Protein Is Essential for Malaria Parasite Transmission.

Mature gametocytes of Plasmodium falciparum display a banana (falciform) shape conferred by a complex array of subpellicular microtubules (SPMT) associated with the inner membrane complex (IMC). Microtubule-associated proteins (MAPs) define MT populations and modulate interaction with pellicular components. Several MAPs have been identified in Toxoplasma gondii, and homologues can be found in the genomes of Plasmodium species, but the function of these proteins for asexual and sexual development of malaria parasites is still unknown. Here, we identified a novel subpellicular MAP, termed SPM3, that is conserved within the genus Plasmodium, especially within the subgenus Laverania, but absent in other Apicomplexa. Conditional knockdown and targeted gene disruption of Pfspm3 in Plasmodium falciparum cause severe morphological defects during gametocytogenesis, leading to round, nonfalciform gametocytes with an aberrant SPMT pattern. In contrast, Pbspm3 knockout in Plasmodium berghei, a species with round gametocytes, caused no defect in gametocytogenesis, but sporozoites displayed an aberrant motility and a dramatic defect in invasion of salivary glands, leading to a decreased efficiency in transmission. Electron microscopy revealed a dissociation of the SPMT from the IMC in Pbspm3 knockout parasites, suggesting a function of SPM3 in anchoring MTs to the IMC. Overall, our results highlight SPM3 as a pellicular component with essential functions for malaria parasite transmission. IMPORTANCE A key structural feature driving the transition between different life cycle stages of the malaria parasite is the unique three-membrane pellicle, consisting of the parasite plasma membrane (PPM) and a double membrane structure underlying the PPM termed the inner membrane complex (IMC). Additionally, there are numerous linearly arranged intramembranous particles (IMPs) linked to the IMC, which likely link the IMC to the subpellicular microtubule cytoskeleton. Here, we identified, localized, and characterized a novel subpellicular microtubule-associated protein unique to the genus Plasmodium. The knockout of this protein in the human-pathogenic species P. falciparum resulted in malformed gametocytes and aberrant microtubules. We confirmed the microtubule association in the P. berghei rodent malaria homologue and show that its knockout results in a perturbed microtubule architecture, aberrant sporozoite motility, and decreased transmission efficiency.

Freeze-Fracture Electron Microscopy for Extracellular Vesicle Analysis.

Extracellular vesicles (EVs) are membrane-limited structures released from the cells into the extracellular space and are implicated in intercellular communication. EVs consist of three populations of vesicles, namely microvesicles (MVs), exosomes, and apoptotic bodies. The limiting membrane of EVs is crucially involved in the interactions with the recipient cells, which could lead to the transfer of biologically active molecules to the recipient cells and, consequently, affect their behavior. The freeze-fracture electron microscopy technique is used to study the internal organization of biological membranes. Here, we present a protocol for MV isolation from cultured cancerous urothelial cells and the freeze-fracture of MVs in the steps of rapid freezing, fracturing, making and cleaning the replicas, and analyzing them with transmission electron microscopy. The results show that the protocol for isolation yields a homogenous population of EVs, which correspond to the shape and size of MVs. Intramembrane particles are found mainly in the protoplasmic face of the limiting membrane. Hence, freeze-fracture is the technique of choice to characterize the MVs' diameter, shape, and distribution of membrane proteins. The presented protocol is applicable to other populations of EVs.

First Responders to Hyperosmotic Stress in Murine Astrocytes: Connexin 43 Gap Junctions Are Subject to an Immediate Ultrastructural Reorganization.

Simple SummaryGap junctions are intercellular channels that provide the means for direct transport of small molecules, ions, and water between connected cells. With these functions, gap junctions are essential for the maintenance of astrocytic homeostasis and of particular importance in the context of pathophysiological disbalances. These include the hyperosmolar hyperglycemic syndrome or the pathology after brain trauma. We demonstrate that short-term hyperosmolarity reduces intercellular communication via gap junctions. These functional changes coincide with the transformation of gap junction ultrastructure as evidenced by freeze-fracture replica immunolabeling and transmission electron microscopy. The hyperosmolarity-induced immediate changes in the ultrastructural assembly of connexons, the protein constituents of gap junction channels, have not been described in astrocytes before and are revealing the coherence of structure and function in gap junctions. Phosphorylation of Connexin 43, the main gap junction protein in astrocytes, at amino acid 368 (Serine) might link the two.In a short-term model of hyperosmotic stress, primary murine astrocytes were stimulated with a hyperosmolar sucrose solution for five minutes. Astrocytic gap junctions, which are mainly composed of Connexin (Cx) 43, displayed immediate ultrastructural changes, demonstrated by freeze–fracture replica immunogold labeling: their area, perimeter, and distance of intramembrane particles increased, whereas particle numbers per area decreased. Ultrastructural changes were, however, not accompanied by changes in Cx43 mRNA expression. In contrast, transcription of the gap junction regulator zonula occludens (ZO) protein 1 significantly increased, whereas its protein expression was unaffected. Phosphorylation of Serine (S) 368 of the Cx43 C–terminus has previously been associated with gap junction disassembly and reduction in gap junction communication. Hyperosmolar sucrose treatment led to enhanced phosphorylation of Cx43S368 and was accompanied by inhibition of gap junctional intercellular communication, demonstrated by a scrape loading-dye transfer assay. Taken together, Cx43 gap junctions are fast reacting elements in response to hyperosmolar challenges and can therefore be considered as one of the first responders to hyperosmolarity. In this process, phosphorylation of Cx43S368 was associated with disassembly of gap junctions and inhibition of their function. Thus, modulation of the gap junction assembly might represent a target in the treatment of brain edema or trauma.

Ultrastructural and Functional Analysis of a Novel Extra-Axonemal Structure in Parasitic Trichomonads.

Trichomonas vaginalis and Tritrichomonas foetus are extracellular flagellated parasites that inhabit humans and other mammals, respectively. In addition to motility, flagella act in a variety of biological processes in different cell types, and extra-axonemal structures (EASs) have been described as fibrillar structures that provide mechanical support and act as metabolic, homeostatic, and sensory platforms in many organisms. It has been assumed that T. vaginalis and T. foetus do not have EASs. However, here, we used complementary electron microscopy techniques to reveal the ultrastructure of EASs in both parasites. Such EASs are thin filaments (3–5 nm diameter) running longitudinally along the axonemes and surrounded by the flagellar membrane, forming prominent flagellar swellings. We observed that the formation of EAS increases after parasite adhesion on the host cells, fibronectin, and precationized surfaces. A high number of rosettes, clusters of intramembrane particles that have been proposed as sensorial structures, and microvesicles protruding from the membrane were observed in the EASs. Our observations demonstrate that T. vaginalis and T. foetus can connect to themselves by EASs present in flagella. The protein VPS32, a member of the ESCRT-III complex crucial for diverse membrane remodeling events, the pinching off and release of microvesicles, was found in the surface as well as in microvesicles protruding from EASs. Moreover, we demonstrated that the formation of EAS also increases in parasites overexpressing VPS32 and that T. vaginalis-VPS32 parasites showed greater motility in semisolid agar. These results provide valuable data about the role of the flagellar EASs in the cell-to-cell communication and pathogenesis of these extracellular parasites.

An Alveolata secretory machinery adapted to parasite host cell invasion.

Apicomplexa are unicellular eukaryotes and obligate intracellular parasites, including Plasmodium (the causative agent of malaria) and Toxoplasma (one of the most widespread zoonotic pathogens). Rhoptries, one of their specialized secretory organelles, undergo regulated exocytosis during invasion1. Rhoptry proteins are injected directly into the host cell to support invasion and subversion of host immune function2. The mechanism by which they are discharged is unclear and appears distinct from those in bacteria, yeast, animals and plants. Here, we show that rhoptry secretion in Apicomplexa shares structural and genetic elements with the exocytic machinery of ciliates, their free-living relatives. Rhoptry exocytosis depends on intramembranous particles in the shape of a rosette embedded into the plasma membrane of the parasite apex. Formation of this rosette requires multiple non-discharge (Nd) proteins conserved and restricted to Ciliata, Dinoflagellata and Apicomplexa that together constitute the superphylum Alveolata. We identified Nd6 at the site of exocytosis in association with an apical vesicle. Sandwiched between the rosette and the tip of the rhoptry, this vesicle appears as a central element of the rhoptry secretion machine. Our results describe a conserved secretion system that was adapted to provide defence for free-living unicellular eukaryotes and host cell injection in intracellular parasites.

Galactose as novel target against Acanthamoeba cysts.

Galactose as novel target against Acanthamoeba cysts.

Cellulose fibrils formation and organisation of cytoskeleton during encystment are essential for Acanthamoeba cyst wall architecture

Acanthamoebae success as human pathogens is largely due to the highly resistant cysts which represent a crucial problem in treatment of Acanthamoeba infections. Hence, the study of cyst wall composition and encystment play an important role in finding new therapeutic strategies. For the first time, we detected high activity of cytoskeletal elements – microtubular networks and filamentous actin, in late phases of encystment. Cellulose fibrils – the main components of endocyst were demonstrated in inter-cystic space, and finally in the ectocyst, hereby proving the presence of cellulose in both layers of the cyst wall. We detected clustering of intramembranous particles (IMPs) and their density alterations in cytoplasmic membrane during encystment. We propose a hypothesis that in the phase of endocyst formation, the IMP clusters represent cellulose microfibril terminal complexes involved in cellulose synthesis that after cyst wall completion are reduced. Cyst wall impermeability, due largely to a complex polysaccharide (glycans, mainly cellulose) has been shown to be responsible for Acanthamoeba biocide resistance and cellulose biosynthesis pathway is suggested to be a potential target in treatment of Acanthamoeba infections. Disruption of this pathway would affect the synthesis of cyst wall and reduce considerably the resistance to chemotherapeutic agents.

Effect of jasplakinolide and cytochalasin D on cortical elements involved in the gliding motility of the eugregarine Gregarina garnhami (Apicomplexa)

Since apicomplexans represent exclusively parasitic unicellular organisms with medical and economic impacts, the principles of their motility have been studied intensively. By contrast, the movement in apicomplexan basal groups, such as gregarines, remains to be elucidated. The present study focuses on Gregarina garnhami parasitising the digestive tract of the locust Schistocerca gregaria, and investigates the involvement of cytoskeletal elements (the ectoplasmic network and myonemes) and the secretion of mucosubstances during eugregarine gliding motility. Combined microscopic analyses were used to verify the role of actin filaments and membranes’ organisation in G. garnhami motility. A freeze-etching analysis of membranes revealed the size, density, and arrangement of intramembranous particles along with the distribution and size of pores and ducts. Experimental assays using actin-modifying drugs (jasplakinolide, cytochalasin D) confirmed that actin most likely plays a role in cell motility, principally in its filamentous form (=F-actin). Myonemes, localised in the border between the ectoplasm and endoplasm, correspond to the concentric bundles of F-actin. Microscopic analyses confirmed that changes in gamonts motility corresponding to the changes in the organisation and density of myonemes and the ectoplasmic network in drug-treated cells, suggesting that these structures might serve as contractile elements facilitating gliding motility in G. garnhami.

Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of <100 nm in diameter in the plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane.

Some Ultrastructural Features of the Planktonic Freshwater Ciliate Limnostrombidium viride (Alveolata, Ciliophora, Oligotrichida) and Improved Diagnoses of Oligotrich Taxa.

The first transmission and scanning electron microscopical studies in combination with freeze-fracture technology have disclosed some important morphological and ultrastructural features in the freshwater oligotrichid Limnostrombidium viride. (I) The dikinetids (paired basal bodies) of the girdle kinety have a club-shaped cilium associated only with each left basal body. The electron-dense (paraflagellar) body on one side of its "9×2+2"-axoneme and the regular array of intramembranous particles indicate a sensory, perhaps photoreceptor function of these club-shaped cilia. (II) The stichomonad endoral membrane is proximally covered by a cytoplasmic fold and distally by multiple membranous layers. Thus entirely covered, the endoral is probably no longer involved in food capture; nonetheless, its associated microtubules might stabilise the cytopharynx. (III) Instead of a contractile vacuole, a horizontal ring-canal with supposed osmoregulatory function occurs. (IV) The extrusive nature of the trichites is not only observed in electron micrographs, but the attachment sites of these organelles also display a rosette of "8+1"-particles in the P-face of freeze-fracture replicas typical for ciliate extrusomes. (V) The neoformation organelle, the subsurface tube in which stomatogenesis takes place, shows short basal bodies and normal axonemes about 1 μm long. It is accompanied by numerous membrane vesicles, which might provide membrane material for the outgrowing cilia.

Freeze fracture: new avenues for the ultrastructural analysis of cells in vitro.

The ultrastructural analysis of biological membranes by freeze fracture has a 60-year tradition. In this review, we summarize the benefits of the freeze-fracture technique and review special structures analyzed by freeze fracture and by combined freeze-fracture replica immunogold labeling (FRIL) of cell cultures. In principle, every cellular membrane whether of cell suspensions, mono- or bilayers of cell cultures can be analyzed in freeze fracture. The combination of freeze fracture and immunogold labeling of the replica allows the ultrastructural identification of protein assemblies in combination with the molecular identification of their constituent proteins using specific antibodies. The analysis of fractured and labeled intramembrane particles enables determination of the arrangement and organization of proteins within the membrane due to the high resolution of the transmission electron microscope. Because of cell-specific ultrastructural features such as square arrays, identification of cell types can be performed in parallel. This review is aimed at presenting the possibilities of freeze fracture and FRIL in the high-resolution ultrastructural analysis of membrane proteins and their assembly in naïve, transfected or otherwise treated cultured cells. At the interface of molecular approaches and morphology, the application of FRIL in genetically modified cells provides a novel and intriguing aspect for their analysis.

A novel imaging method revealed phosphatidylinositol 3,5-bisphosphate-rich domains in the endosome/lysosome membrane

ABSTRACTWe 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.

KV1 channels identified in rodent myelinated axons, linked to Cx29 in innermost myelin: support for electrically active myelin in mammalian saltatory conduction.

Saltatory conduction in mammalian myelinated axons was thought to be well understood before recent discoveries revealed unexpected subcellular distributions and molecular identities of the K(+)-conductance pathways that provide for rapid axonal repolarization. In this study, we visualize, identify, localize, quantify, and ultrastructurally characterize axonal KV1.1/KV1.2 channels in sciatic nerves of rodents. With the use of light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling electron microscopy, KV1.1/KV1.2 channels are localized to three anatomically and compositionally distinct domains in the internodal axolemmas of large myelinated axons, where they form densely packed "rosettes" of 9-nm intramembrane particles. These axolemmal KV1.1/KV1.2 rosettes are precisely aligned with and ultrastructurally coupled to connexin29 (Cx29) channels, also in matching rosettes, in the surrounding juxtaparanodal myelin collars and along the inner mesaxon. As >98% of transmembrane proteins large enough to represent ion channels in these specialized domains, ∼500,000 KV1.1/KV1.2 channels define the paired juxtaparanodal regions as exclusive membrane domains for the voltage-gated K(+)conductance that underlies rapid axonal repolarization in mammals. The 1:1 molecular linkage of KV1 channels to Cx29 channels in the apposed juxtaparanodal collars, plus their linkage to an additional 250,000-400,000 Cx29 channels along each inner mesaxon in every large-diameter myelinated axon examined, supports previously proposed K(+)conductance directly from juxtaparanodal axoplasm into juxtaparanodal myeloplasm in mammalian axons. With neither Cx29 protein nor myelin rosettes detectable in frog myelinated axons, these data showing axon-to-myelin linkage by abundant KV1/Cx29 channels in rodent axons support renewed consideration of an electrically active role for myelin in increasing both saltatory conduction velocity and maximum propagation frequency in mammalian myelinated axons.

Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes.

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 p40(phox) 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.

Chemical-Free Technique to Study the Ultrastructure of Primary Cilium.

A primary cilium is a hair-like structure with a width of approximately 200 nm. Over the past few decades, the main challenge in the study of the ultrastructure of cilia has been the high sensitivity of cilia to chemical fixation, which is required for many imaging techniques. In this report, we demonstrate a combined high-pressure freezing (HPF) and freeze-fracture transmission electron microscopy (FFTEM) technique to examine the ultrastructure of a cilium. Our objective is to develop an optimal high-resolution imaging approach that preserves cilia structures in their best natural form without alteration of cilia morphology by chemical fixation interference. Our results showed that a cilium has a swelling-like structure (termed bulb), which was previously considered a fixation artifact. The intramembrane particles observed via HPF/FFTEM indicated the presence of integral membrane proteins and soluble matrix proteins along the ciliary bulb, which is part of an integral structure within the ciliary membrane. We propose that HPF/FFTEM is an important and more suitable chemical-free method to study the ultrastructure of primary cilia.

Eisosome Ultrastructure and Evolution in Fungi, Microalgae, and Lichens.

Eisosomes are among the few remaining eukaryotic cellular differentations that lack a defined function(s). These trough-shaped invaginations of the plasma membrane have largely been studied in Saccharomyces cerevisiae, in which their associated proteins, including two BAR domain proteins, have been identified, and homologues have been found throughout the fungal radiation. Using quick-freeze deep-etch electron microscopy to generate high-resolution replicas of membrane fracture faces without the use of chemical fixation, we report that eisosomes are also present in a subset of red and green microalgae as well as in the cysts of the ciliate Euplotes. Eisosome assembly is closely correlated with both the presence and the nature of cell walls. Microalgal eisosomes vary extensively in topology and internal organization. Unlike fungi, their convex fracture faces can carry lineage-specific arrays of intramembranous particles, and their concave fracture faces usually display fine striations, also seen in fungi, that are pitched at lineage-specific angles and, in some cases, adopt a broad-banded patterning. The conserved genes that encode fungal eisosome-associated proteins are not found in sequenced algal genomes, but we identified genes encoding two algal lineage-specific families of predicted BAR domain proteins, called Green-BAR and Red-BAR, that are candidate eisosome organizers. We propose a model for eisosome formation wherein (i) positively charged recognition patches first establish contact with target membrane regions and (ii) a (partial) unwinding of the coiled-coil conformation of the BAR domains then allows interactions between the hydrophobic faces of their amphipathic helices and the lipid phase of the inner membrane leaflet, generating the striated patterns.

Isolation of Chloroplast Inner and Outer Envelope Membranes

[Abstract] The chloroplast is an important organelle found in plant cells that conduct photosynthesis. It is enclosed by a pair of closely spaced membranes, the double-membrane envelope, consisting of the inner membrane bounding the matrix or stroma and the outer membrane in contact with the cytoplasm. Like many bio-membranes, the chloroplast envelope plays an important role in mediating the complex interactions between the chloroplast and the cytoplasm. The envelope is also the site of various biosynthetic reactions, including the formation of the galactolipids, which are the major components of both envelope and the thylakoid membranes. The inner and outer envelope membranes have differences in both structure and function. For example, the outer membrane exhibits lower density of intramembranous particles than the inner membrane dose, suggesting that the protein content of the outer membrane is low. Also, the outer membrane is nonspecifically permeable to low molecular weight compounds, whereas the inner is impermeable to such compounds and contains several translocator systems for the transport of metabolites. To prepare the envelope membranes, it is necessary to isolate intact chloroplasts first. Then the inner and outer envelope membranes are separated by: 1) the protease-treatment method and 2) the centrifuge method which based on the fact that the outer envelope is lighter and the inner membrane heavier. Both methods need to isolate the intact chloroplasts firstly. However, the centrifugal separation can get the pure inner and outer envelope preparations, which therefore are suitable to the subsequent analyses. Also, the centrifuge method can avoid the destruction of inner envelope polypeptides during the protease treatment, because some of the protease may gain access to the inner membrane. Moreover, the centrifuge method is easy to operate and to get the complete enveloped that contain less of the adhesion regions of the outer and inner envelope membranes. Here we describe a reliable method for isolation of the inner and outer envelope membranes of the chloroplasts from tobacco, which is the plant that relatively not easy to use for envelope isolation.

Target- and input-dependent organization of AMPA and NMDA receptors in synaptic connections of the cochlear nucleus.

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.

Molecular ultrastructure of the urothelial surface: insights from a combination of various microscopic techniques.

The urothelium forms the blood-urine barrier, which depends on the complex organization of transmembrane proteins, uroplakins, in the apical plasma membrane of umbrella cells. Uroplakins compose 16 nm intramembrane particles, which are assembled into urothelial plaques. Here we present an integrated survey on the molecular ultrastructure of urothelial plaques in normal umbrella cells with advanced microscopic techniques. We analyzed the ultrastructure and performed measurements of urothelial plaques in the normal mouse urothelium. We used field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), transmission electron microscopy (TEM) on immunolabeled ultrathin sections (immuno-TEM), and freeze-fracture replicas (FRIL). We performed immunolabeling of uroplakins for scanning electron microscopy (immuno-FESEM). All microscopic techniques revealed a variability of urothelial plaque diameters ranging from 332 to 1179 nm. All immunolabeling techniques confirmed the presence of uroplakins in urothelial plaques. FRIL showed the association of uroplakins with 16 nm intramembrane particles and their organization into plaques. Using different microscopic techniques and applied qualitative and quantitative evaluation, new insights into the urothelial apical surface molecular ultrastructure have emerged and may hopefully provide a timely impulse for many ongoing studies. The combination of various microscopic techniques used in this study shows how these techniques complement one another. The described advantages and disadvantages of each technique should be considered for future studies of molecular and structural membrane specializations in other cells and tissues.