Clusters Of Intramembrane Particles Research Articles

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.

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.

Quantitative Localization of Cav2.1 (P/Q-Type) Voltage-Dependent Calcium Channels in Purkinje Cells: Somatodendritic Gradient and Distinct Somatic Coclustering with Calcium-Activated Potassium Channels

P/Q-type voltage-dependent calcium channels play key roles in transmitter release, integration of dendritic signals, generation of dendritic spikes, and gene expression. High intracellular calcium concentration transient produced by these channels is restricted to tens to hundreds of nanometers from the channels. Therefore, precise localization of these channels along the plasma membrane was long sought to decipher how each neuronal cell function is controlled. Here, we analyzed the distribution of Ca(v)2.1 subunit of the P/Q-type channel using highly sensitive SDS-digested freeze-fracture replica labeling in the rat cerebellar Purkinje cells. The labeling efficiency was such that the number of immunogold particles in each parallel fiber active zone was comparable to that of functional channels calculated from previous reports. Two distinct patterns of Ca(v)2.1 distribution, scattered and clustered, were found in Purkinje cells. The scattered Ca(v)2.1 had a somatodendritic gradient with the density of immunogold particles increasing 2.5-fold from soma to distal dendrites. The other population with 74-fold higher density than the scattered particles was found within clusters of intramembrane particles on the P-face of soma and primary dendrites. Both populations of Ca(v)2.1 were found as early as P3 and increased in the second postnatal week to a mature level. Using double immunogold labeling, we found that virtually all of the Ca(v)2.1 clusters were colocalized with two types of calcium-activated potassium channels, BK and SK2, with the nearest neighbor distance of ∼40 nm. Calcium nanodomain created by the opening of Ca(v)2.1 channels likely activates the two channels that limit the extent of depolarization.

Quantitative localisation of synaptic and extrasynaptic GABAA receptor subunits on hippocampal pyramidal cells by freeze-fracture replica immunolabelling.

Hippocampal CA1 pyramidal cells, which receive γ-aminobutyric acid (GABA)ergic input from at least 18 types of presynaptic neuron, express 14 subunits of the pentameric GABA(A) receptor. The relative contribution of any subunit to synaptic and extrasynaptic receptors influences the dynamics of GABA and drug actions. Synaptic receptors mediate phasic GABA-evoked conductance and extrasynaptic receptors contribute to a tonic conductance. We used freeze-fracture replica-immunogold labelling, a sensitive quantitative immunocytochemical method, to detect synaptic and extrasynaptic pools of the alpha1, alpha2 and beta3 subunits. Antibodies to the cytoplasmic loop of the subunits showed immunogold particles concentrated on distinct clusters of intramembrane particles (IMPs) on the cytoplasmic face of the plasma membrane on the somata, dendrites and axon initial segments, with an abrupt decrease in labelling at the edge of the IMP cluster. Neuroligin-2, a GABAergic synapse-specific adhesion molecule, co-labels all beta3 subunit-rich IMP clusters, therefore we considered them synapses. Double-labelling for two subunits showed that virtually all somatic synapses contain the alpha1, alpha2 and beta3 subunits. The extrasynaptic plasma membrane of the somata, dendrites and dendritic spines showed low-density immunolabelling. Synaptic labelling densities on somata for the alpha1, alpha2 and beta3 subunits were 78-132, 94 and 79 times higher than on the extrasynaptic membranes, respectively. As GABAergic synapses occupy 0.72% of the soma surface, the fraction of synaptic labelling was 33-48 (alpha1), 40 (alpha2) and 36 (beta3)% of the total somatic surface immunolabelling. Assuming similar antibody access to all receptors, about 60% of these subunits are in extrasynaptic receptors.

The flagellar attachment zone of Trypanosoma cruzi epimastigote forms

The flagellar attachment zone (FAZ) is an adhesion region of Trypanosoma cruzi epimastigote forms where the flagellum emerges from the flagellar pocket and remains attached to the cell body. This region shows a junctional complex which is formed by a linear series of apposed macular structures that are separated by amorphous material and clusters of intramembranous particles. Two protein groups appear to be important in the FAZ region: a membrane glycoprotein of 72 kDa and several high molecular weight proteins. To gain a better understanding of the FAZ region, we compared wild-type Y strain T. cruzi epimastigotes with a mutant cell in which the 72-kDa surface glycoprotein (Gp72), involved in cell body-flagellum adhesion, had been deleted by target gene replacement. Using immunofluorescence confocal microscopy and electron microscopy techniques to analyze the FAZ region the results suggest that, in the absence of Gp72, other proteins involved in the formation of FAZ remain concentrated in the flagellar pocket region. The analysis of a 3-D reconstruction model of wild-type epimastigotes showed that the endoplasmic reticulum and mitochondrion are in intimate association with FAZ, in contrast to the null mutant cells where the endoplasmic reticulum was not visualized.

Reduced density of intramembranous particle clusters: freeze-fracture study of mdx mouse muscle plasma membrane.

Our previous freeze-fracture study demonstrated the decreased density of intramembranous particles (IMPs) on the protoplasmic (P) face of muscle plasma membranes in mdx mice. However, the molecular mechanism is unknown. In the present freeze-fracture study, we examined whether the reduced P-face IMP density in mdx mice would be caused by depletion of the rosette-like IMP clusters, which are IMP aggregations differing from crystal-like orthogonal arrays (OAs). By comparison with control mice, the P-face plasma membranes of extensor digitorum longus (EDL) and soleus (SOL) muscles of mdx mice demonstrated the following findings: (1) decreased IMP density with subunit particles of OAs and IMP clusters, (2) decreased IMP density without subunit particles of OAs, (3) normal IMP density without subunit particles of OAs and IMP clusters, (4) decreased OA density in EDL muscles and normal OA density in SOL muscles, and (5) decreased IMP cluster densities in both muscles. Thus, the reduced IMP density of P-face muscle plasma membranes in mdx mice may result from the decreased IMP clusters, suggesting the relationship between IMP clusters and the integral membrane proteins is influenced by dystrophin deficiency such as that of dystrophin-associated glycoproteins or other membrane proteins.

The axonal membrane cytoskeletal protein A60 and the development of the spectrin/ankyrin-based neuronal membrane skeleton.

Introduction Among the processes of developing a polarized neuron from an undifferentiated neuronal precursor is the extension of neurites and their differentiation into dendrites and axons. The formation of synaptic contacts, myelin sheaths and nodes of Ranvier lends complexity to the surface of neurons. To generate such diversity and specialization of membrane domains a complex series of events occurs during differentiation, involving targeting of axonal or dendritic components to their destination by specific sorting. transport and placing mechanisms. Once in place, specific transporters, cell adhesion molecules and ion channels must have their tendency to diffuse in the plane of the plasma membrane reduced by locking mechanisms that lower their diffusion coefficients. Furthermore, such factors as the shape and dimensions of the cell (including the diameter of the axon that specifies the conduction speed of the action potential) must be specified. Clues to the mechanisms of each of these processes have begun to emerge in the past 8 to 10 years and recent progress has been very rapid. An emerging concept is that cytoskeletal specializations in the cortical cytoplasm control many events at the plasma membrane, and communicate morphological information from the cytoskeleton in the inner cytoplasm to the plasma membrane. Such cortical cytoskeletal specializations are morphologically obvious at points such as nodes of Ranvier and post-synaptic densities where clusters of intramembrane particles (detected in freeze-fracture analysis of the plasma membrane) can be correlated with the presence of high concentrations of filamentous actin. I Iowever, in addition to these specializations, some components of the cortical, or plasma membrane-associated, cytoskeleton are broadly distributed. It seems that the cytoskeleton has a broad role in dictating membrane structure.

Induction of intramembranous particle clusters in mice with nephrogenic diabetes insipidus.

In mice with hereditary nephrogenic diabetes insipidus (NDI), the inability of vasopressin to increase hydraulic water permeability is reflected in a lack of intramembranous particle (IMP) clusters in apical membranes of inner medullary collecting ducts. The lack arises from anomalously high activity of one or two isozymes of adenosine 3',5'-cyclic monophosphate-phosphodiesterase (cAMP-PDE). We asked whether inhibition of these isozymes with rolipram and cilostamide would raise not only the tissue content of cAMP but also and simultaneously restore IMP clusters. Inner medullary collecting ducts from NDI mice were incubated in vitro. Tissue content of cAMP (fmol of cAMP per bundle) and number of IMP clusters (per 100 microns 2 of principal cell apical membrane) were, respectively: control, 44.8 +/- 13.0 and 4.16 +/- 1.49; arginine vasopressin (AVP), 31.7 +/- 8.0 and 3.98 +/- 1.56; rolipram and cilostamide, 109.7 +/- 21.0 and 58.09 +/- 15.74; and AVP plus rolipram and cilostamide, 305.7 +/- 75 and 48.63 +/- 11.03 (with the last four values showing significant difference from control and AVP only, respectively). In addition, treating NDI mice with rolipram and cilostamide in vivo reduced their high fluid turnover. We conclude that failure by AVP to increase cAMP in cells of collecting ducts, which results from anomalously high activity of one or two specific isozymes of cAMP-PDE, is the major or sole cause for the excretion of hypotonic urine in NDI mice (DI +/+ Severe strain).

Ultrastructure of the electrotonic and chemical components of the lateral‐to‐motor and medial‐to‐motor synapses in crayfish nerve cord

Lateral-to-motor and medial-to-motor synapses in crayfish nerve cords are composed of an electrical and a chemical component. The presynaptic terminals showed localized clusters of synaptic vesicles, electron-dense areas, coated pits, and coated vesicles. In thin sections, active zones were defined by electron-dense regions where synaptic vesicles attached and, in freeze-fracture replicas, by clusters of intramembrane particles localized in bands with vesicle openings on the sides of these bands. The cytoplasmic surface of the postsynaptic membrane opposite the active zones was coated with electron-dense material that in freeze-fracture replicas was seen as an increase in intramembrane particles located in the external leaflet (EF-face). This specialization of the postsynaptic membrane may correspond to the neurochemical receptor. Also, pre- and postsynaptic membranes were separated by a wider extracellular gap than those of adjacent nonsynaptic regions and electrical synapses or gap junctions. Synaptic vesicles were located exclusively at the synaptic regions by means of a cytoskeleton that was different for the electrical and the chemical components. The vesicles associated with the electrical component were anchored to a cytoskeleton composed of a beaded layer of densities located parallel to the membrane. This cytoskeleton maintained the synaptic vesicles separated from the presynaptic membrane by a distance of 13 +/- 2 nm. The synaptic vesicles associated with the chemical component were anchored to electron-dense regions formed by filaments arranged in bundles, anchored to the presynaptic membrane. Vesicles lined both sides positioned to discharge their contents into the extracellular space and to replace the discharged vesicles.

Myocardial sarcolemma in ischemia: a quantitative freeze-fracture study.

Freeze-fracture electron microscopy permits the visualization of the intramembrane particles (IMP). These IMPs are presumably proteins responsible for the main functions of the membrane. Quantitative techniques (Clark-Evan statistics) were applied to determine in a critical manner whether IMP pattern shifts (random, clustered, or ordered) occur under the ischemic conditions (5-45 min with and without reperfusion) and whether this change is related to the experimental condition. In each case three hearts, eight replicas/heart, one area of 0.25 micron 2 of membrane fracture face/replica was measured to give a total of 6 micron 2 of membrane counted for each condition (control vs. ischemic). A mixed effects nested model analysis of variance was performed in each variable. We found that IMP aggregation can be present in some control membranes, but the degree of aggregation was greater and more consistent in membranes made ischemic and followed by reperfusion. Most striking was the significant clustering of IMPs in membranes from hearts ischemic for only 5 min. Reperfusion after only 5 min of ischemia reversed IMP clustering. Functionally at this time there is an increase in K+ concentration in the interstitial space that reaches approximately 15 mM within 10 min and reverses on reperfusion. The structural alteration in IMPs appears to parallel the function in ischemic hearts.

Effects of cytochalasin D on the integrity of the Sertoli cell (blood-testis) barrier.

Ectoplasmic specializations (ES) containing packed actin microfilaments are associated with the numerous parallel rows of occluding junctions which form the Sertoli cell (blood-testis) barrier. To determine if ES regulate the structure of the occluding junctions and/or barrier permeability, we experimentally disrupted ES microfilaments in vivo with intratesticularly injected cytochalasin D (CD). Electron microscopic observations of seminiferous tubules from CD-treated (150-500 microM CD; 0.5-12 hr) animals indicated that ES was absent from regions where the Sertoli cell barrier is located. Seminiferous epithelial sheets from uninjected or vehicle-injected animals (1 DMSO: 1 saline) stained with NBD-phallacidin demonstrated the presence of patterned ES actin surrounding the basolateral regions of adjacent Sertoli cells. After exposure to CD, epithelial sheets exhibited increasingly patchy fluorescence indicating progressive F-actin disruption. Freeze-fracture replicas of CD-injected testes revealed numerous focal alterations in the region of occluding junctions which included disorganization of the parallel arrangement of junctional rows, the presence of free-ending rows, clustering of intramembranous particles (IMPs) between rows, reduction in the number of rows, and loss of IMPs on both the P-face and E-face. Tracer experiments, following CD exposure, were conducted to test the integrity of occluding junctions: lanthanum hydroxide, dextrose, or filipin was added, in separate experiments, to the fixative during perfusion-fixation. In another study, serum containing an antibody against adluminal germ cells was injected intratesticularly, and frozen sections were processed for immunofluorescence study. A final study consisted of simultaneous intratesticular infusions of CD and radiolabelled inulin with subsequent intraluminal and peritubular fluid sampling. In animals which were injected with CD, lanthanum was found to enter the adluminal compartment; fixative made hypertonic by addition of dextrose caused germ cells within the adluminal compartment to shrink and produce exaggerated intercellular spaces; filipin-cholesterol perturbations were present between some Sertoli cell junctional rows and on spermatid plasma membranes; and IgG was detected within the adluminal compartment of many seminiferous tubules. None of these adluminal manifestations was noted in control animals or those which received vehicle. Quantitatively, in the in vivo micropuncture experiments, significantly more radiolabelled inulin entered the lumen of seminiferous tubules from CD-treated animals than from those exposed to vehicle.(ABSTRACT TRUNCATED AT 400 WORDS)

Endosomes from kidney collecting tubule cells contain the vasopressin-sensitive water channel.

The mechanism by which vasopressin rapidly and dramatically increases the water permeability of target epithelial cell membranes is thought to involve a cycle of exo- and endocytosis during which vesicles carrying 'water channels' are successively inserted into, and removed from the apical plasma membrane of epithelial cells. Clusters of intramembranous particles, visible by freeze-fracture electron microscopy and presumed to represent water channels, appear on apical membranes in parallel with increased transepithelial water flow. In the collecting duct, these clusters are located in clathrin-coated pits which are subsequently internalized. There has been no direct evidence, however, that subcellular membranes in vasopressin-sensitive epithelia contain functional water channels. In this report, we have used fluorophores that are sensitive to volume and do not pass through membranes to label and to measure directly the osmotic water permeability of endocytosed vesicles isolated from renal papilla. We present direct evidence that vasopressin induces the appearance of a population of endocytic vesicles whose limiting membranes contain water channels.

Differential effect of Lonidamine on the plasma membrane ultrastructure of normal and leukemic human lymphocytes

The effect of Lonidamine on the plasma membrane ultrastructure of normal and leukemic human peripheral blood lymphocytes (hPBL) was studied by freeze-fracture electron microscopy. Lonidamine induces remarkable changes in the intramembrane particle distribution on both fracture faces of the plasma membrane as well as of the intracytoplasmic membranes. In particular, a dose-dependent clustering of intramembrane particles was observed in all cell types examined, i.e., normal T and B lymphocytes, T cells from acute lymphoblastic leukemia, and B cells from chronic lymphocytic leukemia, though to a different extent. Normal T lymphocytes appear to be the most sensitive to the action of the drug, while corresponding B cells are much less affected. As regards leukemic cells, in T lymphoblasts the ultrastructural membrane changes are lower than in normal T lymphocytes, whereas leukemic B cells show the same low response to Lonidamine treatment as their normal counterpart. Such a differential effect may be explained by the different membrane molecular organization displayed by normal T and B lymphocytes and by normal and leukemic cells. Moreover, the extent of the ultrastructural modifications at the plasma membrane level, correlates well with literature data on the inhibition of the aerobic glycolysis induced by Lonidamine on the different lymphoid cell types. These findings seem to further confirm that cell membranes are the primary targets of Lonidamine action.

Microtubules and the flagellar apparatus in zoospores and cysts of the fungusPhytophthora cinnamomi

A correlated immunofluorescence and ultrastructural study of the microtubular cytoskeleton has been made in zoospores and young cysts ofPhytophthora cinnamomi. Labelling of microtubules using antibodies directed towards tubulin has revealed new details of the arrangement of the flagellar rootlets in these cells, and of the variability that occurs from cell to cell. Most of the variation exists at the distal ends of the rootlets, and may be correlated with differences in cell shape in these regions. The rootlets have the same right and left configuration in all zoospores. The arrangement of the rootlet microtubules at the anterior end of the zoospores raises the possibility that the microtubules on the left hand side of the groove may not comprise an independent rootlet which arises at the basal bodies. The absolute configuration of the flagellar apparatus has been determined from ultrastructural observations of serial sections. In the vicinity of the basal bodies, there is little, if any, variation between individuals, and the structure of the flagellar apparatus is similar to that described for related species of fungi. Two ribbon-like coils surround the central pair of microtubules at the distal tip of the whiplash flagellum, and clusters of intramembranous particles, similar to ciliary plaques, have been found at the bases of both flagella. There are two arrays of microtubules associated with the nucleus in the zoospores. One array lies next to the outer surface of the nuclear envelope, and probably functions in the shaping and positioning of the apex of the nucleus. The nuclear pores in this region are aligned in rows alongside these microtubules. The second array is formed by kinetochore microtubules which extend into a collar-like arrangement of chromatin material around the narrow end of the (interphase) nucleus. During encystment, all flagellar rootlets are internalized when the flagella are detached at the terminal plate. The rootlets arrays are no longer recognizable 5–10 minutes after the commencement of encystment.

Cell junctions in amphioxus (Cephalochordata): A thin section and freeze-fracture study

Tissues from the epidermis, alimentary tract and notochord of the cephalochordate Branchiostoma lanceolatum have been examined in both thin sections and freeze-fracture replicas to ascertain the nature of the intercellular junctions that characterize their cell borders. The columnar epithelial cells from the branchial chamber (pharynx), as well as from the anterior and posterior intestine, all feature cilia and microvilli on their luminal surfaces. However, their lateral surfaces exhibit zonulae adhaerentes only. No gap junctions have been observed, nor any tight junctions (as are a feature of the gut of urochordates and higher vertebrates), nor unequivocal septate junctions (as are typical of the gut of invertebrates). The basal intercellular borders are likewise held together by zonulae adhaerentes while hemidesmosomes occur along the basal surface where the cells abut against the basal lamina. The lateral cell surfaces, where the adhesive junctions occur, at both luminal and basal borders, do not exhibit any specialized arrangement of intramembrane particles (IMPs), as visualized by freeze-fracture. The IMPs are scattered at random over the cell membranes, being particularly prevalent on the P-face. The only distinctive IMPs arrays are those found on the ciliary shafts in the form of ciliary necklaces and IMP clusters. With regard to these ciliary modifications, cephalochordates closely resemble the cells of the branchial tract of ascidians (urochordates). However, the absence of distinct junctions other than zonulae adhaerentes makes them exceptions to the situation generally encountered in both vertebrates and urochordates, as well as in the invertebrates. Infiltration with tracers such as lanthanum corroborates this finding; the lanthanum fills the extracellular spaces between the cells of the intestine since there are no junctions present to restrict its entry or to act even as a partial barrier. Junctions are likewise absent from the membranes of the notochord; the membranes of its lamellae and vesicles exhibit irregular clusters of IMPs which may be related to the association between the membranes and the notochordal filaments. Epidermis and glial cells from the nervous system possess extensive desmosomal-like associations or zonulae adhaerentes, but no other junctional type is obvious in thin sections, apart from very occasional cross-striations deemed by some previous investigators to represent ‘poorly developed’ septate junctions.

A simple physical model for fungicide induced hexagonal clustering of intramembrane particles in the plasmalemma of Ustilago avenae

Freeze fracture electron microscopy is used to study the influence of fungicide (triadimenol) treatment on the plasmalemma of sporidia of Ustilago avenae. The intramembrane particles (IMPs) randomly distributed in untreated samples form large hexagonal clusters after triadimenol treatment. A simple physical model based on length mismatch between lipid bilayer and IMPs is used to describe the interaction between the IMPs in terms of a lipid mediated potential. Using for a rough estimation also data on artificial membranes the fungicide induced ordering effect of the IMPs is discussed. A homogeneous change of lipid bilayer parameters due to the fungicide action is considered as well as a fluid-gel phase transition of the lipid matrix, which might be related to the cluster formation. Further implications of the different possibilities with respect to the mode of action of the fungicide are suggested.

Lack of intramembranous particle clusters in collecting ducts of mice with nephrogenic diabetes insipidus.

We suggested previously, on the basis of indirect evidence, that in two strains of mice with nephrogenic defects of urinary concentration the deficiency arose from an inadequate rise in water permeability of the collecting duct system. In this study we tested the question further by assuming that the frequency of intramembranous particle (IMP) clusters seen by freeze-fracture can be used as a morphological marker of vasopressin-induced water permeability. Three genotypes of mice were studied: 1) DI +/+ Severe, with florid, vasopressin-resistant diabetes insipidus; 2) DI +/+ Nonsevere, with an intermediate deficiency of urinary concentration; and 3) normal, VII +/+ mice. In addition, we examined a group of DI +/+ Severe mice that had been injected with exogenous 1-desamino-8-D-arginine vasopressin (DDAVP) subcutaneously for 3 days. Since the results in this group did not differ from those in untreated DI +/+ Severe mice, all data for this genotype were combined. IMP clusters within luminal membranes of inner medullary collecting duct principal cells were quantified by freeze-fracture electron microscopy. Urinary osmolality and percentage of cells showing clusters were, respectively: 203 +/- 43 mosmol/kg H2O and 0% in DI +/+ Severe mice; 1,133 +/- 86 and 33 +/- 4 in DI +/+ Nonsevere mice; and 2,234 +/- 190 and 52 +/- 5 in VII +/+ animals. With the exception of one animal, there was no overlap of the data, which were significantly different from one another for each variable. We conclude that in DI +/+ Severe mice, both endogenous and exogenous vasopressin are unable to increase the water permeability of medullary collecting ducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Hexagonal clustering of intramembrane particles in the plasmalemma of Ustilago avenae after fungicide treatment

The plasmalemma of sporidia of Ustilago avenae has been investigated by means of the freezeetching technique after treatment with the fungicide, triadimenol (5 μg ml −1, 10 and 17 hr). The control samples are characterized in the exponential and stationary growth phases by homogeneous plasmatic fracture (PF) and extraplasmatic fracture (EF) faces with a random intramembrane particle (IMP) distribution. Treatment with triadimenol induces clusters of similar size IMPs in hexagonal arrangement. In between the flat clusters the plasmalemma is significantly deformed, showing hemispherical pits and protrusions on both membrane fracture halves. In the stationary growth phase no hexagonal clustering of the IMPs is observed under the described conditions.

(Na+ + K+)-ATPase correlated with a major group of intramembrane particles in freeze-fracture replicas of cultured chick myotubes.

Immunofluorescence microscopy with a fluorescein-labeled monoclonal antibody was used to map the distribution of sodium- and potassium-ion stimulated ATPase [( Na,K]-ATPase) on the surface of tissue-cultured chick skeletal muscle. At this level of resolution it appeared that the (Na,K)-ATPase molecules were distributed nearly uniformly over the plasma membrane. These molecules could be cross-linked by use of the monoclonal antibody followed by a second antibody directed against the monoclonal antibody; the resulting fluorescent pattern was a set of small dots (patches) on the muscle surface. This pattern was stable over several hours, and there was little evidence of interiorization or of coalescence of the patches. Myotubes labeled with immunofluorescence were fixed in glutaraldehyde, cryoprotected with glycerin, then fractured and replicated by standard methods. Replicas of the immunofluorescence-labeled myotubes revealed clusters of intramembrane particles (IMP) only when the immunofluorescent images indicated a patching of the (Na,K)-ATPase molecules. Double antibody cross-linking of antigenic sites on myotubes with each of three other monoclonal antibodies to plasma membrane antigens likewise resulted in patched patterns of immunofluorescence, but in none of these cases were clusters of intramembrane particles found in freeze-fracture replicas. In each case it was shown that the (Na,K)-ATPase molecules were not patched. Other control experiments showed that patching of (Na,K)-ATPase molecules did not cause co-patching of one of the other plasma membrane proteins defined by a monoclonal antibody and did not cause detectable co-clustering of acetylcholine receptors. Detailed mapping showed that there was a one-to-one correspondence between immunofluorescent patches related to the (Na,K)-ATPase and clusters of IMP in a freeze-fracture replica of the same cell. We conclude that the intramembrane particles patched by double antibody cross-linkage of the (Na,K)-ATPase are caused by (Na,K)-ATPase molecules in the fracture plane. Quantification of the IMP indicated that the (Na,K)-ATPase-related particles account for up to 50% of particles evident in the replicas, or up to about 400 particles/micrometers2 of plasma membrane. Particles related to the (Na,K)-ATPase were similar to the average particle size and were as heterodisperse in size as the total population of IMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Intense, reversible aggregation of intramembrane particles in non-haemolyzed human erythrocytes A freeze-fracture study

Acridine orange, a strongly cationic, membrane-penetrating dye, caused intense aggregation of intramembrane particles in non-haemolyzed human erythrocytes at 5 mM concentration. Simultaneously with the particle aggregation, large, empty, intramembrane particle-free or particle-depleted vesicles were detached from the red cells. Washing the erythrocytes after Acridine orange treatment resulted in complete disaggregation of the intramembrane particles. Less cationic acridine dyes (9-aminoacridine, 5-aminoacridine and Quinacrine) caused much less conspicuous alterations. Rivanol (ethacrine lactate), on the one hand, caused intramembrane particle aggregation in human red cells as well as ribosome aggregation in rabbit reticulocytes when it was dissolved in lactate-buffered sucrose. On the other, Rivanol dissolved in phosphate-buffered saline failed to induce these alterations. Neuraminidase treatment had no effect on the intensity of Acridine orange-induced particle aggregation, but impeded disaggregation. Our results indicate that, in contrast to previous observations, intense and reversible clustering of intramembrane particles is certainly possible in non-haemolyzed erythrocytes. The intramembrane particle aggregation may be due primarily to perturbation of the inner red cell surface by strongly cationic dyes and the presence of sialic acid residues on the outer red cell surface seems to be essential for the reversibility of the process.