Intramembranous Particles Research Articles

Post-testicular changes in the density and distribution of intramembrane particles of stallion sperm surface domains

Freeze-fracture replicas of stallion spermatozoa, collected from the proximal caput, corpus and cauda epididymides regions, were analyzed by electron microscopy to explore the distribution and density of intramembrane particles (IMP). Conspicuous differences in density and arrangement of the IMP were observed in the different topographical domains of mature and immature spermatozoa. A reduction of IMP, especially remarkable in the post-acrosomal domain, was observed in mature epididymal spermatozoa when compared with samples collected from ductuli efferentes. Some structural species-specific differences were also observed. The significance of these changes has not been determined, but remodeling of membrane components during developmental processes constitutes a fine control mechanism to ensure that key molecules are in the correct membrane position and during an appropriate timeframe to mediate fertilization.

ZO-1 and ZO-2 Independently Determine Where Claudins Are Polymerized in Tight-Junction Strand Formation

A fundamental question in cell and developmental biology is how epithelial cells construct the diffusion barrier allowing them to separate different body compartments. Formation of tight junction (TJ) strands, which are crucial for this barrier, involves the polymerization of claudins, TJ adhesion molecules, in temporal and spatial manners. ZO-1 and ZO-2 are major PDZ-domain-containing TJ proteins and bind directly to claudins, yet their functional roles are poorly understood. We established cultured epithelial cells (1(ko)/2(kd)) in which the expression of ZO-1/ZO-2 was suppressed by homologous recombination and RNA interference, respectively. These cells were well polarized, except for a complete lack of TJs. When exogenously expressed in 1(ko)/2(kd) cells, ZO-1 and ZO-2 were recruited to junctional areas where claudins were polymerized, but truncated ZO-1 (NZO-1) containing only domains PDZ1-3 was not. When NZO-1 was forcibly recruited to lateral membranes and dimerized, claudins were dramatically polymerized. These findings indicate that ZO-1 and ZO-2 can independently determine whether and where claudins are polymerized.

The relationship between intramembranous particles and aquaporin molecules in the plasma membranes of normal rat skeletal muscles: a fracture-label study

The plasma membrane of skeletal muscles contains water channels such as aquaporin 4 (AQP4), aquaporin 3 (AQP3) and aquaporin 7 (AQP7). In dehydrated mice, we have recently reported the altered distribution of the aggregations of intramembranous particles (IMPs), such as orthogonal array (a crystal-like structure) and IMP cluster (a rosette-like structure) on the freeze-fractured skeletal muscle plasma membranes. In this fracture-label study, we first tested whether the orthogonal arrays (OAs) were composed of AQP4 in skeletal muscles and further analyzed the relationship between IMPs including IMP clusters and AQP3 molecules. As a result, many of the gold particles indicating AQP4 was associated with OAs (79%) by our fracture-label technique. On the other hand, approximately 50% of gold particles indicating AQP3 were associated with IMP clusters. Thus we confirmed that the OAs are composed of AQP4 in skeletal muscles, and further demonstrated that some of the IMP clusters are composed of AQP3 and may participate in maintaining osmotic homeostasis in skeletal myofibers. The fracture-label method is useful in investigating the molecular identification of membrane proteins such as AQP3 and AQP4.

Dynamic study of intramembranous particles in human fresh erythrocytes using an “in vitro cryotechnique”

For analyses of dynamic ultrastructures of erythrocyte intramembranous particles (IMPs) in situ, a quick-freezing method was used to stabilize the flow behavior of erythrocytes embedded in vitreous ice. Fresh human blood was jetted at various pressures through artificial tubes, in which the flowing erythrocytes were elongated from biconcave discoid shapes to elliptical ones, and quickly frozen in liquid isopentane-propane cryogen (-193 degrees C). They were freeze-fractured using a scalpel in liquid nitrogen, and routinely prepared for replica membranes. Many IMPs were observed on the protoplasmic freeze-fracture face (P-face) of the erythrocyte membranes. Some control erythrocytes under nonflowing or stationary conditions showed IMPs with their random distribution. However, other jetted erythrocytes under flowing conditions showed variously sized IMPs with much closer distribution. They were also arranged into parallel rows in some parts, and aggregated together. This quick-freezing method enabled for the first time the visualization of time-dependent topology and the molecular alteration of IMPs in dynamically flowing erythrocytes.

2P154 Intra-membrane particles of Na^+/K^+-ATPase observed by quick-freezing replica technique(34. Membrane protein,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

2P154 Intra-membrane particles of Na^+/K^+-ATPase observed by quick-freezing replica technique(34. Membrane protein,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

A Novel Calcium-Independent Peripheral Membrane-Bound Form of Annexin B12

Annexins are soluble proteins that can interact with membranes in a Ca2+-dependent manner. Recent studies have shown that they can also undergo Ca2+-independent membrane interactions that are modulated by pH and phospholipid composition. Here, we investigated the structural changes that occurred during Ca2+-independent interaction of annexin B12 with phospholipid vesicles as a function of pH. Electron paramagnetic resonance analysis of a helical hairpin encompassing the D and E helices in the second repeat of the protein showed that this region refolded and formed a continuous amphipathic alpha helix following Ca2+-independent binding to membranes at mildly acidic pH. At pH 4.0, this helix assumed a transmembrane topography, but at pH approximately 5.0-5.5, it was peripheral and approximately parallel to the membrane. The peripheral form was reversibly converted into the transmembrane form by lowering the pH and vice versa. Furthermore, analysis of vesicles incubated with annexin B12 using freeze-fracture electron microscopy methods showed classical intramembrane particles at pH 4.0 but none at pH 5.3. Together, these data raise the possibility that the peripheral-bound form of annexin B12 could act as a kinetic intermediate in the formation of the transmembrane form of the protein.

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.

Structural Characterization of Na,K-ATPase from Shark Rectal Glands by Extensive Trypsinization

Extensive trypsinization of Na,K-ATPase from the salt gland of Squalus acanthias removes about half of the extramembranous protein mass of the alpha-subunit, while leaving the beta-subunit intact. Sequence analysis and epitope recognition of the remaining alpha-peptides show that transmembrane segments M1/M2 and M3/M4 are present when trypsinization is performed in either NaCl or RbCl. The M5/M6 segment and the intact 19-kDa peptide (M7-M10) are detected in Rb-trypsinized membranes but not in Na-trypsinized membranes. The L7/L8 loop is associated with Na-trypsinized membranes, indicating the presence of an M7/M8 or M8/M9 fragment. Freeze-fracture electron microscopy of both Rb- and Na-trypsinized membranes reveals intramembranous particles that indicate a retained cluster of peptides, even in the absence of an intact 19-kDa fragment. The rotational diffusion of covalently spin-labeled trypsinized complexes is studied in the presence of poly(ethylene glycol) or glycerol by using saturation transfer electron spin resonance. Rotational correlation times in aqueous poly(ethylene glycol) are longer than in glycerol solutions of the same viscosity and increase nonlinearly with the viscosity of the suspending medium, indicating that poly(ethylene glycol) induces aggregation of the tryptic peptides (and beta-subunit) within the membrane. The aggregates of enzyme trypsinized in the presence of NaCl are larger than those for enzyme trypsinized in RbCl, at both low and high aqueous viscosities. Similarities in mobility for native and Rb-trypsinized enzymes suggest either a change in average orientation of the spin-label upon trypsinization or that trypsinization leads to a reorganized protein structure that is more prone to aggregation.

Explorative statistical analysis of planar point processes in microscopy

Basic methods of explorative statistical analysis for stationary and isotropic planar point processes are briefly and informally reviewed. At the explorative level, planar point patterns may be characterized in terms of the intensity, the K-function and the pair correlation function. These second-order functions enable one to classify a given point process as completely random, clustering or repulsive. The repulsive behaviour may be quantified by an estimate of the hard-core distance. In the exploratory approach, the statistics are essentially free from model assumptions. Second-order spatial functions have been estimated to characterize genuine planar point processes in the macroscopic domain, for example in forestry, geography and epidemiology. For light microscopy and transmission electron microscopy, two situations are distinguished, which may be summarized as the genuine planar case and the stereological case. In the genuine planar case, a direct interpretation of the results of spatial statistics is feasible. Here, monolayers in cell culture, intramembranous particles on freeze fracture specimens and amacrine cells of the retina are mentioned as examples. In the stereological case, point patterns are generated by sections through 3D structures. Here the observed point patterns may arise as the centres of sectional profiles of particles, or as centres of sectional profiles of spatial fibre processes. In both situations, exploratory spatial point process statistics allow a quantitative characterization of sectional images for the purposes of group comparisons and classification. Moreover, for spatial fibre processes it has recently been shown that the observed pair correlation function of the centres of the fibre profiles is an estimate of the reduced pair correlation function of the fibre process in 3D. Hence for fibre processes a stereological interpretation of point process statistics obtained from sections is an additional option.

The structural bases of long-term anabiosis in non-spore-forming bacteria

Peculiarities of the structural organization in non-spore-forming bacteria associated with long-term anabiosis were revealed both in laboratory cultures and in natural populations isolated from 1–3-Myr-old Eastern Siberian permafrost and tundra soil. Different advanced methods were used, including (a) high-resolution electron microscopy; (b) simulation of in situ conditions in the laboratory by varying the composition of growth medium and cultivation conditions; (c) low-temperature fractionation to isolate and concentrate microbial cells from natural soils; (d) comparative morphological analysis of microbial cells in model cultures and natural soils (in situ). Under laboratory conditions, the intense formation of resting cells by representatives of various taxa of eubacteria and halophilic archaea occurred in 2–9-month-old cultures grown in carbon-, nitrogen-, or phosphorus-limited media, in starved cell suspensions in the presence of sodium silicate, or on soil agar. Among resting cells, we revealed cystlike forms having a complicated structure and common features. These included a thick capsule; a thickened and multiprofile cell wall; the presence of large intramembrane particles on PF- and EF-fracture surfaces; fine-grained or lumpy cytoplasm; and a condensed nucleoid. The general morphological properties, ultrastructural organization, physiological features of cystlike cells, and their ability to germinate under the appropriate conditions suggest the existence of constitutive dormancy in non-spore-forming bacteria. It was found that the majority of microorganisms in permafrost and tundra soil are cystlike cells, very similar to those in laboratory cultures. Anabiotic (resting) cystlike cells are responsible for the survival of non-spore-formers in extreme Earth habitats and may be regarded as possible analogs of extraterrestrial forms of microbial life.

Amphiphile-induced tubular budding of the bilayer membrane

Amphiphile-induced tubular budding of the erythrocyte membrane was studied using transmission electron microscopy. No chiral patterns of the intramembraneous particles were found, either on the cylindrical buds, or on the tubular nanoexovesicles. In agreement with these observations, the tubular budding may be explained by in-plane ordering of anisotropic membrane inclusions in the buds where the difference between the principal membrane curvatures is very large. In contrast to previously reported theories, no direct external mechanical force is needed to explain tubular budding of the bilayer membrane.

Alterations of molecular architectures in skeletal muscle plasma membranes of dehydrated and water-loaded mice

It is likely that orthogonal arrays (OAs) and caveolae seen on the replicas of freeze-fractured muscle plasma membranes are involved in maintaining osmotic homeostasis. Therefore, using the freeze-fracture technique, we examined the ultrastructural changes in OAs and caveolae of the skeletal muscle plasma membrane of dehydrated and water-loaded mice. In the muscle plasma membranes of 6 dehydrated and 6 water-loaded mice, caveolar distribution was not altered, and the densities of caveolae and OAs did not show statistically significant differences when compared with those in 6 control mice, although the skeletal muscles of water-loaded mice sometimes had muscle plasma membranes with extremely numerous OAs. In contrast, the muscle plasma membranes of dehydrated mice often revealed changes in the distribution of OAs, which existed in a group at the confined area of the muscle plasma membranes and were frequently accompanied by the aggregations of intramembranous particles (IMPs) around OAs. Thus, on the basis of the present study, we suggest that OAs in skeletal muscles as well as those in brain may play an important role in maintaining osmotic homeostasis of these organs under abnormal water balance.

Cryo-SEM and subsequent TEM examinations of identical neural tissue specimen

Low temperature scanning electron microscopy of frozen-fractured specimens under cryo-protecting, non-dehydrating, and non-etching “wet” conditions, that is, direct cryo-SEM, was followed by transmission electron microscopy (TEM) with the same neural tissue specimens. In comparison to replica TEM, direct cryo-SEM can obtain images with a smooth gradation of contrast. The major advantage of direct cryo-SEM combined with TEM was that time was saved in SEM preparation. It had a high potentiality at a wide-range survey of multi-dimensional specimen structures with less-artifacts. Because the specimens were prepared as quickly as possible under “wet” conditions, the target structures could be examined under lower through higher magnifications. In the present study, neuronal and glial elements, such as plasma membranes and cell organelles that include the synaptic vesicles, were localized on the fractured surface. In subsequent TEM examination, it was confirmed that the underlying internal structures could be further characterized from cytological as well as molecular biological aspects. In addition, direct cryo-SEM distinctively demonstrated small intra-membrane particles (ca. 10 nm in diameter). However, due to electron lucency, they could not be confirmed in the re-processed TEM specimens. Applying the present protocol, stereological and internal architectural examinations of the neural tissues have been simultaneously conducted at ultra-fine levels.

Segment-specific expression of tight junction proteins, claudin-2 and -10, in the rat epididymal epithelium

Tight junctions are known to be related to the variance by segment of the luminal fluid microenvironment of the epididymis. We examined the expression of claudins that regulate the paracellular permeability of ions, solutes, and water through tight junctions in the rat epididymis. RT-PCR analysis showed that claudin-2 and -10 were expressed in a segment-specific manner among fifteen claudins examined. Immunofluorescence microscopy demonstrated that both claudins showed developmental stage- and segment-specific subcellular localizations in the epididymal epithelium. Claudin-2 was detected principally in the apical junctional region in the initial segment at postnatal day 7 and week 10 but lost its expression in all the distal segments including the caput, corpus, and cauda. Claudin-10 was detected principally in the apical junctional region in all segments on postnatal day 7 but only in the initial segment--especially along the entire lateral membrane--at 10 weeks. In freeze fracture electron microscopy, well-developed intramembranous particle strands were observed on the P face and complementary grooves with a few particles on the E face at 10 weeks. Many particles were occasionally observed on the grooves in the E face in the initial segment and distal caput. Tight junction strands were continuous and impermeable to lanthanum nitrate on postnatal day 7, suggesting the establishment of functional tight junctions. These segment-specific expressions of claudin-2 and -10 may contribute to creating the specific luminal fluid microenvironment which is necessary for the transport, maturation, and storage of spermatozoa.

Medich giant platelet disorder: a unique α granule deficiency I. Structural abnormalities

Human platelet granule deficiency disorders include the gray platelet syndrome (GPS), α δ storage pool deficiency, the Hermansky–Pudlak syndrome and the White platelet syndrome. The present study describes a patient with a lifelong history of easy bleeding, thrombocytopenia and giant platelets. Her cells were found to have normal numbers of dense bodies, but a markedly decreased number of α granules. Many platelets had no α granules and resembled the gray platelets of patients with GPS. However, the empty vacuoles without granule contents that fill the cytoplasm of GPS platelets were not present in significant numbers in her platelets. In addition to the decrease in α granules the patients platelets contained membranous inclusions resembling cigars or scrolls. Usually, only one scroll open at each end was present, but many platelets contained two and some as many as five. Freeze-fracture revealed an absence of intramembranous particles in many layers of the scrolls. They occur in no other human platelet disorder, but are common in platelets from the Wistar–Furth rat. Thus, the patient is a unique variant of human platelet granule deficiency disorders unlike any described previously.

Specificity of membrane specializations in mechanoreceptors of birds—A freeze-etching study

The detailed knowledge of the molecular process of mechanotransduction is still an unsolved question. The investigation of the intramembranous structure of the cutaneous mechanoreceptors may play an important role in elucidating this problem. In this relation, Herbst sensory corpuscles in ducks were studied for the first time using the freeze-etching and thin sectioning techniques. Herbst corpuscles have the basic structural components valid for most of the encapsulated mechanoreceptors in mammals: a capsule made of perineural cells, a lamellar complex of modified Schwann cells, surrounding the non-myelinated part of the receptor nerve fiber and its ending. Freeze-etching replicas reveal that the plasmalemmae of the capsule cells, modified Schwann cells and axolemmae of parts of the nerve fiber differ in both density and pattern of distribution of intramembranous particles (IMPs) as well as IMP size. On all the plasmalemmae the IMP density is higher on the P-face (2000–3300 µm−2) than the respective E-face (800–1500 µm−2). The axolemma of the ending of the receptor nerve fiber expresses higher density of IMPs than its shaft. The mean IMP size for all the plasmalemmae varies between 5.5 and 7.5 nm. Many tight junctions occur between the capsule cells. These results indicate that the non-myelinated axolemma as well as the plasmalemmae of other components of Herbst corpuscles are specialized in terms of content and distribution of IMPs. The IMPs may represent various kinds of mechanosensitive channel proteins or related membrane-bound proteins participating in the process of mechanotransduction.

Differences in intramembrane particle distribution in young and old human erythrocytes.

The distribution of intramembrane particles in human erythrocytes was studied by freeze-fracture on young and old cells and compared to that obtained after ATP depletion or following addition of a clustering agent. It was shown that intramembrane particles became aggregated and the mean particle density increased as the cells aged. Likewise, both particle aggregation and increased density were found in young cells after moderate ATP depletion. In contrast, mean particle density was markedly reduced in both cell types after exhaustive depletion. Paradoxically, Zn treatment led to decreased particle density in young cells, whilst producing the opposite effect in aged cells. The results suggest that their low ATP content may account for the increased particle density of senescent cells.

Membrane organization and function of M1 and M23 isoforms of aquaporin-4 in epithelial cells.

Aquaporin-4 (AQP4) water channels exist as heterotetramers of M1 and M23 splice variants and appear to be present in orthogonal arrays of intramembraneous particles (OAPs) visualized by freeze-fracture microscopy. We report that AQP4 forms OAPs in rat gastric parietal cells but not in parietal cells from the mouse or kangaroo rat. Furthermore, the organization of principal cell OAPs in Brattleboro rat kidney is perturbed by vasopressin (arginine vasopressin). Membranes of LLC-PK(1) cells expressing M23-AQP4 showed large, abundant OAPs, but none were detectable in cells expressing M1-AQP4. Measurements of osmotic swelling of transfected LLC-PK(1) cells using videomicroscopy, gave osmotic water permeability coefficient (P(f)) values (in cm/s) of 0.018 (M1-AQP4), 0.019 (M23-AQP4), and 0.003 (control). Quantitative immunoblot and immunofluorescence showed an eightfold greater expression of M1- over M23-AQP4 in the cell lines, suggesting that single-channel p(f) (cm(3)/s) is much greater for the M23 variant. Somatic fusion of M1- and M23-AQP4 cells (P(f) = 0.028 cm/s) yielded OAPs that were fewer and smaller than in M23 cells alone, and M1-to-M23 expression ratios ( approximately 1:4) normalized to AQP4 in M1 or M23 cells indicated a reduced single-channel p(f) for the M23 variant. Expression of an M23-AQP4-Ser(111E) mutant produced approximately 1.5-fold greater single-channel p(f) and OAPs that were up to 2.5-fold larger than wild-type M23-AQP4 OAPs, suggesting that a putative PKA phosphorylation site Ser(111) is involved in OAP formation. We conclude that the higher-order organization of AQP4 in OAPs increases single-channel osmotic water permeability by one order of magnitude and that differential cellular expression levels of the two isoforms could regulate this organization.

Cold-induced imbibition damage of lettuce embryos: a study using cryo-scanning electron microscopy

The impact of rehydration on a multicellular organism was studied in lettuce (Lactuca sativaL.) embryos, using cryo-scanning electron microscopy (cryo-SEM). Naked embryos were sensitive to imbibitional stress, whereas embryos with an intact, thick-walled endosperm were not. Imbibitional injury to naked embryos was mainly confined to the outer 2–3 cell layers of the axis. These cells failed to swell and appeared poorly hydrated. Deeper layers were not affected even after extended periods of cold rehydration. The proportion of damaged cells (6–7% of total) roughly corresponded with the additional K+that gradually leached from the embryos. Damaged embryos were able to survive the loss of their surface layers and form adventitious roots. The swelling of inner tissues caused the dead surface layers to rupture into patches. Plasma membranes in dried embryos showed normal bilayer structure with a homogeneous distribution of intra-membrane particles (IMPs), also after non-injurious rehydration. Imbibitionally damaged plasma membranes showed many irregularities, such as globular insertions, that probably resulted from malfusions in the ruptured membrane, but the IMPs were still randomly distributed.

A study of lipid secretion from the lichen symbionts, ascomycetous fungus Myelochroa leucotyliza and green alga Trebouxia sp.

Lichens are symbionts of fungi and algae. Although wild fungi secrete lipids to form crystals, those grown in culture either alone or with algae do not secrete enough lipids to be crystallized. To investigate the mode of lipid secretion, we stimulated fungi cultured alone to form crystals. (1) The fungi had serpentine invaginations on the P-faces. These were formed as a consequence of secretory granule exocytosis. (2) Fungi cultured alone in normally used medium had on their P-faces intramembrane particle-cleared parts that also showed a serpentine configuration. (3) After the medium was fortified by further addition of glucose, the fungi cultured alone produced multiple lipid bodies and secretory granules, though no crystals were formed. (4) After the addition of filtered algal culture medium into the fungal culture medium that had been fortified, the fungi grown under this condition showed extracellular crystals. As fungi also showed extensive exocytotic activity, protein secretion seemed essential prior to lipid secretion. The place with no intramembrane particles was postulated to be the site through which lipids penetrate to the outside. (5) As we had identified crystals as atranorin, we made atranorin-containing liposome. The liposome released atranorin to the aqueous phase by the addition of albumin or albumin-like proteins.