Myelinating glia of earthworm giant axons: thermally induced intramembranous changes

The median and lateral giant axons in the ventral nerve cord of the earthworm Lumbricus terrestris are ensheathed by extensive spiral glial cell wrappings which resemble vertebrate myelin. The other, smaller, axons are encompassed by attenuated glial processes, as is typical of invertebrates. The fine structural details of the glial cells have been studied in thin sections and in replicas produced by freeze-fracturing where the intramembranous particle (IMP) populations within the lipid bilayer are visible. These consist of both low-profile IMPs as well as prominent ones 6-8 nm in diameter, scattered at random over the lipid interface in the myelinating glia. The larger IMPs on both P and E faces number about 80/mum2 at 16 degrees C in contrast to the IMP density of 400/mum2 in the other glial membranes. After acclimation to 5, 16 and 26 degrees C, the loose myelin glial membranes show variations in the density of their larger IMP population; in animals acclimated over 3 or more weeks to 5 degrees C, the number of these IMPs is significantly (P less than 0.001) less per unit area than in animals acclimated to 16 or 26 degrees C. The size of the particles at 5 degrees C is significantly (P less than 0.001) smaller than those at 16 or 26 degrees C. When animals are subjected to a sudden differential in ambient temperature, from 26 or 16 to 5 degrees C, or from 5 to 26 degrees C, and their giant axons with encompassing glia are fixed and frozen 30 min after this temperature change, the IMP population of the glial membranes remaining does not appear to alter. The differences in the IMP population of the myelinating glial membranes at different temperatures may reflect the extent to which they insulate and/or influence the velocity of impulse propagation.     

Strategies for shotgun identification of integral membrane proteins by tandem mass spectrometry

Integral membrane proteins (IMPs) are difficult to identify, mainly for two reasons: the hydrophobicity of IMPs and their low abundance. Sample preparation is a key component in the large-scale identification of IMPs. In this review, we survey strategies for shotgun identification of IMPs by MS/MS. We will discuss enrichment, solubilization, separation, and digestion of IMPs, and data analysis for membrane proteomics.     

Improvement of hydrophobic integral membrane protein identification by mild performic acid oxidation-assisted digestion

Integral membrane proteins (IMPs) are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of IMPs make them difficult to analyze. In proteomic analyses, hydrophobic peptides including transmembrane domains are often underrepresented, and this reduces the sequence coverage and reliability of the identified IMPs. Here we report a new strategy, mild performic acid oxidation treatment (mPAOT), for improvement of IMP identification. In the mPAOT strategy, the hydrophobicity of IMPs is significantly decreased by oxidizing their methionine and cysteine residues with performic acid, thereby improving the solubility and enzymolysis of these proteins. The application of the mPAOT strategy to the analysis of IMPs from human nasopharyngeal carcinoma CNE1 cell line demonstrated that many IMPs, including those with high hydrophobicity, could be reliably identified.     

Freeze-fracture studies on unmyelinated axolemma of rat cervical sympathetic trunk: correlation with saxitoxin binding

The density and diameter distributions of intramembranous particles (IMPs) within unmyelinated axolemma from rat cervical sympathetic trunk were examined with freeze-fracture electron microscopy. The axolemma displays a highly asymmetrical partitioning of IMPs with ca. 1200 IMPs microns-2 on P-faces and ca. 100 IMPs microns-2 on E-faces. Particle sizes (diameters) are unimodally distributed on both fracture faces, with a range from 2.4 nm to 15.6 nm. Approximately 16% of the particles on P-faces and 28% of particles on E-faces are of a large (greater than 9.6 nm) diameter. On both fracture faces, the IMPs appear to be randomly distributed; no aggregations of particles were observed. The results indicate that there are ca. 230 large IMPs microns-2 of unmyelinated axolemma from rat cervical sympathetic trunk. The density of these IMPs is similar to the density of saxitoxin binding sites on unmyelinated axolemma from rat cervical sympathetic trunk (Pellegrino et al. 1984 (Brain Res. 305, 357-360)), which suggests that many of the large diameter particles may be the morphological correlate of voltage-sensitive Na+ channels.     

Detection of plasma membrane cholesterol by filipin during microvillogenesis and ciliogenesis in quail oviduct

Using filipin as a probe for the presence of membrane cholesterol, the evolution of cholesterol distribution in the apical plasma membrane was studied during estrogen-induced ciliogenesis in quail oviduct and compared with the distribution of intramembrane particles (IMPs). Ciliary growth is preceded by the first step of microvillus differentiation. Microvilli emerge in membrane domains rich in IMPs and devoid of filipin-cholesterol (f-c) complexes. However growing microvillus membrane shows f-c complexes. During ciliary growth, microvilli lengthen from 0.5 to 2 microns, indicating that the microvillar membrane is not a membrane reservoir for ciliogenesis. During ciliary growth, the characteristic ciliary necklace IMP rows appear progressively at the base of cilia. The first IMP row is organized in a membrane circlet lacking of f-c complexes, whereas the new shaft membrane in the middle of the circlet exhibits numerous complexes. These two different domains of the cilia keep their specificity during ciliary growth. Only the ciliary tip shows fewer complexes than the shaft membrane. The apical membrane of differentiated ciliated cells is thus composed of various domains, the ciliary shaft full of f-c complexes and poor in IMPs, the ciliary necklace is devoid of f-c complexes and rich in IMPs, the microvilli membrane is rich in both IMPs and f-c complexes, and the interciliary membrane is poor in both f-c complexes and IMPs, whereas the undifferentiated cells exhibit an apical membrane in which f-c complexes and IMPs are distributed homogeneously.     

Disruption and reformation of the acetylcholine receptor clusters of cultured rat myotubes occur in two distinct stages

We have examined the redistribution of acetylcholine receptor (AChR) intramembrane particles (IMPs) when AChR clusters of cultured rat myotubes are experimentally disrupted and allowed to reform. In control myotubes, the AChR IMPs are evenly distributed within the AChR domains of cluster membrane. Shortly after addition of azide to disrupt clusters, IMPs become unevenly scattered, with some microaggregation. After longer treatment, IMPs are depleted from AChR domains with no further change in IMP distribution. Contact domains of clusters are relatively poor in IMPs both before and after cluster dispersal. Upon visualization with fluorescent alpha-bungarotoxin, some AChR in azide-treated samples appear as small, bright spots. These spots do not correspond to microaggregates seen in freeze-fracture replicas, and probably represent receptors that have been internalized. The internalization rate is insufficient to account completely for the loss of IMPs from clusters, however. During reformation of AChR clusters upon removal of azide, IMP concentration in receptor domains increases. At early stages of reformation, IMPs appear in small groups containing compact microaggregates. At later times, AChR domains enlarge and IMPs within them assume the evenly spaced distribution characteristic of control clusters. These observations suggest that the disruption of clusters is accompanied by mobilization of AChR from a fixed array, allowing AChR IMPs to diffuse away from the clusters, to form microaggregates, and to become internalized. Cluster reformation appears to be the reverse of this process. Our results are thus consistent with a two-step model for AChR clustering, in which the concentration of IMPs into a small membrane region precedes their rearrangement into evenly spaced sites.     

Distribution of intramembranous particles and filipin-sterol complexes in mouse sperm membranes: polyene antibiotic filipin treatment

The distribution of intramembranous particles (IMPs) and membrane filipin-sterol complexes (FSC) was examined ultrastructurally in mouse spermatozoa from the male reproductive tract and ejaculates. IMPs were qualitatively analyzed on freeze-fracture replicas of glutaraldehyde-fixed tissue, while membrane FSC were quantitatively analyzed on replicas of filipin-treated cells. The distribution pattern of IMPs of mouse spermatozoa was fundamentally similar to that of other mammalian spermatozoa. 1) In the head, the plasma membrane had a heterogeneous population density, e.g., few IMPs on the acrosomal region, particularly few on the marginal segment, and somewhat regularly arranged IMPs on the postacrosomal region. The acrosomal membrane had many IMPs in hexagonal arrays. The nuclear membrane had many IMPs on the P-face, few IMPs on the variegated E-face, and an intense population density on the P-face of the basal plate. 2) In the neck, the plasma membrane had many IMPs with square arrangements of small IMPs in some areas on the P-face; the redundant nuclear membrane had a few IMPs on both P- and E-faces. 3) In the tail, the plasma membrane had diagonal rows of IMPs in some areas amongst larger IMPs on the middle piece, while it had "zippers" composed of IMPs running parallel to the axis on the principal piece. The distribution of sperm membrane FSC may be summarized as follows: 1) In the head, the acrosomal plasma membrane, which was heavily labeled with filipin, had much more FSC in the equatorial segment than in the marginal segment throughout the study. The postacrosomal plasma membrane generally had no FSC, but some sperm in ejaculates were slightly positive to filipin. The acrosomal membranes (both outer and inner) had no FSC. The nuclear membrane in the main part of the head had less FSC in vas deferens and ejaculated sperm than in the epididymal sperm. The nuclear membrane on the basal plate had no FSC. 2) In the neck, the plasma membrane had little FSC. The redundant nuclear envelope had scattered FSC with a higher incidence in the epididymal sperm than in those from the vas deferens and ejaculates. The membrane scroll, which was elongated from the extreme caudal end of the redundant nuclear envelope, had abundant FSC in the vas deferens and ejaculated sperm. 3) The tail plasma membrane (both middle and principal piece), which was weakly labeled with filipin, had less FSC in sperm from the vas deferens and ejaculates than in those from the epididymis. The limiting membrane covering the mitochondria had no FSC.     

Modeling of the structural features of integral-membrane proteins reverse-environment prediction of integral membrane protein structure (REPIMPS)

The Profiles-3D application, an inverse-folding methodology appropriate for water-soluble proteins, has been modified to allow the determination of structural properties of integral-membrane proteins (IMPs) and for testing the validity of solved and model structures of IMPs. The modification, known as reverse-environment prediction of integral membrane protein structure (REPIMPS), takes into account the fact that exposed areas of side chains for many residues in IMPs are in contact with lipid and not the aqueous phase. This (1) allows lipid-exposed residues to be classified into the correct physicochemical environment class, (2) significantly improves compatibility scores for IMPs whose structures have been solved, and (3) reduces the possibility of rejecting a three-dimensional structure for an IMP because the presence of lipid was not included. Validation tests of REPIMPS showed that it (1) can locate the transmembrane domain of IMPs with single transmembrane helices more frequently than a range of other methodologies, (2) can rotationally orient transmembrane helices with respect to the lipid environment and surrounding helices in IMPs with multiple transmembrane helices, and (3) has the potential to accurately locate transmembrane domains in IMPs with multiple transmembrane helices. We conclude that correcting for the presence of the lipid environment surrounding the transmembrane segments of IMPs is an essential step for reasonable modeling and verification of the three-dimensional structures of these proteins.     

Effect of Temporal Expression of Integral Membrane Proteins by Baculovirus Expression Vector System

Integral membrane proteins (IMPs) are popular target for drugs, but their resolved structures have been overlooked when compared with cytosolic proteins. The main reason is that IMPs usually need intensive post-translational modifications and they are bound to membranes, which increase the complexity of purifying or crystalizing them. Although different expression systems are used to express IMPs, baculovirus is considered one of the most successful expression systems for those proteins. Despite that, there are always unknown discrepancies in the level of IMPs expression in the baculovirus expression system. Retrospective studies have shown that expression of an immunoglobulin (anti-Chymase mouse monoclonal IgG1) driven by vp39 promoter was more efficient compared to its expression under polyhedrin (polh) promoter; however, this conclusion was not tested on different IMPs to generalize such a conclusion. In this study, the expression of eight different IMPs has been compared under vp39 and polh promoters of Autographa californica nucleopolyhedrovirus. Although different IMPs have shown different patterns of expression, the expression driven by vp39 promoter was found to be generally more efficient than the polh promoter.     

Quality control of integral membrane proteins

Integral membrane proteins (IMPs) are essential components of the plasma and organellar membranes of the eukaryotic cell. Non-native IMPs, which can arise as a result of mutations, errors during biosynthesis or cellular stress, can disrupt these membranes and potentially lead to cell death. To protect against this outcome, the cell possesses quality control (QC) systems that detect and dispose of non-native IMPs from cellular membranes. Recent studies suggest that recognition of non-native IMPs by the QC machinery is correlated with the thermodynamic stability of these proteins. Consistent with this, small molecules known as chemical and pharmacological chaperones have been identified that stabilize non-native IMPs and enable them to evade QC. These findings have far-reaching implications for treating human diseases caused by defective IMPs.     

A study of metabolic cooperation with established myoblast cell lines.

The present study was undertaken to test the action of ConA on the distribution of intramembranous particles (IMPs) and on the reassembly of junctional contacts in isolated and reaggregated embryonic neuronal and glial cells. The lectin ConA causes all embryonic cells to aggregate in unorganized cell patterns. ConA does not alter the distribution of IMPs but it inhibits the formation of the zonula occludens (ZO) by preventing the alignment and fusion of IMPs or by inducing them to become arranged in bizarre arrays. The possible relationship between ConA receptor sites and the IMPs is discussed. From a morphological viewpoint the aggregation of embryonic cells influenced by lectin is distinctly different from the normal processes of cell adhesion, cell sorting and establishment of intercellular contacts.     

Flexibility in targeting and insertion during bacterial membrane protein biogenesis

The biogenesis of Escherichia coli inner membrane proteins (IMPs) is assisted by targeting and insertion factors such as the signal recognition particle (SRP), the Sec-translocon and YidC with translocation of (large) periplasmic domains energized by SecA and the proton motive force (pmf). The use of these factors and forces is probably primarily determined by specific structural features of an IMP. To analyze these features we have engineered a set of model IMPs based on endogenous E. coli IMPs known to follow distinct targeting and insertion pathways. The modified model IMPs were analyzed for altered routing using an in vivo protease mapping approach. The data suggest a facultative use of different combinations of factors.     

Freeze-fracture analysis of phloem structure in plant tissue cultures. II. The sieve element plasma membrane

Sieve element plasma membranes reveal a unique distribution of intramembrane particles (IMPs) in tissue cultures fixed and cyroprotected prior to freeze-fracturing. Sieve element IMPs are smaller than those found in the plasma membranes of callus parenchyma cells from these same cultures. The PF/EF ratio of plasma membrane IMPs is 9.6 for parenchyma cells and 1.21 for sieve elements. The increased binding of IMPs to the sieve element E face may be related to the role of membrane proteins in the loading of sucrose and other molecules by these cells. The enlargement of the cell wall at the site of sieve area pores creates complementary ridges and depressions in the E and P fracture faces of sieve element plasma membranes. No alteration of IMP density is seen at the sieve area pore site.     

Solubilization of native integral membrane proteins in aqueous buffer by noncovalent chelation with monomethoxy poly(ethylene glycol) (mPEG) polymers

Highly hydrophobic integral membrane proteins (IMPs)are typically purified in excess detergent media, often resulting in rapid inactivation and denaturation of the protein. One promising approach to solve this problem is to couple hydrophilic polymers, such as monomethoxypolyethylene glycol (mPEG) to IMPs under mild conditions in place of detergents. However, the broad application of this approach is hampered by poor reaction efficiencies, low tolerance of detergent stabilized membrane proteins to reaction conditions, and a lack of proper site-specific reversible approaches. Here, we have developed a straightforward, efficient, and mild approach to site-specific noncovalent binding of long-chain polymers to recombinant IMPs. This method uses the hexa-histidine tag (His-Tag) often used for purification of recombinant proteins as an attachment site for mPEGs. Solubility studies performed using five different IMPs confirmed that all tested mPEG-bound IMPs were completely soluble and stable in detergent free aqueous buffer compared to their precipitated native proteins under the identical circumstances. Activity assays and circular dichroism (CD) spectroscopy confirmed the structural integrity of modified IMPs.     

Sperm morphology and distribution of intramembranous particles in the sperm heads of selected freshwater teleosts

The morphology of spermatozoa and the distribution of intramembranous particles (IMPs) in sperm-head membranes in teleostean fish were examined ultrastructurally to clarify the presence of characteristic arrays (parallelogram or hexagon in packing) of IMPs. The following four species of fish were used: goldfish (Carassius auratus), loach (Misgurnus anguillicaudatus), flat bitterling (Acheilognatus rhombeus), sweetfish (Plecoglossus altivelis). It was demonstrated that spermatozoa of all these fish were devoid of an acrosomal structure in the anterior portion of the head. Spermatozoa had round heads in goldfish, loach and flat bitterling. Two centrioles (proximal centriole and basal body) were present and located adjacent to each other in all fish. The characteristic arrays of IMPs were found in spermatozoa of goldfish and flat bitterling. IMPs were more numerous on the P-face than on the E-face in all species. The present work showed that the characteristic arrays of IMPs were not common structures in spermatozoa of teleostean fish.     

The Structure of Multiple Polypeptide Domains Determines the Signal Recognition Particle Targeting Requirement of Escherichia coli Inner Membrane Proteins

The signal recognition particle (SRP) targeting pathway is required for the efficient insertion of many polytopic inner membrane proteins (IMPs) into the Escherichia coli inner membrane, but in the absence of SRP protein export proceeds normally. To define the properties of IMPs that impose SRP dependence, we analyzed the targeting requirements of bitopic IMPs that are structurally intermediate between exported proteins and polytopic IMPs. We found that disruption of the SRP pathway inhibited the insertion of only a subset of bitopic IMPs. Studies on a model bitopic AcrB-alkaline phosphatase fusion protein (AcrB 265-AP) showed that the SRP requirement for efficient insertion correlated with the presence of a large periplasmic domain (P1). As previously reported, perturbation of the SRP pathway also affected the insertion of a polytopic AcrB-AP fusion. Even exhaustive SRP depletion, however, failed to block the insertion of any AcrB derivative by more than 50%. Taken together, these data suggest that many proteins that are normally targeted by SRP can utilize alternative targeting pathways and that the structure of both hydrophilic and membrane-spanning domains determines the degree to which the biogenesis of a protein is SRP dependent.     

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.     

Colloidal iron hydroxide staining of surface carbohydrates after glycerol treatment of uterine epithelial cells

The structural relationships between intramembraneous particles (IMPs) and surface carbohydrates has been studied in uterine epithelial cells with a colloidal iron hydroxide (CIH) technique. To aggregate IMPs, glycerol treatment of unfixed cells was used and this treatment also caused some patching of CIH deposits on the cell surface. We conclude that some of the CIH receptors may be the surface expression of the IMPs.     

Structural differentiation of membranes involved in the secretion of polysaccharide slime by root cap cells of cress (Lepidium sativum L.)

The peripheral secretion tissue of the root cap of Lepidium sativum L. was investigated by electronmicroscopy and freeze-fracturing in order to study structural changes of membranes involved in the secretion process of polysaccharide slime. Exocytosis of slime-transporting vesicles occurs chiefly in the distal region of the anticlinal cell walls. The protoplasmic fracture face (PF) of the plasmalemma of this region is characterized by a high number of homogenously distributed intramembranous particles (IMPs) interrupted by areas nearly free of IMPs. Near such areas slime-transporting vesicles are found to be underlying the plasma membrane. It can be concluded that areas poor in particles are prospective sites for membrane fusion. During the formation of slime-transporting vesicles, the number of IMPs undergoes a striking change in the PF of dictyosome membranes and their derivatives. It is high in dictyosome cisternae and remarkably lower in the budding region at the periphery of the cisternae. Slime-transporting vesicles are as poor in IMPs as the areas of the plasmalemma. Microvesicles rich in IMPs are observed in the surroundings of dictyosomes. The results indicate that in the plasmalemma and in membranes of the Golgi apparatus special classes of proteins — recognizable as IMPs — are displaced laterally into adjacent membrane regions. Since the exoplasmic fracture face (EF) of these membranes is principally poor in particles, it can be concluded that membrane fusion occurs in areas characterized by a high quantity of lipid molecules. It is obvious that the Golgi apparatus regulates the molecular composition of the plasma membrane by selection of specific membrane components. The drastic membrane transformation during the formation of slime-transporting vesicles in the Golgi apparatus causes the enrichment of dictyosome membranes by IMPs, whereas the plasma membrane probably is enriched by lipids. The structural differentiations in both the plasma membrane and in Golgi membranes are discussed in relation to membrane transformation, membrane flow, membrane fusion, and recycling of membrane constituents.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face - IMP intramembranous particle