On the heterogeneous glycosylation of the membranes of the trans Golgi network in rabbit luteal cells

Summary In rabbit luteal cells the transmost element (G₂) of the Golgi apparatus bears cytochemical resemblances to the limiting membrane of lysosomes and its was suggested that lysosomal membranes may originate from the above element. But in the normal Golgi apparatus it cannot be made out whether the considered molecules are indeed membrane bound. Perfusing the rabbit ovary with buffer containing monensin or ammonium chloride allowed to vesiculate the trans Golgi network (G₂–G₁) selectively. Controls showed a well-preserved ultrastructure.Parts of the limiting membrane of the vacuoles derived from the transmost reticulum (G₂) were spiny coated and carried an osmiophilic inner layer. They also showed a heavy precipitate for acid phosphatase (AcPase) and were strongly stained with phosphotungstic acid (PTA) at low pH. By neutralizing the acidic groups, involved in the PTA-staining, it was possible to show that the same membranes were more heavily glycosylated. The MvB's and the limiting membrane of lysosomes showed the same staining characteristics. The other membrane domains revealed a gradient in PTA staining and in AcPase activity.It is concluded that the trans Golgi network (G₂–G₁) is an acidic compartment. The presence of differentially glycosylated membranes reveals a sorting mechanism for membranous components. The highly glycosylated membrane streches seem to be involved in endocytosis and in the formation of lysosomal membranes.     

High resolution analysis of the formation of cellulose synthesizing complexes inVaucheria hamata

Summary Ultrastructure and assembly of cellulose terminal synthesizing complexes (terminal complexes, TCs) in the algaVaucheria hamata (Waltz) were investigated by high resolution analytical techniques for freeze-fracture replication.Vaucheria TCs consist of many diagonal rows of subunits located on the inner leaflet of the plasma membrane. Each row contains about 10–18 subunits. The subunits themselves are rectangular, approx. 7×3.5 nm, and each has a single elliptical hole which may be the site of a single glucan chain polymerization. The subunits are connected with extremely small filaments (0.3–0.5 nm). Connections are more extensive in a direction parallel to the subunit rows and less extensive perpendicular to them. Nascent TC subunits are found to be packed within globules (15–20 nm in diameter) which are larger than typical intramembranous particles (IMPS are 10–11 nm in diameter) distributed in the plasma membrane. The subunits in the globule, which may be a zymogenic precursor of the TC, are generally exhibited in the form of doublets. Approximately 6 doublets are connected to a center core with small filaments. The globules are inserted into the plasma membrane together with IMPS by the fusion of cytoplasmic (Golgi derived) vesicles. Two or three globules attach to each other, unfold, and expand to form the first subunit rows of the TC on the inner leaflet of the plasma membrane. More globules attach to the structure and unfold until the nascent TC consists of a few rows of subunits. These rows are arranged almost parallel to each other. Two formation centers of subunits appear at both ends of an elongating TC. New subunits carried by the globules are added at each of these centers to create new rows until the elongating TC structure is completed. On the basis of this study, a model of TC assembly and early initiation of microfibril formation inVaucheria is proposed.Abbreviations IMPS intramembranous particles - MF microfibril - TC terminal complex     

Further investigations on structural and catalytic properties of O2 evolving preparations from tobacco and two chlorophyll deficient tobacco mutants

Previous investigations (Specht, S., Pistorius, E.K. and Schmid, G.H.: Photosynthesis Res. 13, 47–56, 1987) of Photosystem II membranes from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contain normally stacked thylakoid membranes and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked or essentially unstacked thylakoids with occasional membrane doublings, have been extended by using monospecific antisera raised against the three extrinsic polypeptides of 33,21 and 16 kDa. The results show that all three peptides are synthesized as well in wild type tobacco as in the two mutants to about the same level and that they are present in thylakoid membranes of all three plants. However, in the mutants the 16 and 21 kDa peptides (but not the 33 kDa peptide) are easily lost during solubilization of Photosystem II membranes. In the absence of the 16 and 21 kDa peptide Photosystem II membranes from the mutants have a higher O₂ evolving activity without addition of CaCl₂ than the wild type Photosystem II membranes. On the other hand, after removal of the 33 kDa peptide no significant differences in the binding of Mn could be detected among the three plants. The results also show that reaction center complexes from wild type tobacco and the mutant Su/su are almost identical to the Triton-solubilized Photosystem II membranes from the mutant Su/su var. Aurea.Abbreviations PS photosystem - chl chlorophyll - LHCP light harvesting chlorophyll a/b protein complex - WT wild type - OEE1, OEE2 and OEE3 oxygen evolution enhancing complex of 29–36 kDa, 21–24 kDa and 16–18 kDa, respectively     

The morphology of multivesicular bodies in soybean protoplasts and their role in endocytosis

Summary Multivesicular bodies (MVBs) in soybean protoplasts are distinct organelles (generally 250–500 nm in diameter) consisting of a limiting membrane and a number of smaller internal vesicles (generally 40–100 nm in diameter). MVBs of soybean protoplasts are morphologically similar to MVBs of animal cell systems. They can have tubular protuberances which extend from the main body of the organelle and a lamellar plaque on the cytoplasmic surface of their limiting membrane. In addition, the internal vesicles can be labeled by a zinc iodide-osmium tetroxide postfixation and may form via invagination of the limiting membrane.The MVBs of soybean protoplasts are a major compartment in the endocytotic pathway. They accumulate, over time, exogenously applied cationized ferritin and may deliver it to the major lysosomal or lytic compartment of the plant cell, namely, the vacuoles.     

Insolubility and redistribution of GPI-anchored proteins at the cell surface after detergent treatment.

A diverse set of cell surface eukaryotic proteins including receptors, enzymes, and adhesion molecules have a glycosylphosphoinositol-lipid (GPI) modification at the carboxy-terminal end that serves as their sole means of membrane anchoring. These GPI-anchored proteins are poorly solubilized in nonionic detergent such as Triton X-100. In addition these detergent-insoluble complexes from plasma membranes are significantly enriched in several cytoplasmic proteins including nonreceptor-type tyrosine kinases and caveolin/VIP-21, a component of the striated coat of caveolae. These observations have suggested that the detergent-insoluble complexes represent purified caveolar membrane preparations. However, we have recently shown by immunofluorescence and electron microscopy that GPI-anchored proteins are diffusely distributed at the cell surface but may be enriched in caveolae only after cross-linking. Although caveolae occupy only a small fraction of the cell surface (< 4%), almost all of the GPI-anchored protein at the cell surface becomes incorporated into detergent-insoluble low-density complexes. In this paper we show that upon detergent treatment the GPI-anchored proteins are redistributed into a significantly more clustered distribution in the remaining membranous structures. These results show that GPI-anchored proteins are intrinsically detergent-insoluble in the milieu of the plasma membrane, and their co-purification with caveolin is not reflective of their native distribution. These results also indicate that the association of caveolae, GPI-anchored proteins, and signalling proteins must be critically re-examined.     

The high resolution ultrastructure of the periodicity and architecture of lipid-retained and extracted lung multilamellar body laminations

The three-dimensional aspect of rat and monkey lung multilamellar bodies was demonstrated in lipid retained thin sections. The glutaraldehyde and urea lipid retention embedment and an Epon 812 resin polar dehydrant procedure were utilized to retain lamellar lipids for precise morphological study. The unextracted multilamellar bodies were found to conform to a general, though complex, threedimensional structure. A model that demonstrated that structure was derived. Freezeetch and extracted material were shown to support the model. Mature multilamellar bodies were from 1.2–1.6 μ in diameter and were 1.0–1.6 μ high. Each body contained a matrix core that included from 2–25 vesicular bodies and was in contact with the limiting membrane at the matrix plate. Most bodies had from 25–70 lamellae attached for 360 ° to the projection plate. Microtubules were seen in communication with the matrix core. When sectioned in longitudinal section, lamellae projected from the base plate and coursed parallel to the limiting membrane of the top half of the body. Any cross-section produced circular lamellae without apparent attachment. Oblique sections sometimes produced both ‘stacked’ and ‘circular’ lamellae. Four postulates of multilamellar body formation were discussed in light of these findings.     

Structural differences between the resting eggs of Brachionus plicatilis and Brachionus rotundiformis (Rotifers, Brachionidae): an electron microscopic study

Surface topography and layering of resting eggs of Brachionus plicatilis and B. rotundiformis are described, based on scanning and transmission electron microscope studies. In B. plicatilis, the resting eggs are spherical with smooth wavy ridges on the surface, whereas these ridges are small and condensed in B. rotundiformis. The distribution of pores on the egg surface clearly distinguishes the two species.Ultrastructure of the egg membranes of both species varies greatly with regard to size, shape and sculpturing of each membrane. The alveolar and dense sublayers, which constitute the outer egg membrane (S1) are very thick (10–12 µm) in B. rotundiformis compared to B. plicatilis (4–5 µm). Thus, each species has a characteristic surface and membrane architecture. The functional roles of these membranes, during the resting phase of rotifers, are discussed.     

Postsynaptic membranes in the electric tissue of Narcine: III. Isolation and characterization

Postsynaptic membranes in homogenates of the electric tissue of Narcine were identified by labelling nicotinic acetylcholine receptors in the membranes with radioactive alpha-bungarotoxin. Various media and centrifugation conditions were examined in an attempt to obtain highly purified postsynaptic membranes. The main criterion for purification was approach towards the specific activity of the pure receptor protein, 9–10 nmol toxin-sites/mg protein. Isolation of tissue microsomes with Tris buffer, EDTA and the protease inhibitor phenylmethylsulfonylfluoride (PMSF), conditions which preserve the receptor molecules optimally, yielded about 50 % of the tissue toxin-sites, 5 % of the protein, 4 % of the ATPase and less than 2 % of the acetylcholinesterase (AChE). Further separation of vesiculated membranes in continuous density gradients of sucrose showed that the major contaminants of postsynaptic membrane vesicles were damaged mitochondria and tubular vesicles of dorsal electroplaque membranes rich in ATPase. Mitochondria were effectively removed from homogenates by ‘differential’ centrifugation, and ATPase-rich vesicles could be largely removed by causing their agglutination with calcium ions, or by controlled proteolysis in the absence of PMSF. Partially purified postsynaptic membranes were obtained having about 7 nmol toxin-sites/mg membrane protein. Further purification appears possible by affinity techniques.     

Effect of phosphatidylserine on the basal and GABA-activated Cl− permeation across single nerve membranes from rabbit Deiters' neurons

The permeation of labeled Cl^– ions across single plasma membranes from Deiter's neurons has been studied in the presence of various concentrations of phosphatidylserine (PS) on their extracellular side. PS reduces significantly basal Cl^– permeation only at 10^–5 M on the membrane exterior. No effect was found at other concentrations. GABA activable³⁶Cl^– permeation is heavily reduced and almost abolished at 10^–11–10^–5 M phosphatidylserine. This exogenous phosphatidylserine effect is difficult to interpret in relation to the function of the endogenous phospholipid. However, it may be involved in the epileptogenic effect in vivo of exogenous phosphatidylserine administration to rats.     

Observations on the ultrastructure of nerve cells in the brain of the planarian,Dugesia gonocephala

Summary The submicroscopic structure of the nerve cells in the planarian brain was studied. Close similarities with neurons of other invertebrates were noted. In the cytoplasm of the planarian nerve cells there are at least three types of vesicular inclusions: 1) Clear vesicles (200–800 Å in epon embedded tissue) similar in morphological appearance to classical synaptic vesicles. These have generally some content of extremely low density but occasionally a dense core. 2) Dense vesicles (400–1,200 Å in epon embedded tissue) containing highly osmiophilic granules. Between the limiting membrane of the vesicle and the granule there is always a clear rim of variable width. These vesicles closely resemble synaptic vesicles described in vertebrate adrenergic endings. 3) Neurosecretory vesicles (600–1,300 Å in Vestopal embedded tissue) similar to elementary granules observed in neurosecretory systems in vertebrates and invertebrates. All three vesicle types have the same mode of origin from the Golgi membranes. All are present in the nerve cell processes of the neuropil as well as in the perikarya. Any given perikaryon or axon contains only one of the three vesicle types. All of these vesicles are considered to be discharged into the axons from their site of origin within the perikaryon.     

AP1/2β-mediated exocytosis of tapetum-specific transporters is required for pollen development in Arabidopsis thaliana

AP-1 and AP-2 adaptor protein (AP) complexes mediate clathrin-dependent trafficking at the trans-Golgi network (TGN) and the plasma membrane, respectively. Whereas AP-1 is required for trafficking to plasma membrane and vacuoles, AP-2 mediates endocytosis. These AP complexes consist of four subunits (adaptins): two large subunits (β1 and γ for AP-1 and β2 and α for AP-2), a medium subunit μ, and a small subunit σ. In general, adaptins are unique to each AP complex, with the exception of β subunits that are shared by AP-1 and AP-2 in some invertebrates. Here, we show that the two putative Arabidopsis thaliana AP1/2β adaptins co-assemble with both AP-1 and AP-2 subunits and regulate exocytosis and endocytosis in root cells, consistent with their dual localization at the TGN and plasma membrane. Deletion of both β adaptins is lethal in plants. We identified a critical role of β adaptins in pollen wall formation and reproduction, involving the regulation of membrane trafficking in the tapetum and pollen germination. In tapetal cells, β adaptins localize almost exclusively to the TGN and mediate exocytosis of the plasma membrane transporters such as ATP-binding cassette (ABC)G9 and ABCG16. This study highlights the essential role of AP1/2β adaptins in plants and their specialized roles in specific cell types.

Arabidopsis AP1/2β adaptins are shared by the AP-1 and AP-2 complexes and required for pollen development by mediating the trafficking of ABCG transporters in tapetal cells.

IN A NUTSHELL Background: Adaptor protein (AP) complexes are critical for the recruitment of cargo proteins during vesicle trafficking. AP-1 and AP-2 mediate clathrin-dependent trafficking at the trans-Golgi network (TGN) and the plasma membrane, respectively. Whereas AP-1 regulates trafficking to the plasma membrane (exocytosis) and vacuole, AP-2 mediates endocytosis. These AP complexes consist of four subunits (adaptins): two large subunits (β1 and γ for AP-1, β2, and α for AP-2), a medium subunit μ, and a small subunit σ. The general roles of some AP-1 and AP-2 adaptins in vegetative and reproductive development have been characterized in plants. However, the function of the large β subunits and whether they are shared by the two AP-1 and AP-2 complexes in plants is currently unknown. Questions: Are β adaptins shared by AP-1 and AP-2 complexes in Arabidopsis thaliana? What function do they play in plant development? Findings: We found that the two putative Arabidopsis AP1/2β adaptins co-assemble with both AP-1 and AP-2 subunits and regulate exocytosis and endocytosis in root cells, consistent with their dual localization to the TGN and the plasma membrane. However, in tapetal cells of developing anthers, AP1/2β adaptins localize almost exclusively to TGN. Mutations in AP1/2β adaptins result in collapsed pollen grains with abnormal walls and reduced pollen germination due to impaired exocytosis of the tapetum-specific plasma membrane transporters ABCG9 and ABCG16, highlighting the essential role of AP1/2β adaptins in plants and their specialized roles in specific cell types. Next steps: We will investigate the mechanism by which AP1/2β adaptins recognize cargo proteins and their role in female gametophyte and embryonic development.     

Fine structural studies on the reabsorption of colloid and fusion of colloid droplets in thyroid glands of TSH-treated mice

Summary The mechanism of the luminal colloid reabsorption and the fate of reabsorbed colloid droplets were studied ultracytochemically in epithelial cells of thyroid cells of TSH-treated mice. The luminal colloid is reabsorbed by micropinocytosis as well as phagocytosis into the follicle epithelial cell. Almost all the pinocytotic pits and vesicles are coated and often closely associated with actin filaments demonstrated by use of heavy meromyosin (HMM). This suggests the involvement of the actin filament system in making and transporting coated vesicles for micropinocytosis of the luminal colloid. Freeze-fracture images show aggregates of intramembrane particles on the P-face of the small depressions corresponding to the initial site for coated pits.The reabsorbed colloid droplets fuse with one another and with lysosomes. At the initial stage of this fusion, the limiting membranes of adjoining droplets fuse in a limited area to become pentalaminar, and then become trilaminar. Eventually, the membranes at the fusion point disappear, and the contents of both droplets become continuous. Freeze-fracture images reveal the disappearance of the intramembrane particles at the initial site where the fusion occurs.Examination of thin-sectioned tissue treated by rapid-freeze substitution fixation, shows clearly delineated cell organelles, and the rounded mitochondria have a characteristically high electron-dense matrix. Just beneath the limiting membrane of each colloid droplet, there always exists a low electron-dense layer about 10 nm thickness. The lysosomes are sometimes seen wrapped around the colloid droplet.This study was supported by grants (No. 56370002, No. 00544016) from the Japan Ministry of Education     

Cytochemical studies on the localization and functional properties of calcium in anterior pituitary cells

The localization of calcium and its functional properties in anterior pituitary cells were studied using a potassium pyroantimonate technique. In all kinds of secretory cells, the precipitates of the calcium-pyroantimonate complex were distributed on the limiting membrane of the secretory granule. They were present also in the cytoplasmic matrix, the mitochondrial matrix, small smooth vesicles, coated vesicles, and in the nuclear euchromatin area. The precipitates were usually seen at the contact region between the limiting membranes of two adjacent secretory granules, or between the granule limiting membrane and the plasma membrane. When the tissues were incubated in the medium containing A23187 (10 microM) for 5 min, the deposits on the granule limiting membrane were increased in number and those on the mitochondrial matrix were decreased; the reaction products almost disappeared on the limiting membranes of the secretory granules after membrane fusion following single or multigranular exocytosis induced by A23187-treatment. In addition, small vesicles in the capillary endothelium contained reaction precipitates. Based on these results we propose a hypothetical model for the relationship between the localization of calcium and secretory activity.     

Fine structure of the autonomic nerves of the rabbit myometrium

Summary The fine structure of the preterminal nerve fibers of the rabbit myometrial smooth muscle was studied using potassium permanganate fixation or glutaraldehyde fixation with postosmification. The preterminal fibers were mostly formed by 2–10 axons enveloped by Schwann cells. Two kinds of axons and axon terminals were found. (1) Adrenergic axons, which contained many small, granular vesicles (diameter 300–600 Å) and large granular vesicles (diameter 700–1200 Å) which represented ca. 2% of the total count of the vesicles. (2) Nonadrenergic axons, which contained small agranular vesicles (diameter 300–600 Å) and large granular vesicles (diameter 700–1200 Å). Both types of axons formed preterminal varicosities along their course. The real terminal varicosities, representing the anatomical end of the axons, were usually larger than the preterminal ones and showed close contact to the plasma membranes of the smooth muscle cells. Both adrenergic and nonadrenergic terminals were found close to the smooth muscle cells, but a gap of at least 2000 Å was always present between the two cell membranes. The axons and preterminal varicosities of both types of nerves were in intimate contact with each other within the preterminal nerve fiber. Axo-axonal interactions between the two types of axons are possible in the rabbit myometrium. The relative proportion of the nonadrenergic axons from the total was about one fourth.     

1-Anilino-8-naphthalenesulfonate: A fluorescent probe of ion and ionophore transport kinetics and trans-membrane asymmetry

Summary The kinetics of the transport of the 1-anilino-8-naphthalenesulfonate (ANS^–, an anionic fluorescent probe of the membrane surface) across phospholipid vesicle membranes have been studied using a stopped-flow rapid kinetic technique. The method has been used to gain detailed information about the mechanism of transport of this probe and to study ionophore-mediated cation transport across the membrane. The technique has also been exploited to study differences between the inside and outside surfaces of vesicles containing phosphatidyl choline (PC).The following is a summary of the major conclusions of this study. (a) Binding of ANS^– on the outside surface occurs within times shorter than 100 sec while permeation occurs in the time range 5–100 sec. (b) Net transport of ANS^– occurs with cotransport of alkali cations. (c) The transport rate is maximal in the region of the crystalline to liquidcrystalline phase transition, and the increase correlates with changes in the degree of aggregation of the vesicles. (d) Incorporation of phosphatidic acid (PA), phosphatidyl ethanolamine (PE) or cholesterol into PC membranes decreases the rate of ANS^– transport. (e) Neutral ionophores (I) of the valinomycin type increase ANS^– permeability in the presence of alkali cations (M ⁺) by a mechanism involving the transport of a ternaryI–M ⁺-ANS^– complex. The equilibrium constants for formation of these complexes and their rate constants for their permeation are presented. The maximal turnover number for ANS^– transport by valinomycin in dimyristoyl PC vesicles at 35°C was 46 per sec. (f) The partitioning of the ionophore between the aqueous and membrane phases and the rate of transfer of an ionophore from one membrane have been determined in kinetic experiments. (g) A method is described for the detection ofI–M ⁺ complexes on the membrane surface by their enhancement effects on ANS^– fluorescence at temperature below the phase transition temperature on monolayer vesicles. The apparent stability constants for severalI–M ⁺ complexes are given. (h) Analysis of the effect of ionic strength on the ANS^– binding to the inside outside surfaces indicates that the electrostatic surface potential (at fixed ionic strength and surface change) is larger for the inside surface than for the outside surface. (i) Analysis of the dependence of the maximal ANS^– binding for the inside and outside surfaces of vesicles made from PC and a variable mole fraction of PA, PE or cholesterol indicate that the latter three are located preferentially on the inside surface.     

Unique properties of the camel erythrocyte membrane: II. Organization of membrane proteins

Camel erythrocyte membranes are distinguished by some unique properties of stability and composition. Notable is their abundance in proteins (protein: lipid ratio of 3 : 1). Membrane proteins of camel erythrocytes were compared with those of human erythrocytes, which have been intensively investigated. Proteins were extracted with various aqueous media (EDTA, alkaline or high ionic strength) and with ionic and non-ionic detergents and were analyzed by gel electrophoresis. In membranes of camel erythrocytes, the peripheral proteins constitute, proportionally, a much smaller fraction of total proteins than in the human erythrocyte, while their distribution is identical per unit of surface area. The camel erythrocyte membrane is particularly rich in integral proteins and in intramembranous particles. The proteins in this membrane are more closely organized than in the human system, as revealed by crosslinking and freeze-etching studies. It is proposed that protein-protein interaction of integral proteins, presumably constituting an “integral skeleton”, is a dominant structural feature stabilizing the camel erythrocyte membrane.     

In Vitro Generation from the trans-Golgi Network of Coatomer-Coated Vesicles Containing Sialylated Vesicular Stomatitis Virus-G Protein

We describe an in vitro system in which post-Golgi vesicles containing metabolically labeled, sialylated, vesicular stomatitis virus (VSV) G protein molecules (VSV-G) are produced from the trans-Golgi network (TGN) of an isolated Golgi membrane fraction. This fraction is prepared from VSV-infected Madin–Darby canine kidney (MDCK) cells in which the ³⁵S-labeled viral envelope glycoprotein was allowed to accumulate in the trans-Golgi network during a prolonged incubation at 20°C. The vesicles produced in this system are separated from the remnant Golgi membranes by differential centrifugation or by velocity sedimentation in a sucrose gradient. Vesicle production, quantified as the percentage of labeled VSV-G released from the Golgi membranes, is optimal at 37°C and does not occur below 20°C. It requires GTP and the small GTP-binding protein Arf (ADP-ribosylation factor), as well as coat protein type I (COPI) coat components (coatomer) and vesicle scission factors—one of which corresponds to the phosphatidylinositol transfer protein (PITP). Formation of the vesicles does not require GTP hydrolysis which, however, is necessary for their uncoating. Thus, vesicles generated in the presence of the nonhydrolyzable GTP analogs, GTPγS or GMP–PNP, retain a coatomer coat visible in the electron microscope, sediment more rapidly in sucrose density gradients than those generated with ATP or GTP, and can be captured with anticoatomerantibodies. The process of coatomer-coated vesicle formation from the TGN can be dissected into two distinct sequential phases, corresponding to coat assembly/bud formation and vesicle scission. The first phase is completed when Golgi fractions are incubated with cytosolic proteins and nonhydrolyzable GTP analogs at 20°C. The scission phase, which leads to vesicle release, takes place when coated Golgi membranes, recovered after phase I, are incubated at higher temperatures in the presence of cytosolic proteins. The scission phase does not take place if protein kinase C inhibitors are added during the first phase, even though these inhibitors do not prevent membrane coating and bud formation. The phosphorylating activity of a protein kinase C, however, plays no role in vesicle formation, since this process does not require ATP.     

Substructure of inner mitochondrial membranes of myocardial cells as shown by cryo-ultramicrotomy

Summary The substructure of the inner mitochondrial membranes has been studied by cryo-ultramicrotomy under conditions during which denaturation of proteins by treatment with chemical solutes has been totally avoided. In such preparations, the inner membrane has a substructure consisting of globular subunits. These subunits have an average diameter of ca. 20Å–ca. 62Å and are fairly regularly spaced. Intracristal space is absent in the unstained, freeze-dried preparations, whereas a space of ca 40Å is seen in preparations lightly treated by OsO₄-vapour. It is concluded that the subunits of the inner mitochondrial membranes probably consist either of single protein molecules or of complexes of protein molecules.This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanities     

Redistribution and fate of colchicine-induced alkaline phosphatase in rat hepatocytes: possible formation of autophagosomes whose membrane is derived from excess plasma membrane

The redistribution and fate of colchicine-induced alkaline phosphatase (ALPase) in rat hepatocytes were investigated by electron microscopic enzyme cytochemistry and biochemistry. ALPase activity markedly increased in rat hepatocytes after colchicine treatment (2.0 mg/kg body weight, intraperitoneal injection). At 20–24 h after colchicine treatment, the liver showed the highest activity of ALPase. Thereafter, ALPase activity decreased and returned to normal levels at 48 h. In normal hepatocytes from control rats, ALPase activity was seen only on the bile canalicular membrane. However, at 20–24 h after colchicine treatment, colchicine-induced ALPase was redistributed in the sinusoidal and lateral (basolateral) membranes as well as in the bile canalicular membrane. At 30–36 h after colchicine treatment, ALPase activity on the basolateral membrane gradually decreased. In contrast, ALPase in the bile canalicular membrane increased along with the enlargement of bile canaliculi, suggesting that ALPase in the basolateral membrane had been transported to the bile canalicular membrane. Furthermore, ALPase-positive vesicles, cisternae and autophagosome-like structures were frequently seen in the cytoplasm. ALPase was also positive in some lysosomal membranes. ALPase in hepatocytes at 48 h after colchicine treatment returned to almost the same location as in control hepatocytes. Altogether, it is suggested that excessively induced ALPase is at least partially retrieved by invagination of the bile canalicular membrane and then transported to lysosomes for degradation. In addition, this study indicates that excess plasma membrane might be a possible origin of autophagosomal membrane.     

Calcium ions in the presence of exogenous phosphatidylserine interfere with GABA diffusion through the Deiters' neuron membrane

Diffusion of GABA through the plasma membrane of GABA-acceptive neurons might be a mechanism of importance for the termination of its synaptic action. In the present investigation we studied the effects of phosphatidylserine (PS) (10^–4–10^–3 M), Ca²⁺ 2 mM and PS+2 mM Ca²⁺ on such a process. The method involved the use of single microdissected Deiters' membranes which were put between two small microchambers in order to study the passage of GABA across the membrane. The results show that whereas PS and Ca²⁺ by themselves have no effect on such a process, PS+2 mM CaCl₂ give a significant, although slight, inhibition. The hypothesis that Calcium ion + PS effect is due to a disturbance of the interaction between GABA and endogenous PS molecules of the membrane is discussed.