Membrane structure of caveolae and isolated caveolin-rich vesicles

 Caveolae are specialized invaginated domains of the plasma membrane. Using freeze-fracture electron microscopy, the shape of caveolae and the distribution of intramembrane particles (integral membrane proteins) were analyzed. The caveolar membrane is highly curved and forms flask-like invaginations with a diameter of 80–120 nm with an open porus of 30–50 nm in diameter. The fracture faces of caveolar membranes are nearly free of intramembrane particles. Protein particles in a circular arrangement surrounding the caveolar opening were found on plasma membrane fracture faces. For isolation of caveolin-enriched membrane vesicles, the method of Triton X-100 solubilization, as well as a detergent-free isolation method, was used. The caveolin-rich vesicles had an average size of between 100 and 200 nm. No striated coat could be detected on the surface of isolated caveolin-rich vesicles. Areas of clustered intramembrane particles were found frequently on membrane fracture faces of caveolin-rich vesicles. The shape of these membrane protein clusters is often ring-like with a diameter of 30–50 nm. Membrane openings were found to be present in the caveolin-rich membrane vesicles, mostly localized in the areas of the clustered membrane proteins. Immunogold labeling of caveolin showed that the protein is a component within the membrane protein clusters and is not randomly distributed on the membrane of caveolin-rich vesicles. Accepted: 16 September 1998     

Regular arrays of intramembranous particles in the plasmalemma of guard cell and mesophyll cell protoplasts of Vicia faba

Freeze fractures of the plasmalemma membranes of guard-cell and mesophyll protoplasts of Vicia faba demonstrate that the inner monolayer of the plasmalemma is compartmentalized into areas with distinct, highly organized structures. Between areas of intramembranous particles dispersed randomly on a relatively smooth fracture face, membrane domains showing an extremely regular planar, hexagonal array of particles are interspersed. The dimensions of these hexagonal lattices are about 0.5 m in diameter, the center-to-center spacing is about 22 nm, and the particle size is about 9 nm. The particle in the hexagonal arrays are accompanied by complementary pits in the opposite monolayer fracture of the plasmalemma membrane.The freeze-fracture preparation was performed by using an improved Leybold Bioetch device which provides a sufficiently high cooling rate and allows the omission of cryoprotectants, like glycerol.Presented by H. Schnabl on the Workshop on Plant Membrane Transport, Toronto, Canada, July 1979     

Close-packing of plasma membrane particles during wall regeneration by isolated higher plant protoplasts—fact or artefact?

Summary Freeze-fracture preparations of protoplasts isolated from cell suspension cultures and leaf mesophyll tissue have been examined by transmission electron microscopy. During the first 72 hours of cell wall regeneration, the 8–10nm intramembraneous particles were randomly distributed on both the protoplasmic and extracellular fracture faces of the plasma membranes of protoplasts frozen and fractured in the culture medium without glutaraldehyde fixation or cryoprotection. Incubation of living protoplasts in culture medium containing 20% v/v glycerol as cryoprotectant prior to freezing without fixation caused deformation of the plasma membrane in the form of protrusions accompanied by particle aggregation on the protoplasmic fracture face of the membrane. Intramembraneous particle aggregation was not observed in protoplasts fixed in glutaraldehyde prior to incubation in medium containing glycerol. The aggregation of particles into hexagonal close packed arrays and elongate chains is discussed in relation to a previous report in the literature of the possible involvement of intramembraneous particle complexes in microfibril formation by isolated higher plant protoplasts.     

Detection of distinct endocytotic and phagocytotic activities in epithelial cells (pinacocytes) of freshwater sponges (Porifera, Spongillidae)

 Light and electron microscopical investigations using externally applied fluorescent and gold-labeled markers have revealed the existence of distinct endocytotic and phagocytotic activities in basal epithelial cells (pinacocytes) of the freshwater sponges Spongilla lacustris and Ephydatia es) of the f. The total rate of endocytotic membrane uptake, ascertained by the application of the cationic lipid probe TMA-DPH, was found to be 3.2% of the cell surface area/h. A typical fluid-phase endocytosis, demonstrated by the use of the water-soluble membrane-impermeable tracers BCECF-dextran and LY-CH, participates in endocytotic activity at a rate of 0.7% of the cell surface area/h and results in the formation of endosomes measuring 0.8–1 μm in diameter. Moreover, the application of labeled BSA succeeded in the detection of a receptor-mediated endocytosis amounting to a concentration-dependent uptake of 2.3–2.8% of the cell surface area/h. Coated pits and coated vesicles conveying the adsorbed BSA measure 0.3 μm in diameter and are covered on the cytoplasmic face with a clathrin-like protein (HC, 180 kDa; LC, 30 kDa). To facilitate phagocytotic activities, a series of fluorescent–labeled and chemically treated particles such as bacteria or latex beads have been successfully employed. Accordingly, the measured values of phagocytic membrane uptake between 1 and 8% of the cell surface area/h depend on the variety of size as well as the chemical nature of the different bioparticles and clearly point to phagocytosis as a key mechanism for providing freshwater sponges with nourishment. Accepted: 3 April 1997     

Chitin synthetase activity is bound to chitosomes anf to the plasma membrane in protoplasts of Saccharomyces cerevisiae

The sub-cellular distribution of chitin synthetase was studied in homogenates of Saccharomyces cerevisiae protoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacoular proteinases. After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40–110 nm; bouyant density (d) = 1.146 g/cm³). Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density (d = 1.203 g/cm³) and by the presence of the vanadate-sensitive ATPase activity. Moreover, after surface labeling of protoplasts with ³H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic. Activation of the chitosome fraction produces microfibrils 100–250 nm in length. Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae.     

Organization of filaments underneath the plasma membrane of developing chicken skeletal muscle cells in vitro revealed by the freeze-dry and rotary replica method

Summary Cytoskeletal organization and its association with plasma membranes in embryonic chick skeletal muscle cells in vitro was studied by the freeze-drying and rotary-shadowing method of physically ruptured cells. The cytoskeletal filaments underlying the plasma membranes were sparse in myogenic cells at the stage when cells exhibited great lipid fluidity in plasma membranes (fusion competent mononucleated myoblasts and recently fused young myotubes). Myotubes at more advanced stages of development possessed a highly interconnected dense filamentous network just underneath the cell membrane. This subsarcolemmal network was composed predominantly of 8–10 nm filaments; they were identified as actin filaments because of their decoration with myosin subfragment-1. Fine fibrils having a diameter of 3–5 nm were found on the protoplasmic surface of the plasmalemma at both the early and advanced stages of development. They were associated with the subsarcolemmal cytoskeletal filaments. Short 2–5 nm cross-linking filaments were occasionally seen between filaments in the subsarcolemmal network. We conclude that, although the subsarcolemmal cytoskeletal network contains many actin filaments, this domain appears to play some role in preserving the cell shape in the form of the membrane skeleton rather than membrane mobility.     

天麻大型细胞消化蜜环菌过程中溶酶体小泡的作用

蜜环菌(Armillaria mellea Fr.)菌丝由天麻(Gastrodia elata Bl.)皮层细胞经纹孔侵入大型细胞。初期大型细胞的原生质膜凹陷,同时细胞壁产生乳突状加厚阻止菌丝侵入。当菌丝侵入大型细胞以后,凹陷的质膜将菌丝紧密包围,大量由单位膜围成的小泡聚集在其周围。随后这些小泡的膜与质膜融合并将其内含物释放到菌丝周围的空间中,凹陷质膜逐渐膨大成为一个包围菌丝的消化泡。小泡和消化泡中均具酸性磷酸酶活性反应产物,证实其分别相当于植物溶酶体系统中的初级和次级溶酶体。菌丝在消化泡中被彻底消化。     

Role of Lysosomal Vesicles in the Digestive Process of Armillaria mellea by the Large Cells of Gastrodia elata

The hyphae of Armillaria mellea Fr. invade the large ceils of Gastrodia elata BI. Through the wall pits of cortical cells. During early stage the plasmalemma of large cell invaginates and the cell wall forms papillary thickenings to restrain the hyphae from invading. When a hypha enters a large cell, it is encircled tightly by the invaginated plasmalemma which is surrounded by a large number of vesicles coated by a unit membrane. As these vesicles fusing with their membranes to the plasmalemma and discharging their contents into the space around the hypha, the space lined by the invaginated plasmalemma enlarges gradually and becomes a digestive vacuole in which a hypha is completely digested. Reaction product form acid phosphatase activities in the vesicles and digestive vacuoles testifies that the vesicles and digestive vacuoles are identical with primary and secondary lysosomes of plant lysosomal system respectively.     

Structural Features of Membrane Fusion between Influenza Virus and Liposome as Revealed by Quick-Freezing Electron Microscopy

The structure of membrane fusion intermediates between the A/PR/8(H1N1) strain of influenza virus and a liposome composed of egg phosphatidylcholine, cholesterol, and glycophorin was studied using quick-freezing electron microscopy. Fusion by viral hemagglutinin protein was induced at pH 5.0 and 23°C. After a 19-s incubation under these conditions, small protrusions with a diameter of 10–20 nm were found on the fractured convex faces of the liposomal membranes, and small pits complementary to the protrusions were found on the concave faces. The protrusions and pits corresponded to fractured parts of outward bendings of the lipid bilayer or “microprotrusions of the lipid bilayer.” At the loci of the protrusions and pits, liposomal membranes had local contacts with viral membranes. In many cases both the protrusions and the pits were aligned in regular polygonal arrangements, which were thought to reflect the array of hemagglutinin spikes on the viral surface. These structures were induced only when the medium was acidic with the virus present. Based on these observations, it was concluded that the microprotrusions of the lipid bilayer are induced by hemagglutinin protein. Furthermore, morphological evidence for the formation of the “initial fusion pore” at the microprotrusion was obtained. The protrusion on the convex face sometimes had a tiny hole with a diameter of <4 nm in the center. The pits transformed into narrow membrane connections <10 nm in width, bridging viruses and liposomes. The structures of the fusion pore and fusion neck with larger sizes were also observed, indicating growth of the protrusions and pits to distinct fusion sites. We propose that the microprotrusion of the lipid bilayer is a fusion intermediate induced by hemagglutinin protein, and suggest that the extraordinarily high curvature of this membrane structure is a clue to the onset of fusion. The possible architecture of the fusion intermediate is discussed with regard to the localization of intramembrane particles at the microprotrusion.     

Assembly of Cellulose Synthesizing Complexes on the Plasma Membrane of Boodlea coacta

The assembly of cellulose synthesizing complexes (terminal complexes,TCs) on the plasma membrane of Boodlea coacta was investigatedduring the formation of both the matrix-rich layer (MRL) andfibril-rich layers (FRLs) of cell walls. The TCs appeared tobe located mostly within the outer leaflet of the plasma membrane,and were observed as elliptical protrusions consisting of manyparticles of about 9 nm in diameter. Their length varied from100 to 500 nm (average, 220 nm) during MRL formation and from100 to 860 nm (average, 360 nm) during FRL formation. A correlationwas found between the length of TCs and the microfibril widthin both MRL and FRL. On the E-face of the plasma membrane, numerous round protrusions(30–130 nm in diameter), consisting of many particles,8–10 nm in diameter, were also present. Their densitywas greater during FRL formation than during MRL formation.Some of these structures larger than 100 nm were associatedwith microfibril impressions and some appeared to be bound tothe TCs. These protrusions increased in number with Calcofluortreatment but decreased in number when the dye was removed fromthe culture medium. Thus, the TCs may be assembled from massesof particles aggregated on the outer surface of the plasma membrane,and may grow longer by incorporation of these masses. The appearanceof the longer TCs during FRL formation is probably due to thegreater density of these masses. (Received May 1, 1985; Accepted August 16, 1985)     

Plasma Membrane Protein Clusters Appear in CFTR-Expressing Xenopus Laevis Oocytes after cAMP Stimulation

Membrane trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is supposed to be an important mechanism controlled by the intracellular messenger cAMP. This has been shown with fluorescence techniques, electron microscopy and membrane capacitance measurements. In order to visualize protein insertion we applied atomic force microscopy (AFM) to inside-out oriented plasma membrane patches of CFTR-expressing Xenopus laevis oocytes before and after cAMP-stimulation. In a first step, oocytes injected with CFTR-cRNA were voltage-clamped, verifying successful CFTR expression. Water-injected oocytes served as controls. Then, plasma membrane patches were excised, placed (inside out) on glass and scanned by AFM. Before cAMP-stimulation plasma membranes of both water-injected and CFTR-expressing oocytes contained about 200 proteins per μm². Molecular protein masses were estimated from molecular volumes measured by AFM. Before cAMP-stimulation, protein distribution showed a peak value of 11 nm protein height corresponding to 475 kDa. During cAMP-stimulation with 1 mm isobutylmethylxanthine (IBMX) plasma membrane protein density increased in water-injected oocytes to 700 proteins per μm² while the peak value shifted to 7 nm protein height corresponding to 95 kDa. In contrast, CFTR-expressing oocytes showed after cAMP-stimulation about 400 proteins per μm² while protein distribution exhibited two peak values, one peak at 10 nm protein height corresponding to 275 kDa and another one at 14 nm corresponding to 750 kDa. They could represent heteromeric protein clusters associated with CFTR. In conclusion, we visualized plasma membrane protein insertion upon cAMP-stimulation and quantified protein distribution with AFM at molecular level. We propose that CFTR causes clustering of plasma membrane proteins. Received: 11 September 2000/Revised: 13 December 2000     

The turnover of cell surface proteins of carrot protoplasts

The covalent modification of cell surface proteins with N-hydroxysuccinimide esters of biotin was used to develop a strategy for following the turnover of proteins on the surface of carrot (Daucus carota L.) protoplasts. A biotinylation/internalisation assay was established which enabled the turnover of cell surface proteins to be examined by biochemical and immunocytochemical techniques. The detection of biotinylated proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting indicated that a variety of proteins on the surface of the protoplasts were covalently modified. Immunolocalisation of biotinylated proteins in protoplasts directly after their derivatisation, demonstrated that the proteins were initially restricted to the cell surface. Incubation of biotinylated protoplasts at 25 °C for 1 h resulted in the detection of biotin-labelled proteins on the cell surface and intracellularly. A small proportion of these proteins was associated with coated pits, the Golgi apparatus and vacuolar compartments. Biochemical analysis of internalised proteins revealed that a polypeptide of approximate M_r 100 000 was internalised by the protoplasts. Immunolabelling of a biotinylated protein of M_r 100 000 by an antibody raised against an isoform of a tobacco plasma-membrane H⁺-ATPase, strongly suggests that the plasma-membrane H⁺-ATPase is internalised by carrot protoplasts. The implications of these results are discussed within the context of endocytosis in plants. Received: 13 July 1998 / Accepted: 11 November 1998     

Proteins in Intercellular Washing Fluids from Leaves of Barley (Hordeum vulgare L.)

Primary leaves of barley were detached, infiltrated with variousbuffers, and centrifuged to yield ‘intercellular washingfluid’ (IWF). Effective pH control of the IWF was obtainedonly with Tris, among all buffers tried. In these liquids, upto 30 proteins were detected by gradient gel electrophoresis.Intracellular protein from injured cells at the cut ends ofleaves was present in IWF but did not contribute significantlyto the total protein recovered in this liquid. The yield ofprotein in the IWF depended on the buffer used for infiltrationand on the concentration of the buffer. Higher concentrationsof buffer yielded more protein. In other experiments leaves were infiltrated with Tris, centrifuged,and then infiltrated a second time with this buffer containingvarious concentrations of the zwitterionic detergent CHAPS,a sulphobetaine derivative of cholate. Gel electrophoresis ofthe IWF obtained after the second centrifugation revealed protein‘bands’ not detected when the detergent had beenomitted from the infiltration buffer. The electrophoretic patternsof protein ‘bands’ in the gels differed dependingon the CHAPS concentration used for infiltration. The effect of CHAPS on plasmalemma integrity was studied byobserving infiltrated tissue with the electron microscope andby treating isolated protoplasts with the detergent. After infiltrationwith CHAPS at 0.6 mM or 2.0 mM no plasmalemma breaks were detectedin leaves, and isolated protoplasts survived exposure to CHAPSat these concentrations for 2 h without bursting. Evidently,CHAPS at these low concentrations did not destroy the integrityof the plasmalemma; the additional protein recovered in theIWF under these conditions probably originated in the cell wall.Infiltration of leaves with 6.0 mM CHAPS resulted in breaksof the plasmalemma, in tissue collapse and leaf tip necrosis.Isolated protoplasts burst within minutes after being exposedto CHAPS at this concentration. Key words: Cell wall permeability, Intercellular space, Detergent, CHAPS, Protoplasts     

Behavior of silkworm yolk protein on phospholipid membranes

During early development, the plasma membrane of silkworm (Bombyx mori) eggs undergoes a superficial cleavage that separates the blastodermal protoplasm and the yolk. To test whether the blastoderm absorbs yolk through the plasma membrane in B. mori, we studied the interaction of phospholipid membranes and yolk using a phospholipid planar bilayer membrane (PBM) and liposomes. In addition, egg-specific protein (ESP; 225 kDa), a yolk protein that is specific to B. mori eggs, was collected by fractionating the eggs. Liposomes were mixed with either B. mori yolk or ESP, and observed under an electron microscope. This showed that the phospholipid membrane was spanned by fine particles 10-20 nm in diameter. Both yolk and ESP caused the PBM to become extraordinarily leaky, with a membrane potential of −70 mV for yolk and −198 mV for ESP. These results suggest that although it is a water-soluble protein, ESP permeates the phospholipid membrane without the help of enzymes.     

A Freeze-Fracture Study of the Cortex of Xenopus laevis Eggs

The organization of the cortex of Xenopus laevis eggs was investigated by freeze-fracture electron microscopy. The cortical endoplasmic reticulum (CER) formed a network surrounding and interconnecting the cortical granules. It formed junctions with the plasma membrane and was confluent with the ER in subcortical regions. Intramembranous particles (IMP₁) were only present in the P face of the CER, the E face being apparently devoid of pits and particles. Arrays of densely packed IMP₁, having a mean diameter of 17 nm, were restricted to the microvillar region of the plasma membrane. The cortical granule membrane also contained IMP₁ (mean diameter, 21 nm) that were sparsely and randomly distributed. Several types of cortical granule seemed to exist based on an analysis of the distribution of the different IMP sizes.     

Insulin-producing cells from human adipose tissue-derived mesenchymal stem cells detected by atomic force microscope

We successfully differentiated human adipose tissue-derived mesenchymal stem cells (haMSCs) into insulin-producing cells (IPCs) in vitro and did not use any insulin which might be absorbed by cells during in vitro culture. Expression of insulin gene was massively increased by 28,000-fold at day 12 compared with haMSCs (P < 0.05). IPCs could secrete insulin after glucose was stimulated. The higher the concentration of glucose, the more production of insulin was noted. We reported AFM images of IPCs for the first time. AFM images showed that the sizes of cells were similar to each other, and all IPC surface had a porous structure in the cytoplasm area. In sugar-free group, the size of holes was similar (diameter, 1,086.98 ± 156.70 nm; depth, 185.22 ± 52.14 nm). In higher sugar-stimulated group, there were more holes with bigger diameter and smaller depth. (diameter, 3,183.65 ± 2,229.18 nm; depth 109.42 ± 56.26 nm, P < 0.05). We found that the hole diameter and depth could change with the concentration of glucose in media. Concurrently, laser scanning confocal microscopy images indicated that cortical actin network beneath plasma membrane in IPCs was dense and continuous. After glucose stimulation, we found the actin web depolymerized and became discontinuous in IPCs. We speculated that diameter augmentation of holes located in the cytoplasm area in IPCs was one manifestation of excytosis increase.     

Photosynthetic performance of regenerated protoplasts from disintegrated cells of <Emphasis Type="Italic">Bryopsis hypnoides</Emphasis> (Chlorophyta)

The photosynthetic performances of regenerated protoplasts of Bryopsis hypnoides, which were incubated in seawater for 1, 6, 12, and 24 h, were studied using chlorophyll (Chl) fluorescence and oxygen measurements. Results showed that for the regenerated protoplasts, the pigment content, the ratios of photosynthetic rate to respiration rate, the maximal photosystem II (PSII) quantum yield (F_v/F_m), and the effective PSII quantum yield (Φ_PSII) decreased gradually along with the regeneration progress, indicated that during 24 h of regeneration there was a remarkable reduction in PSII activity of those newly formed protoplasts. We assumed that during the cultivation progress the regenerated protoplasts had different photosynthetic vigor, with only some of them able to germinate and develop into mature thalli. The above results only reflected the photosynthetic features of the regenerated protoplasts at each time point as a whole, rather than the actual photosynthetic activity of individual aggregations. Further investigation suggested a relationship between the size of regenerated protoplasts and their viability. The results showed that the middle-sized group (diameter 20–60 μm) retained the largest number of protoplasts for 24 h of growth. The changes in F_v/F_m and Φ_PSII of the four groups of differently sized protoplasts (i.e. < 20, 20–60, 60–100, and > 100 μm) revealed that the protoplasts 20–60 μm in diameter had the highest potential activity of the photosynthetic light energy absorption and conversion for several hours.     

Invaginations in plant cell protoplasts containing mycoplasma-like bodies (MLO)

MLO containing invaginations were found in protoplasts of phloem parenchyma cells in symptomless young leaves ofRibes houghtonianum Jancz. infected with a yellows disease. The invaginations originate between the cell wall and plasmalemma, usually at plasmodesmata, and change apparently into superficial vesicles in the protoplast; they are entirely or partially limited by host plasmalemma. The formations mentioned occur in parenchyma cells which contain normal organelles. Sometimes they are divided by a smooth membrane system enclosing MLO. Besides MLO the invaginations contain in some cases slimy fibrils resembling the P-protein in sieve tubes. The MLO bodies seen in invaginations have usually a diameter of 50–250 nm and their plasmalemma (unit membrane) is identical with the plasmalemma of MLO bodies occurring in sieve tubes. However, only few MLO bodies in invaginations are electron dense, so that they resemble naturally degenerated forms of MLO. Similar MLO containing invaginations were formerly described from some leafhoppers transmitting MLO.     

Hydrogen fluoride effects on plasma membrane composition and ATPase activity in needles of white pine (Pinus strobus) seedlings pretreated with 12 h photoperiod

 Eastern white pine (Pinus strobus L.) seedlings were pretreated with 12 h photoperiod to induce dormancy. Dormant plants were fumigated with 0.5 ppb (0.4 μg m^–3) or 2.0 ppb (1.6 μg m^–3) hydrogen fluoride (HF) for 2 – 28 days. Plasma membranes were isolated from needles of treated and control seedlings to determine their chemical composition and ATPase activity. For all analyses, only those plants which did not show needle necrosis were selected. The amount of plasma membrane phospholipid expressed on a plasma membrane protein basis was higher after 2 days in the 0.5 ppb HF treatment as compared to controls. After 2 days of 2.0 ppb HF treatment as well as after 8 and 28 days of both HF treatments phospholipid to protein ratios in fluoride treated seedlings were lower as compared to control levels. A decrease in sterol levels could be observed after 2 days in both HF treatments. A large increase in the ratio of sterols to proteins was observed in plasma membranes of eastern white pine seedlings treated with 0.5 ppb HF for 28 days. Increased sterol to phospholipid ratios were observed after 8 and 28 days in 0.5 ppb and after 2 and 8 days of 2.0 ppb HF treatment. A decrease in ATPase activity was observed after 8 days with both fluoride treatments. Drastic increase of ATPase activity was observed after 28 days of HF treated plants. Observed changes of sterol and phospholipid levels after only 2 days of fumigation suggest early fluoride effects on plasma membrane composition during plant dormancy. Received: 25 October 1995 / Accepted: 24 May 1996     

Membrane differentiations of an absorptive epithelium covering embryonic trophotaeniae in a goodeid teleost

Summary The trophotaenial absorptive cells (TACs) in goodeid embryos facilitate nutrient absorption during prolonged periods of intraovarian gestation. In a study of membrane differentiations associated with solute and ligand transfer in the trophotaeniae of Xenotoca eiseni, embryos were incubated in vivo with cationized ferritin (CF) prior to freeze-cleaving. This exposure to high concentrations of an adsorptive ligand was meant to induce swelling of the endosomal compartment. Macromolecular trafficking in TACs occurs via an apical endocytic complex consisting of plasma membrane invaginations, a large population of small vesicles, uniformly thick apical tubules, and endosomes. Freeze-fracture replicas showed that the microvillar plasma membrane P-face of TACs was studded with intramembrane particles (IMPs) at a fairly high density, whereas that of the cell surface proper contained a distinctly lower density and the tubulovesicular endocytic pits contained almost no IMPs. The majority of small vesicles and apical tubules in a near surface position displayed P-fracture faces with only a few odd IMPs, indicating that membrane, shuttling between the apical plasma membrane and intracellular sorting organelles, obviously does not carry along many large-sized integral membrane proteins. The distended endosomal compartment had many P-face-associated particles primarily clustered into patches. Specializations of the lateral plasma membrane included 4–8 tight junctional strands, relatively large complements of gap junction proteins, and numerous plaques of desmosomal membrane particles. A system of lamellar cisternae underlay the lateral cell surface that was in continuity with the intraepithelial space by numerous tubular canals, giving rise to an intracellular amplification of the basolateral plasma membrane. Their outward openings appeared as tiny pits on the cytoplasmic faces of freeze-cleaved cell membrane. The density of IMPs on the P-faces of the surface plasma membrane was apparently lower than that on its invaginated lamellar complex. Hence, it is concluded that the mobility of integral membrane proteins in the plane of the membrane may be hampered in movement across the surface pores.Supported by the Deutsche Forschungsgemeinschaft (Schi 268/1-1)