Marginal plates in hepatic peroxisomes of Ichthyophis glutinosus (Amphibia: Gymnophiona)

Summary The ultrastructure of hepatic peroxisomes was investigated in Ichthyophis glutinosus (Amphibia: Gymnophiona), employing perfusion fixation and the diaminobenzidine (DAB) technique for the visualization of catalase. The majority of peroxisomes is circular or rod-shaped, although elongated particles occasionally occur. They contain a finely granular matrix, lightly stained after the DAB procedure. Their mean diameter is approximately 0.25 m. Serial sections reveal that the circular and rod-shaped peroxisomal profiles are cross and oblique sections of highly tortuous, tubular organelles exceeding 2 m in length.In addition to tubular profiles, elongated, rectangular particles, as well as straight dumbbell-shaped organelles with distinct marginal plates are observed. They range from 900 to 1650 nm in length (mean = 1200 nm). In the flattened, thin central portion of the dumbbell-shaped particle, the peroxisomal membranes form a cisterna enclosing one or two uniformly thick marginal plates, which display a definite substructure with a periodicity of 10 nm.These findings indicate that peroxisomes in the liver of Ichthyophis exhibit a complex organization. It is suggested that the organelles undergo a specific differentiation process, morphologically characterized by the formation of enlarged segments of unusual shape.This study was supported by grants from the Deutsche Forschungsgemeinschaft, Fa 146/1-2 and Sto 75/9     

Immunoelectron microscopic localization of the isozymes of L-alpha-hydroxyacid oxidase in renal peroxisomes of beef and sheep: evidence of distinct intraorganellar subcompartmentation

L-alpha-hydroxyacid oxidase (HAOX), a peroxisomal marker enzyme in mammals, exists in two isozymic forms, HAOX A (EC 1.1.3.1) and HAOX B (EC 1.3.4.2), which differ in their substrate specificity. In rat tissues HAOX A is found exclusively in hepatocyte peroxisomes and HAOX B in renal peroxisomes. Recently we found enzymatic evidence that highly purified peroxisome preparations from beef and sheep kidney cortex contain both isozymes. In situ, the peroxisomes in the proximal tubule cells of both species exhibit peculiar angular outlines apparently due to the presence of multiple marginal plates. Marginal plates are plate-like crystalline matrix inclusions which are apposed to the inner aspect of the peroxisomal membrane. In this study monospecific antibodies against HAOX A and B proteins purified from rat liver and kidney, respectively, were raised in rabbits and used to study the intraorganellar localization of each isozyme in beef and sheep kidney cortex peroxisomes. Incubation of ultra-thin sections of LR White-embedded tissue with anti-HAOX A or B followed by protein A-gold revealed that in both species HAOX A is present diffusely in the peroxisomal matrix, whereas HAOX B is localized almost exclusively in the membrane associated marginal plates. This is the first report on the in situ immunocytochemical characterization of marginal plates, which are the most common inclusions in the matrix of renal peroxisomes.     

Purification of marginal plates from bovine renal peroxisomes: identification with L-alpha-hydroxyacid oxidase B

The matrix of mammalian peroxisomes frequently contains crystalline inclusions. The most common inclusions are membrane associated plate-like "marginal plates" of hitherto unknown nature in renal peroxisomes and central polytubular "cores" composed of urate oxidase in hepatic peroxisomes. In bovine kidney, peroxisomes of proximal tubules exhibit peculiar angular shapes that are caused by multiple marginal plates (Zaar, K., and H.D. Fahimi. 1990. Cell Tissue Res. 260:409-414). Enriched or highly purified peroxisome preparations from this source were used to purify and characterize marginal plates. By SDS-PAGE, one major polypeptide of Mr 33,500 was observed that corresponded to the marginal plate protein. This polypeptide was identified by its enzymatic activity as well as by immunoblotting and preembedding immunocytochemistry as the isozyme B of L-alpha-hydroxyacid oxidase (EC 1.4.3.2). Morphologically, marginal plates were revealed to consist of rectangular straight-edged sheets, exhibiting a defined crystalline lattice structure. The sheets apparently are composed of a single layer of protomers which associate laterally to form a plate-like structure. As deduced from the negative staining results and the additional information of the thickness of marginal plates, each protomer seems to consist of eight subunits forming a cube-like array. The tendency of L-alpha-hydroxyacid oxidase B to self-associate in vitro (Philips, D.R., J.A. Duley, D.J. Fennell, and R.S. Holmes. 1976. Biochim. Biophys. Acta. 427:679-687) corresponds to the mode of association of cubical protomers to form the so-called marginal plates in renal peroxisomes.     

A comparison of receptive and non-receptive plasma membrane areas of photoreceptor cells in the leech,Hirudo medicinalis

Summary Microvillar (receptive) and external (non-receptive) portions of the plasmalemma of photoreceptor cells of Hirudo were compared electron microscopically in thin sections and freeze-fracture replicas. A morphometric approximation showed that the surface area of the microvillar membrane is about 19 times larger than that of the external membrane. The microvillar membrane most probably undergoes extensive membrane turnover. In both segments of the membrane the particles associated with the P- and the E-fracture faces are randomly distributed except at some specific sites. The particles adhere predominantly to the P-faces. The particle densities on the fracture faces of the microvillar membrane differ from those of the external membrane. The P-face particles of the external membrane appear to be larger than those of the microvillar membrane. It is suggested that the P-face particles of the microvillar membrane represent sites where the photopigment is incorporated into the membrane. The distinguishing structural features correspond to the functional differences postulated for both portions of the plasma membrane.     

Contributions of the immunogold technique to investigation of the biology of peroxisomes

 The immunogold labeling technique has been extremely useful in investigation of the structure and function of peroxisomes. In this report a few examples of the application of this technique with significant implications in the field are briefly reviewed. The problem of extraperoxisomal catalase, the subject of long controversy between the biochemists and cytochemists, was settled with the immunogold technique, which unequivocally revealed the presence of that enzyme not only in the cytoplasm, but also in the euchromatin region of nucleus, in addition to peroxisomes. On the other hand, lactate dehydrogenase, a typical cytoplasmic protein, has also been shown recently to be present in peroxisomes and to be involved in the reoxidation of NADH produced by the peroxisomal β-oxidation system. The immunogold technique has revealed several distinct compartments in the matrix of mammalian peroxisomes: urate oxidase in the crystalline cores, α-hydroxy acid oxidase B in the marginal plates and d-amino acid oxidase in a non-crystaline condensed region of matrix. The specific alterations of peroxisomal proteins are reflected in their immunolabeling density with gold particles. Quantitation of gold-label by automatic image analysis has revealed that the induction of lipid β-oxidation enzyme proteins by diverse hypolipidemic drugs is initiated and more pronounced in the pericentral region of the liver lobule. Finally, immunogold labeling with an antibody to 70 kDa peroxisomal membrane protein has identified a novel class of small peroxisomes that initially incorporate radioactive amino acids more efficiently than regular peroxisomes and thus may represent early stages in the biogenesis of peroxisomes. Accepted: May 5, 1996     

Freeze-fracture study of rat liver peroxisomes: evidence for an induction of intramembrane particles by agents stimulating peroxisomal proliferation

The membrane ultrastructure of isolated rat liver peroxisomes has been observed by rapid freezing and freeze-fracture techniques. Unidirectional and rotary shadowing allows a clear visualization of the intramembrane particles (IMPs) on both the protoplasmic fracture (PF) leaflet and the endoplasmic fracture (EF) leaflet and reveals an asymmetric distribution of IMPs. Both fracture faces were uniformly studded by IMPs, and the frequency was about seven times higher on the P face (2322 per 1.0 micron2) than on the E face (322 per 1.0 micron2). Administration of the peroxisomal proliferator clofibrate (ethyl-p-chlorophenoxyisobutyrate) induced a marked increase in the frequency of IMPs on both the P face (2.2-fold) and the E face (1.7-fold). The average size decreased (P less than 0.001) from 45.7 +/- 16.5 nm2 to 35.2 +/- 10.8 nm2 on the P face. A similar increase in the frequency of IMPs was observed on the P face (1.8-fold) and the E face (1.8-fold) of peroxisomes from rats fed a semisynthetic diet containing 20% (w/w) of partially hydrogenated fish oil. The average size increased (P less than 0.001) from 36.6 +/- 19.7 to 50.0 +/- 23.5 nm2 on the E face. This study demonstrates alterations both in frequency and size distribution of IMPs in liver peroxisomal membranes on exposure of rats to agents known to induce peroxisomal proliferation. The increase in frequency of IMPs was as expected from the observed increase in one of the major integral membrane polypeptides, with apparent molecular mass of 69 (or 70) kDa, in proliferating rat liver peroxisomes.     

Permeability properties of peroxisomal membranes from yeasts

We have studied the permeability properties of intact peroxisomes and purified peroxisomal membranes from two methylotrophic yeasts. After incorporation of sucrose and dextran in proteoliposomes composed of asolectin and peroxisomal membranes isolated from the yeasts Hansenula polymorpha and Candida boidinii a selective leakage of sucrose occurred indicating that the peroxisomal membranes were permeable to small molecules. Since the permeability of yeast peroxisomal membranes in vitro may be due to the isolation procedure employed, the osmotic stability of peroxisomes was tested during incubations of intact protoplasts in hypotonic media. Mild osmotic swelling of the protoplasts also resulted in swelling of the peroxisomes present in these cells but not in a release of their matrix proteins. The latter was only observed when the integrity of the cells was disturbed due to disruption of the cell membrane during further lowering of the concentration of the osmotic stabilizer. Stability tests with purified peroxisomes indicated that this leak of matrix proteins was not associated with the permeability to sucrose. Various attempts to mimic the in vivo situation and generate a proton motive force across the peroxisomal membranes in order to influence the permeability properties failed. Two different proton pumps were used for this purpose namely bacteriorhodopsin (BR) and reaction center-light-harvesting complex I (RCLH_I complex). After introduction of BR into the membrane of intact peroxisomes generation of a pH-gradient was not or barely detectable. Since this pump readily generated a pH-gradient in pure liposomes, these results strengthened the initial observations on the leakiness of the peroxisomal membrane fragments. Generation of a membrane potential () was also not observed when RCLH_I complex was introduced into vesicles of purified peroxisomal membranes. The significance of the observed permeability of isolated yeast peroxisomal membranes to small molecules with respect to current and future in vitro import studies is discussed.Abbreviations CL cardiolinin - PE phosphatidylethanolamine - PC phosphatidylcholine - MES 2-(N-Morpholino)ethanesulfonic acid - R₁₈ octadecyl Rhodamine B Chloride - SUVs small unilamellar vesicles - RCLH_I-complex reaction center-light-harvesting complex I - BR bacteriorhodopsin - DCCD N,N-dicyclohexylcarbodiimide     

Stable and transient expression of chimeric peroxisomal membrane proteins induces an independent "zippering" of peroxisomes and an endoplasmic reticulum subdomain

Peroxisomal ascorbate peroxidase (APX) (EC 1.11.1.11) was shown recently to sort through a subdomain of the ER (peroxisomal endoplasmic reticulum; pER), and in certain cases, alter the distribution and/or morphology of peroxisomes and pER when overexpressed transiently in Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cells. Our goal was to gain insight into the dynamics of peroxisomal membrane protein sorting by characterizing the structure and formation of reorganized peroxisomes and pER. Specifically, we test directly the hypothesis that the observed phenomenon is due to the oligomerization of cytosol-facing, membrane-bound polypeptides. a process referred to as membrane "zippering". Results from differential detergent permeabilization experiments confirmed that peroxisomal APX is a C-terminal "tail-anchored" (Cmatrix-Ncytosol) membrane protein with a majority of the polypeptide facing the cytosol. Transient expression of several APX chimeras whose passenger polypeptides can form dimers or trimers resulted in the progressive formation of "globular" peroxisomes and circular pER membranes. Stable expression of the trimer-capable fusion protein yielded suspension cultures that reproducibly maintained a high degree of peroxisomal globules but relatively few detectable pER membranes. Electron micrographs revealed that the globules consisted of numerous individual peroxisomes, seemingly in direct contact with other peroxisomes and/or mitochondria. These peroxisomal clusters or aggregates were not observed in cells transiently expressing monomeric versions of APX. These findings indicate that the progressive, independent "zippering" of peroxisomes and pER is due to the post-sorting oligomerization of monomeric, cytosol-facing polypeptides that are integrally inserted into the membranes of "like" organelles. The dynamics of this process are discussed, especially with respect to the involvement of the microtubule cytoskeleton.     

Walsby's square bacterium: fine structure of an orthogonal procaryote.

The "square" bacterium, first described by Walsby from brine collected at the Red Sea shore [A. E. Walsby, Nature (London) 283:69-71, 1980] was examined by electron microscopy. The cells appeared as flat rectangular boxes in scanning electron micrographs. In sections and freeze-fracture preparation, the edges looked more rounded. The thickness apparently remains constant as the cells grow and divide. Their sides were a few micrometers long, but the cells were only 0.25 micrometers thick. They showed typical procaryote structure, with a regular cell wall and a gas vacuole fine structure similar to that of other halophilic procaryotes. The inner fracture faces of the cell membrane showed a much denser population of intramembrane particles than the outer fracture faces, but no patches of purple membrane, despite the presence of bacteriorhodospin-like pigment in the cell suspension. Morphologically identical cells have been found in brine from Baja California, Mexico.     

Topographical localization of peroxisomal acyl-CoA ligases: differential localization of palmitoyl-CoA and lignoceroyl-CoA ligases

We found that peroxisomal lignoceroyl-CoA ligase, like palmitoyl-CoA ligase, is present in the peroxisomal membrane whereas the peroxisomal beta-oxidation enzyme system is localized in the matrix. To further define the role of peroxisomal acyl-CoA ligases (membrane component) in providing acyl-CoA for peroxisomal beta-oxidation, we examined the transverse topographical localization of enzymatic sites of palmitoyl-CoA and lignoceroyl-CoA ligases in the peroxisomal membranes. The disruption of peroxisomes by various techniques resulted in the release of a "latent" pool of lignoceroyl-CoA ligase activity while palmitoyl-CoA ligase activity remained the same. Proteolytic enzyme treatment inhibited palmitoyl-CoA ligase activity in intact peroxisomes but had no effect on lignoceroyl-CoA ligase activity. Lignoceroyl-CoA ligase activity was inhibited only if peroxisomes were disrupted with detergent before trypsin treatment. Antibodies to palmitoyl-CoA ligase and to peroxisomal membrane proteins (PMP) inhibited palmitoyl-CoA ligase in intact peroxisomes, and no pool of "latent" activity appeared when antibody-treated peroxisomes were disrupted with detergent. On the other hand, disruption of PMP antibody-treated peroxisomes with detergent resulted in the appearance of a "latent" pool of lignoceroyl-CoA ligase activity. These results demonstrate that the enzymatic site of palmitoyl-CoA ligase is on the cytoplasmic surface whereas that for lignoceroyl-CoA ligase is on the luminal surface of peroxisomal membranes. This implies that palmitoyl-CoA is synthesized on the cytoplasmic surface and is then transferred to the matrix through the peroxisomal membrane for beta-oxidation in the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of intramembranous particles and filipin-sterol complexes in the spermatid and spermatozoon of Culex quinquefasciatus (Culicidae).

A freeze-fracture study was carried out on spermatid and spermatozoon of the mosquito Culex quinquefasciatus. In the spermatid plasma membrane few and randomly distributed intramembranous particles were observed. In the spermatozoon the density of intramembranous particles was higher on the P- than on the E-fracture face of the plasma membrane. Two populations of particles were observed. Large particles (about 15 nm in diameter) are regularly arranged in double rows as a zipper-line, longitudinally oriented in relation to the main cell axis. These strands of particles were observed in the posterior head region, mainly associated with the E-fracture face. Filipin was used to analyse the presence and distribution of cholesterol in thin sections and freeze-fracture replicas. Filipin-sterol complexes were not homogeneously distributed throughout the spermatozoon plasma membrane. They were more abundant on the P-fracture face of the membrane lining the nuclear region. The results obtained show that Culex spermatozoon differs from those of other species in that its plasma membrane exhibits only a membrane domain, the zipper-line, localized in the postacrosomal region.     

Immunocytochemical localization of L-α-hydroxyacid oxidase in dense bar of dumb-bell-shaped peroxisomes of monkey kidney

Localization of the B of L-hydroxyacid oxidase (HOX-B) in monkey kidney peroxisomes was investigated by immunoelectron microscopic techniques. Kidneys of Japanese monkeys,Macaca fuscata, were fixed with 4% paraformaldehyde+0.25% glutaraldehyde and embedded in LR White resin. Thin sections were stained for HOX-B and catalase by the immunogold technique. HOX-B was localized in the marginal plates of normal peroxisomes and the dense bar of dumb-bellshaped peroxisomes. Catalase was detected in the matrix of normal peroxisomes and in the terminal dilatations of dumb-bell-shaped peroxisomes. There were no gold particles indicating presence of catalase associated with the marginal plates or with the dense bars. Immunoblot analysis of monkey kidney homogenate showed that HOX-B has a molecular mass of 42 kDa that was slightly larger than that of rat kidney HOX-B (39 kDa). The results show that the dense bar of dumb-bell-shaped peroxisomes in monkey kidney is composed of at least HOX-B and is a variation of the marginal plates.     

The behavior of peroxisomes in vitro: mammalian peroxisomes are osmotically sensitive particles

It has been known for a long time that mammalian peroxisomes are extremely fragile in vitro. Changes in the morphological appearance and leakage of proteins from purified particles demonstrate that peroxisomes are damaged during isolation. However, some properties of purified peroxisomes, e.g., the latency of catalase, imply that their membranes are not disrupted. In the current study, we tried to ascertain the mechanism of this unusual behavior of peroxisomes in vitro. Biochemical and morphological examination of isolated peroxisomes subjected to sonication or to freezing and thawing showed that the membrane of the particles seals after disruption, restoring permeability properties. Transient damage of the membrane leads to the formation of peroxisomal "ghosts" containing nucleoid but nearly devoid of matrix proteins. The rate of leakage of matrix proteins from broken particles depended inversely on their molecular size. The effect of polyethylene glycols on peroxisomal integrity indicated that these particles are osmotically sensitive. Peroxisomes suffered an osmotic lysis during isolation that was resistant to commonly used low-molecular-mass osmoprotectors, e.g., sucrose. Damage to peroxisomes was partially prevented by applying more "bulky" osmoprotectors, e.g., polyethylene glycol 1500. A method was developed for the isolation of highly purified and nearly intact peroxisomes from rat liver by using polyethylene glycol 1500 as an osmoprotector. osmolarity; cell fractionation; isolation of organelles     

The development of membrane specializations in the receptor-bipolar-horizontal cell synapse of the chick embryo retina

Retinae of chick embryos and chicks one to six weeks after hatching were examined in ultrathin sections and in freeze-etch specimens. The development of the synaptic contacts between receptor cells and bipolar cells starts at the end of the second week of incubation with the enclosure of the dendritic prolongations, invaginating receptor terminals accompanied by the appearance of electron dense material at the synaptic contact sites. Subsequently receptor terminals become filled with synaptic vesicles which surround the synaptic lamellae that appear on the 16th day of incubation. The application of the freeze-fracture technique demonstrates that the differentiation of the synaptic membranes continues into the first week post hatching. E-fracture faces of the presynaptic membranes are characterized by crater-like structures, called synaptopores. Their number is rather small during incubation and increases after hatching. In the P-fracture faces of the dendrites, which are enclosed by the receptor terminals, small particle aggregations appear on the 16th day of incubation. These small particle clusters increase by the apposition of further particles which become arranged in lines and bring out a lattice-like aspect. This arrangement of particles in the inner part of the cell membrane is the morphological expression of the maturation process. The significance of these aggregations as a postsynaptic receptor for neurotransmitters in excitatory cells is discussed.     

Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha

The localization of methanol oxidase activity in cells of methanol-limited chemostat cultures of the yeast Hansenula polymorpha has been studied with different cytochemical staining techniques. The methods were based on enzymatic or chemical trapping of the hydrogen peroxide produced by the enzyme during aerobic incubations of whole cells in methanol-containing media. The results showed that methanol-dependent hydrogen peroxide production in either fixed or unfixed cells exclusively occurred in peroxisomes, which characteristically develop during growth of this yeast on methanol. Apart from methanol oxidase and catalase, the typical peroxisomal enzymes d-aminoacid oxidase and l--hydroxyacid oxidase were also found to be located in the peroxisomes. Urate oxidase was not detected in these organelles. Phase-contrast microscopy of living cells revealed the occurrence of peroxisomes which were cubic of form. This unusual shape was also observed in thin sections examined by electron microscopy. The contents of the peroxisomes showed, after various fixation procedures, a completely crystalline or striated substructure. It is suggested that this substructure might represent the in vivo organization structure of the peroxisomal enzymes.     

Organization of the cell membrane inEuglena

Summary The cell membrane ofEuglena gracilis has been investigated with the freeze-fracture technique. When split, this membrane produces two fracture faces which are striking in their non-complementarity. The P fracture face is covered with a high density of 110 Å (average diameter) particles, while the E face is made up of a complex series of striations occurring at regular angles to the pellicle ridges which encircle the organism. Under certain conditions, however, the structure of the P fracture face assumes a more ordered configuration, and striations are visible on this fracture face which are precisely complementary to those observed on the E face. These observations suggest that the cortical cell membrane ofEuglena may be organized along the lines of a two dimensional crystal. However, this pattern of organization is restricted to the cortical region of the cell membrane; as the membrane invaginates near the anterior end of the cell the fracture faces change abruptly, and organization more typical of other cell membranes is observed. This invagination forms an extensive reservoir in the anterior of the cell, and the membrane bounding it is distinctly fluid in structure, with clear examples of endo- and exocytosis observable. These differences suggest that the cell membrane inEuglena is divided into two distinct but contiguous regions, each specialized with regard to structure and function.     

Changes to the integral membrane protein composition of mouse liver peroxisomes in response to the peroxisome proliferators clofibrate,Wy-14,643 and Di(2-ethyl-hexyl)phthalate

Peroxisomes were purified from livers of control mice and from mice treated with three agents which induce proliferation of hepatic peroxisomes — namely two structurally unrelated hypolipidemic drugs, clofibrate (ethyl--p-chlorophenoxyisobutyrate) and Wy-14,643 (4-chloro-6[2,3-xylidino)-2-pyrimidinylthio] acetic acid), and a plasticizer, DEHP (di-(2-ethylhexyl)phthalate).Membranes were isolated from these purified peroxisomes and analysed by SDS-polyacrylamide gel electrophoresis. All membranes which were tested, displayed two predominant integral membrane proteins of apparent molecular weights of 68 kDa and 70 kDa respectively, as well as a number of minor components. Treatment of animals with clofibrate, Wy-14,643 and DEHP was observed to result in each case in an increased proportion of the 70 kDa protein in the peroxisomal membranes. These treatments also resulted in increased peroxisomal fatty acid oxidation in livers and an increase in the proportion of catalase activity in the cytosolic fraction of liver cells.These results have been discussed in relation to alterations in the molecular composition of the membranes, the mechanisms of peroxisome proliferation and the inducibility of peroxisomal membrane proteins.     

Freeze-fracture studies on barley plastid membranes. VI. Location of the P700-chlorophyll a-protein 1

The photosystem I mutant of barley, viridis-zb63, which totally lacks the P700-chlorophyll a-protein 1 was characterised by rotary shadowed, freeze-fracture electron microscopy. Objective measurements were made of particle density and size distribution for all four fracture faces, and compared with values for wild-type. A highly significant reduction in the size of the PFu particles was found, which could be attributed to the loss of a population of large particles from the mutant PFu face. A corresponding loss of EFu pits was also observed. It is concluded that the photosystem I reaction centre and two or three ancillary polypeptides are located in the large PFu particles which account for two-thirds of the total, and that these particles span the membrane. Since no differences were seen on the PFs and EFs faces, there was no evidence for the localisation of any photosystem I in appressed granal membranes, supporting the concept of extreme lateral heterogeneity. A model is presented of the localisation of different functional polypeptide units to different freeze-fracture particles within the membrane. A peculiar feature of viridis-zb63 thylakoids was the presence of EFs particle arrays in vivo.     

Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.     

MEMBRANE FUSION IN A MODEL SYSTEM : Mucocyst Secretion in Tetrahymena

The freeze-fracture, freeze-etch technique can be employed to reveal new details of the process of fusion of two unit membranes For this study, mucocyst discharge in Tetrahymena pyriformis provides a model system with certain general implications The undischarged mature mucocyst is a saclike, membrane-bound, secretory vesicle containing crystalline material The organelle tip finds its way toward a special site, a rosette of 150 Å diameter particles within the plasma membrane. To match this site, the mucocyst membrane forms an annulus of 110 Å diameter particles, above whose inner edge the rosette particles sit. Discharge of some mucocysts is triggered by fixation. As discharge proceeds, the organelle becomes spherical and its content changes from crystalline to amorphous. The cytoplasm between the two matching membrane sites is squeezed away and the membranes fuse Steps in membrane reorganization can be reconstructed from changes in rosette appearance in the fracture faces. First, a depression in the rosette—the fusion pocket—forms. The rosette particles spread at the lip as the pocket deepens and enlarges from 60 to 200 nm. The annulus particles then become visible at the lip, indicating completed fusion of the A fracture faces of mucocyst and plasma membranes The remaining B faces of the two membranes have opposite polarities When the content of the mucocyst is released, the edges of these faces join so that the unit membrane runs uninterruptedly around the lip and into the pocket.