Distribution Of Intramembranous Particles Research Articles

Parasite-Host-Cell Interactions of Eimeria papillata Sporozoites with Cultured Cells A Freeze-Fracture Study

Summary The relationship between the sporozoite of Eimeria papillata and the host cell was studied by comparing the frequency and distribution of intramembranous particles (IMP) in their respective plasmalemmas before and after invasion. The entering sporozoite invaginates the host cell membrane during entry and this forms the boundary of the vacuole that encloses the parasite. Freeze-fracture replicas of this parasitophorous vacuole (PV) membrane showed a 3 fold decrease in IMP on the P- face (400 ± 215) and E-face (100 ± 29) when compared to the uninfected cell membrane (1369 ± 124 on the P-face and 395 ± 79 on the E-face). Studies of other coccidia and Plasmodium suggest that this considerable modification of the membrane appears to be the result of addition of proteins and lipids which are contributed by the invading parasite. The P- and E-faces of the plasmalemma of the free or internalized sporozoite was virtually unchanged.

Freeze-fracture characterization of cell types at the surface of the taste organ of the frog,Rana esculenta

The ultrastructure and distribution of intramembrane particles in the chemoreceptor surface of the frog taste organ have been studied by means of freeze-fracture. Sustentacular, wing, mucous cells and two different types of putative taste cells were found to reach the free surface of this chemoreceptor. Each of these cell types was characterized by a different pattern and density of intramembrane particles in the free surface. Wing cells displayed a relatively low number of large intramembrane particles (11.1 +/- 1.4 nm in diameter). Particles of similar size were also present in a much higher concentration in the membrane of cylinder-ending putative taste cells. In microvilli-ending putative taste cells, mucous cells, and sustentacular cells, small intramembrane particles were observed (6.8 +/- 0.78, 6.9 +/- 1.3, 7.2 +/- 0.7 nm in diameter, respectively). The density of these particles was higher in the sustentacular cells than in the other two cell types. These data provide evidence that there are two morphologically distinct types of putative taste cells in the frog taste organ, demonstrating that they are characterized by different pattern of intramembrane particles in their free surface. Furthermore, the present results support previous findings indicating that wing and sustentacular elements represent two different cell types.

Cytoskeleton-dependent surface blebbing induced by the polar solvent N-methylformamide

In vivo and in vitro studies performed on the polar solvent N-methytformamide (NMF), as well as on its association with chemotherapeutic agents or X rays, have clearly demonstrated that this compound is capable of inducing changes in biological characteristics of tumor cells, e.g., cell differentiation. However, the mechanism of action of NMF is far from being elucidated. Hence, in order to better clarify such a mechanism an in vitro study was carried out by using mouse fibroblasts in primary culture (MEF) and human melanoma cultured cells (M14). Results obtained by immunocytochemical and ultrastructural methods with doses of NMF ranging from 0.1 to 7% are reported here. As a general rule, a different sensitivity (in terms of cytopathologic changes induced by NMF) was found between the cell types considered. In fact, melanoma cells appeared to be highly susceptible to the action of the drug, undergoing severe morphological modifications represented mainly by a reversible dose and time-dependent cell rounding and surface blebbing. In contrast, NMF-induced injury in MEF cells was characterized mainly by a simple retraction of the cell body. A cytochemical analysis of the expression of certain membrane antigens (e.g., glycoproteins, epidermal growth factor receptor, B 2 microglobulin) in NMF-treated M14 cells undergoing blebbing was also carried out. A randomly distributed labeling of such molecules was observed. Accordingly, freeze-fracturing electron microscopic analysis also displayed a random distribution of intramembrane particles over the plasma membrane. When subcellular changes induced by the drug were investigated, a remarkable modification of cytoskeletal components was detected in both cell types. In particular, cross-linked actin microfilament bundles were easily observed in NMF-exposed MEF cells. Finally, when different experimental conditions which perturb calcium ion homeostasis or restore protein thiol group reduced state were analyzed, a noticeable impairment of the blebbing phenomenon was observed. Thus, a target effect of NMF on the microfilament system, probably leading, in turn, to several subcellular changes and cell surface blebbing, can be hypothesized. Such a cytoskeletal element-dependent cytopathology appears to be related to changes of the oxidized state of such molecules as well as to calcium ion perturbations.

Intramembrane particles distribution in the naturally synchronous stage of Physarum polycephalum

Comparative ultrastructural investigations of the Physarum polycephalum intramembrane particles in the plasma membrane at different stages of the cycle has provided valuable information in relation to possible changes that occur in the plasma membrane of higher organisms. In addition, it gives insight on how plasma membrane changes correlate with gene expression and gene regulation in eukaryotes. In this report Freeze-fracture-etched techniques were utilized to study the arrangements of intramembrane particles (IMP) distribution and density at eight hours of the naturally synchronous plasmodial stage.

In situ freeze-fracture of monolayer cell cultures grown on a permeable support.

The growth of cultured epithelial cells on permeable supports allows increased cell differentiation and the assessment of a variety of transcellular and paracellular transport processes. The need to assess the corresponding ultrastructural characteristics of these cells under identical conditions prompted this laboratory to develop a reliable method for producing freeze-fracture replicas of these cultures. Sections of filter inserts with the cell-side facing up are placed between layers of polyvinyl alcohol with a strip of mylar positioned on the layer of polyvinyl alcohol. Following freezing, the monolayer is fractured by lifting the mylar strip from the assembly. The result is a consistent fracture of the apical membrane sufficient for analysis of tight junction sealing strands, microvilli distribution, and intramembranous particle (IMP) distribution between apical and lateral membrane domains. This method utilizes standard equipment and readily available materials and, most importantly, allows the freeze-fracture and replication of an undisturbed cell monolayer.

Evidence for the transport of opsin in the connecting cilium and basal rod outer segment in rat retina: rapid-freeze, deep-etch and horseradish peroxidase labelling studies.

In order to clarify the pathway of opsin transport in the connecting cilium and basal rod outer segment, we examined rat rod cells by a rapid-freeze and deep-etch technique and also examined the uptake of horseradish peroxidase into isolated retina. The distribution of intramembrane particles on the P-face of the cilium indicated that the ciliary plasma membrane has similar opsin content to the basal rod outer segment plasma membrane. Dilated cisternae were detected below the stack of disk membranes at the basal rod outer segment in fresh retina. The fine structure of the P-face and true surface of these cisternae was identical to that of the disk membrane. Uptake of horseradish peroxidase was detected in the cisternae or in both cisternae and most basal disk, indicating that the cisternae are formed prior to the disk membrane. In the distal part of connecting cilium, we found axially oriented infoldings on the P-face of the plasma membrane, and subplasmalemmal tubules or cisternae adjacent and parallel to them. Such subplasmalemmal membranes were labeled by exogenous horseradish peroxidase, suggesting that the infoldings are invaginating plasma membrane. These results may indicate that opsin molecules are conveyed on the ciliary plasma membrane, and that this opsin-rich plasma membrane is internalized in the distal connecting cilium to form dilated cisternae, which subsequently change to the disk membranes.

Freeze-fracture analysis of the effects of intermediates of the phosphatidylinositol cycle on fusion of rough endoplasmic reticulum membranes

While searching for the identity of the effector of the putative GTP-binding protein involved in fusion of rough endoplasmic reticulum (RER) cell-free incubation conditions were found permitting fusion in a GTP-independent manner. Membrane fusion was obtained using medium required to study synthesis of phosphatidylinositol (PI). We now report on the effects of various co-factors and intermediates of the PI cycle on the interaction of rough microsomes. By freeze-fracture, fusion of rough microsomes was defined as the appearance of fracture-planes of membrane larger than those of unincubated membrane. Cytosine triphosphate (CTP, 3 mM) in the presence of 2 mM MnCl 2 was most effective in stimulating fusion. Guanosine triphosphate (GTP) at the same concentration, could substitute for CTP to stimulate fusion, ATP, ITP, UTP and guanosine 5′-[γ-thio]triphosphate (GTPγS) could not. When combined together in the same medium CTP potentiated the effect of GTP. Arachidonic acid (20 μg/ml) also stimulated fusion in the presence of MnCl 2. This led to the appearance of large fracture-planes of membrane with a heterogeneous distribution of intramembranous particles. Other saturated fatty acids at the same concentration did not stimulate fusion. Phosphatidylinositol (PI, 50 μg) and 2 mM MnCl 2 had a similar effect as arachidonic acid and MnCl 2 in stimulating fusion. The PI effect was largely augmented in the presence of CTP. Our results are consistent with the concept that metabolism of phospholipids may modulate GTP-dependent fusion of RER membranes.

A freeze-fracture study of isolates of the genusPhytomonas

Freeze-fracture replicas were used to analyze the organization of the plasma membrane of different isolates of the genusPhytomonas. Isolates fromEuphorbia hyssopifolia, E. characias, andE. pinea, P. francai fromManihot esculenta, andP. davidi were studied. We observed that each isolate showed a distinct distribution of intramembranous particles between the extracellular (E) and protoplasmic (P) faces.

Freeze-fracture evidence for a novel restricting junction at the blood-brain barrier of the cuttlefish Sepia officinalis.

The blood-brain barrier in the cuttlefish Sepia officinalis has been studied with the freeze-fracture technique. Previous thin-section electron microscopy showed that a restricting junction is formed between perivascular glial processes in microvessels and venous vessels, and between pericytes in arterial vessels; the restriction appeared not to be a classical zonula occludens or septate junction. In freeze-fracture replicas from brain optic and vertical lobe, endothelial cells, pericytes and perivascular glia could be recognized by their morphology and relation to the vascular lumen. In microvessels, endothelial and pericyte membranes showed sparse but uniform distribution of P-face intramembranous particles, with no particular particle aggregations. Perivascular glial membranes had a higher density of intramembranous particles but again no particle alignments characteristic of known restricting junctions were seen, although clusterings of intramembranous particles resembling gap junctions were present. In larger venous vessels, the perivascular glial layer showed a multilamellated organization, but again no arrays of intramembranous particles were detected, although this should be a favourable site for visualization of the restricting junctions. The walls of arterial vessels showed collagen deposits and cell processes with apparent intracellular myofilamentous profiles, but no intramembranous junctional particle arrays. It is concluded that the junctional zone observed in thin section electron microscopy is not associated with aligned aggregations of intramembranous particles detectable in freeze-fracture replicas, strengthening the evidence that this is a novel type of restricting junction.

Changes in the distribution of filipin-sterol complexes in the boar sperm head plasma membrane during epididymal maturation and in the uterus.

The distribution of filipin-sterol complexes (FSCs) and intramembranous particles (IMPs) in the plasma membrane of the late spermatid of the boar and of the sperm obtained from the epididymides, ejaculates, and uterus 2 hours after mating was examined by a freeze-fracture replica technique. In the late spermatid, the FSC density was found to be very low. A majority of the FSCs in the acrosomal plasma membrane (APM) appeared as protuberances on the E face in the epididymal, ejaculate, and uterine sperm. The density of the FSCs in the principal segment (PS) of the APM was 291 +/- 44 FSC/microns2 (mean +/- standard deviation, S.D.), 322 +/- 41 FSC/microns2 and 355 +/- 31 FSC/microns2 in the caput, corpus, and cauda epididymidis, respectively. In comparison with the cauda epididymal sperm, the FSC density gradually decreased in the PS of the ejaculated (277 +/- 39 FSC/microns2) and uterine sperm (243 +/- 50 FSC/microns2). The reduction was especially remarkable in the equatorial segment (ES), where the density of FSCs in ejaculated and uterine sperm decreased to about half and less than half of that in the cauda epididymal sperm, respectively. Large (13 nm) and small (8 nm) IMPs were distributed evenly and densely in the P face of the APM in the late spermatid, epididymal, and ejaculated sperm. In the uterine sperm, IMP-free areas were observed in the P face of the plasma membrane, a feature thought to represent one of the capacitation changes of the boar sperm.

Effects of retinyl acetate on surface morphology and intramembrane particle distribution in the plasma membrane of 10T1/2 cells

Scanning electron microscopy and freeze fracture electron microscopy were used to characterize membrane ultrastructural differences between parental, C3H/10T1/2, and carcinogen-initiated, INIT C3H/10T1/2, cells and treatments with retinyl acetate. The intramembranous particle distribution on the E-face was detected and quantitated by the methods of automated image analysis to obtain statistically meaningful numerical characteristics of intramembranous particle size and density. Subtle differences were found when no differences were apparent by light microscopy or by scanning electron microscopy. Initial retinyl acetate treatment caused a significant increase of the intramembranous particle size in parental cells. Intramembranous particle density increased for retinyl acetate treatment in parental and INIT cells and in INIT cells previously maintained but withheld from retinyl acetate. Intramembranous particle distribution analysis includes the interparticle distance of nearest neighbors and the randomness of the distribution by the differential density distribution function, which compares the observed sample to Poisson modified for particle size. These measures show that the three cell groups that have been treated with retinyl acetate have a more even distribution of intramembranous particles than was found for untreated parental cells. The relationship between the freeze fracture morphology and the biological responses to retinyl acetate treatment is discussed.

An electron-microscope and freeze-fracture study of the egg cortex of Brachydanio rerio

We have examined the cortex of the teleost (Brachydanio rerio) egg before and during exocytosis of cortical granules by scanning, transmission, and freeze-fracture electron microscopy. In the unactivated egg, the P-face of the plasma membrane exhibits a random distribution of intramembranous particles, showing a density of 959/micron2 and an average diameter of 8 nm. Particles over P- and E-faces of the membranes of cortical granules are substantially larger and display a significantly lower density. An anastomosing cortical endoplasmic reticulum forms close associations with both the plasma membrane of the egg and the membranes of cortical granules. Exocytosis begins with cortical granules pushing up beneath the plasma membrane to form dome-shaped swellings, coupled with an apparent clearing of particles from the site of contact between the apposed membranes. A depression in the particle-free plasma membrane appears to mark sites of fusion and pore formation between cortical granules and plasma membranes. Profiles of exocytotic vesicles undergo a predictable sequence of morphological change, but maintain their identity in the egg surface during this transformation. Coated vesicles form at sites of cortical granule breakdown. Differences in particle density between cortical granules and egg plasma membranes persist during transformation of the exocytotic profiles. This suggests that constituents of the 2 membrane domains remain segregated and do not intermix rapidly, lending support to the view that the process of membrane retrieval is selective (i.e., cortical granule membrane is removed).

Periaxonal ensheathment of lobster giant nerve fibres as revealed by freeze-fracture and lanthanum penetration

Sheath structure and permeability have been studied in the nerve fibres of lobster (Panulirus argus) walking limbs, in particular the individually ensheathed larger giant fibres, 100-150 microns in diameter, of which there are five or six in a peripheral bundle. They are easily distinguished and can be separated from neighbouring fibre bundles in which smaller giant axons (65-80 microns diameter) and many axons of much smaller diameter (5-15 microns) are ensheathed together. Each of the larger giant axons is enveloped by a Schwann cell layer outside of which is a multilayered sheath consisting of one-cell thick belts of flattened cells and interleaved zones of collagen fibrils and extracellular matrix. The cells in each belt lack basal lamina and, after freeze-fracture, as well as in thin sections, exhibit intercellular gap junctions and incomplete, fascia type, tight junctions; their most striking aspect is an exceedingly large number of exo-endocytic profiles. Permeability to lanthanum chloride in the bathing medium studied before or during fixation both in intact nerves and in nerves with surgically breached (slit) epineurium showed penetration of lanthanum tracer between the cells around the giant fibres, but the electron-dense tracer was excluded from the Schwann cell layer and the periaxonal space unless the epineurium had been slit. The extent of lanthanum diffusion was evaluated by transmission electron microscopy of thin sections and confirmed by X-ray microanalysis (EDAX) of comparable selected areas in such sections. The results indicate structural similarities but distinct permeability differences between the multilayered sheath surrounding the lobster giant axons and the vertebrate nerve perineurium. Other ultrastructural details provided by the freeze-fracture replicas concern the distribution of intramembrane particles in the axolemma and the Schwann and sheath cell membranes.

Intramembranous particle distribution and filipin binding in dysplastic canine retina

We examined dysplastic canine retina for changes in intramembranous particle (IMP) density and filipin binding to sterols. Differences in IMP density were identified in incipiently dysplastic fetal retina and also in the degree of filipin binding near the onset of the dysplastic process. The data suggest that there are temporal differences in IMPs and filipin-sterol complexes that may be related to the formation of retinal folds and disorganized dysplastic retina proliferation.

Morphology of mammalian sperm membranes during differentiation, maturation, and capacitation

The mammalian spermatozoon is a highly polarized cell whose surface membrane can be divided into five functionally, structurally, and biochemically distinct domains. These domains are formed during spermatogenesis, continue to be modified during passage through the epididymis, and are further refined in the female reproductive tract. The integrity of these domains appears to be necessary for the sperm to perform its function--fusion with the egg and subsequent fertilization. The domains can be identified morphologically by their surface contours and texture, the content, distribution, and organization of intramembranous particles after freeze-fracture, and by the density of surface and cytoplasmic electron-dense coatings in thin sections. By using a variety of labels that stain carbohydrates (lectins), lipids (filipin and polymyxin B), and monoclonal antibodies to specific membrane constituents, the biochemical composition of these contiguous membrane regions has also been partly elucidated. We review here what is known about the structure, composition, and behavior of each membrane domain in the mature sperm and include some information regarding domain formation during spermatogenesis. The sperm is an excellent model system to study the creation and maintenance of cell polarity, granule exocytosis, and fertilization. Hopefully this review will provide impetus for future studies aimed more directly at addressing the relationship of its morphology to its functions.

Changes in intercellular junctions during peripheral nerve regeneration in insects

Peripheral nerves of the adult cockroach have been cut and the changes in glial cells followed during the subsequent process of regeneration. After three to four weeks of regrowth, the severed tips of nerves were examined by freeze-fracture to assess the state of intercellular junctions between the perineurial sheath cells as well as the underlying glial cells. Both pleated septate and gap junctions were found in the immature state; their intramembranous particle (IMP) distribution was characteristic of junctions in the process of assembly, since the IMPs were irregularly and loosely arrayed in contrast with the parallel septate junctional IMP rows and gap junctional plaques found in the fully regenerated or control tissues. These junctional stages resembled those occurring in developing embryonic or metamorphosing insect tissues.

FREEZE‐FRACTURE STUDY OF THE SINGLE MEMBRANE BETWEEN HOST CELL AND ENDOCYTOBIONT IN THE DINOFLAGELLATESGLENODINIUM FOLIACEUMANDPERIDINIUM BALTICUM1

ABSTRACTThe dinoflagellatesGlenodinium foliaceumStein andPeridinium balticum(Levander) Lemmermann harbor a chrysophytic endocytobiont which is bounded by only a single membrane. This unique membrane is of particular interest because it could correspond to an intermediate stage in the evolution of “complex” plastids found in many Plastids of this type are surrounded by three or membranes instead of the usual two. With freeze‐fracture techniques, we show that the single membrane inP. balticumhas a pronounced polarity with respect to the distribution of intramembrane particles (IMPs) on the two corresponding fracture faces. The inner face exhibited more IMPs than the outer. We suggest that this stdedness identifies the separating membrane as the plasma membrane of the endocytobiont. A symbiontophoric vacuole with a separate membrane apparently is lacking. In the endocytobiosis ofG. foliaccum,the single membrane separating host and endocylobiont exhibits a symmetrical particle partition. Nevertheless, from the size distribution of the IMPs it appears likely that this membrane, too, corresponds to the plasma membrane of the symbiont.

Evidence for early changes in membrane structure during post‐harvest development of cut carnation (Dianthus caryophyllus L.) flowers

SUMMARYFreeze‐fracture electron microscopy and X‐ray diffraction have indicated that there are marked alterations in membrane structure prior to the initiation of increased ethylene production and petal inrolling during post‐harvest development of cut carnation flowers. In replicas of petals from partially open flowers (stage 1 of post harvest development), an even distribution of intramembranous particles, which is characteristic of normal membranes, was evident on the fracture plane of the plasma membrane. By contrast, endoplasmic reticulum membranes had begun to develop intramembranous particle‐free areas indicative of gel phase lipid domains. By stage 2 of development, when the flowers were fully open, large areas devoid of intramembranous particles were apparent on the plasma membrane as well. The presence of gel phase lipid was confirmed by wide‐angle X‐ray diffraction of isolated microsomes. Imaginations reflecting microvesiculation were also discernible in the fracture planes of plasma membrane at stage 2 of development and vesicles lying adjacent to the cell surface were evident in the cytoplasm. These observations collectively indicate that there are deteriorative alterations in membrane structure during the very early stages of post‐harvest development in cut carnation flowers. They also provide an explanation for the previous finding that carnation petals become leaky well before morphological symptoms of senescence such as petal inrolling are apparent.

Cold-induced ultrastructural changes in bull and boar sperm plasma membranes

The effect of low temperatures on the ultrastructure of the plasma membrane of bull and boar spermatozoa was investigated. Cold-induced changes in the organization of sperm plasma membrane components were demonstrated by the use of fast-freezing combined with freeze-fracture electron microscopy. This preparation technique ensures fixation without artifacts. At 38 °C bull and boar spermatozoa exhibited a random distribution of intramembranous particles over the plasma membrane of both head and tail. Exposure to 0 °C resulted in redistribution of the intramembranous particles: on the head and principal piece of bull spermatozoa and on the principal piece of boar spermatozoa, particle-free areas were observed, whereas on the boar sperm head, particle aggregates were present. The original particle distribution was restored upon rewarming of bull and boar spermatozoa to 38 °C, as well as after freezing and thawing of bull spermatozoa. Dilution of bull and boar semen into Tris-dilution buffer and Beltsville Thaw Solution-dilution buffer, respectively, could not prevent cold-induced redistribution of intramembranous particles. The observed particle reorganization upon cooling was interpreted as the result of lateral phase separation in the plasma membrane. Species-dependent differences in cold-induced ultrastructural changes were considered to be determined by lipid composition and asymmetry of the plasma membrane, and might be related to differences in cold resistance between species.

Endo-exocytotic images and changes in synaptic transmission induced by diamide at a cholinergic junction

Small tissue fragments excised from the electric organ of Torpedo marmorata were treated with diamide, a penetrating thiol oxidizing agent, until synaptic transmission was blocked. At this stage, we found an unexpected number of exo-endocytotic images in the presynaptic plasmalemma. Omega-shaped profiles, some of them coated, were seen in thin sections of fixed tissue and pits opened in the P-face of the presynaptic membrane in freeze-fracture replicas from rapidly-frozen preparations. Diamide-treated specimens were frozen at 1 ms time intervals before, during and after a single electrical stimulus. This stimulation did not result in a further increase in the density of presynaptic pits, not in any change affecting the density or size distribution of intramembrane particles. This result is in contrast with what is observed in untreated specimens where transmission of a nerve impulse is accompanied by a momentary rise in the number of large particles. The density of synaptic vesicles—especially that of a subpopulation of small size vesicles—transiently increased within the first 2 h of diamide treatment. During the first stages of intoxication, diamide prolonged the time course of postsynaptic potentials—both spontaneous and evoked—probably by altering the gating properties of receptors (acetylcholinesterase activity was not impaired). Later on, all evoked responses were blocked. The spontaneous transmitter release greatly increased, first in the form of quantal miniature potentials. These then subsided whereas a class of very small potentials was generated at a high frequency. Also under the action of diamide, 45 calcium progressively accumulated in the tissue but the number of synaptic vesicles containing calcium deposits was reduced. It is concluded that diamide causes a marked increase in the number of exo-endocytotic images in the presynaptic membrane, suppresses quantal but not subquantal release, and interferes with calcium sequestration in and extrusion from terminals.