Number Of Intramembrane Particles Research Articles

Endosymbiosis in Trypanosomatids as a Model to Study Cell Evolution

Some trypanosomatids harbor a symbiotic bacterium, which maintains a close association with the host, constituting an excellent model to study organelle origin and cellular evolution. Molecular data show that all endosymbiontharboring trypanosomatids are grouped together in a single phylogenetic branch. According to rRNA sequences, symbionts of different species are similar, being classified in the ß division of Proteobacteria, thus suggesting that a single evolutionary event gave rise to the symbiosis in the Trypanosomatidae family. The bacterium is enclosed by two unit membranes and presents a reduced peptidoglycan layer, which is essential for cell division and morphological maintenance. Regarding the protein composition, the number of intramembrane particles in the endosymbiont envelope is similar to that described for Gram-negative bacteria. Lipid analyses of purified endosymbionts show absence of sterols and indicate phosphatidylcholine as a major component of the envelope, as described in other intracellular bacteria. The presence of the endosymbiont is associated with distinct ultrastructural and physico-chemical alterations in the trypanosomatid and influences the protozoan interaction with the insect host and mammalian cells. Symbiont-containing trypanosomatids are able to infect and to replicate inside fibroblasts and macrophages, whose microbicidal activity was deactivated by HIV-1 infection. The symbiosis in trypanosomatids is characterized by intensive metabolic exchanges; the bacterium expresses enzymes and metabolic precursors that complete essential biosynthetic pathways of the protozoan. Conversely, the symbiont is capable of obtaining part of the required energetic molecules from the host glycosomes. Taken together data suggest that endosymbiosis in trypanosomatids represents an interesting model to study cell evolution.

Ultrastructure of rhoptry development in Plasmodium falciparum erythrocytic schizonts.

Prior to the separation of merozoites from the Plasmodium falciparum schizont, various stage-specific organelles are synthesized and assembled within each merozoite bud. The apical ends of the merozoites are initiated close to the ends of endomitotic spindles. At each of these sites, the nuclear membrane forms coated vesicles, and a single discoidal or cup-like Golgi cisterna appears. Reconstruction from serial sections indicates that this structure receives vesicles from the nuclear envelope and in turn gives off coated vesicles to generate the apical secretory organelles. Rhoptries first form as spheroidal structures and grow by progressive fusion of small vesicles around their margins. As each rhoptry develops, 2 distinctive regions separate within it, an apical reticular zone with electron-lucent areas separated by cords of granular material, and a more homogenously granular basal region. The apical part elongates into the duct, with evidence for further vesicular fusion at the duct apex. The rounded rhoptry base becomes progressively more densely packed to form a spheroidal mass, and compaction also occurs in the duct. Typically, one rhoptry matures before the other. Cryofractured rhoptry membranes show asymmetry in the sizes and numbers of intramembranous particles at the internally- and externally-directed fracture faces.

Freeze-fracture and cytochemical evidence for structural and functional alteration in the axolemma and myelin sheath of adult guinea pig optic nerve fibers after stretch injury.

Recent work in animal models of human diffuse axonal injury has generated the hypothesis that, rather than there being physical disruption of the axolemma at the time of injury, a pertubation of the membrane occurs, which leads, over time, to a dysfunction of the physiology of the axolemmal. This dysfunction is posited to lead to a disruption of ionic homeostasis within the injured axon, leading to secondary axotomy some hours after the initial insult. We decided to test the hypothesis that membrane pump/ion channel activity or function is compromised and this would be reflected in structural changes within the axolemma and myelin sheath. We used freeze fracture and cytochemical techniques to provide evidence for change in membrane structure and the activity of membrane pumps after nondisruptive axonal injury in the adult guinea pig optic nerve. Within 10 min of injury, structural changes occurred in the distribution and number of intramembranous particles (IMPs) in the internodal axolemma. By 4 h, there was novel labeling for Ca-ATPase membrane pump activity at the same site. There was loss of IMPs from the nodal axolemma extending over several hours after injury. There was loss of both membrane pump Ca-ATPase and p-nitro-phenylphosphatase (p-NPPase) activity of the node. There was loss of ecto-Ca-ATPase activity but increased labeling for p-NPPase activity at sites of dissociation of compacted myelin. Quantitative freeze-fracture demonstrated statistically significant changes in membrane structure. We provide support for the hypothesis that structural and functional changes occur in the axolemma and myelin sheath at nondisruptive axonal injury.

A new organotypic culture method to study the actions of steroid hormones on the nervous system.

A new organotypic culture method for growing slices of nervous system tissue, based on the use of hyaluronic acid as a growth supporting milieu, is described. This method allows cultures derived from either fetuses or newborns to grow and develop with markedly reduced amounts of added serum. Organotypic cultures from fetal rat hypothalamus were exposed to 17 beta estradiol and compared to control cultures exposed to the ethanol vehicle. When exposed to estradiol, cultures showed an outgrowth of thick nerve fibers that was accompanied by an elevation in the number of microtubules present in the neuronal processes, an increment in the number of synapses, and an increased morphological differentiation of synaptic terminals. Freeze-fracture analysis of neuronal membranes from estradiol-treated cultures revealed a significant increase in the number of exoendocytotic images and a decrease in the number of intramembranous particles. Estradiol's effects parallel those found in in vivo studies, indicating that hyaluronic acid-based organotypic cultures represent an appropriate model to study hormonal influences on the developing nervous system.

Morpho-functional modifications of human syncytiotrophoblast plasma membrane during pregnancy induced hypertension.

A decrease of Na+/K(+)-ATPase activity has been reported in syncytiotrophoblast plasma membrane (SPM) obtained from pregnancy induced hypertensive (PIH) women. The aim of the present work was to verify if the reported modifications in activity are due to a decreased number of enzymatic molecules or to a conformational change of the enzyme itself. Morphological studies were performed in order to better understand the relations between the enzymatic protein and the lipid bilayer. Kinetic studies were also performed. SPM obtained from PIH showed: i) an increased affinity of Na+/K(+)-ATPase for ouabain binding, ii) a significant change in the maximum velocity of the enzyme, iii) a higher distribution factor (DF) of intramembrane particles (IMPs) in the exoplasmic face of the membrane, iv) a decreased mean diameter of IMPs both in the protoplasmic and exoplasmic faces, v) a decreased number of IMPs in the exoplasmic face. In conclusion, a conformational modification seems to be at the basis of the decreased Na+/K(+)-ATPase activity during PIH as suggested by binding, ultrastructural and kinetic data herein reported.

The role of estradiol and progesterone in phased synaptic remodelling of the rat arcuate nucleus

During the estrous cycle there is a phasic synaptic remodelling in the hypothalamic arcuate nucleus, consisting in a loss and regain of axo-somatic synapses during the 48 h period between the morning of proestrus and the morning of metestrus. Synaptic changes are accompanied by cyclic modifications in the number of intramembrane particles in the plasma membrane of arcuate neuronal somas. To test the effect of the ovarian steroids on arcuate axo-somatic synapses we treated castrated females either with oil vehicle, 17β-estradiol, progesterone, or a combination of estradiol and progesterone, and observed them for 48 h. The number of axo-somatic synaptic profiles showed a 33% fall by 24 h after estradiol treatment and returned to control levels by 48 h. The effect of estradiol on axo-somatic synapses was accompanied by a marked and reversible modification of the number of intramembrane particles in the plasma membrane of arcuate neuronal somas. Progesterone alone did not affect the number of axo-somatic synaptic profiles nor the number of intramembrane particles, but when administered together with estradiol, blocked the effects of estradiol on neuronal membrane and synapses.

Verapamil reverses the ultrastructural alterations in the plasma membrane induced by drug resistance

Two P388 cell sublines with different levels of resistance to daunomycin (DNM), P388/20 and P388/100 cells (∼ 20- and 100-fold resistance, respectively), undergo a significant (∼ 2-fold) increase in the number of intramembrane particles (IMPS) present at their plasma membrane, as compared to that exhibited by the parental, drug-sensitive P388 (P388/S) cell line. Regardless of the level of resistance, incubation of drug-resistant cells with verapamil, a well known reverting agent of anthracycline resistance, restores the morphology of the plasma membrane in these cells, yielding a pattern in which the number and size distribution of IMPs at both leaflets of the bilayer, become undistinguishable from those displayed by drug-sensitive celIs. Furthermore, verapamil did not affect the ultrastructural organization of the plasma membrane of drug-sensitive cells. It is possible that the alterations in the structural organization of the plasma membrane of the antineoplastic-resistant tumor cells, might represent a reliable ‘marker’ for early diagnosis of drug resistance.

Early changes in plasma membrane in vitro by treatment with chemical carcinogen.

To examine early changes in the cell membrane caused by treatment with 9.10-dimethyl-1.2-benzanthracene (DMBA), rat lingual epithelium in vitro was observed by means of electrophysiolo-gical, freeze-fracture and whole mount electron microscopic methods. Membrane potential showed a transient decrease at 6 h after the treatment but returned to almost the same level as that of the control group 1 day later. Membrane resistance decreased rapidly at 6 h after the treatment but increased at 12 h. Transient decrease of membrane resistance and membrane potential occurred prior to the decrease in coupling ratio at 6 h after treatment with DMBA. In freeze-fracture replicas, after 6 h of treatment with DMBA, the number of intramembranous particles (IMPs) was less than that of the control. This decrease was especially pronounced on the P-face. After 12 h treatment with DMBA, the number of IMPs increased by 20-40 % more than the control value. This increase was especially noticeable on the E-face, which was 70 % of the control value. At 3 days and thereafter, the number of IMPs decreased again and remained approximately the same as that of the control, though the Eface values remained larger than those of the control. Treatment with DMBA had a marked effect on the cytoskeletal organization. Treatment with DMBA caused morphologic changes as early as 6 h. These early changes might represent triggers for subsequent metabolic cellular abnormalities. The affected cells which were treated with DMBA appear to differ from normal cells through accelerated-abnormal cell membrane and cytoskeletal turnover.

Rabbit corneal epithelial wound repair: tight junction reformation.

Previous studies have shown that the corneal epithelium will close a limbus to limbus scrape wound in four to five days, but requires 10 days to become firmly attached to the stroma. In order to determine if the restoration of the corneal epithelial barrier follows a similar sequence, we have used freeze-fracture to study tight junction reformation in a rabbit epithelial wound model. Dutch-belted rabbit corneal epithelium was removed with a limbus to limbus scrape wound and sampled from 0 to 30 days post-wounding. A minimum of 3 animals from each time point were processed for electron microscopy and freeze-fracture. Freeze-fracture showed that the cells at the wound margin had a reduction in the number of intramembrane particles on their apical surface. In areas adjacent to the wound edge, fragments of tight junctions were first observed on two days post-wounding specimens. The junctions became progressively more complex in the area behind the wound edge until wound closure at four days when the junctions were also present in the central region of the cornea. The maturation of the junctions continued and at five days after surgery they resembled control junctions. This sequence suggests that the the establishment of morphologically mature tight junctions may be necessary before the corneal epithelium can firmly reattach to the stroma.

Monomeric state and Ca2+ transport by sarcoplasmic reticulum Ca2(+)-ATPase, reconstituted with an excess of phospholipid.

The structural basis for Ca2+ transport was examined in vesicles reconstituted with an excess of phospholipid by a cholate dialysis procedure. Unincorporated protein and vesicles with a relatively high protein content were removed by sucrose density centrifugation (3-12%), leaving a fraction of lipid-rich vesicles (lipid to protein weight ratio 800-900:1) with a high coupling ratio (1.0) and transport capacity (25 mumol/mg protein, after Ca-phosphate loading). Freeze-fracture analysis showed that the reconstituted vesicles had a remarkably narrow size distribution (diameter 794 +/- 77 A (S.D.], suitable for stereological analysis. Intramembranous particles were dispersed and occurred with a low frequency in the fractured shells, also before sucrose fractionation. It was calculated that the number of intramembranous particles corresponded to the number of Ca2(+)-ATPase polypeptide/vesicle. A ratio of unity between particles and polypeptide chains was also obtained from the density of particle distribution on flat surfaces of fused vesicles, prepared by sucrose fractionation. The size of the particles formed a broad distribution, having a peak value around 60-67 A, both in the reconstituted preparation and sarcoplasmic reticulum vesicles. No evidence for protein-protein interactions was found in chemical cross-linking experiments. It is concluded that the intramembranous particles in the reconstituted preparations are referable to monomeric Ca2(+)-ATPase which is capable of transporting Ca2+ inside the vesicles. The implications of the observations for the associational state of Ca2(+)-ATPase at high protein concentration are considered in relation to previous ultrastructural investigations of membranous Ca2(+)-ATPase in native and two-dimensional-crystalline forms.

Estradiol induces rapid remodelling of plasma membranes in developing rat cerebrocortical neurons in culture

Exo-endocytotic images and intramembrane particles were quantitatively assessed in freeze-fracture replicas from the plasma membrane of dispersed fetal rat cortical neurons (day 16 gestation) grown for 24 days in culture. The addition of 10 −10 M 17β-estradiol to the culture medium resulted in a significant increase in the numerical density of exo-endocytotic images within 1 min. A further increase of the number of exo-endocytotic images associated to a significant decrease in the number of intramembrane particles was observed in cells exposed for 10 min to 17β-estradiol. Similar results were observed when the cells were exposed to 17β-estradiol for 17 days. No effects on exo-endocytotic images and intramembrane particles were observed when 17α-estradiol was added, instead of 17β-estradiol, to the cultures. These results indicate that physiological levels of 17β-estradiol can have rapid effects upon the ultrastructure of the neuronal membrane of developing cerebrocortical neurons.

Plasma Membrane Domains and the Site of Sperm Entrance in Discoglossus pictus (Anura) Eggs: (amphibians/egg/plasma membrane/freeze-fracture).

Freeze-fracture quantitative analysis reveals three different plasma membrane (PM) domains in the unfertilized egg of the anuran Discoglossus pictus. One of these is specific to the sperm entrance site (D1). where the plasma membrane shows a larger number of intramembranous particles (IMP) than the rest of the egg surface. Such an increment is due to a markedly higher number of the IMPs anchored to the P-face. The two other domains (D2 and D3) are characterized by a lower IMP density at the P-face with respect to D1. The IMP density decreased within 10 min after fertilization by about 33% in all domains observed, probably due to the insertion of new membrane through exocytosis. The possibility that the IMPs located in D1 may represent putative plasma membrane proteins playing a role in sperm-egg interaction and/or in egg activation is discussed.

Unmasking of cryptic natural killer (NK) cell recognition sites on chronic lymphocytic leukemia lymphocytes.

The sensitivity of chronic lymphocytic leukemia (CLL) lymphocytes to attack by natural killer (NK) cells has remained questionable. To clarify this issue, freshly isolated lymphocytes of 37 patients with B-CLL, five with WDLL and two with HCL, were tested with a standard cytotoxicity assay with NK cells from normal donors. All these targets were resistant to cytolysis by the effectors. Freeze-fracture analysis of CLL cell plasma membranes revealed that they have a larger number of intramembranous particles (IMP) associated with the external leaflet (E-face) than have normal lymphocytes. Unlike other neoplastic cells, exposure of CLL lymphocytes to phorbol esters or treatment with neuraminidase did not render them vulnerable to attack by NK cells, nor did 5 days of culture have an effect. Incubation of CLL lymphocytes with anti-Ig-mu (24-72 hr) or with 0.1% pepsin (15 min) resulted in 15% and 27% cytolysis, respectively. B-lymphocytes from the blood of healthy donors were not killed when treated similarly: These data establish that freshly isolated B-CLL lymphocytes are resistant to NK cytolysis but that in contrast to normal B-cells, they possess cryptic NK-recognition structures, which may be uncovered by surface modulation.

Electrophysiology and ultrastructure of cultured human proximal tubule cells

The present study was undertaken to determine if cultures of human proximal tubule cells would retain in vivo properties inherent to this segment in the intact nephron. Ussing chamber studies demonstrated that these cultured cells generated transepithelial potential differences of approximately -2.0 mV and resistances of 0.310 K omega.cm2, supporting the concept that the proximal tubule is a "leaky" epithelium. The electrical properties did not change when the cells were exposed to amiloride (10(-4) M), but did respond to acetazolamide (10(-4) M), consistent with responses known to occur in proximal tubules. Ultrastructural analysis of these cells demonstrated features indicative of proximal tubule cells. When grown on permeable supports, where apical and basolateral growth medium compartments were maintained separate from each other, the cells were noted to undergo increased differentiation with morphological evidence of cell polarity. Freeze fracture analysis demonstrated well-formed tight junction strands and segregation of unique numbers of intramembranous particles in apical, lateral, and basal membranes. The replicas also demonstrated the presence of aggregates though to represent gap junctions, structures which occur exclusively in the proximal segment of the human nephron. These observations provide evidence that this human cell culture model originates from the human proximal tubule and retains, in culture, many of the properties associated with proximal tubule cell function and structure in vivo.

Chronic ethanol consumption affects filipin-cholesterol complexes and intramembranous particles of synaptosomes of rat brain cortex.

To assess the effect of ethanol on the planar distribution of cholesterol as well as on the surface architecture of presynaptic terminals of rats, synaptosomes isolated from cerebral cortex of rats chronically exposed to alcohol were incubated with filipin, a cytochemical marker for beta-hydroxycholesterol, and analyzed using both conventional (qualitative and quantitative) and freeze-fracture electron microscopy. Synaptosomes incubated in the absence of filipin were used as cytochemical controls. Biochemical determination indicates a 12% increase of cholesterol in synaptosomal membranes from alcohol treated rats. This increase was confirmed by a significant increment in the number of filipin-cholesterol complexes. Synaptosomes of treated rats showed a reduction in the total number of synaptic vesicles (SV) as well as a decrease in the density and total number of intramembranous particles (IMP) per synaptosome. In control rats, most synaptosomal IMP were distributed in clusters whereas in those of rats exposed to alcohol they were distributed at random. These changes in distribution of IMP were also observed in presynaptic terminals analyzed "in situ." These findings indicate that ethanol acts on the presynaptic terminals. The variations in cholesterol content as well as in the density and distribution of IMP appear to be related to alcohol-induced changes in the physicochemical properties of components of the synaptosomal membrane.

Freeze-fracture studies on Pneumocystis carinii. III. Fine structure of the precyst and the cyst.

The ultrastructure of the precyst and cyst of Pneumocystis carinii was investigated by the freeze-fracture method. The most interesting point in this study is the alteration in the number of intramembranous particles (IMPs) of the plasma membrane during the formation of intracystic bodies. The IMP density of delimiting membrane originating from the precyst plasma membrane, which later becomes the plasma membrane of the intracystic body, was low. In contrast, that of the plasma membrane of the intracystic body was as high as that of a typical trophozoite. These results suggest that the protein content of the plasma membrane increases rapidly when the delimiting membranes fuse and intracystic bodies are organized. The plasma membrane of the ruptured cyst, on the other hand, showed lower density and heterogeneous distribution of IMPs. Since all other stages of P. carinii showed homogeneous distribution of IMPs, the denaturation of the plasma membrane after excystation of intracystic bodies is indicated.

Effects of monensin and ouabain on the cationized ferritin uptake in the marginal cells of the guinea pig stria vascularis

The uptake of cationized ferritin (CF) by the empty vesicles in the apical cytoplasm of the marginal cells of the stria vascularis and the effects of monensin and ouabain on the CF uptake were electron microscopically and morphometrically studied. When 20μg/ml CF with high (145mM) or low (15mM) Na ion concentrations were applied to the cochlea for 10 min, CF particles were found in clusters, scattered on the free-surface plasma membrane facing the endolymph, and in the caveolae of the marginal cells. They were also observed in the empty vesicles, coated vesicles and multivesicular bodies, but not in the Golgi apparatus, endoplasmic reticulum and intercellular space between cells constituting the stria vascularis. The P-face profile of the vesicles was similar to that of the caveolae with regard to the number of intramembranous particles. Monensin, which inhibits receptor-mediated endocytosis, reduced the number of CF-containing vesicles under high sodium ion concentration. These findings suggest that the empty vesicles are associated with the free-surface plasma membrane, and that they ate involved in the efficient exchange of ions between the endolymph and the marginal cells. Ouabain repressed the uptake of CF, lowering the endocochlear potential and altering the normal intracellular environment of the marginal cells. Therefore, the dynamics of the empty vesicles may be related with the sodium-pump in the basolateral infolded plasma membrane of these cells.

Daily changes of structure, function and rhodopsin content in the compound eye of the crab Hemigrapsus sanguineus.

The compound eye of the crab hemigrapsus sanguineus undergoes daily changes in morphology as determined by light and electron microscopy, both in the quantity of chromophore substances studied by HPLC and in visual sensitivity as shown by electrophysiological techniques. 1. At a temperature of 20 degrees C, the rhabdom occupation ratio (ROR) of an ommatidial retinula was 11.6% (maximum) at midnight, 8.0 times larger than the minimum value at midday (1.4%). 2. Observations by freeze-fracture revealed that the densities of intra-membranous particles (9-11 nm in diameter) of rhabdomeric membrane were ca. 2000/microns 2 and ca. 3000/microns 2 for night and daytime compound eyes, respectively. 3. Screening pigment granules migrated longitudinally and aggregated at night, but dispersed during the day. Reflecting pigment granules migrate transversally in the proximal half of the reticula layer i.e. cytoplasmic extensions containing reflecting pigment granules squeeze between neighbouring retinula cells causing optical isolation (Fig. 4). Thus the screening pigment granules within the retinula cells show longitudinal migration and radial movement so that the daytime rhabdoms are closely surrounded by the pigment granules. 4. At 20 degrees C, the total amount of chromophore of the visual pigment (11-cis and all-trans-retinal) was 1.4 times larger at night than during the day i.e. 46.6 pmol/eye at midnight and 33.2 pmol/eye at midday. Calculations of the total surface area of rhabdomeric membrane, total number of intra-membranous particles in rhabdomeric membrane and the total number of chromophore molecules in a compound eye, indicate that a considerable amount of chromophore-protein complex exists outside the rhabdom during the day. 5. The change in rhabdom size and quantity of chromophore were highly dependent on temperature. At 10 degrees C both rhabdom size and amount of chromophore stayed close to daytime levels throughout the 24 hours. 6. The intracellularly determined relative sensitivity of the dark adapted night eye to a point source of light was about twice as high as the dark-adapted day eye. Most of the increase in the sensitivity is attributed primarily to the effect of reflecting pigment migration around the basement membrane and, secondarily, to the changes in the amount and properties of the photoreceptive membrane. The results form the basis of a detailed discussion as to how an apposition eye can function possibly as a night-eye.

Changes in the plasma membrane of regenerating protoplasts of Candida albicans as revealed by freeze-fracture electron microscopy.

Modifications occurring in the plasma membrane and their relationship to newly synthesized microfibrils were examined in regenerating protoplasts of Candida albicans by freeze-fracture electron microscopy. Freshly prepared protoplasts showed no residual wall material, and long invaginations covered the surface of the plasma membrane. Analysis of the external face (E-face) of the plasma membrane showed a significant decrease in the number of intramembranous particles (IMP) in comparison with the original cells. After 40 min incubation in regeneration medium, newly synthesized microfibrils which seemed to originate from protrusions in the plasma membrane were observed. The plasma membrane showed important modifications with respect to IMP. After 3 h 45 min, the cells were covered by an abnormal wall which showed isolated fibrils partially embedded in the matrix material. The plasma membrane of these partially regenerated protoplasts was similar to that of original cells. After 8 h, regeneration of the protoplasts seemed to be complete as no differences from the original cells were detected in the plasma membrane or the wall. Calcofluor white altered the deposition of wall polymers during regeneration, but did not modify the plasma membrane of the protoplasts.

Loss of intercalated membrane particles by treatment with phorbols.

Because brief exposure to phorbol esters renders normal cells vulnerable to deformation and cytolysis by lymphocytes, it was postulated that these tumor promoters might cause a hitherto unrecognized physical alteration in membrane architecture. To investigate this possibility, four tissue culture cell lines (K-562 erythroleukemia cells, melanoma cells, N1121 adult fibroblasts, and normal fetal fibroblasts) and three blood cell types (lymphocytes, monocytes, and platelets) were subjected to freeze-fracture analysis before and after brief treatment with phorbol myristate acetate. Phorbol myristate acetate caused a 50% reduction of intramembranous particles associated with the external leaflet (E face) of the plasma membrane of every cell except platelets. In contrast, no change in size or number of intramembranous particles associated with the protoplasmic membrane leaflet (P face) was evident. Since the platelet membrane is known to be turned "inside out," as regards the partition coefficient of the intramembranous particles, the disparity between the results obtained with platelets and other cells may serve to determine the nature of intramembranous particles affected by phorbols. Also, since phorbols affect primarily glycolipids and/or glycoproteins anchored in the external membrane leaflet, these findings may provide a useful tool for future exploration of membrane structure.