Freeze-fracture study of the trophozoite and the cyst of Entamoeba histolytica

The fine structure of the trophozoite and cyst of Entamoeba histolytica from the stool of a patient was compared using the freeze-fracture method. The intramembranous particles (IMP's) were heterogeneously distributed on the plasma membrane of the trophozoite and their density was 1139 +/- 105/micron 2 on the P face. Particle-rich depressions and linear particle arrays, reported by other investigators on cultured trophozoites, were also observed on the P face while on the E face such special particle arrangement was not recognized. Particle-free, small protrusions were frequently observed on the P face of the trophozoite membrane. The existence of these protrusions is a new finding. In the cyst, the IMP's were also distributed heterogeneously on both the P and E faces of the plasma membrane. The density of the IMP's, however, was much lower than in the trophozoite: 6 +/- 2/micron 2 on the P face and averaging less than 1/micron 2 on the E face. In freeze-fracture images, the plasma membrane of the cyst showed a variety of configurations from smooth to uneven or ridged surfaces. These morphological alterations of the plasma membrane may be attributed to the aging of the cyst. The thick wall of the cyst had a filamentous tri- or tetra-lamellar structure. The cytoplasm of the cyst was similar in structure to that of the trophozoite and the diameter of the nuclear pores was equal in both trophozoites and cysts.     

Freeze-Fracture Localization of Filipin-Sterol Complexes in Plasma- and Cyto-Membranes of Pneumocystis carinii1

The polyene antibiotic, filipin, was used as the probe for demonstrating sterols in the freeze-fractured plasma- and cytomembranes of Pneumocystis carinii. The distribution of filipin-sterol complexes was homogeneous on the plasma membrane throughout all developmental stages from trophozoite to cyst; however, the density of the complexes gradually decreased with the progress of development. In the trophozoite, the density of the complexes was 485 ± 42/μm² on the P face and 341 ± 27/μm² on the E face. It was 249 ± 50 on the P face and 132 ± 48 on the E face in the precyst and 138 ± 24 and 59 ± 20, respectively, in the cyst. The membranes of nucleus, mitochondria, and small round bodies showed more or fewer complexes while no complexes were found in the membranes of one endoplasmic reticulum. In nuclear and mitochondrial membranes, some small scattered clusters of complexes were observed. Two types of vacuoles were distinguished: one having many complexes in its membrane and the other having none at all.     

Freeze-fracture studies on Pneumocystis carinii. I. Structural alteration of the pellicle during the development from trophozoite to cyst

Pneumocystis carinii has generally been distinguished in three developmental stages, namely, trophozoite, precyst and cyst. The fine structure of the pellicle--the plasma membrane and the outer layer existing outside this plasma membrane--of each stage was studied by freeze-fracture technique. By this technique, P. carinii was cleaved through the cytoplasm or through the hydrophobic region of the plasma membrane, and the cross-fractured face of the outer layer was revealed on the replicas. The outer layer, which is electron-dense in the thin section, consisted of numerous fine granules about 15 nm in diameter in freeze-fracture images, whereas the electron-lucent middle layer which appeared in the precyst and cyst was less granular. Measurement of the intramembranous particles (IMP) also was carried out. The number of IMP per square micrometer of the plasma membrane of the trophozoite was 1,512 +/- 125 on the P face and 417 +/- 44 on the E face. In the precyst, the IMP density decreased, and 1,037 +/- 56 on the P face and 262 +/- 22 on the E face. In the cyst, it further decreased, nd 875 +/- 59 and 150 +/- 20 respectively. It is generally assumed that the density of IMP is related to the physiological activity of the cell membrane, so that the present results obtained in P. carinii suggest that the trophozoite is the most active stage, and that metabolic activity of the pellicle gradually decreases with the progress of development to the precyst then to the cyst.     

Freeze-Fracture Study of the Trophozoite and the Cyst of Entamoeba histolytica

ABSTRACT The fine structure of the trophozoite and cyst of Entamoeba histolytica from the stool of a patient was compared using the freeze-fracture method. The intramembranous particles (IMP's) were heterogeneously distributed on the plasma membrane of the trophozoite and their density was 1139 ± 105/μm² on the P face and 27 ± 9/μm² on the E face. Particle-rich depressions and linear particle arrays, reported by other investigators on cultured trophozoites, were also observed on the P face while on the E face such special particle arrangement was not recognized. Particle-free, small protrusions were frequently observed on the P face of the trophozoite membrane. The existence of these protrusions is a new finding. In the cyst, the IMP's were also distributed heterogeneously on both the P and E faces of the plasma membrane. The density of the IMP's, however, was much lower than in the trophozoite: 6 ± 2/μm² on the P face and averaging less than 1/μm² on the E face. In freeze-fracture images, the plasma membrane of the cyst showed a variety of configurations from smooth to uneven or ridged surfaces. These morphological alterations of the plasma membrane may be attributed to the aging of the cyst. The thick wall of the cyst had a filamentous tri- or tetra-lamellar structure. The cytoplasm of the cyst was similar in structure to that of the trophozoite and the diameter of the nuclear pores was equal in both trophozoites and cysts.     

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.     

Reduction of intramembranous particles in the periacrosomal plasma membrane of boar spermatozoa during in vitro capacitation: a statistical study

Membrane remodeling in the periacrosomal plasma membrane (PAPM) of boar spermatozoa during incubation in capacitation medium was examined by the freeze-fracture technique. In the preservation medium (PM) group, the major small (about 8 nm) intramembranous particles (IMP) and the minor large (> 10 nm) IMP were distributed evenly in the PAPM. The IMP-free area increased during capacitation. To correct the IMP-free area, arithmetically redistributed (ARD)-IMP density was used for statistical analysis. In the PM group, the mean density +/- SD of large IMP was 379 +/- 64 and 266 +/- 58/microm2, and that of small IMP was 1450 +/- 155 and 672 +/- 252/microm2 in protoplasmic (P) and external (E) faces, respectively. During capacitation, the significant (P < 0.01) reduction of large IMP density was encountered only in the E face of a few incubation groups, while that of the small IMP density occurred in the P face by 2 h. Consequently, reduction of the total IMP density of both faces was not significant in the large IMP, but it was significant (P < 0.01) in the small IMP. One-fifth of the total small IMP density reduced by 2 h. Filipin-sterol complexes (FSC) were numerous in the PAPM, and FSC-free areas also increased during capacitation. The mechanism of IMP-free area formation and the behavior of the small IMP in the PAPM during capacitation were discussed in relation to membrane stability.     

Freeze-fracture study of Blastocystis hominis

The ultrastructure of Blastocystis hominis was investigated by the freeze-fracture method. Freeze-fracture replicas of the membranes of B. hominis and its organelles were studied with special regard to the density and distribution of the intramembranous particles (IMP's). On all membrane replicas, the concentration of IMP's on the protoplasmic face (P face) invariably was greater than on the exoplasmic face (E face). On the P face, IMP's were heterogeneously distributed in dense aggregates, alternating with particle-free, smooth surface areas. Occasionally, small depressions and protrusions were observed in these areas. On the membrane of the central vacuole, invaginations into the vacuole were frequently observed within the smooth surface regions. Since most of the granules in the central vacuoles had no IMP's, it seems likely that the intervacuolar granules were formed from these invaginations of the vacuole membrane. The width of the intermembrane space between the inner and outer membranes of the nuclear envelope was uneven, with regions of relative narrowness interspersed with regions of expansion. Nuclear pores were localized within the narrow portions of this space. A nucleus, apparently in the process of dividing, was observed enclosed within an intact outer membrane. Division of the outer membrane would then result in the formation of two discrete nuclei.     

The ultrastructure of the cyst wall of Giardia lamblia

Giardiasis is the most common human protozoal infection. In their cystic phase, giardias are protected from the environment by a filamentous cyst wall made up of carbohydrates, proteins, and by two outer membranes separated from the plasma membrane of the parasite by a peripheral space. The present transmission electron microscope observations of G. lamblia cysts of human origin suggest that the extracellular peritrophic space originates from the growth, elongation, and fusion of large cytoplasmic vacuoles. As the large clear vacuoles grew in size, flattening against the inner face of the plasma membrane, they formed a single vacuole that surrounded the body of the parasite, eventually forming two outer membranes. In mature Giardia cysts, the original plasma membrane of the trophozoite becomes the outermost membrane of the cyst wall (CM1). The large vacuoles form a second membrane surrounding the cyst (CM2), and also form a third membrane (CM3), that becomes the new plasma membrane of the trophozoite. During excystation CM1 and CM2 attach to each other and fragment, leaving abundant membrane residues in the peritrophic space. Knowledge of the biochemical composition and functional properties of the complex outer membranous system of G. lamblia cysts here described will be of use to understand the survival of Giardia cysts in the environment, a major factor responsible for the high prevalence of giardiasis worldwide.     

Freeze-Fracture Study of Blastocystis hominis1

The ultrastructure of Blastocystis hominis was investigated by the freeze-fracture method. Freeze-fracture replicas of the membranes of B. hominis and its organelles were studied with special regard to the density and distribution of the intramembranous particles (IMF's). On all membrane replicas, the concentration of IMF's on the protoplasmic face (P face) invariably was greater than on the exoplasmic face (E face). On the P face, IMP's were heterogeneously distributed in dense aggregates, alternating with particle-free, smooth surface areas. Occasionally, small depressions and protrusions were observed in these areas. On the membrane of the central vacuole, invaginations into the vacuole were frequently observed within the smooth surface regions. Since most of the granules in the central vacuoles had no IMF's, it seems likely that the intervacuolar granules were formed from these invaginations of the vacuole membrane. The width of the intermembrane space between the inner and outer membranes of the nuclear envelope was uneven, with regions of relative narrowness interspersed with regions of expansion. Nuclear pores were localized within the narrow portions of this space. A nucleus, apparently in the process of dividing, was observed enclosed within an intact outer membrane. Division of the outer membrane would then result in the formation of two discrete nuclei.     

Filipin-cholesterol complexes in plasma membranes and cell junctions of Tenebrio molitor epidermis

The polyene antibiotic filipin combines with cholesterol in membranes to form complexes that are readily identifiable in the electron microscope. The distribution of filipin-cholesterol (FC) complexes is most easily studied by freeze-fracture. Larval epidermis of Tenebrio molitor (Insecta, Coleoptera) was maintained in vitro for 48 hr, since the electrophysiological properties of the cells are best characterized under these conditions. The cells were fixed in buffered 3.0% glutaraldehyde at RT for 15 min, transferred to fresh fixative containing 1% DMSO and filipin (final concentration; 0.5 mg/ml) for 3 hr RT. Control cells were treated in fixative containing 1% DMSO only. In freeze fracture replicas, FC complexes appear on the plasma membrane as large circular protrusions measuring 26.5 +/- 6.8 nm (x +/- s.d.) n = 50, in diameter and 17.1 +/- 2.8 nm, n = 50, in height and 11.7 +/- 2.6 nm, n = 25, in depth. Protrusions are about two times more frequent on the E face while pits are several times more frequent on the P face. FC complexes are most abundant (greater than 50/mu m2) on the basal membrane surface of the cells but are excluded from regions of hemidesmosomal plaques that anchor the cells to the basal lamina. FC complexes are also abundant on the apical surfaces of the cells where cuticle secretion occurs. In the lateral regions below the junctional belt, FC complexes are less numerous but often appear to increase in frequency in a graded fashion away from the junctional region. The septate junctions are relatively free of FC complexes except in regions where they open to form islands. These islands often contain gap junctions but the FC complexes rarely invade the particle domains of the gap junctions. Single FC complexes were seen in three out of a total of 97 gap junctions. Exposure of the epidermis to 20-hydroxyecdysone for 24 hr in vitro did not induce the appearance of FC complexes within the cell junctions.     

A freeze fracture study of the developing tegumental outer membrane of Schistosoma mansoni

The freeze fracture technique has been used to quantify changes in the integral components of the double outer membrane of Schistosoma mansoni during the 6-week period of development within the mouse. The intramembraneous particle (IMP) density on the P1 face begins to rise within 6 h of host penetration, reaches a maximum at day 4 and then falls rapidly after day 9, so that it is at a low level between 3 and 6 weeks. The E1 face IMP density follows the same course as that of the P1 face except that maximum particle density is recorded on day 1 and the counts begin to fall on day 5. The IMP density on the P2 face remains at a consistently low level throughout development. The E2 face IMP density rises gradually to a peak at day 4, when the parasites have migrated to the lungs, and remains thereafter at a similar level, so that by 6 weeks the E2 face has a higher IMP density than the other three fracture faces. The E2 face IMP show a marked increase in size on day 4. Morphological studies indicate that a different type of inclusion body makes a transient appearance in the tegument of the lung worms, and immunocytochemical techniques show the lung worms to be nonimmunogenic. It is suggested, therefore, that the E2 face IMP may represent complexes of parasite antigens and acquired host antigens. The tegumental membranes of cultured specimens have also been examined by freeze fracturing and the IMP densities compared with those obtained from in vivo parasites; the cultured schistosomula have a lower E2 face particle density than the in vivo specimens.     

Nuclear surface complex as observed with the high resolution scanning electron microscope. Visualization of the membrane surfaces of the neclear envelope and the nuclear cortex from Xenopus laevis oocytes

The nuclear envelope and associated structures from Xenopus laevis oocytes (stage VI) have been examined with the high resolution scanning electron microscope (SEM). The features of the inner and outer surfaces of the nuclear surface complex were revealed by manual isolation , whereas the membranes facing the perinuclear space (the space between the inner and outer nuclear membranes) were observed by fracturing the nuclear envelope in this plane and splaying the corresponding regions apart. Pore complexes were observed on all four membrane surfaces of this double-membraned structure. The densely packed pore complexes (55/micron2) are often clustered into triplets with shared walls (outer diameter = 90 nm; inner diameter = 25 nm; wall thickness = aproximately 30 nm), and project aproximately 20 nm above each membrane except where they are flush with the innermost surface. The pore complex appears to be an aggregate of four 30-nm subunits. The nuclear cortex, a fibrous layer (300 nm thickness) associated with the inner surface of the nuclear envelope, has been revealed by rapid fixation. This cortical layer is interrupted by funnel-shaped intranuclear channels (120-640 nm diam) which narrow towards the pore complexes. Chains of particles, arranged in spirals, are inserted into these intranuclear channels. The fibers associated with the innermost face of the nuclear envelope can be extraced with 0.6 MKI to reveal the pore complexes. A model of the nuclear surface complex, compiled from the visualization of all the membrane faces and the nuclear cortex, demonstrates relations between the intranuclear channels (3.2/micron2) and the numerous pore complexes, and the possibility of their role in nucleocytoplasmic interactions.     

大豆根瘤细菌周膜的冷冻复型研究

用冷冻复型电镜技术研究了中国丰收１１号大豆根瘤中的细菌周膜。细菌周膜的断裂面上有颗粒状物质,但Ｐ面和Ｅ面有所不同,前者颗粒密度较大。即使都在Ｐ面或Ｅ面上,不同的细菌或同一细菌不同部位的颗粒密度也不一样。在细菌周膜与细菌细胞壁之间有一个环形腔隙,腔的大小随细菌和细菌部位不同而异。腔中不仅有泡状和管状结构,有时也有类寄主细胞质物质。细菌周膜表面有近似半球形或嵴形隆起,它们可能是腔中管泡状结构压迫细菌周     

Freeze fracture studies on the interaction between the malaria parasite and the host erythrocyte in Plasmodium knowlesi infections.

The freeze fracture technique has been used to study the internal cyto-architecture of the surface membranes of the parasite and erythrocyte in Plasmodium knowlesi infections. Six fracture faces, derived from the plasma membrane and 2 pellicular membranes, have been identified at the surface of the free merozoite. The apposed leaflets of the 2 pellicular membranes show the characteristic features of E fracture faces, a result compatible with the view that the pellicular membranes line a potential cisterna. There is evidence to suggest that there may be changes in the distribution and density of the integral proteins in the merozoite plasma membrane at invasion. Furthermore, vesicles consisting of stacked membranes occur within and around the erythrocyte invagination at invasion; it is suggested that these vesicles are released from the merozoite rhoptries. Formation of the parasitophorous vacuole is accompanied by dramatic changes in the density and distribution of intra-membraneous particles (IMP) in the vacuolar membrane. Initially there is a great reduction in particle numbers, but subsequently the particles reappear and show reversed polarity. The possible causes and implications of these changes are discussed. The intra-erythrocytic parasite synthesizes new transmembrane proteins as development proceeds, and the trophozoite and schizont stages of development are characterized by the appearance of circular, particle-free regions in the parasite plasmalemma. There is a decrease in the density of transmembrane proteins in the erythrocyte plasma membrane during parasite maturation, and the P face IMP show the characteristic features of aggregation.     

Formation of the Giardia Cyst Wall: Studies on Extracellular Assembly Using Immunogold Labeling and High Resolution Field Emission SEM

Encystment of the intestinal protozoan, Giardia, is a key step in the life cycle that enables this parasite to be transmitted from host to host via either fecal oral, waterborne, or foodborne transmission. The process of encystment was studied by localizing cyst wall specific antigens with immunofluorescence for light microscopy and immunogold staining for field emission scanning electron microscopy. Chronological sampling of Giardia cultures stimulated with endogenous bile permitted identification of an intracellular and extracellular phase in cyst wall formation, a process which required a total of 14-16 h. The intracellular phase lasted for 8-10 h, while the extracellular phase, involved the appearance of cyst wall antigen on the trophozoite membrane, and the assembly of the filamentous layer, a process requiring an additional 4-6 h for completion of mature cysts. The extracellular phase was initiated with the appearance of cyst wall antigen on small protrusions of the trophozoite membrane (-15 nm), which became enlarged with time to caplike structures ranging up to 100 nm in diameter. Caplike structures involved with filament growth were detected over the entire surface of the trophozoite including the adhesive disc and flagella. Encysting cells rounded up, lost attachment to the substratum, and became enclosed in a layer of filaments. Late stages in encystment included a “tailed” cyst in which flagella were not fully retracted into the cyst. Clusters of cysts were seen in which filaments at the surface of one cyst were connected with the surface of adjacent cysts or the “tailed” processes of adjacent cysts, suggesting that the growth of cyst wall filaments may be at the terminal end. In conclusion, the process of encystment has been shown to consist of two morphologically different stages (intracellular and extracellular) which requires 16 h for completion. Further investigation of the extracellular stage with regard to assembly of the filamentous layer of the cyst wall may lead to innovative methods for interfering with production of an intact functional cyst wall, and thereby, regulation of viable Giardia cyst release from the host.     

Differentiation of food vacuolar membranes during endocytosis in Tetrahymena

The origin and differentiation of Tetrahymena pyriformis food vacuolar membranes has been studied by freeze-fracture electron microscopy. By measuring the temperature needed to induce the onset of lipid phase separation (as inferred by the appearance of particle-free regions in replicas) and calculating the changes in average intramembrane particle distribution, a distinct modification of the vacuolar membrane could be observed from the time of its formation from disk-shaped vesicles to its maturation before egestion of its indigestible contents. Whereas the nascent vacuolar membrane first showed signs of phase separation at 9 degrees C, this temperature rose to 14 degrees C in the completed vacuole and then, after lysosomal fusion, eventually declined to 12 degrees C. The average membrane particle density on the PF face increased from 761 +/- 219 to 1,625 +/- 350 per micron 2 during membrane differentiation. Like other membranes of the cell, the vacuolar membrane underwent adaptive changes in its physical properties in cells maintained for several hours at low temperature. This exposure to low temperature caused an equal effect in vacuoles formed before, during, or after the temperature shift-down. Normal changes in the properties of the vacuolar membrane may have some bearing on its programmed sequence of fusion reactions.     

Changes in cell surface primary cilia and microvilli concurrent with measurements of fluid flow across the rabbit corneal endothelium ex vivo

Primary cilia and microvilli have been reported on the mammalian rabbit corneal endothelium but their relationship to cell function is undefined. Six corneas from healthy 2 kg female albino rabbits were glutaraldehyde-fixed post mortem (15:00 h) or twelve corneal stroma-endothelial preparations incubated at 37 degrees C under an applied hydrostatic pressure of 20 cm H2O for 4 h prior to fixation. The corneal endothelium was assessed by quantitative scanning electron microscopy. Cells fixed immediately post mortem were decorated with small stubby microvilli (average 21 +/- 13/100 micron 2), and only 25% of the cells were decorated with primary cilia having an average length of 2.44 +/- 1.56 microns. Following 4 h ex vivo incubation with a phosphate-buffered Ringer solution, conspicuous microvilli developed to an average density of 40 +/- 19/100 micron 2 and primary cilia were found on 12% of the cells, having on average length of 2.27 +/- 1.38 microns. Following 4 h incubation in a bicarbonate-buffered Ringer solution, small stubby microvilli developed to a density of 49 +/- 18/100 micron 2, and 40% of the cells showed primary cilia with an average length of 4.31 +/- 1.93 microns; the net trans-endothelial fluid flow in the latter set was 60% greater. These studies indicate that the primary cilia on corneal endothelial cells might be responsive to fluid flow, but that mild mechanical and/or chemical stress could also be the cause of the change since the elaboration of primary cilia can be accompanied by microvilli as well.     

Distribution of Intramembrane Particles and Its Changes during the Acrosome Reaction in Spermatozoa of the Japanese Abalone

The distribution of intramembrane particles in the plasma and acrosomal membranes of sperm of the Japanese abalone, Haliotis discus , and its changes during the acrosome reaction were studied by the freeze-fracture replica technique. The P face of the plasma membrane covering the acrosome has sparse membrane particles except in the apical region, which includes the trigger and 'truncated cone' regions. Large particles with an average diameter of 10 nm are located in this apical region. The E face of the plasma membrane has only a few particles. On the outer acrosomal membrane, many particles are randomly distributed throughout the P face, but only a small number of particles are found on the E face. Numerous particles on the P face of the inner acrosomal membrane show a regular arrangement as a dense lattice or with a concentric circular pattern. The initial change in the acrosome reaction is clearance of membrane particles from both the P and E faces of the plasma and outer acrosomal membranes around the apical region, where fusion of the two membranes occurs. As the acrosomal process elongates, the dense arrangement of particles on the inner acrosomal membrane changes via a loose lattice arrangement to a patchy distribution with particle-free areas. Then the arrangement is further disorganized becoming a sparse, random distribution.     

A chloroplast membrane lacking Photosystem II. Thylakoid stacking in the absence of the Photosystem II particle

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.     

A chloroplast membrane lacking photosystem II. Thylakoid stacking in the absence of the photosystem II particle.

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.