Ultrastructure of chytridiomycete and oomycete zoospores using spray-freeze fixation

Summary The ultrastructure of zoospores of several zoosporic fungi was examined using a modified cryofixation technique. An atomizer was used to spray a zoospore suspension into the cold propane reservoir of a conventional plunge freeze-substitution apparatus. Spray-freeze fixation and freeze-substitution of zoospores porvided better fixation of vacuolar structures, membranes and the extracellular coat than that obtained with chemical fixation. The overall shape of cryofixed spores was closer to that seen in living zoospores. Two types of vacuoles were seen in cryofixed zoospores ofMonoblepharella andChytridium. One type of vacuole contained electron-opaque material within the lumen while the other type had no visible internal material in the lumen and appeared to be part of the water expulsion vacuole complex. Coated pits and coated vesicles were observed associated with both the water expulsion vacuoles and the plasma membrane inMonoblepharella andPhytophthora, suggesting that endocytosis of the plasma membrane and expulsion vacuoles is part of membrane recycling during osmoregulatory events. An extracellular coat was seen on the outer surface of cryofixed zoospores ofMonoblepharella sp.,Chytridium confervae andPhytophthora palmivora without the use of carbohydrate-specific stains. The spray-freeze method gave good and reproducible fixation of the wall-less spores in quantities greater than those obtained in previously described zoospore cryofixation studies. The technique is potentially useful for cell suspensions in that freeze damage from excess water is limited.Abbreviations ddH₂O deionized distilled water - PME Pipes/MgCl2/EGTA buffer - WEV water expulsion vacuole     

On the ultrastructural organization of Trypanosoma cruzi using cryopreparation methods and electron tomography

The structural organization of Trypanosoma cruzi has been intensely investigated by different microscopy techniques. At the electron microscopy level, bi-dimensional analysis of thin sections of chemically fixed cells has been one of the most commonly used techniques, despite the known potential of generating artifacts during chemical fixation and the subsequent steps of sample preparation. In contrast, more sophisticated and elaborate techniques, such as cryofixation followed by freeze substitution that are known to preserve the samples in a more close-to-native state, have not been widely applied to T. cruzi. In addition, the 3D characterization of such cells has been carried out mostly using 3D reconstruction from serial sections, currently considered a low resolution technique when compared to electron tomography (ET). In this work, we re-visited the 3D ultrastructure of T. cruzi using a combination of two approaches: (1) analysis of both conventionally processed and cryofixed and freeze substituted cells and (2) 3D reconstruction of large volumes by serial electron tomography. The analysis of high-pressure frozen and freeze substituted parasites showed novel characteristics in a number of intracellular structures, both in their structure and content. Organelles generally showed a smooth and regular morphology in some cases presenting a characteristic electron dense content. Ribosomes and new microtubule sets showed an unexpected localization in the cell body. The improved preservation and imaging in 3D of T. cruzi cells using cryopreparation techniques has revealed some novel aspects of the ultrastructural organization of this parasite.     

High pressure freezing and freeze substitution improve immunolabeling of S-locus specific glycoproteins in the stigma papillae ofBrassica

Summary High pressure freezing and freeze substitution methods significantly improve the antigenic preservation of S-locus specific glycoproteins (SLSG). The SLSG, which are implicated in the incompatibility response, are localized over the cell wall and cytoplasm. Labeling in the cytoplasm is mainly associated with dictyosomes and rough endoplasmic reticulum. Quantitative analysis show that in cryofixed papillae the labeling was enhanced by approximately 45% over the cell wall and approximately 90% over the dictyosomes compared to chemically fixed papillae.     

The ultrastructure of plasma and thylakoid membrane vesicles from the fresh water cyanobacteriumAnacystis nidulans adapted to salinity

Summary Photoautotrophically growing cultures of the fresh water cyanobacteriumAnacystis nidulans adapted to the presence of 0.4–0.5 M NaCl (about sea water level) with a lag phase of two days after which time the growth rate reassumed 80–90% of the control. Plasma and thylakoid membranes were separated from cell-free extracts of French pressure cell treatedAnacystis nidulans by discontinuous sucrose density gradient centrifugation and purified by repeated recentrifugation on fresh gradients. Identity of the plasma and thylakoid membrane fractions was confirmed by labeling of intact cells with impermeant protein markers prior to breakage and membrane isolation. Electron microscopy revealed that each type of membrane was obtained in the form of closed and perfectly spherical vesicles. Major changes in structure and function of the plasma membranes (and, to a much lesser extent, of the thylakoid membranes) were found to accompany the adaptation process. On the average, diameters of plasma membrane vesicles from salt adapted cells were only one-third of the diameters of corresponding vesicles from control cells. By contrast, the diameters of thylakoid membrane vesicles were the same in both cases.Freeze-etching the cells and counting the number of membrane-intercalating particles on both protoplasmic and exoplasmic fracture faces of plasma and thylakoid membranes indicated a roughly 50% increase of the particle density in plasma membranes during the adaptation process while that in thylakoid membranes was unaffected. Comparison between particle densities on isolated membranes and those on corresponding whole cell membranes permitted an estimate as to the percentage of inside-out and right-side-out vesicles. Stereometric measurement of particle sizes suggested that two distinct sub-populations of the particles in the plasma membranes increased during the adaptation process, tentatively correlated to the cytochrome oxidase and sodium-proton antiporter, respectively. The effects of salt adaptation described in this paper were fully reversed upon withdrawal of the additional NaCl from the growth medium (deadaptation). Moreover, they were not observed when the NaCl was replaced by KCl.Abbreviations CM cytoplasmic or plasma membrane - ICM intracytoplasmic or thylakoid membrane - EF exoplasmic fracture face - PF protoplasmic fracture face - DABS diazobenzosulfonate; Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulfonate - PMSF phenylmethylsulfonylfluoride Dedicated to the memory of Professor Oswald Kiermayer     

Improved ultrastructural preservation ofPetunia andBrassica ovules and embryo sacs by high pressure freezing and freeze substitution

Summary In order to improve the ultrastructural preservation of the female gametophyte ofPetunia x hybrida andBrassica napus we tested several cryofixation techniques and compared the results with those of conventional chemical fixation methods. Ovules fixed with glutaraldehyde and osmium tetroxide in the presence or absence of potassium ferrocyanide showed poor cell morphological and ultrastructural preservation. In ovules cryo-fixed by plunging into liquid propane, the cell morphology was well preserved. However, at the ultrastructural level structure-distorting ice crystals were detected in all tissues. Due to the large size of the ovules, cryofixation by plunging in liquid propane is not adequate for ultrastructural studies. In contrast,P. x hybrida andB. napus ovules cryo-fixed by high pressure freezing showed improved cell morphological as well as ultrastructural preservation of the embryo sac and the surrounding integumentary tissues. The contrast of the cellular membranes after freeze substitution with 2% osmium tetroxide and 0.1% uranyl acetate in dry acetone was high. At the ultrastructural level, the most prominent improvements were: straight plasma membranes which were appressed to the cell walls; turgid appearing organelles with smooth surface contours; minimal extraction of cytoplasmic and extracellular substances. In contrast to the chemically fixed ovules, in high pressure frozen ovules numerous microtubules and multivesicular bodies could be distinguished.     

In situ molecular identification of the plastid omega3 fatty acid desaturase FAD7 from soybean: evidence of thylakoid membrane localization

omega3 fatty acid desaturases are the enzymes responsible for the synthesis of trienoic fatty acids in plants. These enzymes have been mainly investigated using molecular, biochemical, and genetic approaches but very little is known about their subcellular distribution in plant cells. In this work, the precise subcellular localization of the omega3 desaturase FAD7 was elucidated by immunofluorescence and immunogold labeling using a monospecific GmFAD7 polyclonal antibody in soybean (Glycine max) photoautotrophic cell suspension cultures. Confocal analysis revealed the localization of the GmFAD7 protein within the chloroplast; i.e. signals from FAD7 and chlorophyll autofluorescence showed specific colocalization. Immunogold labeling was pursued on cryofixed and freeze-substituted samples for convenient preservation of antigenicity and ultrastructure of membrane subcompartments. Our data revealed that the FAD7 protein was preferentially localized in the thylakoid membranes. Biochemical fractionation of purified chloroplasts and western analysis of the subfractions further confirmed these results. These findings suggest that not only the envelope, but also the thylakoid membranes could be sites of lipid desaturation in higher plants.     

Cellular membrane contrast and contrast differentiation with osmium triazole and tetrazole complexes

Summary Addition of heterocyclic nitrogen compounds to the classical osmium tetroxide postfixation medium, applied after glutaraldehyde fixation, results in enhanced membrane contrast in ultrathin sections of liver tissue. The addition of similar compounds to potassium osmate solutions, results in contrast differences in some cellular membranes. The membranes of the rough endoplasmic reticulum, the nuclear envelope and the plasma membrane acquire contrast, while the mitochondrial membranes do not. The apolar regions of membranes are contrasted when osmium tetroxide is combined with heterocyclic nitrogen compounds, whereas the polar regions are contrasted by combinations of potassium osmate with these compounds. This polar membrane contrast is probably due to the presence of an amino-group in the heterocyclic nitrogen compounds. Compounds without the amino-group do not contrast membranes, although the glycogen is contrasted.X-ray microanalysis served to establish the relative osmium content in contrasted glycogen, and showed that such nitrogen compounds play a role in complexation of cations in aldehyde-fixed tissues. Electron spectroscopy for chemical analysis (ESCA) measurements of isolated muscle glycogen show that after treatment with various osmium tetroxide or potassium osmate solutions, hexavalent and quadrivalent osmium species are present in the glycogen. The presence of (heterocyclic) nitrogen compounds in such solutions stabilizes certain osmium valency species, and this may account for the contrast observed.     

The ultrastructure of the cell surface and plasma membrane of exponential and stationary phase cells of Schizosaccharomyces pombe,grown in different media

In order to study the ultrastructure of the cell surface and plasma membrane of Schizosaccharomyces pombe as a function of growth conditions we investigated exponential and stationary phase cells grown in rich and minimal medium.Electron microscopic preparation techniques based on rapid cryofixation (without cryoprotectants) were used. The intramembraneous aspects of the plasma membrane were described by freeze fracturing. For the first time the dynamic surface structures could be directly analyzed by freeze drying in the scanning electron microscope and in thin section of freeze substituted samples. This preparation techniques reveal hair-like structures on the surface of yeast cells. The hairs of cells grown in the rich medium are longer than those grown in the minimal medium. A mutant defective in the structure of a cell surface galactomannoprotein (acid phosphatase) reveals (under conditions of maximal acid phosphatase expression) a cell surface structure that differs from the wild type. It is likely that the hairs represent the peripheral galactomannan layer or part of it.On the membrane fracture faces the number, shape, distribution and state of aggregation of the intramembraneous particles are different between membranes of growing and non-growing cells and between cells grown under different physiological conditions. In the minimal medium corresponding periodical structures on the plasmic and exoplasmic fracture faces were observed, which clearly differ between exponential and stationary phase cells. The number, length and depth of plasma membrane invaginations increase as the cells go from the exponential phase to the stationary phase. Short and flattened invaginations are filled with thin periodic structures.     

Ultrastructure and permeability characteristics of the membranes of mucous granules of the hagfish

Mucous granules secreted by the slime glands of the hagfish. Eptatretus stouti, were studied after ultrarapid cryofixation and freeze substitution in diverse media (osmium tetroxide in acetone; several aqueous glutaraldehyde-based media with or without osmium). Only freeze substitution with osmium tetroxide-acetone preserved the granules intact, allowing visualization of its single unit membrane. Tests of the rupture or stability of freshly secreted mucous granules in sea water and other aqueous media showed the membranes of the granules are permeable to all inorganic cations tested, ranging in relative mass from ammonium to barium. They are permeable to the univalent anions chloride, nitrate, and bicarbonate, but not to the di- or trivalent anions sulfate, phosphate, and citrate. Moreover, in solutions where nonpenetrant anions were present, rupture occurred if the osmotic pressure was below a critical level (about 800 mOsmol/l). The structural and permeability characteristics of the granules account for the explosive speed with which they rupture, releasing their mucous contents, on contact with sea water.     

Ultrastructure of ascospore delimitation in freeze substituted samples ofAscodesmis nigricans (Pezizales)

Summary Freeze substitution proved to be a valuable technique for studying the early stages of ascosporogenesis inAscodesmis nigricans. Our observations indicate that the ascus vesicle originated from the ascus plasma membrane. Invaginations of the plasma membrane produced ascus vesicle initials consisting of two closely spaced unit membranes. The appearance of the outer leaflet of each of these membranes was identical to that of the inner leaflet of the ascus plasma membrane. Apparent points of continuity between ascus vesicle initials and the plasma membrane were observed. Ascus vesicle initials accumulated in the ascus cytoplasm near the plasma membrane and then coalesced to form the ascus vesicle, a peripheral, cylinder-like structure consisting of two closely spaced unit membranes that extended from the ascus apex to the ascus base. The ascus vesicle then became invaginated in a number of regions and subsequently gave rise to eight sheet-like segments, or ascosporedelimiting membranes, that encircled uninucleate segments of cytoplasm forming ascospore initials. Like the ascus vesicle, each ascospore-delimiting membrane consisted of two closely spaced unit membranes, the inner of which became the ascospore plasma membrane. The ascospore wall then developed between the spore plasma membrane and the outer membrane. Many details of ascospore maturation were clearly visible in freeze substituted samples.     

Ultrastructure of the membrane systems in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803

Summary. Among prokaryotes, cyanobacteria are unique in having highly differentiated internal membrane systems. Like other Gram-negative bacteria, cyanobacteria such as Synechocystis sp. strain PCC 6803 have a cell envelope consisting of a plasma membrane, peptidoglycan layer, and outer membrane. In addition, these organisms have an internal system of thylakoid membranes where the electron transfer reactions of photosynthesis and respiration occur. A long-standing controversy concerning the cellular ultrastructures of these organisms has been whether the thylakoid membranes exist inside the cell as separate compartments, or if they have physical continuity with the plasma membrane. Advances in cellular preservation protocols as well as in image acquisition and manipulation techniques have facilitated a new examination of this topic. We have used a combination of electron microscopy techniques, including freeze-etched as well as freeze-substituted preparations, in conjunction with computer-aided image processing to generate highly detailed images of the membrane systems in Synechocystis cells. We show that the thylakoid membranes are in fact physically discontinuous from the plasma membrane in this cyanobacterium. Thylakoid membranes in Synechocystis sp. strain PCC 6803 thus represent bona fide intracellular organelles, the first example of such compartments in prokaryotic cells. Supplementary material to this paper is available in electronic form at Correspondence and reprints: Department of Biology, CB1137, Washington University, St. Louis, MO 63130, U.S.A.     

Changes of some physical properties of isolated and purified plasma and thylakoid membrane vesicles from the freshwater cyanobacterium Synechococcus 6301 (Anacystis nidulans) during adaptation to salinity

Photoautotrophically growing cultures of the freshwater cyanobacterium Anacystis nidulans (Synechococcus sp.) became adapted to the presence of 0.4-0.5 M NaCl in the growth medium (about seawater level) with a lag phase of 2 days after which time the growth rate resumed at 80-90% of the control. Major changes in structure and function of the plasma membranes (and, to a much lesser extent, of the thylakoid membranes) were found to accompany the adaptation process. Plasma and thylakoid membranes were separated from crude cell-free extracts of French pressure cell-treated Anacystis by discontinuous sucrose density gradient centrifugation and purified by repeated recentrifugation on fresh gradients. Concentrations of copper, iron, calcium, and magnesium ions were determined by inductively coupled plasma atomic emission spectrometry with EDTA-washed and dialyzed membrane preparations; salt adaptation was found to increase (decrease) the concentration of membrane-bound calcium in plasma (thylakoid) membranes, qualitatively reciprocal results being obtained for magnesium. Levels of plasma membrane-bound copper and iron roughly tripled during the adaptation process; by contrast, corresponding effects on thylakoid membranes were negligible. The size of the membrane vesicles was measured by quasi-elastic laser light-scattering and the electric surface charge of the membranes was measured by laser Doppler velocimetry. Salt adaptation decreased the mean diameter of plasma membrane vesicles to a much higher extent than that of thylakoid membrane vesicles. Overall surface charge densities of resting vesicles were only slightly affected by the salt treatment as was also seen from titration of the electrophoretic mobility of the vesicles with electrolytes. Yet, induction of (photosynthetic or respiratory) electron transport provoked a charge separation across the membrane which was easily measurable in terms of electrophoretic mobility. The results will be discussed with particular emphasis on the stimulated cytochrome c oxidase activity of plasma (but not thylakoid) membranes from salt-adapted cells compared to control cells and also with respect to the decreased ion permeability of the plasma membrane of salt grown cells.     

Membrane histochemistry of Physarum polycephalum myxamoebae

Myxamoebae of Physarum polycephalum are the uninucleate, haploid stage of the organism. Histochemical studies were undertaken to characterize intracellular and plasma membranes, and to provide a basis for assaying subcellular fractions for enrichment in plasma membranes. Lead salts deposition techniques were employed for hydrolytic enzymes. Alcian blue-ruthenium red, osmium tetroxide-potassium ferrocyanide, and phosphotungstic acid-chromic acid stains were evaluated for specificity for plasma membranes. Glucose 6-phosphatase was localized in endoplasmic reticulum, Golgi apparatus, and perinuclear space. 5'-Nucleotidase was localized in food vacuoles, chromatin, and plasmalemma. Acid phosphatase was in food vacuoles and Golgi apparatus. Alkaline phosphatase was in food vacuoles and endoplasmic reticulum. We conclude that none of the above enzymes is suitable as a cytochemical marker for plasma membranes of Physarum myxamoebae, but recommend instead staining ultrathin sections of membrane pellets with phosphotungstic acid-chromic acid, which stains plasma membranes selectively.     

TRANSFER OF PROTEINS FROM THE CHLOROPLAST TO VACUOLES IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA): A PATHWAY FOR DEGRADATION

Several chloroplast proteins were detected by immunoelectron microscopy within dense granules in cytoplasmic vacuoles in the alga Chlamydomonas reinhardtii Dangeard. Transfer from chloroplast to vacuoles of two major, pulse-labeled polypeptides, the large subunit of rubisco and the α subunit of ATPase, which are synthesized on chloroplast ribosomes, was demonstrated by the recovery of these polypeptides in vacuolar granules over a several-hour time period. The ultrastructure of cryofixed algal cells was examined to search for structures that would provide insight into the transfer of chloroplast proteins to vacuoles. Micrographs showed that the two membranes of the envelope were appressed, with no detectable intermembrane space, over most of the chloroplast surface. Protrusions of the outer membrane of the envelope were occasionally found that enclosed stroma, with particles similar in size to chloroplast ribosomes, but generally not thylakoid membranes. These observations suggest that chloroplast material, especially the stromal phase, was extruded from the chloroplast in membrane-bound structures, which then interacted with Golgi-derived vesicles for degradation of the contents by typical lysosomal activities. A protein normally targeted to vacuoles through the endomembrane system for incorporation into the cell wall was detected in Golgi structures and vacuolar granules but not the chloroplast.     

A cryofixation study of presumptive hygroreceptors on the antennule of a terrestrial isopod

The structure of the apical sensilla on the antennule of the terrestrial isopod Porcellio scaber was examined in cryofixed and freeze substituted (CRF) and chemically fixed and dehydrated (CHF) material. CRF specimens generally showed a preservation superior to CHF material. Only in deeper regions did the tissue show damage from freezing. Each of the 13–22 sensilla contains two sensory cells. In contrast to earlier reports, it was observed that the dendritic segments of these cells are arranged in a unique, concentric manner. In CRF specimens the dendrites reach the tip of the sensilla and border upon the innermost layer of the complicated wall of the peg which is not pierced by pores. Silver-protein and lanthanum failed to penetrate the wall of the sensilla and also did not reach the dendrites via an apical pore, which therefore is regarded as a molting pore. The lymph spaces which, in CHF specimens, are observed around the dendrites and beneath the cuticle within the antennal tip are regarded as artefactual. From similarities in the dendritic structures to insect hygroreceptors and their relationship to the adjoining cuticle it can be assumed that the antennular sensilla in Porcellio are sensitive to humidity changes. Mechanoreception and chemoreception, however, cannot entirely be excluded as possible functions.     

Electron tomography of nascent herpes simplex virus virions

Cells infected with herpes simplex virus type 1 (HSV-1) were conventionally embedded or freeze substituted after high-pressure freezing and stained with uranyl acetate. Electron tomograms of capsids attached to or undergoing envelopment at the inner nuclear membrane (INM), capsids within cytoplasmic vesicles near the nuclear membrane, and extracellular virions revealed the following phenomena. (i) Nucleocapsids undergoing envelopment at the INM, or B capsids abutting the INM, were connected to thickened patches of the INM by fibers 8 to 19 nm in length and < or =5 nm in width. The fibers contacted both fivefold symmetrical vertices (pentons) and sixfold symmetrical faces (hexons) of the nucleocapsid, although relative to the respective frequencies of these subunits in the capsid, fibers engaged pentons more frequently than hexons. (ii) Fibers of similar dimensions bridged the virion envelope and surface of the nucleocapsid in perinuclear virions. (iii) The tegument of perinuclear virions was considerably less dense than that of extracellular virions; connecting fibers were observed in the former case but not in the latter. (iv) The prominent external spikes emanating from the envelope of extracellular virions were absent from perinuclear virions. (v) The virion envelope of perinuclear virions appeared denser and thicker than that of extracellular virions. (vi) Vesicles near, but apparently distinct from, the nuclear membrane in single sections were derived from extensions of the perinuclear space as seen in the electron tomograms. These observations suggest very different mechanisms of tegumentation and envelopment in extracellular compared with perinuclear virions and are consistent with application of the final tegument to unenveloped nucleocapsids in a compartment(s) distinct from the perinuclear space.     

Localization of phycoerythrin at the lumenal surface of the thylakoid membrane in Rhodomonas lens

The thylakoids of cryptomonads are unique in that their lumens are filled with an electron-dense substance postulated to be phycobiliprotein. In this study, we used an antiserum against phycoerythrin (PE) 545 of Rhodomonas lens (gift of R. MacColl, New York State Department of Health, Albany, NY) and protein A-gold immunoelectron microscopy to localize this light-harvesting protein in cryptomonad cells. In sections of whole cells of R. lens labeled with anti-PE 545, the gold particles were not uniformly distributed over the dense thylakoid lumens as expected, but instead were preferentially localized either over or adjacent to the thylakoid membranes. A similar pattern of labeling was observed in cell sections labeled with two different antisera against PE 566 from Cryptomonas ovata. To determine whether PE is localized on the outer or inner side of the membrane, chloroplast fragments were isolated from cells fixed in dilute glutaraldehyde and labeled in vitro with anti-PE 545 followed by protein A-small gold. These thylakoid preparations were then fixed in glutaraldehyde followed by osmium tetroxide, embedded in Spurr, and sections were labeled with anti-PE 545 followed by protein A-large gold. Small gold particles were found only at the broken edges of the thylakoids, associated with the dense material on the lumenal surface of the membrane, whereas large gold particles were distributed along the entire length of the thylakoid membrane. We conclude that PE is located inside the thylakoids of R. lens in close association with the lumenal surface of the thylakoid membrane.     

HIGH LOCALIZATION OF RIBULOSE-1,5-BISPHOSPHATE CARBOXULASE/OXYGENASE IN THE PYRENOIDS OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA), AS REVEALED BY CRYOFIXATION AND IMMUNOGOLD ELECTRON MICROSCOPY

The distribution of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the chloroplasts of the unicellular green alga Chlamydomonas reinhardtii Dangeard was examined using cryotechnique and conventional fixation for immunogold electron microscopy. Both methods provided essentially identical results, although somewhat higher densities of gold particles indicating Rubisco molecules were recognized in the pyrenoids of cryofixed cells. The gold particles were highly concentrated in the pyrenoid matrix within the chloroplasts. Even when considering the vast difference in volume between the pyrenoid and the rest of the Chloroplast, more than 99% of the total Rubisco labeling in the chloroplast was calculated to be present in the pyrenoid matrix. High localization of Rubisco in the pyrenoid matrix was also recognized regardless of cell age, based on immunofluorescence microscopy of the same en bloc samples. These results are inconsistent with a recent immunocytochemical study employing cryotechnique in which more than 90% of the total Rubisco was recognized in the thylakoid region (thylakoid membranes and stroma) of C. reinhardtii cells. Rubisco highly localized in the pyrenoid matrix may take part in active photosynthetic CO₂ fixation and/or the CO₂ concentrating mechanism .     

Unique thylakoid membrane architecture of a unicellular N2-fixing cyanobacterium revealed by electron tomography

Cyanobacteria, descendants of the endosymbiont that gave rise to modern-day chloroplasts, are vital contributors to global biological energy conversion processes. A thorough understanding of the physiology of cyanobacteria requires detailed knowledge of these organisms at the level of cellular architecture and organization. In these prokaryotes, the large membrane protein complexes of the photosynthetic and respiratory electron transport chains function in the intracellular thylakoid membranes. Like plants, the architecture of the thylakoid membranes in cyanobacteria has direct impact on cellular bioenergetics, protein transport, and molecular trafficking. However, whole-cell thylakoid organization in cyanobacteria is not well understood. Here we present, by using electron tomography, an in-depth analysis of the architecture of the thylakoid membranes in a unicellular cyanobacterium, Cyanothece sp. ATCC 51142. Based on the results of three-dimensional tomographic reconstructions of near-entire cells, we determined that the thylakoids in Cyanothece 51142 form a dense and complex network that extends throughout the entire cell. This thylakoid membrane network is formed from the branching and splitting of membranes and encloses a single lumenal space. The entire thylakoid network spirals as a peripheral ring of membranes around the cell, an organization that has not previously been described in a cyanobacterium. Within the thylakoid membrane network are areas of quasi-helical arrangement with similarities to the thylakoid membrane system in chloroplasts. This cyanobacterial thylakoid arrangement is an efficient means of packing a large volume of membranes in the cell while optimizing intracellular transport and trafficking.