Localization of cytoskeletal proteins in Pneumocystis carinii by immuno-electron microscopy

Pneumocystis carinii causes serious pulmonary infection in immunosuppressed patients. This study was undertaken to observe the cytoskeletal proteins of P. carinii by immuno-electron microscopy. P. carinii infection was experimentally induced by immunosuppression of Sprague-Dawley rats for seven weeks, and their lungs were used for the observations of this study. The gold particles localized actin, tropomyosin, and tubulin. The actin was irregularly scattered in the cytoplasm of the trophic forms but was much more concentrated in the inner space of the cell wall of the cystic forms called the inner electron-lucent layer. No significant amount of tropomyosin was observed in either trophic forms or cystic forms. The tubulin was distributed along the peripheral cytoplasm and filopodia of both the trophic and cystic forms rather than in the inner side of the cytoplasm. Particularly, in the cystic forms, the amount of tubulin was increased and located mainly in the inner electron-lucent layer of the cell wall where the actin was concentrated as well. The results of this study showed that the cell wall of P. carinii cystic forms is a structure whose inner side is rich in actin and tubulin. The location of the actin and tubulin in P. carinii suggests that the main role of these proteins is an involvement in the protection of cystic forms from the outside environment by maintaining rigidity of the cystic forms.     

Oligomerization state of MIP proteins expressed in Xenopus oocytes as revealed by freeze-fracture electron-microscopy analysis

The MIP (major intrinsic protein) family is a widespread family of membrane proteins exhibiting two major types of channel properties: aquaporins and solute facilitators. In the present study, freeze-fracture electron microscopy was used to investigate the oligomerization state of two MIP proteins heterologously expressed in the plasma membrane of Xenopus laevis oocytes: AQPcic, an aquaporin from the insect Cicadella viridis, and GlpF, a glycerol facilitator from Escherichia coli. Swelling assays performed on oocytes 48 and 72 h following cRNA microinjections showed that these proteins were functionally expressed. Particle density determinations indicated that expression of proteins is related to an increase in particle density on the P fracture face of oocyte plasma membranes. Statistical analysis of particle sizes was performed on protoplasmic fracture faces of the plasma membrane of oocytes expressing AQPcic and GlpF 72 h after cRNA microinjections. Compared to control oocytes, AQPcic-expressing oocytes exhibited a specific population of particles with a mean diameter of 8.7 +/- 0.1 nm. This value is consistent with the previously reported tetrameric organization of AQPcic. In addition, AQPcic particles aggregate and form orthogonal arrays similar to those observed in native membranes of C. viridis, consisting of homotetramers of AQPcic. On the protoplasmic fracture face of oocytes expressing GlpF, the particle density is increased by 4.1-fold and the mean diameter of specifically added particles is 5.8 +/- 0.1 nm. This value fits with a monomer of the 28-kDa GlpF protein plus the platinum-carbon layer. These results clearly demonstrate that GlpF is a monomer when functionally expressed in plasma membranes of Xenopus oocytes and therefore emphasize the key role of the oligomerization state of MIP proteins with respect to their function.     

Ultrastructural characterization of the delimiting membranes of isolated autophagosomes and amphisomes by freeze-fracture electron microscopy

The delimiting membranes of isolated autophagosomes from rat liver had extremely few transmembrane proteins, as indicated by the paucity of intramembrane particles in freeze-fracture images (about 20 particles/microm2, whereas isolated lysosomes had about 2000 particles/microm2). The autophagosomes also appeared to lack peripheral surface membrane proteins, since attempts to surface-biotinylate intact autophagosomes only yielded biotinylation of proteins from contaminating damaged mitochondria. All the membrane layers of multilamellar autophagosomes were equally particle-poor; the same was true of the autophagosome-forming, sequestering membrane complexes (phagophores). Isolated amphisomes (vacuoles formed by fusion between autophagosomes and endosomes) had more intramembrane particles than the autophagosomes (about 90 particles/microm2), and freeze-fracture images of these organelles frequently showed particle-rich endosomes fusing with particle-poor or particle-free autophagosomes. The appearence of multiple particle clusters suggested that a single autophagic vacuole could undergo multiple fusions with endosomes. Only the outermost membrane of bi- or multilamellar autophagic vacuoles appeared to engage in such fusions.     

Assemblages of simian virus 40 capsid proteins and viral DNA visualized by electron microscopy

SV40 assembles in the nucleus by addition of capsid proteins to the minichromosome. The VP15VP2/3 capsomer is composed of a pentamer of the major protein VP1 complexed with a monomer of a minor protein, VP2 or VP3. In the capsid, the capsomers are bound together via their flexible carboxy-terminal arms. Our previous studies suggested that the capsomers are recruited to the packaging signal ses via avid interaction with Sp1. During assembly Sp1 is displaced, allowing chromatin compaction. Here we investigated the interactions in vitro of VP1(5)VP2/3 capsomers with the entire SV40 genome, using mutant VP1 deleted in the carboxy-arm that cannot assemble, but retains DNA-binding capacity. EM revealed that VP1(5)VP2/3 complexes bind non-specifically at random locations around the DNA. Sp1 was absent from mature virions. The findings suggest that multiple capsomers attach simultaneously to the viral genome, increasing their local concentration, facilitating rapid, concerted assembly reaction and removal of Sp1.     

Pseudomonas oleovorans as a tool in bioconversions of hydrocarbons: growth,morphology and conversion characteristics in different two-phase systems

The bioconversion of hydrocarbons by Pseudomonas oleovorans has been studied in two-phase systems. In these systems, the hydrocarbon substrate is present in sufficient amounts to form the bulk apolar phase. High cell densities (up to 20 mg dry mass per ml water phase) are reached when the apolar phase consists of n-octane, 1-octene or 1-decene. There is considerable cell damage after incubation for 50–70 h. Loss of cell viability and membrane damage as observed by freeze-fracture electron microscopy correlate with a loss of hydrocarbon oxidation, measured as the conversion of 1-octene to 1,2-epoxyoctane. The final yield of oxidized hydrocarbon in the apolar substrate phase can be increased substantially by replacing the damaged cells with freshly grown cells. Yields up to 150 mg 1,2-epoxyoctane per ml 1-octene and up to 20–25 mg 1,2-epoxyoctane per ml culture were obtained with four cycles of the cell renewal procedure. Several other substrates in addition to octene were tested in the optimized two-phase system. Of these, 1-decene was converted into (R)-1,2-epoxydecane with an optical purity of 60%, while allylbenzene was converted into chiral 1,2-epoxy-3-phenylpropane. Some of the future applications of the conversion products are discussed.     

The integrative role of cryo electron microscopy in molecular and cellular structural biology

After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy (cryo‐EM) is now heading off at unprecedented speed towards high‐resolution analysis of biological objects of various sizes. This ‘revolution in resolution’ is happening largely thanks to new developments of new‐generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on complementary metal oxide semiconductor devices. Combined with advanced image processing and 3D reconstruction, the cryo‐EM analysis of nucleoprotein complexes can provide unprecedented insights at molecular and atomic levels and address regulatory mechanisms in the cell. These advances reinforce the integrative role of cryo‐EM in synergy with other methods such as X‐ray crystallography, fluorescence imaging or focussed‐ion beam milling as exemplified here by some recent studies from our laboratory on ribosomes, viruses, chromatin and nuclear receptors. Such multi‐scale and multi‐resolution approaches allow integrating molecular and cellular levels when applied to purified or in situ macromolecular complexes, thus illustrating the trend of the field towards cellular structural biology.     

Different phosphatidylinositol 3-phosphate asymmetries in yeast and mammalian autophagosomes revealed by a new electron microscopy technique

Phosphatidylinositol 3-phosphate (PtdIns3P) is a phospholipid essential for autophagy, but the detailed distribution of PtdIns3P in the membrane of autophagosomes, autophagic bodies, and other organelles is unclear due to technical difficulties. In the present study, we examined PtdIns3P distribution in autophagic membranes with an electron microscopy method called the quick-freeze freeze-fracture replica labeling method (QF-FRL), which can define the distribution of membrane lipids at the nanometer scale. In this method, membranes are split into 2 leaflets so that membrane asymmetry, i.e., differences between the 2 leaflets, can be defined unambiguously. As a result, PtdIns3P in the yeast autophagosome was found to exist much more abundantly in the lumenal leaflet (i.e., the leaflet facing the space between the outer and inner autophagosomal membranes) than in the cytoplasmic leaflet. In contrast, PtdIns3P in the mammalian autophagosome was confined to the cytoplasmic leaflet, showing an opposite asymmetry from that found in yeast. In yeast deleted for 2 cytoplasmic PtdIns3P phosphatases, Ymr1 and Sjl3, PtdIns3P distributed in an equivalent density in the 2 leaflets of the autophagosome membrane, suggesting that the asymmetry in wild-type yeast is generated as a result of unilateral PtdIns3P hydrolysis. The contrasting PtdIns3P distribution revealed in the present study suggested that formation of autophagic membranes may proceed in different ways in yeast and mammals.     

Different phosphatidylinositol 3-phosphate asymmetries in yeast and mammalian autophagosomes revealed by a new electron microscopy technique

Phosphatidylinositol 3-phosphate (PtdIns3P) is a phospholipid essential for autophagy, but the detailed distribution of PtdIns3P in the membrane of autophagosomes, autophagic bodies, and other organelles is unclear due to technical difficulties. In the present study, we examined PtdIns3P distribution in autophagic membranes with an electron microscopy method called the quick-freeze freeze-fracture replica labeling method (QF-FRL), which can define the distribution of membrane lipids at the nanometer scale. In this method, membranes are split into 2 leaflets so that membrane asymmetry, i.e., differences between the 2 leaflets, can be defined unambiguously. As a result, PtdIns3P in the yeast autophagosome was found to exist much more abundantly in the lumenal leaflet (i.e., the leaflet facing the space between the outer and inner autophagosomal membranes) than in the cytoplasmic leaflet. In contrast, PtdIns3P in the mammalian autophagosome was confined to the cytoplasmic leaflet, showing an opposite asymmetry from that found in yeast. In yeast deleted for 2 cytoplasmic PtdIns3P phosphatases, Ymr1 and Sjl3, PtdIns3P distributed in an equivalent density in the 2 leaflets of the autophagosome membrane, suggesting that the asymmetry in wild-type yeast is generated as a result of unilateral PtdIns3P hydrolysis. The contrasting PtdIns3P distribution revealed in the present study suggested that formation of autophagic membranes may proceed in different ways in yeast and mammals.     

Crystallisation of CP43, a chlorophyll binding protein of photosystem II: an electron microscopy analysis of molecular packing

Microcrystals of the chlorophyll binding protein, CP43, isolated from spinach thylakoid membranes have been studied by electron microscopy both in negative stain and in vitreous ice. Image analyses of three characteristic views show that the crystals are built of five different layers perpendicular to the c-axis. Each layer consists of different orientations of the CP43 protein. The unit cell derived from the end-on view (looking down the c-axis) shows an angle of 120 degrees, suggesting a threefold rotational symmetry. Both negative staining and cryo data are consistent with a hexagonal crystal lattice. Interpretation of the arrangement of the CP43 protein within this crystal lattice can be made based on 8- and 9-A electron crystallographic structures previously published that provide a model for the organisation of the transmembrane helices of CP43. Overall the analysis presented is consistent with X-ray diffraction data obtained from larger CP43 crystals and forms a framework on which to base further structural studies of this chlorophyll binding protein.     

Characterizing proteins in their cellular environment: Examples of recent advances in quantitative fluorescence microscopy

Quantitative characterization of protein interactions, both intramolecular and intermolecular, is crucial in understanding the mechanisms and regulation of their function. In recent years, it has become possible to obtain such information on protein systems in live cells, from bacteria to mammalian cell lines. This review discusses recent advances in measuring protein folding, absolute concentration, oligomerization, diffusion, transport, and organization at super‐resolution.     

Further cellular investigation of the human hepatoblastoma-derived cell line HepG2: Morphology and immunocytochemical studies of hepatic-secreted proteins

Summary The hepatoblastoma cell line HepG2 has been a matter of many investigations; most of them include biochemical studies of lipoprotein and other hepatic protein metabolism. However, the accurate cellular features of these cells have not been emphasized. We studied the cellular histologic, histochemical, and ultrastructural characteristics of this cell line. In addition, we investigated by immunoenzymatic methods the cellular biosynthesis of several proteins: apolipoproteins-AI,-B,-D, and-E, albumin, alpha-fetoprotein, transferrin, alpha-1-antitrypsin, C-reactive protein, fibronectin, and collagens I, III and IV. The rates of accumulation, in the medium of HepG2 cells, of albumin, alpha-1-antitrypsin, transferrin, and alpha-fetoprotein were 13.2±1.9; 4.9±1.5; 3.2±0.4; and 10.7±1.7 μg/10⁶ cells/24 h, respectively. Our results show that HepG2 cells exhibited most cellular features of normal human hepatocytes. Bile canaliculi as well as Golgi apparatus complexes were particularly developed. Except for the C-reactive protein, HepG2 cells have all retained the ability to synthesize hepatic proteins but with some variable intensity from cell to cell. This hepatoblastoma cell line seems to represent a useful tool in the understanding of hepatic protein biosynthesis, particularly for the investigation on the secretory pathway of plasma proteins.     

Cell to aperture interaction in patch-clamp chips visualized by fluorescence microscopy and focused-ion beam sections

Patch-clamp is an important method to monitor the electrophysiological activity of cells and the role of pharmacological compounds on specific ion channel proteins. In recent years, planar patch-clamp chips have been developed as a higher throughput approach to the established glass-pipette method. However, proper conditions to optimize the high resistance cell-to-probe seals required to measure the small currents resulting from ion channel activity are still the subject of conjecture. Here, we report on the design of multiple-aperture (sieve) chips to rapidly facilitate assessment of cell-to-aperture interactions in statistically significant numbers. We propose a method to pre-screen the quality of seals based on a dye loading protocol through apertures in the chip and subsequent evaluation with fluorescence confocal microscopy. We also show the first scanning electron micrograph of a focused ion beam section of a cell in a patch-clamp chip aperture.     

Tracking protein turnover and degradation by microscopy: photo-switchable versus time-encoded fluorescent proteins

Expanded fluorescent protein techniques employing photo-switchable andfluorescent timer proteins have become important tools in biological research.These tools allow researchers to address a major challenge in cell anddevelopmental biology, namely obtaining kinetic information about the processesthat determine the distribution and abundance of proteins in cells and tissues.This knowledge is often essential for the comprehensive understanding of abiological process, and/or required to determine the precise point ofinterference following an experimental perturbation.     

Lesions induced by complement in vitro on the protoscoleces of Echinococcus multilocularis: a study by electron microscopy.

Changes in the ultrastructure of the tegument of Echinococcus multilocularis protoscoleces during complement-mediated lysis in vitro was studied using transmission and scanning electron microscopy. It was found that the total disintegration of protoscoleces by complement proceeds through formation of ‘tegumental bubbles’ and disruption of the external plasma membrane. This sequence of events was evident in the appearance of numerous loose membrane fragments and vesicles, the lifting of the external unit membrane of the microtriches and the release of organelles from the distal cytoplasm. Subsequent events, such as the appearance of a ‘fuzzy’ coat and disruption of the basement membrane, were probably due to autolysis.     

Discovery of a new RNA-containing nuclear structure in UVC-induced apoptotic cells by integrated laser electron microscopy

Background information. Treatment of cells with UVC radiation leads to the formation of DNA cross‐links which, if not repaired, can lead to apoptosis. γ‐H2AX and cleaved caspase 3 are proteins formed during UVC‐induced DNA damage and apoptosis respectively. The present study sets out to identify early morphological markers of apoptosis using a new method of correlative microscopy, ILEM (integrated laser electron microscopy). Cleaved caspase 3 and γ‐H2AX were immunofluorescently labelled to mark the cells of interest. These cells were subsequently searched in the fluorescence mode of the ILEM and further analysed at high resolution with TEM (transmission electron microscopy). Results. Following the treatment of HUVECs (human umbilical vein endothelial cells) with UVC radiation, in the majority of the cells γ‐H2AX was formed, whereas only in a subset of cells caspase 3 was activated. In severely damaged cells with high levels of γ‐H2AX a round, electron‐dense nuclear structure was found, which was hitherto not identified in UV‐stressed cells. This structure exists only in nuclei of cells containing cleaved caspase 3 and is present during all stages of the apoptotic process. Energy‐loss imaging showed that the nuclear structure accumulates phosphorus, indicating that it is rich in nucleic acids. Because the nuclear structure did not label for DNA and was not affected by regressive EDTA treatment, it is suggested that the UV‐induced nuclear structure contains a high amount of RNA. Conclusions. Because the UV‐induced nuclear structure was only found in cells labelled for cleaved caspase 3 it is proposed as an electron microscopic marker for all stages of apoptosis. Such a marker will especially facilitate the screening for early apoptotic cells, which lack the well‐known hallmarks of apoptosis within a cell population. It also raises new questions on the mechanisms involved in the UV‐induced apoptotic pathway.     

Identification of proteins in Encephalitozoon intestinalis, a microsporidian pathogen of immunocompromised humans: an immunoblotting and immunocytochemical study

Microsporidia are unicellular and obligate intracellular spore-forming parasites. The spore inoculates the host cell with its non-motile infectious content, the sporoplasm, by way of the polar tube--the typical invasive apparatus of the microsporidian spore. Molecules involved in host cell invasion were investigated in Encephalitozoon intestinalis. Mouse polyclonal and monoclonal antibodies were raised against spore proteins and their reactivity was tested by Western-blotting and immunolocalization techniques, including electron and confocal microscopy. The antibodies thus generated could be divided into two major groups. One group reacted to the surface of the parasite at different developmental stages, mostly presporous stages and mature spores, whereas the other group recognized the polar tube. Of the antibodies reacting to the spore wall, one identified an exospore protein at 125 kDa while all others recognized a major doublet at 55-60 kDa, and minor proteins present at the surface of sporogonic stages and in the endospore. All antibodies recognizing spore wall proteins reacted also to the material forming septa in the parasitophorous vacuole. A major polar tube protein at 60 kDa was identified by another group of antibodies.     

Frozen in time: a new method using cryo-scanning electron microscopy to visualize root-fungal interactions

A new method of sample preparation for cryo-scanning electron microscopy was used to visualize internal infection of wheat (Triticum aestivum) roots by the pathogenic fungus Rhizoctonia solani AG-8. The new method retained fungal hyphae and root cells in situ in disintegrating root tissues, thus avoiding the distortions that can be introduced by conventional preparation by chemical fixation, dehydration and embedding. Infected roots frozen in liquid nitrogen were cryo-planed and etched (sublimed) at -80 degrees C for a critical length of time (up to 9 min) in the microscope column to reveal plant and fungal structures in three dimensions. Root and fungal structures were well preserved irrespective of infection severity. Root and hyphal cell walls were clearly seen and hyphal architecture within and between root cells was preserved. This rapid method permits three-dimensional in situ visualization of fungal invasion within roots and has broad application for examination of diseases caused by other necrotrophic fungi.     

Fluorescent proteins in poplar: a useful tool to study promoter function and protein localization

The jellyfish (Aequorea victoria) green fluorescent protein (GFP) and its variants (CFP [cyan] and YFP [yellow]) were successfully used as a vital marker system for the transformation of hybrid poplar (Populus tremula x P. alba). Our results show that, in this woody plant, fluorescent proteins can be expressed: (i) transiently in protoplasts after PEG-mediated transformation, as well as in leaf cells after particle bombardment, and (ii) stably in callus cells and plants after Agrobacterium-mediated transformation. For these studies, we constructed vectors permitting easy recloning of any promoter fragments of interest. Confocal laser scanning microscopy was used both for visualization and differentiation between the different colours of the GFP variants and autofluorescence of chlorophyll and lignified xylem vessels. Peroxisomes were chosen as target organelles for GFP translocation via the peroxisomal targeting sequence PTS1 because this allowed us to concentrate the fluorochrome in the small volume of a few peroxisomes, giving a bright fluorescence over background noise.     

Anisotropy and anharmonicity of atomic fluctuations in proteins: analysis of a molecular dynamics simulation

Positional probability density functions (pdf) for the atomic fluctuations are determined from a molecular dynamics simulation for hen egg-white lysozyme. Most atoms are found to have motions that are highly anisotropic but only slightly anharmonic. The largest deviations from harmonic motion are in the direction of the largest rms fluctuations in the local principal axis frame. Backbone atoms tend to be more nearly harmonic than sidechain atoms. The atoms with the largest anharmonicities tend to have pdfs with multiple peaks, each of which is close to harmonic. Several model pdfs are evaluated on the basis of how well they fit probability densities from the dynamics simulations when parameterized in terms of the moments of the distribution. Gram-Charlier and Edgeworth perturbation expansions, which have been successful in describing the motions of small molecules in crystals, are shown to be inadequate for the distributions found in the dynamics of proteins. Multipeaked distribution functions are found to be more appropriate.     

The musculature of Squatinella rostrum (Milne, 1886) (Rotifera: Lepadellidae) as revealed by confocal laser scanning microscopy with additional new data on its trophi and overall morphology

Wilts, E.F., Wulfken, D., Ahlrichs, W.H. and Martínez Arbizu, P. 2012. The musculature of Squatinella rostrum (Milne, 1886) (Rotifera: Lepadellidae) as revealed by confocal laser scanning microscopy with additional new data on its trophi and overall morphology.—Acta Zoologica (Stockholm) 93 : 14–27. The monogonont rotifer Squatinella rostrum was investigated with light, scanning electron and confocal laser scanning microscopy to reveal new morphological data on its inner and outer anatomy. In total, the visualized somatic musculature displays five paired longitudinal muscles (musculi longitudinales I–V) and nine circular muscles (musculi circulares I–IX). Compared to other species, S. rostrum is characterized by the absence of several longitudinal and circular muscles (e.g. musculus longitudinalis capitis, corona sphincter and pars coronalis). A reconstruction of the mastax musculature revealed a total number of seven paired and two unpaired mastax muscles. Possibly homologous somatic and mastax muscles in other, thus far investigated rotifers are discussed. Moreover, we provide a phylogenetic evaluation of the revealed morphological characters and suggest possible autapomorphic characters supporting Squatinella and Lepadellidae. Finally, we refer to some striking similarities in the morphology, ecology and way of movement of Squatinella and Bryceella that may indicate a closer relationship of both taxa.