Freeze-fractured Cells Research Articles

The proteorhodopsins of the dinoflagellate Oxyrrhis marina: ultrastructure and localization by immunofluorescence light microscopy and immunoelectron microscopy

At least 7 proteorhodopsin sequences of Oxyrrhis marina were recently proven in bands obtained by sucrose density gradient centrifugation, and MS analyses revealed that the bands consisted almost of pure, native proteorhodopsins (Rhiel et al. 2020). The proteorhodopsin fractions, i.e., bands B2, B3, and B4 were subjected to transmission electron microscopy. Negative staining revealed that band B2 consisted most likely of monomeric/oligomeric proteorhodopsins with particle dimensions of about 6 nm. Negative staining, freeze-fracture, and cryo-transmission electron microscopy revealed that bands B3 and B4 consisted of vesicular, sheet-like, and cup-shaped structures which all seemed to be composed of protein. Frequently, ring-like protein aggregates were registered at higher magnifications. They measured about 4 nm in diameter with a tiny hole of 1.5 nm in the middle. The bands B2, B3, and B4 were pooled and used to raise an antiserum. Immunoelectron microscopy resulted in intense labeling of the isolated structures. Immunofluorescence light microscopy of formaldehyde-fixed Oxyrrhis cells resulted in intense labeling of the cell periphery. Some cell internal structures became labeled, too. Immunoelectron microscopy of freeze-fractured cells revealed that most likely the membranes of the amphiesmal vesicles were labeled at the cell periphery, while the cell internal label seemed to originate from the food vacuoles.

LIGHT-INDUCED OIL GLOBULE MIGRATION IN HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE).

Astaxanthin-rich oil globules in Haematococcus pluvialis display rapid light-induced peripheral migration that is unique to this organism and serves to protect the photosynthetic system from excessive light. We observed rapid light-induced peripheral migration that is associated with chlorophyll fluorescence quenching, whereas the recovery was slow. A simple assay to follow globule migration, based on chlorophyll fluorescence level has been developed. Globule migration was induced by high intensity blue light, but not by high intensity red light. The electron transport inhibitor dichlorophenyl-dimethylurea did not inhibit globule migration, whereas the quinone analog (dibromo-methyl-isopropylbenzoquinone), induced globule migration even at low light. Actin microfilament-directed toxins, such as cytochalasin B and latrunculin A, inhibited the light-induced globule migration, whereas toxins against microtubules were ineffective. Electron microscopic (EM) imaging confirmed the cytoplasmic localization and peripheral migration of globules upon exposure to very high light (VHL). Scanning EM of freeze-fractured cells also revealed globules within cytoplasmic bridges traversing the chloroplast, presumably representing the pathway of migration. Close alignments of globules with endoplasmic reticulum (ER) membranes were also observed following VHL illumination. We propose that light-induced globule migration is regulated by the redox state of the photosynthetic electron transport system. Possible mechanisms of actin-based globule migration are discussed.

MeV‐energy probe SIMS imaging of major components in washed and fractured animal cells

AbstractMolecular images of pretreated animal cells were measured by secondary ion mass spectrometry (SIMS) with MeV‐energy ion beam. SIMS analysis was performed for cell surfaces washed with buffers not only at high vacuum, but also at several tens Pa by using an orthogonal acceleration time of flight mass spectrometer (oaTOFMS) system. Moreover, molecular images of major components from washed and freeze fractured cells were obtained using MeV‐energy probe SIMS. Copyright © 2010 John Wiley & Sons, Ltd.

Exploring the nuclear envelope of herpes simplex virus 1-infected cells by high-resolution microscopy.

Herpesviruses are composed of capsid, tegument, and envelope. Capsids assemble in the nucleus and exit the nucleus by budding at the inner nuclear membrane, acquiring tegument and the envelope. This study focuses on the changes of the nuclear envelope during herpes simplex virus 1 (HSV-1) infection in HeLa and Vero cells by employing preparation techniques at ambient and low temperatures for high-resolution scanning and transmission electron microscopy and confocal laser scanning microscopy. Cryo-field emission scanning electron microscopy of freeze-fractured cells showed for the first time budding of capsids at the nuclear envelope at the third dimension with high activity at 10 h and low activity at 15 h of incubation. The mean number of pores was significantly lower, and the mean interpore distance and the mean interpore area were significantly larger than those for mock-infected cells 15 h after inoculation. Forty-five percent of nuclear pores in HSV-1-infected cells were dilated to more than 140 nm. Nuclear material containing capsids protrude through them into the cytoplasm. Examination of in situ preparations after dry fracturing revealed significant enlargements of the nuclear pore diameter and of the nuclear pore central channel in HSV-1-infected cells compared to mock-infected cells. The demonstration of nucleoporins by confocal microscopy also revealed fewer pores but focal enhancement of fluorescence signals in HSV-1-infected cells, whereas Western blots showed no loss of nucleoporins from cells. The data suggest that infection with HSV-1 alters the number, size, and architecture of nuclear pores without a loss of nucleoporins from altered nuclear pore complexes.

Lattice supports single bilayer

The spherical membrane surrounding the poxvirus genome begins to form as an open, cup-like structure. Some have argued that the edge of the cup must be composed of two closely apposed membranes (imagine a collapsed vesicle); otherwise, the hydrophobic edges of a single membrane would be exposed to the cytoplasm. But on page 269, John Heuser confirms that poxvirus has only a single membrane bilayer. He finds that the growing membrane is stabilized by a proteinaceous lattice.Heuser imaged freeze-fractured cells infected with poxvirus. Freeze-fracturing tends to split membranes randomly along bilayer planes, thus revealing all the bilayers that are present. As only one viral membrane plane was revealed, and no known membrane is resistant to this technique, Heuser concluded that the virus has only one bilayer. Figure A tight honeycomb lattice (shown in 3D) covers the growing poxvirus membrane. The EM images also revealed a honeycomb-shaped lattice on the outer surface of the growing viral membrane. In older studies, the lattice appeared only as spikes on the membrane. Using deep-etch EM, Heuser was able to preserve and view the entire face of the lattice. Like clathrin, the lattice curves the membrane it surrounds. It may also stabilize the hydrophobic edges of the incomplete membrane within the cytoplasm. Indeed, others have found that preventing the spikes from associating with the nacent viral membrane causes it to collapse. This is the mechanism of action of rifampicin—an antibiotic that aggregates the lattice protein and blocks poxvirus DNA encapsulation.

In situ observation of freeze-fractured and deep-etched red blood cells with a high-vacuum low-temperature atomic force microscope

A high-vacuum low-temperature atomic force microscope (AFM) for the direct observation of freeze-fracture samples has been developed. This AFM has a freeze-fracture mechanism inside the vacuum chamber. With this AFM it is possible to observe the fractured surface directly without both fabricating a replica and exposure to the ambient atmosphere. Both sandwich and knife fracture methods have been achieved to obtain freeze-fracture surfaces and after deep etching. A fine structure of the fractured red blood cell membrane has been observed using both methods. These are relatively quick and easy methods for the observation of freeze-fracture surfaces without introducing replica artifacts.

Imaging of freeze-fractured cells with in situ fluorescence and time-of-flight secondary ion mass spectrometry.

Bioanalytical imaging techniques have been employed to investigate cellular composition at the single-cell and subcellular regimes. Four imaging modes have been performed sequentially in situ to demonstrate the utility of a more integrated approach to imaging cells. The combination of bright-field, scanning ion, and fluorescence microscopy complements TOF-SIMS imaging of native biomolecules. Bright-field microscopy provides a blurred visualization of cells in frozen-hydrated samples, while scanning ion imaging provides a morphological view of freeze-fractured cells after TOF-SIMS analysis is completed. With the use of selective fluorescent labels, fluorescence microscopy allows single mammalian cells to be located in the complex ice matrix of freeze-fractured samples, a task that has not been routine with either bright-field or TOF-SIMS. A fluorescent label, DiI (m/z 834), that does not interfere with the mass spectra of membrane phosphatidylcholine, has been chosen for fluorescence and TOF-SIMS imaging of membrane phospholipids. In this paper, in situ fluorescence microscopy allows the distinction of single cells from ice and other sample debris, previously not possible with bright-field or scanning ion imaging. Once cells are located, TOF-SIMS imaging reveals the localization of membrane lipids, even in the membrane of a single 15-microm rat pheochromocytoma cell. The utility of mapping lipids in the membranes of single cells using this integrated approach will provide more understanding of the functional role of specific lipids in functions of cellular membranes.

Trophozoites of Giardia lamblia may have a Golgi-like structure.

Trophozoites of the primitive protozoan Giardia lamblia have been considered as cells which do not present the Golgi complex. Using C(6)-NBD ceramide, which has been shown to label the Golgi complex of mammalian cells, labelling of the perinuclear region of G. lamblia was observed by confocal laser scanning microscopy. Transmission electron microscopy of thin sections and of replicas of freeze-fractured cells revealed the presence of concentric perinuclear membranes resembling the Golgi complex.

Trophozoites of Giardia lamblia may have a Golgi-like structure

Trophozoites of the primitive protozoan Giardia lamblia have been considered as cells which do not present the Golgi complex. Using C 6-NBD ceramide, which has been shown to label the Golgi complex of mammalian cells, labelling of the perinuclear region of G. lamblia was observed by confocal laser scanning microscopy. Transmission electron microscopy of thin sections and of replicas of freeze-fractured cells revealed the presence of concentric perinuclear membranes resembling the Golgi complex.

Viral Glycoproteins Accumulate in Newly Formed Annulate Lamellae following Infection of Lymphoid Cells by Human Herpesvirus 6

Ultrastructural analysis of HSB-2 T-lymphoid cells and human cord blood mononuclear cells infected with human herpesvirus 6 revealed the presence, in the cell cytoplasm, of annulate lamellae (AL), which were absent in uninfected cells. Time course analysis of the appearance of AL following viral infection showed that no AL were visible within the first 72 h postinfection and that their formation correlated with the expression of the late viral glycoprotein gp116. The requirement of active viral replication for AL neoformation was further confirmed by experiments using inactivated virus or performed in presence of the viral DNA polymerase inhibitor phosphonoacetic acid. Both conventional electron microscopic examination and immunogold fracture labeling with anti-endoplasmic reticulum antibodies indicated a close relationship of AL with the endoplasmic reticulum and nuclear membranes. However, when the freeze-fractured cells were immunogold labeled with an anti-gp116 monoclonal antibody, AL membranes were densely labeled, whereas nuclear membranes and endoplasmic reticulum cisternae appeared virtually unlabeled, showing that viral envelope glycoproteins selectively accumulate in AL. In addition, gold labeling with Helix pomatia lectin and wheat germ agglutinin indicated that AL cisternae, similar to cis-Golgi membranes, contain intermediate, but not terminal, forms of glycoconjugates. Taken together, these results suggest that in this cell-virus system, AL function as a viral glycoprotein storage compartment and as a putative site of O-glycosylation.

Further characterization and in situ localization of chain-like aggregates of the gliding bacteria Myxococcus fulvus and Myxococcus xanthus.

For the first time, chain-like aggregates, called "strands," have been enriched from crude cell wall preparations of liquid-grown vegetative cells of two strains of Myxococcus xanthus. These strands are highly isomorphic to macromolecular structures, previously described for Myxococcus fulvus (Lünsdorf and Reichenbach, J. Gen. Microbiol. 135:1633-1641, 1989). The strands are morphologically composed of ring elements, consisting of six or more peripheral protein masses and possibly three small central masses. The ring elements are linked by two parallel strings of filamentous proteins, called elongated elements, which keep the ring elements at a constant distance. The overall dimensions of the ring elements are 16.6 +/- 1.0 nm (n = 55) for M. xanthus Mx x48 and 16.4 +/- 1.5 nm (n = 37) for M. xanthus DK 1622. The distance between the ring elements, as a measure of the length of the elongated elements, is 16.6 +/- 1.1 nm (n = 59) for strain Mx x48 and 15.5 +/- 0.6 nm (n = 41) for strain DK 1622. Characteristically, the strands and oligomeric forms thereof show a strict association with the outer membrane. In situ studies of freeze-fractured cells of M. fulvus showed ring elements, isomorphic to those described for M. xanthus, within the periplasm; they appeared in parallel rows just below the outer membrane but not in direct contact with the cytoplasmic membrane. A three-dimensional model summarizes the morphological data. It is hypothesized that the chain-like strands, as building blocks of a more complex belt-like continuum, represent the peripheral part of the gliding machinery, which transforms membrane potential energy into mechanical work.

Freeze-fracture studies in the brown algaAsteronema rhodochortonoides

The gross structure of the cell wall and the organization of the plasmalemma of the filamentous brown algaAsteronema rhodochortonoides were examined in replicas of freeze-fractured cells. The protoplasmic fracture face (PF) of the plasmalemma, apart from the single particles, exhibits two particular particle complexes, i.e., single linear arrays of closely packed particles, and well defined particle pentads. The former display a consistent relationship with the ends of microfibril imprints and therefore are considered as terminal complexes (TCs). They seem to be composed of subunits, each one consisting of two particles. The average diameter of the particles is 7 nm. The number of the subunits forming the TCs varies between 2 and 40. Short TCs, consisting of 3–5 subunits were also found on the PF of dictyosome vesicles, a fact suggesting the involvement of the Golgi apparatus in exocytosis of preformed TC portions. The occurrence, distribution and size of the TCs appear to be related to the developmental stage of the cell. A large number of TCs occur in actively growing cells, while a few or no TCs are found in differentiated cells. The pentads are rectangular structures consisting of five particles, four in the corners and one in the centre. Their dimensions are very constant, but their occurrence and distribution varies. They occur in young developing cells where TCs are few or absent, but were also observed in areas showing many TCs. In differentiated cells no pentads were found. Pentad-like structures were rarely observed on the PF of dictyosome vesicles or cisternae. The observations support the hypothesis that pentads are involved in the synthesis of matrix polysaccharides, which are the major components of brown algal cell wall and their synthesis begins before that of cellulose.

Image analysis of the chromatin organization in the nuclear domains of freeze fractured hepatocytes and lymphocytes

The complex organization of the interphase nucleus can be analyzed, by way of thin sectioning and also freeze-fracture. This approach has previously been utilized in association with image analysis to quantitatively describe the organization of isolated rat liver nuclei and nuclear matrices. The main nuclear domains which, in section, present marked differences due to their electron-density, can be identified in replicas with more complex procedures, based on the quantitative evaluation of the number of particles per unit area and mainly by using image analysis. A quantitative analysis of the nuclear substructures has been performed by way of image analysis on in situ nuclei of freeze-fractured cells presenting marked differences in the heterochromatin quantity, such as hepatocytes and lymphocytes. The replicated nuclear particles have been classified according to their diameter and the obtained histograms have been quantitatively evaluated. The nuclear domains, heterochromatin, interchromatin, nucleolus, present characteristic ratios among the three main classes of particles; that is, ribonucleoproteins, solenoid filaments and solenoid fibre aggregates. The typical patterns of the nuclear domains can be further stressed by selecting a single class of particles and by examining its topographic localization. While interchromatin and nucleolar domains present a similar quantitative pattern in hepatocytes and lymphocytes, the heterochromatin of lymphocytes contains a significative higher percentage of solenoid aggregates than that of hepatocytes.

Morphological and elemental integrity of freeze‐fractured, freeze‐dried cultured cells during ion microscopic analysis

The effects of progressive ion beam bombardment on freeze-fractured, freeze-dried cultured cells during ion microscopic (SIMS) analysis were studied with scanning electron microscopy (SEM) and ion microscopy. The freeze-fracture, freeze-dry sample preparation method was generally found to preserve cell morphology to a level far exceeding the spatial resolution of the ion microscope, with splitting at the nuclear envelope being the most commonly observed artefact. SEM monitoring of surface topography of an NRK-49F fibroblast after various ion bombardment doses showed relatively uniform erosion of cellular material, with some apparent selective retention of small cytoplasmic granules. Prolonged bombardment produced no detectable lateral elemental translocation. 41K+/24Mg+ signal ratios from Swiss 3T3 fibroblasts and RBL rat basophilic leukaemia cells were shown to vary generally by less than 10% during the course of extended ion bombardment. GM0415 human skin fibroblasts containing engorged lysosomes characteristic of Hurler's Syndrome were used to evaluate the effects of ion bombardment during a typical analysis session, where ion images of 39K+, 23Na+, 40Ca+ and 24Mg+ are sequentially recorded. This cell line was chosen as a worst-case system, because these cells are often thinly spread and possess extreme surface topography. Thin cell edges were shown sometimes to sputter away during analysis, giving misleadingly low ion signals from these regions in some 24Mg+ micrographs. Various non-uniform sputtering phenomena occurring in the submicrometre spatial domain had little or no measurable impact on local intensities in ion micrographs, indicating that freeze-dried, freeze-fractured cells are sampled in a sufficiently uniform fashion that quantitative ion microscopic evaluations of intracellular elemental levels in the general cytoplasmic or nuclear regions are feasible.

Regulation of mitochondrial hexokinase in cultured HT 29 human cancer cells. An ultrastructural and biochemical study

The involvement of the mitochondrial bound hexokinase in aerobic glycolysis was investigated in two subpopulations of the HT 29 human colon cancer cell line: a poorly differentiated one with high aerobic lactate production (referred as undifferentiated or standard cells), and an enterocyte-like differentiated one with lower lactate production (referred as differentiated or Glc − cells). After mild digitonin treatment, 85% of the total cellular hexokinase activity remained in the particulate fraction in both cell types. In both cases mitochondria appeared to be tightly coupled but the Glc − cells exhibited a significantly higher oxidation rate in the presence of glucose. Electron microscopy of freeze-fractured cells revealed the absence of contacts between the two limiting mitochondrial membranes in the highly glycolytic standard cells, whereas the contacts were present in the Glc − cells. Furthermore, we investigated the functional relationship between bound hexokinase (as hexokinase-porin complex) and the inner compartment of mitochondria isolated from standard and Glc − HT 29 cells. In contrast to the differentiated cells the hexokinase in undifferentiated standard cells was not functionally coupled to the oxidative phosphorylation. This suggests that the high rate of lactate formation in neoplastic cells is not caused by an increase of particulate hexokinase activity but rather by a disregulation of the hexokinase-porin complex caused by the absence of contact sites between the two mitochondrial membranes. In agreement with this interpretation, the hexokinase-porin complex could be completely removed by digitonin treatment in standard HT 29 cells, while this was not possible in mitochondria from Glc − cells.

Hexagonal periodicity in the outer membrane of Bacteroides buccae.

In Bacteroides buccae, a hexagonally arranged periodic structure was found in the outer membrane (OM), in addition to hexagonal lattices present in its external surface layer (S-layer). This crystalline OM protein (COMP) was present as patches on the concave fracture face (the outer leaflet) of the OM in freeze-fractured cells. Occasionally, hexagonally arranged structures could also be seen on the convex fracture face of the OM as 'fingerprints' of the COMP. The OM proteins were isolated and analysed by gel electrophoresis. The major band protein had an apparent molecular mass of 17 kDa. Whether the minor band proteins are also components in the structure of the COMP remains to be elucidated. Other oral Gram-negative anaerobic rods studied did not show any periodicity in their OM.

Molecular cytochemistry of freeze-fractured cells

I will describe four approaches that combine cytochemistry with freeze-fracture: 1) FREEZE-ETCHING; 2) FRACTURE-LABEL; 3) FRACTURE-PERMEATION; and 4) LABEL-FRACTURE. These techniques, in particular fracture-label, involve delicate points of interpretation and numerous validating controls. In the publications listed at the end, these issues have been addressed in detail.1. FREEZE-ETCHING. I developed freeze-etching as a cytochemical approach to prove that membranes were split by freeze-fracture and to show that biological membranes were comprised of a bilayer membrane continuum interrupted by integral membrane proteins.1 - 4 In freeze-etching, the distribution of the marker over the membrane surface exposed by sublimation is compared to that of the intramembrane particles exposed by fracture. It is often required to aggregate the particles into domains larger than the labeling molecules (Fig. 1). This, and the need for freezing in distilled water, severely limits the application of freeze-etching.

Ultrastructure of Methanosarcina mazei

Summary Thin and semi-thin sections and freeze fracture replicas of unfixed, propane jet frozen cells of the methanogenic bacterium Methanosarcina mazei were examined in normal and medium-voltage (200 KV) transmission electron microscopes. A typical unit membrane surrounds the cytoplasm. Exterior to that is a single dark layer attached to the membrane by regular bridges. The “wall” is really a loose matrix of galactose derivatives polymerized into fibrils parallel to the plasma membrane. Cells 2 to 3 months old contain crystals, fibrillar inclusions, lipid-like inclusions, and cylindrical intrusions of the plasma membrane. We are calling these cylinders “exotubes”. On the cytoplasmic surface of their membrane, they are covered by regular hexagonal arrays. The plasma membrane faces of freeze fractured cells resemble those seen in other bacteria, exhibiting a protoplasmic fracture (PF) face with numerous intramembrane particles and an exoplasmic fracture (EF) face with some particle-free areas, many depressions, and only a few intramembrane particles.

Biogenesis and metabolic significance of microbodies in urate-utilizing yeasts.

Growth of Candida famata and Trichosporon cutaneum on uric acid as the sole source of carbon and nitrogen was associated with the development of a number of microbodies in the cells. Cytochemical staining experiments showed that the organelles contained urate oxidase, a key enzyme of uric acid metabolism, and catalase. Transfer of cells, precultured on glucose or glycerol, into uric acid-containing media indicated that these microbodies originated from the organelles, originally present in the inoculum cells, by growth and division. In urate-grown C. famata the microbodies were frequently observed in large clusters; in both organisms they existed in close association with mitochondria and strands of ER. The organelles lacked crystalline inclusions. In freeze-fractured cells their surrounding membranes showed smooth fracture faces. Exposure of urate-grown cells to glucose-excess conditions led to a rapid inactivation of urate oxidase activity but catalase was only slightly inactivated. Glucose-induced enzyme inactivation was not associated with the degradation of the microbodies present in the cells. Similarly, repression of urate oxidase synthesis by ammonium ions also did not lead to the degradation of peroxisomes.

Structural and cytochemical differentiation of membrane elements of the apical membrane of amphibian urinary bladder epithelial cells. A label fracture study.

It is now generally accepted that ADH-induced increase in water permeability in responsive epithelia is associated with the insertion of specific structures in the apical membrane of epithelial cells. Up to now, these structures have only been recognized in freeze-fractured preparations and their chemical nature is still unknown. In this study, we used the label-fracture method (Pinto da Silva and Kan, J. Cell Biol., 99, 1156-1161, 1984) to investigate the distribution of wheat germ agglutinin (WGA) on the luminal plasma membrane of freeze-fractured frog urinary bladder epithelial cells. With label-fracture, the cytochemical markers are seen superimposed with the conventional high resolution image of the E face. Label-fracture of tissue treated for 15 min with WGA and subsequently labeled with colloidal gold coated with ovomucoid showed uniform distribution of gold particles along the exoplasmic fracture face. Stereomicrographs show that the gold label is under the fracture face as it is attached to the outer surface of the membrane. Preincubation of the bladder with WGA for 3 hr induced a segregation of the intramembranous particles of the apical plasma membrane. In this condition, we observed a co-distribution of WGA-gold complexes with the segregated particles on the E face. This indicates that WGA-binding sites are located on glycoproteins which probably comprise the large intramembranous particles dispersed on the exoplasmic faces of freeze-fractured luminal membranes. In contrast, the numerous small intramembrane particles observed on P faces remained evenly distributed even after exposure to WGA and are, therefore, unrelated to WGA receptor sites. After WGA treatment, ADH still induced the formation of aggregates inside the smooth domains. A few WGA-binding sites appeared to be associated to these aggregates.