Imaging the dynamics of intracellular protein translocation by photoconversion of phamret-cybr/ROM.

Cybr/Reduced On-random Motile (ROM) is a scaffold protein, containing a postsynaptic density protein-95/discs-large/ZO-1 (PDZ) domain, a LEU region and a PDZ domain binding region at the C-terminus. In the immune system, Cybr/ROM was found to localize in vesicles and at the plasma membrane, through interactions with cytohesin-1. In this investigation, we reported Cybr/ROM as occurring in vesicles, the cytoplasm and at membrane ruffles of H1299 lung cancer cells. Its localization at the ruffles was dependent on intact actin structures as indicated by latrunculin A treatment, which abrogated ruffle formation and staining of Cybr/ROM at the cells' periphery. Transfection of truncation mutants consisting of either the PDZ or LEU domain showed that the LEU domain of ROM was localized to membrane ruffles, vesicles and the cytoplasm, whereas, the PDZ domain localized to the membrane ruffles and cytoplasm only. There was therefore, domain/molecular segregation of Cybr/ROM in different cellular compartments. Cybr/ROM was subcloned into a plasmid carrying the photoactivation-mediated resonance energy transfer (Phamret) protein. The photoconversion experiments demonstrated the diffusion of ROM from the cytoplasm to the membrane ruffling sites and conversely from membrane ruffles to the cytoplasm. Large variances in the transport velocity of Cybr/ROM in the cytoplasm suggested that its movements were facilitated by other mechanisms in addition to diffusion.

Correlative fluorescence and transmission electron microscopy: an elegant tool to study the actin cytoskeleton of whole-mount (breast) cancer cells.

Elucidating the structure and dynamics of lamellipodia and filopodia in response to different stimuli is a topic of continuing interest in cancer cells as these structures may be attractive targets for therapeutic purposes. Interestingly, a close functional relationship between these actin-rich protrusions and specialized membrane domains has been recently demonstrated. The aim of this study was therefore to investigate the fine organization of these actin-rich structures and examine how they structurally may relate to detergent-resistant membrane (DRM) domains in the MTLn3 EGF/serum starvation model. For this reason, we designed a straightforward and alternative method to study cytoskeleton arrays and their associated structures by means of correlative fluorescence (/laser)- and electron microscopy (CFEM). CFEM on whole mounted breast cancer cells revealed that a lamellipodium is composed of an intricate filamentous actin web organized in various patterns after different treatments. Both actin dots and DRM's were resolved, and were closely interconnected with the surrounding cytoskeleton. Long actin filaments were repeatedly observed extending beyond the leading edge and their density and length varied after different treatments. Furthermore, CFEM also allowed us to demonstrate the close structural association of DRMs with the cytoskeleton in general and the filamentous/dot-like structural complexes in particular, suggesting that they are all functionally linked and consequently may regulate the cell's fingertip dynamics. Finally, electron tomographic modelling on the same CFEM samples confirmed that these extensions are clearly embedded within the cytoskeletal matrix of the lamellipodium.

Sinusoidal obstruction syndrome (SOS): A light and electron microscopy study in human liver.

AIMS: Oxaliplatin is an important chemotherapeutic agent, used in the treatment of hepatic colorectal metastases, and known to induce the sinusoidal obstruction syndrome (SOS). Pathophysiological knowledge concerning SOS is based on a rat model. Therefore, the aim was to perform a comprehensive study of the features of human SOS, using both light microscopy (LM) and electron microscopy (EM). METHODS AND RESULTS: Included were all patients of whom wedge liver biopsies were collected during a partial hepatectomy for colorectal liver metastases, in a 4-year period. The wedge biopsy were perfusion fixated and processed for LM and EM. The SOS lesions were selected by LM and details were studied using EM. Material was available of 30 patients, of whom 28 patients received neo-adjuvant oxaliplatin. Eighteen (64%) of the 28 patients showed SOS lesions, based on microscopy. The lesions consisted of sinusoidal endothelial cell detachment from the space of Disse on EM. In the enlarged space of Disse a variable amount of erythrocytes were located. CONCLUSION: Sinusoidal endothelial cell detachment was present in human SOS, accompanied by enlargement of the space of Disse and erythrocytes in this area. These findings, originally described in a rat model, were now for the first time confirmed in human livers under clinically relevant settings.

Comparison between aortic and sinusoidal liver endothelial cells as targets of hyperacute xenogeneic rejection in the pig to human combination.

Endothelial cells of aortic origin are usually used in vitro as targets of hyperacute xenogeneic rejection, although endothelial cells from organs may have different properties. The sensitivities of aortic and liver endothelial cells to hyperacute xenogeneic rejection were compared in the pig to human combination. Sinusoidal liver endothelial cells were isolated and purified by collagenase perfusion of pig livers, sedimentation on a percoll gradient and selective adherence. Purity and viability of isolated liver endothelial cells after adherence were 85+/-6% and >95%, respectively. Endothelial cells from pig aortae (purity and viability >95%) were isolated by scraping. Immunoblotting analysis of xenoantigens on liver and aortic endothelial cell membranes preparations showed identical patterns. The strongest bands revealed by human IgM were located between 110 and 135 kD, while human IgG detected two major bands at 115 and 75kD. The membrane expression of xenoantigens recognized by human sera, analyzed by flow cytometry, was significantly lower on liver than on aortic endothelial cells (IgM: P=0.0006; IgG: P=0.0009). However, the complement-dependent cytotoxic activity of human sera was the same whether liver (54.5+/-1.4%) or aortic endothelial cells (50.0+/-4.2%) were used as targets. Taken together, those results allow the use of aortic instead of sinusoidal liver endothelial cells in the characterization of pig antigens recognized by human natural antibodies.

Identification of rat pancreatic duct cells by their expression of cytokeratins 7, 19, and 20 in vivo and after isolation and culture.

Cells from the excretory ducts of the pancreas are thought to be capable of differentiating into exocrine and endocrine cells. To study this in rat models, markers must be found to identify the cells under different experimental conditions. We tested antibodies to different cytokeratins (CKs) by immunocytochemical staining on pancreatic tissue sections from normal rats, after partial pancreatectomy, and after isolation and culture of duct fragments. Monoclonal antibodies to human CK7, CK19, and CK20 were found to react specifically on rat pancreas tissue, as shown by Western blotting. CK20 and CK19 were immunocytochemically detected only in cells of the ductal system, from centroacinar cells to main ducts. CK7 was expressed by islets of Langerhans and by duct cells from main, inter-, and intralobular ducts, but not by centroacinar and terminal duct cells. CKs 7, 19, and 20 were also expressed in proliferating duct cells during tissue regeneration and after isolation and different periods of culture. We conclude that CKs 7, 19, and 20 are very useful markers to study the differentiation of rat duct cells under experimental conditions in vivo and in vitro.

New anti-actin drugs in the study of the organization and function of the actin cytoskeleton.

The high degree of structural and molecular complexity of the actin-based cytoskeleton, combined with its ability to reorganize rapidly and locally in response to stimuli, and its force-generating properties, have made it difficult to assess how the different actin structures are assembled in cells, and how they regulate cell behavior. An obvious approach to study the relationships between actin organization, dynamics, and functions is the specific perturbation of actin structures using pharmacological means. Until recently there were only a few agents available that interfered with cellular activities by binding to actin and most of our knowledge concerning the involvement of actin in basic cellular processes was based on the extensive use of the cytochalasins. In recent years we have identified an increasing number of actin-targeted marine natural products, including the latrunculins, jasplakinolides (jaspamides), swinholide A, misakinolide A, halichondramides, and pectenotoxin II, which are discussed in this article. All these marine-sponge-derived compounds are unusual macrolides and can be classified into several major families, each with its own distinct chemical structures. We describe the current state of knowledge concerning the actin-binding properties of these compounds and show that each class of drugs alters the distribution patterns of actin in a unique way, and that even within a chemical class, structurally similar compounds can have different biochemical properties and cellular effects. We also discuss the effects of these new drugs on fenestrae formation in liver endothelial cells as an example of their usefulness as powerful tools to selectively unmask actin-mediated dynamic processes.

Early detection of cytotoxic events between hepatic natural killer cells and colon carcinoma cells as probed with the atomic force microscope.

The atomic force microscope (AFM) is a powerful tool to investigate surface and submembranous structures of living cells under physiological conditions at high resolution. These properties enabled us to study the interaction between live hepatic natural killer (NK) cells, also called pit cells, and colon carcinoma cells in vitro by AFM. In addition, the staining for filamentous actin and DNA was performed and served as a reference, because actin and nuclear observations at the light microscopic level during the cytotoxic interaction between these two cell types have been presented earlier. In this study, we collected evidence that conjugation of hepatic NK cells with CC531s colon carcinoma cells results in a decreased binding of CC531s cells to the substratum as probed with the AFM in contact mode as early as 10 min after cell contact (n = 11). To avoid the lateral forces and smearing artefacts of contact mode AFM, non-contact imaging was performed on hepatic NK/CC531s cell conjugates, resulting in identical observations (n = 3). In contrast, the first cytotoxic signs, as determined with the nuclear staining dye Hoechst 33342, could be observed 3 h after the start of the co-culture. This study illustrates that the AFM can be used to probe early cytotoxic effects of effector to target cell contact in nearby physiological conditions. Other routine cytotoxicity tests detect the first cytotoxic effects after 1.5-3 h co-incubation at the earliest.

AFM imaging of fenestrated liver sinusoidal endothelial cells.

Each microscope with its dedicated sample preparation technique provides the investigator with a specific set of data giving an instrument-determined (or restricted) insight into the structure and function of a tissue, a cell or parts thereof. Stepwise improvements in existing techniques, both instrumental and preparative, can sometimes cross barriers in resolution and image quality. Of course, investigators get really excited when completely new principles of microscopy and imaging are offered in promising new instruments, such as the AFM. The present paper summarizes a first phase of studies on the thin endothelial cells of the liver. It describes the preparation-dependent differences in AFM imaging of these cells after isolation. Special point of interest concerned the dynamics of the fenestrae, thought to filter lipid-carrying particles during their transport from the blood to the liver cells. It also describes the attempts to image the details of these cells when alive in cell cultures. It explains what physical conditions, mainly contributed to the scanning stylus, are thought to play a part in the limitations in imaging these cells. The AFM also offers promising specifications to those interested in cell surface details, such as membrane-associated structures, receptors, coated pits, cellular junctions and molecular aggregations or domains. The AFM also offers nano-manipulation possibilities, strengths and elasticity measurements, force interactions, affinity measurements, stiffness and other physical aspects of membranes and cytoskeleton. The potential for molecular approaches is there. New developments in cantilever construction and computer software promise to bring real time video imaging to the AFM. Home made accessories for the first generation of AFM are now commodities in commercial instruments and make the life of the AFM microscopist easier. Also, the combination of different microscopies, such as AFM and TEM, or AFM and SEM find their way to the market allowing comfortable correlative microscopy.

Imaging fluorescently labeled complexes by means of multidimensional correlative light and transmission electron microscopy: practical considerations.

These days the common ground between structural biology and molecular biology continues to grow thanks to the biomolecular insights offered by correlative microscopy, even though the vision of combining insights from different imaging tools has been around for nearly four decades. The use of correlative imaging methods to dissect the cell's internal structure is progressing faster than ever as shown by the boom in the number of methodological approaches available for correlative microscopy studies, each designed to address a specific scientific question. In this chapter, we will present a relatively straightforward approach to combining information from fluorescence microscopy and electron microscopy at the supramolecular level. The method combines live-cell and/or confocal laser microscopy with classical sample preparation for transmission electron microscopy (TEM), thereby allowing the integration of dynamic details of subcellular processes with insights about the organelles and molecular machinery involved. We illustrate the applicability of this multidimensional correlative microscopy approach on cultured Caco-2 colorectal cancer cells exposed to fluorescently labeled cisplatin, and discuss how these methods can deepen our understanding of key cellular processes, such as drug uptake and cell fate.

Correlative microscopy: providing new understanding in the biomedical and plant sciences.

Correlative microscopy is the application of two or more distinct microscopy techniques to the same region of a sample, generating complementary morphological, structural and chemical information that exceeds what is possible with any single technique. As a variety of complementary microscopy approaches rather than a specific type of instrument, correlative microscopy has blossomed in recent years as researchers have recognised that it is particularly suited to address the intricate questions of the modern biological sciences. Specialised technical developments in sample preparation, imaging methods, visualisation and data analysis have also accelerated the uptake of correlative approaches. In light of these advances, this critical review takes the reader on a journey through recent developments in, and applications of, correlative microscopy, examining its impact in biomedical research and in the field of plant science. This twin emphasis gives a unique perspective into use of correlative microscopy in fields that often advance independently, and highlights the lessons that can be learned from both fields for the future of this important area of research.

An outbreak of granulomatous peritonitis caused by injectable selenium in a flock of Merino sheep.

During meat inspection, unusual pigmented lesions were found in the abdomens of 411 sheep from a flock raised in the Northern Tablelands of New South Wales. In each affected sheep there were multiple discrete, soft, yellow homogeneous plaques beneath the parietal peritoneum and extending into marginating facial planes of the diaphragm and body wall. Microscopically, the lesions consisted of focal granulomatous peritonitis with intracellular acicular refractile golden-brown crystals. Energy dispersive X-ray spectroscopy revealed intralesional barium and selenium, two components of an injectable selenium compound administered to the sheep 6-8 months prior, which contains the yellow pigment, iron oxide. The mechanism of subperitoneal deposition of the compound could not be confirmed, but is presumed to have involved intraperitoneal injection of barium selenate. Meat inspectors and diagnosticians should consider barium selenate injection-site granulomas as a possible explanation for yellow pigmented lesions, especially in livestock from selenium-deficient areas. Animal care providers should be aware that incorrect administration of barium selenate can result in losses from condemnation or downgrading of meat product.

Creating next-generation microscopists: structural and molecular biology at the crossroads.

This paper highlights the importance of advanced microscopy and microanalysis in the pursuit of quality research in the biological and life sciences. With the growing complexity of modern microscopes, there is substantial risk of incorrect use or misinterpretation of data by the inexperienced researcher. This paper emphasizes the need for collaboration between biological microscopists and molecular biologists, within the context of centralized facilities and supported by first-class training, to fully realize the power of these unique instruments in modern biology and to create the next generation of molecular microscopists.

Nanostructural analysis of starch components by atomic force microscopy.

Morphological and structural features of starch from potato (Solanum tuberosa) and rice (Oryza sativa) have been examined using atomic force microscopy. Amylose from potato and rice was observed in aggregated structures, which are suggested to be a result of retrogradation during sample preparation. The degrees of polymerization of amylose from potato and rice starches were calculated from the mean contour lengths of the observed structures to be approximately 1440 and 1860, respectively. Potato amylopectin appeared as a highly branched and extended molecule. Our results show that atomic force microscopy provides a useful method for examining the fine structural features and estimating the dimensions of starch molecules.

A novel structure involved in the formation of liver endothelial cell fenestrae revealed by using the actin inhibitor misakinolide.

Hepatic endothelial fenestrae are dynamic structures that act as a sieving barrier to control the extensive exchange of material between the blood and the liver parenchyma. Alterations in the number or diameter of fenestrae by drugs, hormones, toxins, and diseases can produce serious perturbations in liver function. Previous studies have shown that disassembly of actin by cytochalasin B or latrunculin A caused a remarkable increase in the number of fenestrae and established the importance of the actin cytoskeleton in the numerical dynamics of fenestrae. So far, however, no mechanism or structure has been described to explain the increase in the number of fenestrae. Using the new actin inhibitor misakinolide, we observed a new structure that appears to serve as a fenestrae-forming center in hepatic endothelial cells.

New observations on cytoskeleton and fenestrae in isolated rat liver sinusoidal endothelial cells.

Fenestrae control the exchange of fluids, dissolved compounds and small particles between the blood and the space of Disse, and are primarily limited at one side by parenchymal cells. We recently described a simple and rapid method for the isolation, purification and cultivation of rat liver sinusoidal endothelial cells. With regard to the purity and morphology of liver endothelial cells, a detailed microscopic study was performed. Purity and viability after selective adherence was 74 and 95%, respectively. Liver endothelial cell purity was further enhanced to about 95% during adherence and spreading on collagen after 8 h of culture. Liver endothelial cells isolated by this method provide a viable cell population, enabling the study of structure and function of these cells in vitro. We investigated the cytoskeleton associated with fenestrae and sieve plates of liver endothelial cells. Cultured cells were slightly fixed and treated with cytoskeleton extraction buffer containing 0.1% Triton. Whole mounts of extracted liver endothelial cells were prepared for scanning and transmission electron microscopy. Extracted liver endothelial cells show an integral, intricate cytoskeleton. Sieve plates and fenestrae are clearly delineated by cytoskeleton elements. Fenestrae are surrounded by a filamentous, fenestrae-associated cytoskeleton ring with an average filament thickness of 16 nm. Additionally, sieve plates are surrounded and delineated by microtubuli, which form a network together with additional branching cytoskeletal elements. Microtubuli are sometimes found delineating linear arrangements of fenestrae. In conclusion, liver endothelial cells possess a cytoskeleton, that defines and supports sieve plates and fenestrae. Fenestrae-associated cytoskeleton rings are involved in determining the size of fenestrae. The fenestrae-associated cytoskeleton therefore probably controls the important hepatic function of endothelial filtration.

AFM imaging and elasticity measurements on living rat liver macrophages.

The authors investigated the morphology and the elastic properties of living cultured rat liver macrophages (Kupffer cells) with an atomic force microscope (AFM). Continuous imaging and elasticity mapping of individual cells in physiological buffer was carried out for several hours without damaging the cells as judged by their persistent undisturbed morphology. Dynamic events such as protrusive activity were observed in time course. The importance of the cytoskeleton for the mechanical properties of the cell has been investigated by measuring the cell's elasticity as a function of position. Chemical disassembly of the actin network by applying 10 microgram/ml cytochalasin B decreased the cell's average elastic modulus seven-fold within less than 40 minutes. Treating the cells with 0.1 micrograms/ml latrunculin A resulted in a two-fold decrease in the elastic modulus merely in the perinuclear region after 40 minutes, whereas other parts of the cell were not affected.

Imaging surface and submembranous structures with the atomic force microscope: a study on living cancer cells, fibroblasts and macrophages.

Atomic force microscopy (AFM) has been used to image a wide variety of cells. Fixed and dried-coated, wet-fixed or living cells were investigated. The major advantage of AFM over SEM is the avoidance of vacuum and electrons, whereas imaging can be done at environmental pressure and in aqueous conditions. Evidence of the successful application of AFM in biological imaging is provided by comparing results of AFM with SEM and/or TEM. In this study, we investigated surface and submembranous structures of living and glutaraldehyde-fixed colon carcinoma cells, skin fibroblasts and liver macrophages by AFM. Special attention was paid to the correct conditions for the acquisition of images of the surface of these cells, because quality SEM examinations have already been abundantly presented. AFM imaging of living cells revealed specific structures, such as the cytoskeleton, which were not observed by SEM. Membrane structures, such as ruffles, lamellipodia, microspikes and microvilli, could only clearly be observed after fixing the cells with 0.1% glutaraldehyde. AFM images of living cells were comparable to SEM images of fixed, dried and coated cells, but contained a number of artefacts due to tip-sample interaction. In addition, AFM imaging allowed the visualization of cytoplasmic submembranous structures without the necessity for further preparative steps, allowing us: (i) to follow cytoskeletal changes in fibroblasts under the influence of the microfilament disrupting agent latrunculin A; (ii) to study particle phagocytosis in macrophages. Therefore, in spite of the slow image acquisition of the AFM, the instrument can be used for high-resolution real-time studies of dynamic changes in submembranous structures.

Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells.

Critical point drying (CPD) is a common method of drying biological specimens for scanning electron microscopy (SEM). Drying by evaporation of hexamethyldisilazane (HMDS) has been described as a good alternative. This method, however, is infrequently used. Therefore, we reassessed HMDS drying. Cultured rat hepatic sinusoidal endothelial cells (LEC), possessing fragile fenestrae and sieve plates, were subjected to CPD and HMDS drying and evaluated in the scanning electron microscope, atomic force microscope (AFM) and transmission electron microscope (TEM). We observed no differences between the two methods regarding cellular ultrastructure. In contrast with CPD, HMDS drying takes only a few minutes, less effort, low costs for chemicals and requires no equipment. We conclude that HMDS-dried specimens have equal quality to CPD ones. Furthermore, the method also proved useful for drying whole-mount cells for TEM and AFM.

Insulin-like growth factor-1 acts as a chemoattractant factor for 5T2 multiple myeloma cells.

The chemotactic and growth-stimulatory effect of insulin-like growth factor 1 (IGF-1) was investigated in the experimental mouse 5T2 multiple myeloma (MM) model. Chemotaxis was analyzed by classical checkerboard analysis. Bone marrow fibroblasts-conditioned medium exhibited a chemotactic effect on 5T2 MM cells that could be neutralized by adding a blocking antibody to IGF-1. On the other hand, exogenously added IGF-1 also had a chemotactic effect on the 5T2 MM cells. Moreover, in vitro analysis demonstrated that transmigrated 5T2 MM cells have a higher expression of IGF-1 receptor, both in bone marrow-conditioned medium and in IGF-1-induced chemotaxis, in comparison to cells before migration. When analyzed in vivo, 18 hours after injection of the heterogeneous 5T2 MM population, 5T2 MM cells present in the bone marrow show a higher expression of the IGF-1 receptor than their counterparts before injection. When the proliferative effect of IGF-1 was analyzed, no stimulation was observed, which is in contrast to the influence of bone marrow-conditioned medium and interleukin-6. Our results suggest a causal relationship between the presence of IGF-1 in the bone marrow and the chemotaxis of MM cells to and their subsequent presence in the bone marrow.