Cryo-section immunolabelling of difficult to preserve specimens: advantages of cryofixation, freeze-substitution and rehydration

BACKGROUND INFORMATION: Electron microscopic immunolabelling of ultrathin thawed cryo-sections, according to the method of Tokuyasu, is widely used as a very sensitive high-resolution localization technique. Its main advantages are that antigens remain in a hydrated environment prior to immunolabelling, and that antigen accessibility is improved compared with resin section labelling. However, the quality of structural appearance and antigenicity depends highly on the limitations of the initial conventional chemical fixation step, such as slow diffusion and selective reaction/cross-linking of fixative molecules. RESULTS AND CONCLUSIONS: Cryofixation, instead of conventional chemical fixation, followed by freeze-substitution/chemical fixation, rehydration and further processing for Tokuyasu cryo-sectioning leads to an improved preservation of both ultrastructure and antigenicity. This is especially true for tissues which are difficult to preserve by conventional chemical fixation at ambient temperatures, such as plant material, Drosophila embryos or nematode tissue. In particular labile and highly dynamic structures (for example, microtubules and Golgi apparatus) are remarkably better preserved. These improvements are also valid for light microscopic applications.     

Degradation, Recycling, and Shedding of Trypanosoma brucei Variant Surface Glycoprotein

ABSTRACT. Trypanosoma brucei bloodstream forms express a densely packed surface coat consisting of identical variant surface glycoprotein (VSG) molecules. This surface coat is subject to antigenic variation by sequential expression of different VSG genes and thus enables the cells to escape the mammalian host's specific immune response. VSG turnover was investigated and compared with the antigen switching rate. Living cells were radiochemically labeled with either ¹²⁵I-Bolton-Hunter reagent or ³⁵S-methionine, and immunogold-surface labeled for electron microscopy studies. The fate of labeled VSG was studied during subsequent incubation or cultivation of labeled trypanosomes. Our data show that living cells slowly released VSG into the medium with a shedding rate of 2.2 ± 0.6% h⁻¹ (t_1/2= 33 ± 9 h). In contrast, VSG degradation accounted for only 0.3 ± 0.06% h⁻¹ (t_1/2= 237 ± 45 h) and followed the classical lysosomal pathway as judged by electron microscopy. Since VSG uptake by endocytosis was rather high, our data suggest that most of the endocytosed VSG was recycled to the surface membrane. These results indicate that shedding of VSG at a regular turnover rate is sufficient to remove the old VSG coat within one week, and no increase of the VSG turnover rate seems to be necessary during antigenic variation.     

Antibodies specific for gp40 inhibit cell-cell adhesion by cross-linking the protein on the surface of Dictyostelium purpureum

We have previously suggested a role for gp40 in cell-cell adhesion in Dictyostelium purpureum from the fact that antibodies specific for this protein inhibited adhesion in an in vitro assay [Springer: Dev Biol 133:447–455, 1989]. To further confirm this role mutants lacking the protein were isolated and characterized. To our surprise, the mutants had normal adhesive properties both in vitro and in situ. These results lead us to the conclusion that gp40 is not necessary for the cell-cell adhesions observed and may not be a molecule which directly participates in these adhesions. When studied further, we found that adhesion-inhibitory antibodies were only effective as divalent IgG. Monovalent Fab fragments of the same antibodies could not inhibit adhesion. The inhibitory antibodies also caused the cells to remain rounded and incapable of attaching to plastic surfaces. We conclude that when divalent antibodies specific for gp40 cross-link this protein on the cell surface a cytoskeletal change prevents them from attaching to substratum as well as to other cells, thereby inhibiting cell-cell adhesion. We suggest that an alternative mechanism for inhibition of cell-cell adhesion by divalent antibodies exists and should be considered before proposing a direct role for a protein in adhesion.     

The Plant Nucleoskeleton: Ultrastructural Organization and Identification of NuMA Homologues in the Nuclear Matrix and Mitotic Spindle of Plant Cells

In the present work we investigate the structural organization of the nucleoskeleton ofAllium cepameristematic root cells. Resinless sections reveal for the first time a residual filamentous network in plant nuclei. This network is composed of branched knobbed filaments with associated globular structures, connected to the lamina and to the dense aggregates of different sizes. Results of immunoblotting show that many components of this network are homologues of intermediate filament-type proteins. NuMA, a coiled-coil protein related to intermediate filaments, found in animal cells, can also be detected in this plant nuclear matrix system. Immunofluorescence reveals a diffuse distribution of the animal NuMA homologues in plant nuclear core filaments in interphase. Resinless immunoelectron microscopy further reveals a distribution along the extended filaments and the dense aggregates. During mitosis, in contrast to the accumulation at the poles in animal cells, NuMA homologues in plant onion cells show a diffuse pattern, which may correspond to the spindle matrix. Our data are the first report of the conservation in plants of NuMA proteins, which may be involved in both nuclear and mitotic spindle organizations.     

Systemic induction of H2O2 in pea seedlings pretreated by wounding and exogenous jasmonic acid

Pea seedlings (Pisum sativum L.) were used as materials to test the timings and compartments of hydrogen peroxide (H₂O₂) triggered by wounding and exogenous jasmonic acid (JA). The results showed that H₂O₂ could be systemically induced by wounding and exogenous JA. H₂O₂ increased within 1 h and reached the peak 3–5 h after wounding in either the wounded leaves or the unwounded leaves adjacent to the wounded ones and the inferior leaves far from the wounded ones. After this, H₂O₂ decreased and recovered to the control level 12 h after wounding. The activities of antioxidant enzymes, however, were rapidly increased by wounding. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, could significantly inhibit H₂O₂ burst that was mediated by wounding and exogenous JA. Assay of H₂O₂ subcellular location showed that H₂O₂ in response to wounding and exogenous JA was predominantly accumulated in plasma membrane, cell wall and apoplasmic space. Numerous JA (gold particles) was found via immunogold electron microscopy to be located in cell wall and phloem zones of mesophyll cell after wounding.     

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.     

Advanced Correlative Light/Electron Microscopy: Current Methods and New Developments Using Tokuyasu Cryosections

Microscopy is an essential tool for analysis of cellular structures and function. With the advent of new fluorescent probes and super-resolution light microscopy techniques, the study of dynamic processes in living cells has been greatly facilitated. Fluorescence light microscopy provides analytical, quantitative, and three-dimensional (3D) data with emphasis on analysis of live cells using fluorescent markers. Sample preparation is easy and relatively inexpensive, and the use of appropriate tags provides the ability to track specific proteins of interest. Of course, only electron microscopy (EM) achieves the highest definition in terms of ultrastructure and protein labeling. To fill the gap between light microscopy and EM, correlative light and electron microscopy (CLEM) strategies have been developed. In particular, hybrid techniques based upon immuno-EM provide sensitive protein detection combined with high-resolution information on cell structures and protein localization. By adding the third dimension to EM with electron tomography (ET) combined with rapid freezing, CLEM techniques now provide additional tools for quantitative 3D analysis. Here, we overview the major methods applied and highlight the latest advances in the field of CLEM. We then focus on two selected techniques that use cryosections as substrate for combined biomolecular imaging. Finally, we provide a perspective of future developments in the field. (J Histochem Cytochem 57:1103–1112, 2009)     

Ultrastructural Analysis of Nanogold-labeled Endocytic Compartments of Yeast Saccharomyces cerevisiae Using a Cryosectioning Procedure

Yeast Saccharomyces cerevisiae has been a valuable model organism for the study of the endosomal system of eukaryotic cells. Morphological analyses, however, have been limited because of the lack of specific protein markers and of procedures that lead to a satisfactory ultrastructural resolution. We have recently developed an immunoelectron microscopy (IEM) protocol adapted from the Tokuyasu method to prepare cryosections from mildly fixed yeast. This novel approach allows excellent cell preservation and a unique resolution of the yeast morphology. Here, we present a protocol that combines this procedure with the specific labeling of the various endosomal compartments with positively charged Nanogold. In particular, we show that this new protocol generates excellent results when applied for the examination of early and late endosomes, and of mutants with an endosomal trafficking defect. Importantly, this method is compatible with immunogold labeling of protein markers, and it is consequently appropriate for localization studies of both resident and cargo proteins. This new IEM protocol will be a valuable tool for the large community of scientists using yeast as a model system to investigate the membrane transport and the biogenesis of the endosomal system. (J Histochem Cytochem 57:801–809, 2009)