TEM as an Important Tool to Study Aquatic Microorganisms and their Relationships with Ecological Processes

Microorganisms are critically important for ecological processes in aquatic environments. Bacteria and viruses are key components of the microbial loop and are central for biogeochemical cycles in aquatic ecosystems. Our group has been using transmission electron microscopy (TEM) to study aquatic microorganisms in both natural tropical ecosystems and cultures. In this review, we highlight structural aspects of freshwater bacteria, based on TEM findings that have provided insights into the functional capabilities of these cells in aquatic tropical ecosystems. First, we focus on TEM applied to the study of the ultrastructural diversity and morphological alterations of bacteria in response to environmental stress. Second, we address the relationship between viruses and bacteria in freshwater ecosystems. Third, we demonstrate by TEM that outer membrane vesicles (OMVs), structures associated with cell secretion and cell communication, are released by aquatic bacteria into natural ecosystems and cultures. Thus, TEM has proven to be a powerful technique to study aquatic microorganisms, contributing to the understanding of ecological processes, including regulation of bacterial populations, during different environmental conditions

Contributions of Electron Microscopy to Understand Secretion of Immune Mediators by Human Eosinophils

Mechanisms governing secretion of proteins underlie the biologic activities and functions of human eosinophils, leukocytes of the innate immune system, involved in allergic, inflammatory, and immunoregulatory responses. In response to varied stimuli, eosinophils are recruited from the circulation into inflammatory foci, where they modulate immune responses through the release of granule-derived products. Transmission electron microscopy (TEM) is the only technique that can clearly identify and distinguish between different modes of cell secretion. In this review, we highlight the advances in understanding mechanisms of eosinophil secretion, based on TEM findings, that have been made over the past years and that have provided unprecedented insights into the functional capabilities of these cells

Piecemeal degranulation in human eosinophils: a distinct secretion mechanism underlying inflammatory responses

Secretion is a fundamental cell process underlying different physiological and pathological events. In cells from the human immune system such as eosinophils, secretion of mediators generally occurs by means of piecemeal degranulation, an unconventional secretory pathway characterized by vesicular transport of small packets of materials from the cytoplasmic secretory granules to the cell surface. During piecemeal degranulation in eosinophils, a distinct transport vesicle system, which includes large, pleiomorphic vesiculotubular carriers is mobilized and enables regulated release of granule-stored proteins such as cytokines and major basic protein. Piecemeal degranulation underlies distinct functions of eosinophils as effector and immunoregulatory cells. This review focuses on the structural and functional advances that have been made over the last years concerning the intracellular trafficking and secretion of eosinophil proteins by piecemeal degranulation during inflammatory responses

Host lipid bodies as platforms for intracellular survival of protozoan parasites

Pathogens induce several changes in the host cell signaling and trafficking mechanisms in order to evade and manipulate the immune response. One prominent pathogen-mediated change is the formation of lipid-rich organelles, termed lipid bodies or lipid droplets, in the host cell cytoplasm. Protozoan parasites, which contribute expressively to the burden of infectious diseases worldwide, are able to induce lipid body genesis in non-immune and immune cells, mainly macrophages, key players in the initial resistance to the infection. Under host-parasite interaction, lipid bodies not only accumulate in the host cytoplasm but also relocate around and move into parasitophorous vacuoles. There is increasing evidence that protozoan parasites may target host-derived lipid bodies either for gaining nutrients or for escaping the host immune response. Newly formed, parasite-induced lipid bodies may serve as lipid sources for parasite growth and also produce inflammatory mediators that potentially act in the host immune response deactivation. In this mini review, we summarize current knowledge on the formation and role of host lipid bodies as sites exploited by intracellular protozoan parasites as a strategy to maintain their own survival

Activated Human Eosinophils

Eosinophils are leukocytes which, when stimulated with cytokines or chemokines, become activated and release mediators stored in their dominant population of cytoplasmic granules (termed specific or secondary granules) [1]. The morphology of activated eosinophils is quite distinct. In a range of inflammatory and allergic disorders or upon physiological stimulation, activated eosinophils are generally seen as cells full of granules, but these structures are undergoing progressive losses of their contents with retention of granule outer membranes, a process known as ‘piecemeal degranulation’ [2,3] (fig. 1a). As a result, a mixed population of intact (fig. 1a, asterisks) and enlarged, emptying (fig. 1a, arrows) granules is always visualized in cell sections [3]. Also, a decrease of specific granule numbers can occur [3]. A rare view of an activated eosinophil is shown in figure 1b taken from a hypereosinophilic subject. While a single specific granule (Gr) is seen in the cytoplasm in conjunction with an osmiophilic lipid body (LB), the cell surface shows elaborate projections indicative of activation. Vesiculotubular structures (fig. 1b, arrowheads), termed ‘eosinophil sombrero vesicles’ (EoSV) and recently associated with the eosinophil secretory pathway [4,5] reside in the cytoplasm. This vesicle population is unique to eosinophils and allows the prompt identification of these cells by electron microscopy when specific granules are not present[3]. Shape changes are another hallmark of activated human eosinophils. In parallel with the morphological alterations of specific granules, eosinophils are able to change their morphology in response to different agonists. Figure 2 shows shape changes in eosinophils stimulated by eotaxin, a potent eosinophil activator

Imaging Lipid Bodies Within Leukocytes with Different Light Microscopy Techniques

Lipid bodies, also known as lipid droplets, are present in most eukaryotic cells. In leukocytes, lipid bodies are functionally active organelles with central roles in inflammation and are considered structural markers of inflammatory cells in a range of diseases. The identification of lipid bodies has methodological limitations because lipid bodies dissipate upon drying or dissolve upon fixation and staining with alcohol-based reagents. Here we discuss several techniques to detect and visualize lipid bodies within leukocytes by light microscopy. These techniques include staining with osmium or use of different fluorescent probes such as Nile red, BODIPY, Oil red, P96 and immunofluorescence labeling for adipose differentiation-related protein (ADRP)

Intragranular Vesiculotubular Compartments are Involved in Piecemeal Degranulation by Activated Human Eosinophils

Eosinophils, leukocytes involved in allergic, inflammatory and immunoregulatory responses, have a distinct capacity to rapidly secrete preformed granule-stored proteins through piecemeal degranulation (PMD), a secretion process based on vesicular transport of proteins from within granules for extracellular release. Eosinophil-specific granules contain cytokines and cationic proteins, such as major basic protein (MBP). We evaluated structural mechanisms responsible for mobilizing proteins from within eosinophil granules. Human eosinophils stimulated for 30–60 min with eotaxin, regulated on activation, normal, T-cell expressed and secreted (RANTES) or platelet activating factor exhibited ultrastructural features of PMD (e.g. losses of granule contents) and extensive vesiculotubular networks within emptying granules. Brefeldin A inhibited granule emptying and collapsed intragranular vesiculotubular networks. By immunonanogold ultrastructural labelings, CD63, a tetraspanin membrane protein, was localized within granules and on vesicles outside of granules, and mobilization of MBP into vesicles within and extending from granules was demonstrated. Electron tomography with three dimension reconstructions revealed granule internal membranes to constitute an elaborate tubular network able to sequester and relocate granule products upon stimulation. We provide new insights into PMD and identify eosinophil specific granules as organelles whose internal tubulovesicular networks are important for the capacity of eosinophils to secrete, by vesicular transport, their content of preformed and granule-stored cytokines and cationic proteins