PKCε controls the fusion of secretory vesicles in mast cells in a phosphatidic acid-dependent mode.

PKCε is highly expressed in mast cells and plays a fundamental role in the antigen-triggered activation of the allergic reaction. Although its regulation by diacylglycerols has been described, its regulation by acidic phospholipids and how this regulation leads to the control of downstream vesicle secretion is barely known. Here, we used structural and evolutionary studies to find the molecular mechanism that explains the selectivity of the C1B domain of PKCε by Phosphatidic Acid (PA). This resided in a collection of Arg residues that form a specific rim on the outer surface of the C1B domain, around the diacylglycerol binding cleft. In RBL-2H3 cells, this basic rim allowed the kinase to respond specifically to phosphatidic acid signals that induced its translocation to the plasma membrane and subsequent activation. Further experiments in cells that overexpress PKCε and a mutant of the PA binding site, showed that PA-dependent PKCε activation increased vesicle degranulation in RBL-2H3 cells, and this correlated with increased SNAP23 phosphorylation. Over-expression of PKCε in these cells also induced an increase in the number of docked vesicles containing SNAP23, when stimulated with PA. This accumulation could be attributed to the stabilizing effect of phosphorylation on the formation of the SNARE complex, which ultimately led to increased release of content in the presence of Ca2+ during the fusion process. Therefore, these findings reinforce the importance of PA signaling in the activation of PKCε, which could be an important target to inhibit the exacerbated responses of these cells in the allergic reaction.

Neutrophils: Need for Standardized Nomenclature.

Neutrophils are the most abundant innate immune cell with critical anti-microbial functions. Since the discovery of granulocytes at the end of the nineteenth century, the cells have been given many names including phagocytes, polymorphonuclear neutrophils (PMN), granulocytic myeloid derived suppressor cells (G-MDSC), low density neutrophils (LDN) and tumor associated neutrophils (TANS). This lack of standardized nomenclature for neutrophils suggest that biologically distinct populations of neutrophils exist, particularly in disease, when in fact these may simply be a manifestation of the plasticity of the neutrophil as opposed to unique populations. In this review, we profile the surface markers and granule expression of each stage of granulopoiesis to offer insight into how each stage of maturity may be identified. We also highlight the remarkable surface marker expression profiles between the supposed neutrophil populations.

Automated classification of synaptic vesicles in electron tomograms of C. elegans using machine learning.

Synaptic vesicles (SVs) are a key component of neuronal signaling and fulfil different roles depending on their composition. In electron micrograms of neurites, two types of vesicles can be distinguished by morphological criteria, the classical "clear core" vesicles (CCV) and the typically larger "dense core" vesicles (DCV), with differences in electron density due to their diverse cargos. Compared to CCVs, the precise function of DCVs is less defined. DCVs are known to store neuropeptides, which function as neuronal messengers and modulators [1]. In C. elegans, they play a role in locomotion, dauer formation, egg-laying, and mechano- and chemosensation [2]. Another type of DCVs, also referred to as granulated vesicles, are known to transport Bassoon, Piccolo and further constituents of the presynaptic density in the center of the active zone (AZ), and therefore are important for synaptogenesis [3]. To better understand the role of different types of SVs, we present here a new automated approach to classify vesicles. We combine machine learning with an extension of our previously developed vesicle segmentation workflow, the ImageJ macro 3D ART VeSElecT. With that we reliably distinguish CCVs and DCVs in electron tomograms of C. elegans NMJs using image-based features. Analysis of the underlying ground truth data shows an increased fraction of DCVs as well as a higher mean distance between DCVs and AZs in dauer larvae compared to young adult hermaphrodites. Our machine learning based tools are adaptable and can be applied to study properties of different synaptic vesicle pools in electron tomograms of diverse model organisms.

PC12 cells that lack synaptotagmin I exhibit loss of a subpool of small dense core vesicles.

Neurons communicate by releasing neurotransmitters that are stored in intracellular vesicular compartments. PC12 cells are frequently used as a model secretory cell line that is described to have two subpools of vesicles: small clear vesicles and dense core vesicles. We measured transmitter molecules released from vesicles in NGF-differentiated PC12 cells using carbon-fiber amperometry, and relative diameters of individual vesicles using electron microscopy. Both amperometry and electron micrograph data were analyzed by statistical and machine learning methods for Gaussian mixture models. An electron microscopy size correction algorithm was used to predict and correct for observation bias of vesicle size due to tangential slices through some vesicles. Expectation maximization algorithms were used to perform maximum likelihood estimation for the Gaussian parameters of different populations of vesicles, and were shown to be better than histogram and cumulative distribution function methods for analyzing mixed populations. The Bayesian information criterion was used to determine the most likely number of vesicle subpools observed in the amperometric and electron microscopy data. From this analysis, we show that there are three major subpools, not two, of vesicles stored and released from PC12 cells. The three subpools of vesicles include small clear vesicles and two subpools of dense core vesicles, a small and a large dense core vesicle subpool. Using PC12 cells stably transfected with short-hairpin RNA targeted to synaptotagmin I, an exocytotic Ca(2+) sensor, we show that the presence and release of the small dense core vesicle subpool is dependent on synaptotagmin I. Furthermore, synaptotagmin I also plays a role in the formation and/or maintenance of the small dense core vesicle subpool in PC12 cells.

[Identification and classification of lysozyme-expressing cells in the mouse small intestinal crypt and their correlation with the morphology of secretory granules and labeling density of immunogold].

The four principal epithelial cell lineages (absorptive enterocytes, goblet cells, enteroendocrine cells and Paneth cells) of the adult mouse small intestine derive from multipotent stem cells. Furthermore, the intermediate cells and granule goblet cells are located near the base of crypts of mouse intestine; the former has the characteristics of goblet and Paneth cells and the latter is transformed from the intermediate cells. However, the grounds and the definition for classifing these three cell types (Paneth, intermediate and granule goblet cells) are vague, making it difficult to discuss the structure and a function of those cells. The purpose of this study was to investigate the identification and classification of lysozyme-expressing cells in the mouse small intestinal crypt and their correlation with the morphology of secretory granules and labeling density of immunogold using quantitative immunoelectron microscopy analysis. The results were follows. (1) Paneth cells, intermediate cells and granule goblet cells showed lysozyme immunoreactivity in the electron-dense core of biphasic secretory granules, and therefore lysozyme-exprssing cells were identified in the mouse small intestinal crypt. The sizes of secretory granules were divided into ten groups (every 10%) according to area ratio (core/granule (%)). (2) This distribution of three type cells was classified statistically into "Paneth cell phase": 61% < or = (core/granule (%)), "intermediate cell phase": (core/granule (%)) 21 < or = 60%, "granule goblet cell phase": (core/granule (%)) < or = 20%. (3) Labeling density for lysozyme was commensurate with the size of the central dense core. The Paneth cells had the highest labeling density among the cells. When the transformation from intermediate to granule goblet cell occurred, it happened at the same time that the core of secretory granules gradually shrinks, and the labeling density for lysozyme disappears. (4) The labeling density of immunogold for lysozyme in the small intestine varied at different sites. The labeling density in the Paneth and intermediate cells of the ileal crypt was lower than those of the duodeal and jejunal crypts. (5) In the lysozyme-expressing cells in small intestinal crypt of 2- and 24-month old mouse, the ultrastructure and labeling density did not change.

Neutrophils launch monocyte extravasation by release of granule proteins.

During their journey from the blood stream to sites of inflammation polymorphonuclear leukocytes (PMN) release a wide panoply of granule proteins. Shortly after the PMN efflux, the extravasation of monocytes sets in and recent research provides evidence that the release of PMN granule proteins and monocyte extravasation are causally interrelated. Granule proteins seeded on the endothelium by adherent PMN allow direct activation and subsequent adhesion of monocytes. In addition, PMN granule components enhance the endothelial expression of cell adhesion molecules, efficiently supporting the arrest of monocytes at inflamed vessels. Moreover, granule proteins contribute to the fine tuning of the local chemokine network. Proteolytic modification of chemokines as well as enhancement of local chemokine synthesis lead to increased monocyte extravasation. Finally, PMN granule proteins exert direct chemotactic effects, a mechanism which is of special importance in the early recruitment of inflammatory monocytes. Hence, granule proteins modify the monocyte extravasation cascade in a multifaceted manner ensuring the efficiency of these mechanisms.

Mouse chromaffin cells have two populations of dense core vesicles.

The quantal hypothesis states that neurotransmitter is released in discrete packages, quanta, thought to represent the neurotransmitter content of individual vesicles. If true, then vesicle size should influence quantal size. Although chromaffin cells are generally thought to have a single population of secretory vesicles, our electron microscopy analysis suggested two populations as the size distribution was best described as the sum of two Gaussians. The average volume difference was fivefold. To test whether this difference in volume affected quantal size, neurotransmitter release from permeabilized cells exposed to 100 microM Ca2+ was measured with amperometry. Quantal content was bimodally distributed with both large and small events; the distribution of vesicle sizes predicted by amperometry was extremely similar to those measured with electron microscopy. In addition, each population of events exhibited distinct release kinetics. These results suggest that chromaffin cells have two populations of dense core vesicles (DCV) with unique secretory properties and which may represent two distinct synthetic pathways for DCV biogenesis or alternatively they may represent different stages of biosynthesis.

Requirements for the identification of dense-core granules.

Dense-core granules (DCGs), cytoplasmic organelles competent for regulated exocytosis, show considerable heterogeneity depending upon the specificity of their expressing cells--primarily neurons and neurosecretory cells. DCGs have been mainly identified by detecting their cargo molecules, often members of the granin family, and using conventional electron microscopy and immunocytochemistry. However, by a critical analysis of the various stages of DCG "life" within neurosecretory cells, we have highlighted several specific molecular and functional properties that are common to all these organelles. We propose that these properties be considered as strict requirements for the identification of DCGs.

Immunocytochemical study of the GH cells in the anterior pituitary gland of human fetus II. Anencephalic fetus.

In order to elucidate the effects of hypothalamic regulation on the morphology of GH cells, light and electron microscopic immunocytochemical examinations were carried out comparing GH cells in the anterior pituitary gland of anencephalic fetus with those of normal fetuses. Three types of GH cells were identified in the anterior pituitary gland of anencephalic fetus as well as in the normal fetus. Type-I is a small, round cell containing a few small secretory granules. Type-III is a large, polygonal cell with numerous large secretory granules. Type-II is a polygonal cell with medium-sized secretory granules. The Type-II GH cell was predominant in both anencephalic and normal fetuses. The most striking difference between anencephalic and normal fetuses was the presence of atypical forms of the Type II cell. These were polygonal cells containing secretory granules, which were either immunopositive or immunonegative to anti-human GH (anti-hGH) serum. Furthermore, two other types of GH cells were identified. The somatomammotroph (SM cell) contained GH and PRL in different granules within the same cell. Also, a different type of the GH cell was noted containing two varieties of secretory granules; one was immunolabeled only with anti-hGH and the other was not immunolabeled to either anti-hGH or anti-human PRL (anti-hPRL). From these results, we suggest that an absence of hypothalamic regulation in the anencehpalic does not seriously modify GH cell morphology but induces an altered GH storage pattern in some of the cells.