Identification of Ultrastructural Signatures of Sleep and Wake in the Fly Brain

The cellular consequences of sleep loss are poorly characterized. In the pyramidal neurons of mouse frontal cortex we found that mitochondria and secondary lysosomes occupy a larger proportion of the cytoplasm after chronic sleep restriction compared to sleep, consistent with increased cellular burden due to extended wake. For each morphological parameter the within-animal variance was high, suggesting that the effects of sleep and sleep loss vary greatly among neurons. However, the analysis was based on 4-5 mice/group and a single section/cell. Here, we applied serial block-face scanning electron microscopy to identify signatures of sleep and sleep loss in the Drosophila brain. Stacks of images were acquired and used to obtain full 3D reconstructions of the cytoplasm and nucleus of 263 Kenyon cells from adult flies collected after a night of sleep (S) or after 11 hours (SD11) or 35 hours (SD35) of sleep deprivation (9 flies/group). Relative to S flies, SD35 flies showed increased density of dark clusters of chromatin and of Golgi apparata and a trend increase in the percent of cell volume occupied by mitochondria, consistent with increased need for energy and protein supply during extended wake. Logistic regression models could assign each neuron to the correct experimental group with good accuracy, but in each cell nuclear and cytoplasmic changes were poorly correlated, and within-fly variance was substantial in all experimental groups. Together, these results support the presence of ultrastructural signatures of sleep and sleep loss but underscore the complexity of their effects at the single-cell level.

Lysosomes appear as the auto-fluorescent vacuoles in Dictyostelium discoideum cells

Dictyostelium discoideum cells contain auto-fluorescent vacuoles. To determine the identity of these vacuoles, the fluorescent dye 4-nitro-7-(1-piperazinyl)-2,1,3-benzoxadiazole (NBD-PZ) was used to stain the lysosomes in D. discoideum cells. Neither the auto-fluorescent vacuoles nor lysosomes were observed in D. discoideum cells immediately after they arose from spores or in stationary phase cells. However, both the auto-fluorescent vacuoles and lysosomes were visible in cells that had entered growth phase. Auto-fluorescent vacuoles and lysosomes were also observed in stationary phase cells incubated with chloroquine. When the cells were allowed to phagocytose BioParticles Fluorescent Bacteria (orange fluorescence) for 1 h, orange phagosomes and blue auto-fluorescent vacuoles were observed as independent moieties. However, after an additional 2 h of incubation, we observed vacuoles with mixed fluorescence (orange and blue) in the cells, suggestive of secondary lysosomes. These results suggest that the auto-fluorescent vacuoles in D. discoideum cells are lysosomes.

Feeding biology of carnivore and detritivore Mediterranean pycnogonids

The digestive system of sea spiders (Pycnogonida) presents peculiarities that have not been discussed in the context of their ecology or feeding behaviour. We investigated the digestive system of two Mediterranean species, a carnivorous speciesAmmothella longipesand a detritivorousEndeis spinosa, with special focus on its correlation with behavioural feeding habits. The midgut and hindgut sections did not present significant differences between the two species, but major differences were observed in the foregut, reflecting concordance to their diet and their feeding behaviour. Jaws, setose lips, the structure of the pharyngeal filter and musculature of the proboscis are the main differential elements when comparing feeding habits ofA. longipesandE. spinosa. These elements are responsible for the reduction of the food pulp down to subcellular size. The digestion process observed in the species studied agrees with that observed in other pycnogonid lineages, but differs from most marine arthropods mainly because of the absence of midgut gland cells and the presence of a unique multifunctional type of midgut epithelial cell. Epithelial digestive cells are present in a small ‘resting’ form during starvation periods. During digestion, secretion granules possibly containing zymogen move to their apical border to be secreted to the midgut lumen, secondary lysosomes are formed and intracellular digestion occurs within them. Residual bodies are formed within the epithelial cell and released to the midgut lumen to be transported towards the hindgut. The characteristics of the digestive process of the pycnogonids studied seem to reflect a plesiomorphic state in arthropods.

Francisella tularensis Phagosomal Escape Does Not Require Acidification of the Phagosome

ABSTRACT Following uptake, Francisella tularensis enters a phagosome that acquires limited amounts of lysosome-associated membrane glycoproteins and does not acquire cathepsin D or markers of secondary lysosomes. With additional time after uptake, F. tularensis disrupts its phagosomal membrane and escapes into the cytoplasm. To assess the role of phagosome acidification in phagosome escape, we followed acidification using the vital stain LysoTracker red and acquisition of the proton vacuolar ATPase (vATPase) using immunofluorescence within the first 3 h after uptake of live or killed F. tularensis subsp. holarctica live vaccine strain (LVS) by human macrophages. Whereas 90% of the phagosomes containing killed LVS stained intensely for the vATPase and were acidified, only 20 to 30% of phagosomes containing live LVS stained intensely for the vATPase and were acidified. To determine whether transient acidification might be required for phagosome escape, we assessed the impact on phagosome permeabilization of the proton pump inhibitor bafilomycin A. Using electron microscopy and an adenylate cyclase reporter system, we found that bafilomycin A did not prevent phagosomal permeabilization by F. tularensis LVS or virulent type A strains (F. tularensis subsp. tularensis strain Schu S4 and a recent clinical isolate) or by “F. tularensis subsp. novicida,” indicating that F. tularensis disrupts its phagosomal membrane by a mechanism that does not require acidification.

Developmental competence and ultrastructural changes of heat-stressed mouse early blastocysts produced in vitro

Mouse early blastocysts were exposed to temperatures of 39°C and 41°C for 2 h, respectively, to determine their developmental competence and ultrastructural changes. The results showed that heat stress at 41°C for 2 h, significantly reduced the percentages of expanded and hatched blastocysts, but not at 39°C for 2 h. The average cell numbers in expanded blastocysts, which developed from early blastocysts heat-stressed at temperatures of 39°C and 41°C, were significantly reduced. The average cell numbers in hatched blastocysts subjected to heat stress were no different from those in the control group cultured at 37°C. The mitochondria of the early blastocysts heat-stressed at 39°C for 2 h, were slightly swollen, but they had recovered after culturing at 37°C for 2 h. However, the mitochondria in the blastocysts heat stressed at 41°C for 2 h were severely swollen, and their number increased. The ribosomes shed from the rough endoplasmic reticulum, and the number of secondary lysosomes in the plasma increased. The integrity of desmosomes was disrupted. The space between the nuclear envelope and the perivitelline membrane enlarged. The fibre fraction and the particulate fraction of nucleoli were separated. The heterochromatin in nucleoli was also increased in its quantity. There were some lamellar-shape structures and heterogeneous dense materials exhibiting in the cytoplasm. The ultrastructural changes induced by heat shock at 41°C for 2 h were not reversible. In conclusion, the damage of heat stress to mitochondria, lysosomes, ribosomes and cell nucleus, may be one of the most important factors that inhibit the normal development of mouse early blastocysts.

Autophagy is Increased after Traumatic Brain Injury in Mice and is Partially Inhibited by the Antioxidant γ-glutamylcysteinyl Ethyl Ester

Autophagy is a homeostatic process for recycling of proteins and organelles, induced by nutrient deprivation and regulated by oxygen radicals. Whether autophagy is induced after traumatic brain injury (TBI) is not established. We show that TBI in mice results in increased ultrastructural and biochemical evidence of autophagy. Specifically, autophagosomal vacuoles and secondary lysosomes were frequently observed in cell processes and axons in ipsilateral brain regions by electron microscopy, and lipidated microtubule-associated protein light chain 3, a biochemical footprint of autophagy referred to as LC3 II, was increased at 2 and 24 h after TBI versus controls. Since oxygen radicals are believed to be important in the pathogenesis of TBI and are essential for the process of starvation-induced autophagy in vitro, we also sought to determine if treatment with the antioxidant γ-glutamylcysteinyl ethyl ester (GCEE) reduced autophagy and influenced neurologic outcome after TBI in mice. Treatment with GCEE reduced oxidative stress and partially reduced LC3 II formation in injured brain at 24 h after TBI versus vehicle. Treatment with GCEE also led to partial improvement in behavioral and histologic outcome versus vehicle. Taken together, these data show that autophagy occurs after experimental TBI, and that oxidative stress contributes to overall neuropathology, in part by initiating or influencing autophagy.

Tripeptidyl-peptidase I in health and disease

The lysosomal lumen contains numerous acidic hydrolases involved in the degradation of carbohydrates, lipids, proteins, and nucleic acids, which are basic cell components that turn over continuously within the cell and/or are ingested from outside of the cell. Deficiency in almost any of these hydrolases causes accumulation of the undigested material in secondary lysosomes, which manifests itself as a form of lysosomal storage disorder (LSD). Mutations in tripeptidyl-peptidase I (TPP I) underlie the classic late-infantile form of neuronal ceroid lipofuscinoses (CLN2), the most common neurodegenerative disorders of childhood. TPP I is an aminopeptidase with minor endopeptidase activity and Ser475 serving as an active-site nucleophile. The enzyme is synthesized as a highly glycosylated precursor transported by mannose-6-phosphate receptors to lysosomes, where it undergoes proteolytic maturation. This review summarizes recent progress in understanding of TPP I biology and molecular pathology of the CLN2 disease process, including distribution of the enzyme, its biosynthesis, glycosylation, transport and activation, as well as catalytic mechanisms and their potential implications for pathogenesis and treatment of the underlying disease. Promising data from gene and stem cell therapy in laboratory animals raise hope that CLN2 will be the first neurodegenerative LSD for which causative treatment will become available for humans.

96ULTRASTRUCTURE AND CELL DEATH OF VITRIFIED PORCINE BLASTOCYSTS

Cryopreservation of porcine embryos by the simple open pulled straw (OPS) method was recently reported to result in live offspring (Berthelot et al., 2000, Cryobiology 41; 116–124,) and is evaluated in the present study by light (LM) and transmission electron microscopy (TEM) as well as by TUNEL staining in order to detect morphological and molecular signs of cell death and subsequent regeneration. Blastocysts were collected from gilts on Day 5 (Day 0=1st AI) and were randomly assigned to one of three groups: Fresh controls (FC) were fixed immediately after collection, and vitrified embryos were fixed either immediately after vitrification and warming (V0) or after 24h of culture upon warming after vitrification (V24). In each of the three groups, embryos were fixed and processed for LM/TEM (FC, n=13; V0, n=20; V24, n=18) or TUNEL staining (counter staining with propidium iodide) and confocal laser scanning microscopy (FC, n=32; V0, n=31; V24, n=33). At LM, the FC embryos displayed a well-defined trophoblast (Tb) and inner cell mass (ICM), expanded blastocoele cavity and a narrow or no perivitelline space. In V0 embryos, collapse of the blastocoele cavity and cell swelling was detected. At the TEM level, the V0 embryos showed extensive injuries including a general distension or shrinkage of mitochondria and massive increase in the amount of membrane-bound vesicles, vacuoles and secondary lysosomes. In both FC and V0 embryos, the presence of dead or phagocytozed cells in the ICM and Tb was occasional. A few extruded cells were often noticed in the perivitelline space or in the blastocoele cavity, and such cells ranged from being rather normal to showing typical morphological features of apoptosis. TUNEL staining confirmed the presence of a few apoptotic cells in both groups of embryos. Approximately 2/3 of the V24 embryos had, as evaluated by LM, partially recovered, re-expanded or even hatched whereas the remaining 1/3 had degenerated. At the TEM level, the recovered embryos displayed almost normal blastocyst morphology, except for a widening of the perivitelline space, accumulation of debris, increased electron-lucidity of the ICM and partial distension of mitochondria. The degenerated embryos had disintegrated into a poorly defined mass of cells and debris including cells with either decreased or increased electron-density of the cytoplasm and with abundant degeneration of mitochondria and other organelles. Both recovered and degenerated embryos displayed persistent abundant presence of small membrane-bound vesicles, vacuoles and secondary lysosomes. All V24 embryos displayed increased occurrence of dead or phagocytozed cells in the ICM and Tb as well as increased occurrence of extruded cells showing typical morphological features of apoptosis or secondary necrosis. TUNEL staining confirmed the increased occurrence of apoptotic cells in this group of embryos. In conclusion, immediately after vitrification and warming, porcine embryos displayed severe subcellular damages, but during 24h of culture the majority of the embryos were able to regenerate. Along with the regenerative process, apoptosis became evident. Supported by CRAFT EC contract no. QLK5-CT-2002-70983.