In-Depth Characterization of the Chikungunya Virus Replication Cycle.

The chikungunya virus has spread globally with a remarkably high attack rate. Infection causes arthralgic sequelae that can last for years. Nevertheless, there are no specific drugs or vaccines to contain the virus. Understanding the biology of the virus, such as its replication cycle, is a powerful tool to identify new drugs and comprehend virus-host interactions. Even though the chikungunya virus has been known for a long time (it was first described in 1952), many aspects of the replication cycle remain unclear. Furthermore, part of the cycle is based on observations of other alphaviruses. In this study, we used electron and scanning microscopy, as well as biological assays, to analyze and investigate the stages of the chikungunya virus replication cycle. Based on our data, we found infection cellular activities other than those usually described for the chikungunya virus replication cycle, i.e., we show particles enveloping intracellularly without budding in a membrane-delimited morphogenesis area, and we also observed virion release by membrane protrusions. Our work provides novel details regarding the biology of chikungunya virus and fills gaps in our knowledge of its replication cycle. These findings may contribute to a better understanding of virus-host interactions and support the development of antivirals. IMPORTANCE The understanding of virus biology is essential to containing virus dissemination, and exploring the virus replication cycle is a powerful tool to do this. There are many points in the biology of the chikungunya virus that need to be clarified, especially regarding its replication cycle. Our incomplete understanding of chikungunya virus infection stages is based on studies with other alphaviruses. We systematized the chikungunya virus replication cycle using microscopic imaging in the order of infection stages, as follows: entry, replication, protein synthesis, assembly/morphogenesis, and release. The imaging evidence shows novel points in the replication cycle of enveloping without budding, as well as particle release by cell membrane protrusion.

Plasma Membrane Fluidity: An Environment Thermal Detector in Plants.

The lipid matrix in cell membranes is a dynamic, bidimensional array of amphipathic molecules exhibiting mesomorphism, which contributes to the membrane fluidity changes in response to temperature fluctuation. As sessile organisms, plants must rapidly and accurately respond to environmental thermal variations. However, mechanisms underlying temperature perception in plants are poorly understood. We studied the thermal plasticity of membrane fluidity using three fluorescent probes across a temperature range of -5 to 41 °C in isolated microsomal fraction (MF), vacuolar membrane (VM), and plasma membrane (PM) vesicles from Arabidopsis plants. Results showed that PM were highly fluid and exhibited more phase transitions and hysteresis, while VM and MF lacked such attributes. These findings suggest that PM is an important cell hub with the capacity to rapidly undergo fluidity modifications in response to small changes of temperatures in ranges spanning those experienced in natural habitats. PM fluidity behaves as an ideal temperature detector: it is always present, covers the whole cell, responds quickly and with sensitivity to temperature variations, functions with a cell free-energy cost, and it is physically connected with potential thermal signal transducers to elicit a cell response. It is an optimal alternative for temperature detection selected for the plant kingdom.

The MPO system participates actively in the formation of an oxidative environment produced by neutrophils and activates the antioxidant mechanism of Naegleria fowleri.

Naegleria fowleri produces a fatal disease called primary amebic meningoencephalitis (PAM), which is characterized by an extensive inflammatory reaction in the CNS. It is known that the immune response is orchestrated mainly by neutrophils, which activate several defense mechanisms in the host, including phagocytosis, the release of different enzymes such as myeloperoxidase (MPO), and the production of neutrophil extracellular traps. However, the mechanisms by which amoebas evade the neutrophil response are still unknown. In this study, we analyzed the ability of N. fowleri to respond to the stress exerted by MPO. Interestingly, after the interaction of trophozoites with neutrophils, the amoeba viability was not altered; however, ultrastructural changes were observed. To analyze the influence of MPO against N. fowleri and its participation in free radical production, we evaluated its enzymatic activity, expression, and localization with and without the specific 4-aminobenzoic acid hydrazide inhibitor. The production of oxidizing molecules is the principal mechanism used by neutrophils to eliminate pathogens. In this context, we demonstrated an increase in the production of NO, superoxide anion, and reactive oxygen species; in addition, the overexpression of several antioxidant enzymes present in the trophozoites was quantified. The findings strongly suggest that N. fowleri possesses antioxidant machinery that is activated in response to an oxidative environment, allowing it to evade the neutrophil-mediated immune response, which may contribute to the establishment of PAM.

Altered in vitro muscle differentiation in X-linked myopathy with excessive autophagy.

X-linked myopathy with excessive autophagy (XMEA) is a genetic disease associated with weakness of the proximal muscles. It is caused by mutations in the VMA21 gene, coding for a chaperone that functions in the vacuolar ATPase (v-ATPase) assembly. Mutations associated with lower content of assembled v-ATPases lead to an increase in lysosomal pH, culminating in partial blockage of macroautophagy, with accumulation of vacuoles of undigested content. Here, we studied a 5-year-old boy affected by XMEA, caused by a small indel in the VMA21 gene. Detection of sarcoplasmic Lc3 (also known as MAP1LC3B)-positive vacuoles in his muscle biopsy confirmed an autophagy defect. To understand how autophagy is regulated in XMEA myogenesis, we used patient-derived muscle cells to evaluate autophagy during in vitro muscle differentiation. An increase in lysosomal pH was observed in the patient's cells, compatible with predicted functional defect of his mutation. Additionally, there was an increase in autophagic flux in XMEA myotubes. Interestingly, we observed that differentiation of XMEA myoblasts was altered, with increased myotube formation observed through a higher fusion index, which was not dependent on lysosomal acidification. Moreover, no variation in the expression of myogenic factors nor the presence of regenerating fibers in the patient's muscle were observed. Myoblast fusion is a tightly regulated process; therefore, the uncontrolled fusion of XMEA myoblasts might generate cells that are not as functional as normal muscle cells. Our data provide new evidence on the reason for predominant muscle involvement in the context of the XMEA phenotype.This article has an associated First Person interview with the first author of the paper.

Autophagy and vacuolar biogenesis during the nectary development.

MAIN CONCLUSION: Different autophagy pathways are a driver of vacuolar biogenesis and are development stage specific during the extrafloral nectary development in Citharexylum myrianthum. Plant autophagy plays an important role in various developmental processes such as seed germination, pollen maturation and leaf senescence. However, studies that address the evidence of autophagy and its role in the development of plant glands are scarce and largely restricted to laticifers. Regarding nectary, studies have repeatedly pointed to signs of degradation associated with the end of the secretory cycle, without exploring autophagy. Likewise, the relationship between autophagy and biogenesis of vacuoles remains an unexplored issue. In this study, using conventional and microwave fixation in association with ultracytochemical methods for transmission electron microscopy, we investigated the occurrence of autophagy and its implication in the differentiation of extrafloral nectary in Citharexylum myrianthum (Verbenaceae) under natural conditions, focusing on the vacuole biogenesis. We described a variety of vacuole types associated with the stage of nectary epidermis development, which differs with respect to origin, function and nature of the products to be stored. Three distinct autophagy pathways were detected: macroautophagy, microautophagy (both restricted to the undifferentiated epidermal cells, at the presecretory stage) and megaautophagy (circumscribed to the differentiated epidermal cells, at the postsecretory stage). Our study clearly demonstrated that the vacuole variety and autophagy processes in the nectary epidermal cells are development specific. This study highlights the role of autophagy in vacuole biogenesis and its implications for the development of nectary and opens new venues for future studies on regulation mechanisms for autophagy in plant secretory structures under normal conditions.

Application of CNTF or FGF-2 increases the number of M2-like macrophages after optic nerve injury in adult Rana pipiens.

We have previously shown that a single application of the growth factors ciliary neurotrophic factor (CNTF) or fibroblast growth factor 2 (FGF-2) to the crushed optic nerve of the frog, Rana pipiens, increases the numbers and elongation rate of regenerating retinal ganglion cell axons. Here we investigate the effects of these factors on the numbers and types of macrophages that invade the regeneration zone. In control PBS-treated nerves, many macrophages are present 100 µm distal to the crush site at 1 week after injury; their numbers halve by 2 weeks. A single application of CNTF at the time of injury triples the numbers of macrophages at 1 week, with this increase compared to control being maintained at 2 weeks. Application of FGF-2 is equally effective at 1 week, but the macrophage numbers have fallen to control levels at 2 weeks. Immunostaining with a pan-macrophage marker, ED1, and a marker for M2-like macrophages, Arg-1, showed that the proportion of the putative M2 phenotype remained at approximately 80% with all treatments. Electron microscopy of the macrophages at 1 week shows strong phagocytic activity with all treatments, with many vacuoles containing axon fragments and membrane debris. At 2 weeks with PBS or FGF-2 treatment the remaining macrophages are less phagocytically active, containing mainly lipid inclusions. With CNTF treatment, at 2 weeks many of the more numerous macrophages are still phagocytosing axonal debris, although they also contain lipid inclusions. We conclude that the increase in macrophage influx seen after growth factor application is beneficial for the regenerating axons, probably due to more extensive removal of degenerating distal axons, but also perhaps to secretion of growth-promoting substances.

The Use of Drugs in the Study of Vacuole Morphology and Trafficking to the Vacuole in Arabidopsis thaliana.

Chemical compounds are useful to perturb biological functions in the same way as classical genetic approaches take advantage of mutations at the DNA level to perturb gene function. The use of bioactive chemicals currently called chemical genetic is especially valuable for cell biology. Chemical genetic approaches allow perturbations of cellular processes post-germination in a given time window controlling the severity of the effect by modifying or modulating the dose and/or the period of the treatment. Additionally, compounds can be applied directly to different mutants and translational fluorescent reporters/marker lines, expanding the repertoire of experimental setups addressing cell biology research. In this chapter, we describe standard protocols to visualize vacuole morphology and trafficking to the vacuole and the use of bioactive compounds as a proxy to study these biological processes.

Toxoplasma gondii reorganizes the host cell architecture during spontaneous cyst formation in vitro.

Toxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis, a prevalent infection related to abortion, ocular diseases and encephalitis in immuno-compromised individuals. In the untreatable (and life-long) chronic stage of toxoplasmosis, parasitophorous vacuoles (PVs, containing T. gondii tachyzoites) transform into tissue cysts, containing slow-dividing bradyzoite forms. While acute-stage infection with tachyzoites involves global rearrangement of the host cell cytoplasm, focused on favouring tachyzoite replication, the cytoplasmic architecture of cells infected with cysts had not been described. Here, we characterized (by fluorescence and electron microscopy) the redistribution of host cell structures around T. gondii cysts, using a T. gondii strain (EGS) with high rates of spontaneous cystogenesis in vitro. Microtubules and intermediate filaments (but not actin microfilaments) formed a 'cage' around the cyst, and treatment with taxol (to inhibit microtubule dynamics) favoured cystogenesis. Mitochondria, which appeared adhered to the PV membrane, were less closely associated with the cyst wall. Endoplasmic reticulum (ER) profiles were intimately associated with folds in the cyst wall membrane. However, the Golgi complex was not preferentially localized relative to the cyst, and treatment with tunicamycin or brefeldin A (to disrupt Golgi or ER function, respectively) had no significant effect on cystogenesis. Lysosomes accumulated around cysts, while early and late endosomes were more evenly distributed in the cytoplasm. The endocytosis tracer HRP (but not BSA or transferrin) reached bradyzoites after uptake by infected host cells. These results suggest that T. gondii cysts reorganize the host cell cytoplasm, which may fulfil specific requirements of the chronic stage of infection.

Ultrastructural analysis of salivary glands in a phytophagous stink bug revealed the presence of unexpected muscles.

The exceptional abilities of stink bugs (Hemiptera: Pentatomidae) to colonize a diverse group of plants have been attributed to the feeding behaviors and the functions of the salivary complex of these insects. Here, we describe the ultrastructure of the salivary glands of the Neotropical brown stink bug, Euschistus heros, which is a major component of the pentatomid pest complex on soybeans, Glycine max, in the neotropics. Our results revealed a salivary gland complex consisting of two lobes (i.e., anterior and posterior), with a constriction between them (i.e., the hilum), in which the salivary and accessory gland ducts are inserted. The principal gland epithelium has a single layer of cells lining an enlarged lumen filled with saliva, and these cells are cuboidal, rich in rough endoplasmic reticulum and secretory vesicles, with well-developed nuclei, all of which are typical features of protein-secreting cells. We report, for the first time in insects, the presence of a layer of muscle cells surrounding the columnar hilum epithelium. The accessory salivary gland cells are cuboidal with nuclei containing condensed chromatin and cytoplasm rich in vacuoles and rough endoplasmic reticulum, indicating the potential involvement of these glands in water transport/secretion. The lumen content of each lobe of the principal gland suggests that the lobes produce different compounds. Thus, our results suggest that the E. heros salivary complex might have unconventional mechanisms to mix/release saliva, which might help explain the polyphagous abilities of these insects.

Structure and functioning of oil cavities in the shoot apex of Metrodorea nigra A. St.-Hil. (Rutaceae).

This study investigates the histology and subcellular features of secretory cavities during the development of the shoot apex of Metrodorea nigra A. St.-Hil. in order to better understand the functioning of these glands. This Rutaceae species is a very suitable model for studying secretory cavity life span, since the shoot apex exhibits both dormant and growth stages during its annual cycle. Shoot apices were collected during the dormant and growth stages from populations of M. nigra growing under natural conditions. Materials were processed using standard techniques for light and electron microscopy. The secretory cavities originate under the protodermis, and their initiation is restricted to the early developmental stage of shoot organs, which are protected by a hood-shaped structure. Secretory cavities have a multi-seriate epithelium surrounding a lumen that expands schizolysigenously. Oil production begins before lumen formation. When the shoot apex resumes development after the dormant stage, the glands remain active in oil secretion in the developing shoot apex and fully expanded leaves. The mature epithelial cells are flattened and exhibit very thin walls, large oil bodies, leucoplasts surrounded by endoplasmic reticulum, and mitochondria with unusual morphology. The tangential walls of the epithelial cells facing the lumen undergo continuous peeling. The vacuole extrusion appears to be the primary mode of release oil into the lumen, in an exocytotic way. The continuity of oil secretion is ensured by the replacement of the damaged inner epithelial cells by divisions in the parenchyma layer that surround the oil gland, likely a meristematic sheath.

Phosphatidylinositol 4-phosphate 5-kinases 1 and 2 are involved in the regulation of vacuole morphology during Arabidopsis thaliana pollen development.

The pollen grains arise after meiosis of pollen mother cells within the anthers. A series of complex structural changes follows, generating mature pollen grains capable of performing the double fertilization of the female megasporophyte. Several signaling molecules, including hormones and lipids, have been involved in the regulation and appropriate control of pollen development. Phosphatidylinositol 4-phophate 5-kinases (PIP5K), which catalyze the biosynthesis of the phosphoinositide PtdIns(4,5)P2, are important for tip polar growth of root hairs and pollen tubes, embryo development, vegetative plant growth, and responses to the environment. Here, we report a role of PIP5Ks during microgametogenesis. PIP5K1 and PIP5K2 are expressed during early stages of pollen development and their transcriptional activity respond to auxin in pollen grains. Early male gametophytic lethality to certain grade was observed in both pip5k1(-/-) and pip5k2(-/-) single mutants. The number of pip5k mutant alleles is directly related to the frequency of aborted pollen grains suggesting the two genes are involved in the same function. Indeed PIP5K1 and PIP5K2 are functionally redundant since homozygous double mutants did not render viable pollen grains. The loss of function of PIP5K1 and PIP5K2results in defects in vacuole morphology in pollen at the later stages and epidermal root cells. Our results show that PIP5K1, PIP5K2 and phosphoinositide signaling are important cues for early developmental stages and vacuole formation during microgametogenesis.

The morphological analysis of autophagy in primary skeletal muscle cells infected with Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular protozoan parasite, the causative agent of toxoplasmosis, one of the most widespread zoonoses in the world. During the host immune response, tissue cysts are formed, allowing the maintenance of the parasite within the host cell. Autophagy, a degradation process of cellular components, is critical for cellular homeostasis. Recently, it has been proposed that autophagy participates in host-pathogen interactions. Autophagic inducers (rapamycin or glucose plus serum deprivation) inhibited infection and parasite proliferation in a clinically relevant model of primary skeletal muscle cells (SkMC). The ultrastructural analysis showed in SkMC submitted to autophagic stimuli the presence of structures suggestive of autophagosomes close to the parasitophorous vacuole containing degraded parasites. Fluorescence microscopy results pointed out the increase in LC3 puncta in these cells after incubation with autophagic inducers. In the present study, SkMC autophagy controlled the proliferation of tachyzoites inside the cell, data reinforced by ultrastructural evidences and increased LC3 expression.

The Metalloprotease Mpl Supports Listeria monocytogenes Dissemination through Resolution of Membrane Protrusions into Vacuoles.

Listeria monocytogenes is an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on the mlp-actA-plcB operon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcB or Δmpl strains resulted in the formation of small infection foci. As expected, the ΔplcB strain displayed a strong defect in vacuole escape. However, the Δmpl strain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmpl strain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmpl strain, the ΔactA207-238 strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207-238 strain displayed wild-type actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution.

Arcobacter butzleri survives within trophozoite of Acanthamoeba castellanii.

The survival of three Arcobacter butzleri strains inside Acanthamoeba castellanii was assessed using axenic cultures of A. castellanii that were inoculated with the tested strains and incubated at 26°C under aerobic conditions for 240h. The behavior of bacteria in contact with amoebae was monitored using phase contrast microscopy. The bacterial survival rate within amoebae was assessed through counting colony forming units, using the gentamicin protection assay. All A. butzleri strains were able to survive during 240h within the amoebae, thus suggesting that (i) A. butzleri resists the amoebic digestion processes at least for the analyzed time; (ii) that A. castellanii could serve as an environmental reservoir for this bacterium, probably acting as a transmission vehicle for A. butzleri.

Haemophilus influenzae triggers autophagy in HEp-2 cells.

The MAP-LC3 system regulates the intracellular formation of autophagy-associated vacuoles. These vacuoles contain the LC3 protein; thus it has been utilized as a marker to identify autophagosomes. The aim of our study was to investigate whether Haemophilus influenzae strains and their supernatants could activate autophagy in human larynx carcinoma cell line (HEp-2). We demonstrate that higher expression of the LC3B-II protein was induced, particularly by nontypeable Haemophilus influenzae (NTHi) 49766 and by supernatants, containing <50 kDa proteins, of both strains. Ultrastructural studies demonstrate vacuoles with a double membrane and/or membrane material inside, showing similar features to those of autophagic vacuoles. Together, our findings demonstrate that H. influenzae strains and their supernatants trigger an autophagic process.

Cyclopalladated Compound 7a Induces Apoptosis- and Autophagy-Like Mechanisms in Paracoccidioides and Is a Candidate for Paracoccidioidomycosis Treatment.

Paracoccidioidomycosis (PCM), caused by Paracoccidioides species, is the main cause of death due to systemic mycoses in Brazil and other Latin American countries. Therapeutic options for PCM and other systemic mycoses are limited and time-consuming, and there are high rates of noncompliance, relapses, toxic side effects, and sequelae. Previous work has shown that the cyclopalladated 7a compound is effective in treating several kinds of cancer and parasitic Chagas disease without significant toxicity in animals. Here we show that cyclopalladated 7a inhibited the in vitro growth of Paracoccidioides lutzii Pb01 and P. brasiliensis isolates Pb18 (highly virulent), Pb2, Pb3, and Pb4 (less virulent) in a dose-response manner. Pb18 was the most resistant. Opportunistic Candida albicans and Cryptococcus neoformans were also sensitive. BALB/c mice showed significantly lighter lung fungal burdens when treated twice a day for 20 days with a low cyclopalladated 7a dose of 30 µg/ml/day for 30 days after intratracheal infection with Pb18. Electron microscopy images suggested that apoptosis- and autophagy-like mechanisms are involved in the fungal killing mechanism of cyclopalladated 7a. Pb18 yeast cells incubated with the 7a compound showed remarkable chromatin condensation, DNA degradation, superoxide anion production, and increased metacaspase activity suggestive of apoptosis. Autophagy-related killing mechanisms were suggested by increased autophagic vacuole numbers and acidification, as indicated by an increase in LysoTracker and monodansylcadaverine (MDC) staining in cyclopalladated 7a-treated Pb18 yeast cells. Considering that cyclopalladated 7a is highly tolerated in vivo and affects yeast fungal growth through general apoptosis- and autophagy-like mechanisms, it is a novel promising drug for the treatment of PCM and other mycoses.

Metatranscriptomic analysis of diminutive Thiomargarita-like bacteria ("Candidatus Thiopilula" spp.) from abyssal cold seeps of the Barbados Accretionary Prism.

Large sulfur-oxidizing bacteria in the family Beggiatoaceae are important players in the global sulfur cycle. This group contains members of the well-known genera Beggiatoa, Thioploca, and Thiomargarita but also recently identified and relatively unknown candidate taxa, including "Candidatus Thiopilula" spp. and "Ca. Thiophysa" spp. We discovered a population of "Ca. Thiopilula" spp. colonizing cold seeps near Barbados at a ∼4.7-km water depth. The Barbados population consists of spherical cells that are morphologically similar to Thiomargarita spp., with elemental sulfur inclusions and a central vacuole, but have much smaller cell diameters (5 to 40 µm). Metatranscriptomic analysis revealed that when exposed to anoxic sulfidic conditions, Barbados "Ca. Thiopilula" organisms expressed genes for the oxidation of elemental sulfur and the reduction of nitrogenous compounds, consistent with their vacuolated morphology and intracellular sulfur storage capability. Metatranscriptomic analysis further revealed that anaerobic methane-oxidizing and sulfate-reducing organisms were active in the sediment, which likely provided reduced sulfur substrates for "Ca. Thiopilula" and other sulfur-oxidizing microorganisms in the community. The novel observations of "Ca. Thiopilula" and associated organisms reported here expand our knowledge of the globally distributed and ecologically successful Beggiatoaceae group and thus offer insight into the composition and ecology of deep cold seep microbial communities.

Neurotoxicity of coral snake phospholipases A2 in cultured rat hippocampal neurons.

The neurotoxicity of two secreted Phospholipases A2 from Brazilian coral snake venom in rat primary hippocampal cell culture was investigated. Following exposure to Mlx-8 or Mlx-9 toxins, an increase in free cytosolic Ca(2+) and a reduction in mitochondrial transmembrane potential (ΔΨm) became evident and occurred prior to the morphological changes and cytotoxicity. Exposure of hippocampal neurons to Mlx-8 or Mlx-9 caused a decrease in the cell viability as assessed by MTT and LDH assays. Inspection using fluorescent images and ultrastructural analysis by scanning and transmission electron microscopy showed that multiphase injury is characterized by overlapping cell death phenotypes. Shrinkage, membrane blebbing, chromatin condensation, nucleosomal DNA fragmentation and the formation of apoptotic bodies were observed. The most striking alteration observed in the electron microscopy was the fragmentation and rarefaction of the neuron processes network. Degenerated terminal synapses, cell debris and apoptotic bodies were observed among the fragmented fibers. Numerous large vacuoles as well as swollen mitochondria and dilated Golgi were noted. Necrotic signs such as a large amount of cellular debris and membrane fragmentation were observed mainly when the cells were exposed to highest concentration of the PLA2-neurotoxins. PLA2s exposed cultures showed cytoplasmic vacuoles filled with cell debris, clusters of mitochondria presented mitophagy-like structures that are in accordance to patterns of programmed cell death by autophagy. Finally, we demonstrated that the sPLA2s, Mlx-8 and Mlx-9, isolated from the Micrurus lemniscatus snake venom induce a hybrid cell death with apoptotic, autophagic and necrotic features. Furthermore, this study suggests that the augment in free cytosolic Ca(2+) and mitochondrial dysfunction are involved in the neurotoxicity of Elapid coral snake venom sPLA2s.

Actin, RhoA, and Rab11 participation during encystment in Entamoeba invadens.

In the genus Entamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins during Entamoeba invadens encystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.