Intracellular life of protozoan <i>Toxoplasma gondii</i>: Parasitophorous vacuole establishment and survival strategies

Toxoplasma gondii is a protozoan of worldwide distribution and the agent of toxoplasmosis. It is estimated that 30%–50% of the world population could be infected with this parasite. Although the infection in immunocompetent individuals is mostly asymptomatic, the disease in immunosuppressed and pregnant is a risk condition. As a member of the phylum Apicomplexa, T. gondii has an obligatory intracellular lifestyle; therefore, invading a host cell and establishing it inside a parasitophorous vacuole (PV) are mandatories for the survival of this parasite. The construction of a perfect intracellular niche for T. gondii requires the secretion of an arsenal of proteins from unique secretory organelles. These proteins will remodel the vacuolar environment and the host cell organization and functions, allowing the parasite to access essential nutrients and stay “invisible” inside a host cell. In the present review, we will discuss the main steps involved in the PV formation and its differentiation to tissue cyst, focusing mainly on the strategies employed in the acquisition of nutrients and proteins involved in host cell modification.     

Comparisons of the topographic characteristics and electrical charge distributions among Babesia‐infected erythrocytes and extraerythrocytic merozoites using AFM

Tick‐borne Babesia parasites are responsible for costly diseases worldwide. Improved control and prevention tools are urgently needed, but development of such tools is limited by numerous gaps in knowledge of the parasite–host relationships. We hereby used atomic force microscopy (AFM) and frequency‐modulated Kelvin probe potential microscopy (FM‐KPFM) techniques to compare size, texture, roughness and surface potential of normal and infected Babesia bovis, B. bigemina and B. caballi erythrocytes to better understand the physical properties of these parasites. In addition, AFM and FM‐KPFM allowed a detailed view of extraerythrocytic merozoites revealing shape, topography and surface potential of paired and single parasites. B. bovis‐infected erythrocytes display distinct surface texture and overall roughness compared to noninfected erythrocytes. Interestingly, B. caballi‐infected erythrocytes do not display the surface ridges typical in B. bovis parasites. Observations of extraerythrocytic B. bovis, B. bigemina and B. caballi merozoites using AFM revealed differences in size and shape between these three parasites. Finally, similar to what was previously observed for Plasmodium‐infected erythrocytes, FM‐KPFM images reveal an unequal electric charge distribution, with higher surface potential above the erythrocyte regions that are likely associated with Babesia parasites than over its remainder regions. In addition, the surface potential of paired extraerythrocytic B. bovis Mo7 merozoites revealed an asymmetric potential distribution. These observations may be important to better understand the unique cytoadhesive properties of B. bovis‐infected erythrocytes, and to speculate on the role of differences in the distribution of surface charges in the biology of the parasites.     

Ultrastructural aspects of Cystoisospora belli (syn. Isospora belli) in continuous cell lines

Cystoisospora belli is an opportunistic protozoan that causes human cystoisosporiasis, an infection characterized by diarrhea, steatorrhea, abdominal pain, fever, and weight loss. The lack of animal models susceptible to C. belli, and the difficulty in obtaining clinical samples with fair amounts of oocysts have limited the research pertaining to the basic biology of this parasite. This study aimed to describe the ultrastructure of endogenous stages of C. belli in Monkey Rhesus Kidney Cells (MK2) and Human Ileocecal Adenocarcinoma cells (HCT‐8). Zoites of C. belli exhibited typical morphological features of coccidia, which included a trilaminar pellicle, an apical complex formed by a conoid, polar rings, rhoptries, and micronemes, in addition to dense granules and the endoplasmic reticulum. No crystalloid body was observed but various lipid and amylopectin granules were usually present in the cytoplasm of zoites. We observed a tendency of the endoplasmic reticulum of the host cell to be located near the parasitophorous vacuole membrane. Merozoites were formed by endodyogeny and during replication, the apical complex of the mother cell remained intact. The formation of gametes or oocysts was not observed. The ultrastructural findings of C. belli are further evidence of its proximity to Sarcocystidae family members and corroborate their reclassification as Cystoisospora spp. Microsc. Res. Tech. 77:472–478, 2014. © 2014 Wiley Periodicals, Inc.     

The yeast endocytic membrane transport system

Progress has been made recently in visualizing the structures and organelles responsible for endocytic membrane traffic from the cell surface to the lysosome-like vacuole in Saccharomyces cerevisiae. This, together with the recent discovery of several new membrane trafficking pathways connecting these organelles, has led to a quantum leap in our understanding of the S. cerevisiae endocytic pathway. We now know that although the cortical actin cytoskeleton is required for the internalization step of endocytosis, the internalization event occurs at furrow-like invaginations of the plasma membrane, which are distinct from cortical actin patches. Internalized material is taken into the cell in the form of small (30-50 nm diameter) vesicles and delivered to tubulo-vesicular early endosomes at the cell periphery. Subsequently, the internalized material arrives in multivesicular late endosomes adjacent to the vacuole. Recent microscopy evidence suggests that transfer from late endosomes to the vacuole may involve direct fusion of late endosomes with the vacuole. The visualization of the S. cerevisiae endocytic pathway has revealed similarities to endocytic pathways visualized in higher eukaryotes.     

<i>Coxiella burnetii:</i> living inside the host cell

Coxiella burnetii is an obligate intracellular pathogen and the causative agent of Q fever. In this brief review, we describe how recently described mechanisms help our understanding of C. burnetii invasion and its survival in the host cell by the formation of a replicative niche: the Coxiella-containing vacuole. We describe the actin-associated proteins involved in the internalization of C. burnetii, and we discuss the contribution of diverse degradation pathways of the cell during the formation and stabilization of the Coxiella-containing vacuole.     

Dynamic aspects of the interaction between microtubules and actin filaments

This paper summarizes observations on the motility and behavior of microtubule asters in polymorphonuclear leukocytes under a variety of experimental conditions. These observations suggest that the location and organization of microtubules is influenced, at least in part, by the activity of the cortical actin network. Aster motility may therefore serve as an indicator of the interaction between the centrosomal microtubules and the cell cortex, and it is speculated that this interaction is of importance for leukocyte translocation.     

Cellulose microfibril deposition: coordinated activity at the plant plasma membrane

Plant cell wall production is a membrane-bound process. Cell walls are composed of cellulose microfibrils , embedded inside a matrix of other polysaccharides and glycoproteins. The cell wall matrix is extruded into the existing cell wall by exocytosis. This same process also inserts the cellulose synthase complexes into the plasma membrane. These complexes, the nanomachines that produce the cellulose microfibrils, move inside the plasma membrane leaving the cellulose microfibrils in their wake. Cellulose microfibril angle is an important determinant of cell development and of tissue properties and as such relevant for the industrial use of plant material. Here, we provide an integrated view of the events taking place in the not more than 100 nm deep area in and around the plasma membrane, correlating recent results provided by the distinct field of plant cell biology. We discuss the coordinated activities of exocytosis, endocytosis, and movement of cellulose synthase complexes while producing cellulose microfibrils and the link of these processes to the cortical microtubules.     

Mapping cholesteryl ester analogue uptake and intracellular flow in Paramecium by confocal fluorescence microscopy

In Paramecium primaurelia the uptake and intracellular flow of cholesteryl ester was studied by fluorescence confocal laser scanning optical microscopy and by the fluorescent analogue cholesteryl‐BODIPY^® FL C₁₂ (BODIPY‐CE). The BODIPY FL fluorophore has the characteristic of emitting green fluorescence, which is red‐shifted as the probe concentrates. In cells incubated with 25 µm BODIPY‐CE for 30 s, fluorescence is found in vesicles located around the cytopharynx in the posterior half of the cell. Successively, the lipid is internalized by food vacuoles, the fluorescent vesicles are distributed throughout the cell and the intracellular membranes are labelled. The food vacuole number is maximum after 10–15 min of continuous labelling, then it decreases until no food vacuoles are found in 30‐min fed cells. BODIPY‐CE accumulates in red‐labelled cytoplasmic droplets located in the anterior half of the cell. When food vacuole formation is inhibited by trifluoperazine, fluorescence is found on cellular membranes and in small green‐labelled vesicles at the apical pole. The inhibition of clathrin‐mediated endocytosis does not interfere in P. primaurelia with BODIPY‐CE intracellular flow: intracellular membranes and storage droplets in the cell anterior part are dyed. Conversely, the use of sterol‐binding drugs prevents the lipid accumulation in droplets, stopping the lipid within the cytoplasmic membranes. Furthermore, the cells treated with monensin and cytochalasin B show a labelling of the cellular membranes and lipid droplets, whereas NH₄Cl reduces the lipid storage. Low temperature (4 °C) does not prevent the internalization of BODIPY‐CE that, however, is localized at the cytoplasmic membrane level and does not accumulate in storage droplets. In addition, BODIPY‐CE inhibits phagocytosis, as evidenced by comparing the kinetics of food vacuole formation of control cells, only fed with latex particles, with that of cells fed with latex particles and BODIPY‐CE. In conclusion, this study points out that in P. primaurelia the cholesteryl ester enters the cell via food vacuoles and through the plasma membrane and, inside the cell, it alters cell functions.     

Vacuolar sequestration of fluorescent probes in plant cells: a review

The use of fluorescent probes as indicator and tracer molecules is becoming an important aspect of plant cell biology. In many cases the dye, whether introduced directly into the cytosol or sequestered by the cell from its external environment, is preferentially transferred to the vacuole. In the light of increasing evidence for endocytosis in plant cells, the sequestration of high-molecular-weight fluorescent dextrans and the membrane-impermeant dye Lucifer Yellow-CH into the vacuole has been cited as evidence supporting the presence of a fluid-phase endocytic pathway. In this review we consider these recent reports of vacuolar sequestration in the light of new evidence arising on the mechanisms underlying dye uptake.     

Brief note: Differences in intracellular localization of corn stunt spiroplasmas in magnesium treated maize.

Maize plants infected with Spiroplasma kunkelii show symptoms similar to that of plants in a magnesium-deficient soil, and it has been shown that the spiroplasma alters the plants’ magnesium absorption. In the current study we compared changes associated to either spiroplasma infection, two soil magnesium levels and their combinations. Plant symptoms were recorded and correlated with transmission electron microscopy observations. Plants grown on a high magnesium treatment showed no macroscopical alterations nor organelle ultrastructural alterations, while plants on a low magnesium treatment showed macroscopical vein yellowing and, ultrastructurally, they had most chloroplasts and mitochondrial membranes altered. Infected plants on a low magnesium treatment had an ageing aspect, ultrastructurally showed chloroplasts and mitochondrial alterations similar to those non-infected and grown on a low magnesium treatment, and spiroplasma cells were found in phloem cells, but outside their cytoplasm. Infected plants on a high magnesium treatment showed similar symptoms and ultrastructural alterations as either non-infected plants on the low magnesium treatment or in infected plants on the low magnesium treatment, but differ from them in that the spiroplasma cells were located inside the cytoplasm. Results suggest that magnesium is involved in the plant-pathogen interaction.     

Chemical mapping of the distribution of viruses into infected bacteria with a photothermal method

We show that an infrared spectromicroscopy method based on a photo-thermal effect, is able to localize single viruses as well when they are isolated and when they are located inside the bacteria they have infected. In this latter case, although the topography performed by an AFM cannot image the viruses, the AFMIR is able to do so. In addition, we are able to determine different stages of the bacteria infection.     

Levels of plant cell wall structural organization revealed by atomic force microscopy

We used an atomic force microscope to image cell wall isolated from needles of Serbian spruce tree and that synthesized from cell wall components. We also observed the structure of lignin model polymer (DHP), as a best substitute for the natural lignin. A tendency of aggregate formation was observed in all samples. Cell wall was revealed as a laminated fibrous structure. General organization is similar in both isolated and synthesized cell wall samples, with dominating globular motifs arranged regularly as rods and forming cavities. The synthesized cell wall has a more regular structural organization than isolated cell wall. The dimensions of individual globular aggregates and pores differed between the two samples. DHP showed a similar, regular organization, with globular aggregates and holes. Globules and pores are smaller in size than the corresponding structures in both the isolated and synthesized cell walls. Such modular organization of cell walls may have a physiological role in response to the external mechanical stress caused to plant cells.     

Nanofabrication of cylindrical STEM specimen of InGaAs/GaAs quantum dots for 3D-STEM observation

We demonstrate the multiazimuth observation (360 degrees in principle) of InGaAs/GaAs quantum dots (QDs) by means of a 300 kV scanning transmission electron microscope (STEM), where both cross-sectional and plan-view observations are performed on a single STEM specimen for the first time. A cylindrical specimen with a diameter of 200-300 nm including the QD layer inside along the rotation axis was fabricated by the focused ion beam (FIB) technique, with the application of a newly developed mesa-cutting method to adjust the position and angle of the QD layer precisely. The 360 degrees STEM observation is realized by mounting the cylindrical specimen on a holder equipped with a specimen-rotation mechanism. High potential of 3D-STEM observation is briefly presented by showing high contrast images of QDs, dark field images, and moire fringes with various incident angles.     

Measurement of glutathione levels in intact roots of Arabidopsis

Levels of glutathione were measured for different cell types in roots of intact Arabidopsis seedlings after labelling with monochlorobimane to give fluorescent glutathione S‐bimane (GSB) and imaging using confocal laser scanning microscopy with excitation at 442 nm. Labelling increased to a plateau in most cell types after about 15–20 min and the GSB accumulated rapidly in the vacuole. Formation of GSB in the cytoplasm was not affected by treatment with sodium azide; however, vacuolar transport of GSB was substantially inhibited under these conditions. We infer that vacuolar sequestration was mediated by a tonoplast glutathione S‐conjugate pump. Quantitative estimates of the cytoplasmic glutathione concentration involved correction for the loss in fluorescence signal with depth into the specimen using an empirically determined model derived in situ from a permeabilized root. Correction for the dilution experienced on transport into the vacuole also required an estimate of the amount of cytoplasm present in each cell type. This was achieved in two stages: first, the levels of protein were mapped after fixation, permeabilization and labelling with fluroescein isothiocyanate. Second, the corresponding cytoplasmic volume was determined as 40% for epidermal cells in the elongation zone by manual segmentation of the cytoplasm in serial optical sections. Values of relative cytoplasmic volume for other cells were extrapolated in proportion to their protein content. Using this approach, cytoplasmic glutathione concentrations were found to be 2–3 mm in most cell types. There was a marked difference between the central cells and the neighbouring, rapidly dividing initials, and between the columella cells and the outermost cells of the root cap. In the latter case, the difference was equalized in the presence of azide. This might indicate that additional cell–cell movement and preferential sequestration of GSB can occur during the detoxification process in an intact system.     

On the use of TEM cells for the calibration of power frequency electric field meters

This paper focuses on the performances of TEM cells when used in the calibration of power frequency environmental electric field meters. The spatial non-uniformity of the electric field inside a TEM cell is analyzed through experimental investigations and three-dimensional Boundary Element modeling to evaluate the field experienced by the sensing elements of actual 3D meter probes. The perturbation caused by the probe support is also taken into account. The uncertainty component associated with the spatial non-uniformity in the volume taken up by typical power and low frequency field probes is estimated. The field non-uniformity is also evaluated in relation to the use of TEM cells of reduced size. Finally, the field non-uniformity is exploited to predict the performance of an actual field meter operating in significant field gradients.     

Microscopic and molecular evidence in support of rodent as a reservoir for dissemination of Leishmaniasis

Leishmaniasis is a worldwide public health problem and vector‐borne disease. It is caused by a diverse group of protozoan parasites that belong to the genus Leishmania and transmitted to humans through a bite of an infected female sand fly. Leishmaniasis has attained epidemic proportion in Khyber Pakhtunkhwa and raises serious concern over its management. The present research work was conducted in cutaneous leishmaniasis (CL) prevalent village named Surgul of district Kohat, Khyber Pakhtunkhwa with a focus to investigate whether rodent can act as a source for dissemination of leishmanial species or not. In this context, rodent samples were analyzed via morphological and molecular approaches to unveil prevalence of CL. It was reported that 12.5% of samples were positive for signs of leishmaniasis through microscopy and 18.75% through polymerase chain reaction (PCR). Supporting the findings further, the color character of rodents was also taken into consideration, which shows that light dark colored rodents were more infected (13.3%) compared to brown colored rodents (11.43%). Based on our findings, we speculate that small rodents are a possible reservoir of various leishmanial parasites and play a significant role in zoonosis and maintenance of their species.     

A new freeze-drying device for platinum replica studies of cell surface and cytoskeleton: An example using immunogold-labeled human erythrocytes

We designed and built a freeze-drying device that ensures the protection of the specimens against contaminants during mounting on the cold stage of the freeze-fracture machine, transferring into the vacuum chamber and deep etching. The device consists of a copper cap that covers the specimen and a thermal connection that ensures thermal transfer between the microtome arm and the copper cap. This device was used to study the ultrastructural features of the erythrocyte membrane skeleton and the immunocytchemical localization of spectrin in an “in situ” approach, by freeze drying and platinum rotary shadowing. Human erythrocytes adhered to polylysine-coated coverslips and were broken by a stream of buffer that mimics the intracellular ionic environment (“inside buffer”). The samples were prefixed in periodate-lysine-paraformaaldehyde fixative, labeled with antispectrin 5-nm gold particles, fixed in glutaraldehyde, mordanted in tannic acid, postfixed in OsO₄, repeatedly washed in water, rinsed quickly in 30% ethanol, freeze-dried, and rotary-shadowed. Electron microscopic examination of the replicas revealed the skeletal network on the inner surface of the erythrocyte membrane. Immunocytochemical labeling proved that spectrin represents a fibrillar component of the network. Our data confirm the speculative model of the molecular organization of the erythrocyte skeleton, based on studies on in vitro association of proteic constituents. Both the technique and the device developed by us may lead to a deeper understanding of the spatial organization of the cytoskeletal network of more complex cell types.     

Fine structural analysis of the epithelial cells in the hepatopancreas of Palaemonetes argentinus (Crustacea, Decapoda, Caridea) in intermoult.

The aim of this study is to describe the ultrastructure of the hepatopancreas of P. argentinus in intermoult. P. argentinus hepatopancreas was studied using standard TEM techniques. Each tubule consists of four cellular types: E (embryonic), F (fibrillar), R (resorptive) and B (blister like). E-cells have embryonic features and some of them were found in mitosis. F, R and B cells possess an apical brush border. F-cells have a central or basal nucleus, a conspicuous RER, and dilated Golgi cisternae. R cells show a polar organization of organelles in three areas: apical, with numerous mitochondria and sER tubules, a central area with the nucleus and RER, and a basal area containing a sER-like tubule system and mitochondria. B-cells were observed at different stages of their life cycle. In an early differentiation stage they comprise an apical endocytotic complex and Golgi vesicles. The fusion of endocytotic and Golgi vesicles originates subapical vacuoles. During maturation, a big central vacuole is formed by coalescence of subapical vacuoles. The central vacuole is eliminated by holocrine secretion. The ultrastructure suggests that F-cells synthesize proteins, R-cells storage nutrients and B-cells have a secretory or excretory function, and confirms the independent origin of F, B and R cells from the embryonic cells.     

Ultrastructural characterization of isolated rat Kupffer cells by transmission X-ray microscopy

The ultrastructure of primary cultured rat Kupffer cells was studied using transmission X-ray microscopy as well as transmission electron microscopy. X-ray microscopical images of intact, hydrated Kupffer cells demonstrated structures such as cell nucleus separated by a nuclear membrane and filaments concentrated in the perinuclear area. Within the cytoplasm, a number of vacuoles were visible; some of these were crescent-shaped vacuoles that were half X-ray lucent, half X-ray dense; others were uniformly dense. The number of crescent-shaped vacuoles was predominant. After phagocytosis of haematite particles, enlarged vacuoles containing the ingested material were visible within the cytoplasm of Kupffer cells while crescent-shaped vacuoles were no longer detectable. Densitometric analysis of the two types of vacuole revealed that the X-ray absorption of the uniform vacuole was approximately half that of the dense part of the crescent-shaped vacuoles. This observation led to speculation on the existence of only one type of vacuole in the cytoplasm of Kupffer cells. The different morphological aspects — crescent-shaped versus uniform vacuoles — might be due to different three-dimensional orientation with respect to the image plane. Using transmission electron microscopy, the morphology of vacuoles differed more widely in diameter, density and shape. Two main types of vacuole were identified: electron-lucent and electron-dense. Based on the observation of only one type of vacuole by transmission X-ray microscopy, the different morphological aspects of vacuoles obtained by transmission electron microscopy could be explained by imaging several different sections of a crescent-shaped vacuole. From the present data it can be concluded that transmission X-ray microscopy is a versatile technique that reveals the ultrastructure of intact, unsectioned biological specimens in their aqueous environment, thereby allowing a more comprehensive interpretation of data obtained by transmission electron microscopy.