Structure Of Organelles Research Articles

Effect of vascular endothelial growth factor on remodeling of C6 glioma tissue in vivo

Pathological characteristics and biological behaviors of tumor are considered to result from pathological tissue remodeling regulated by the interaction of factors in the tumor microecosystem (TMES). Vascular endothelial growth factor (VEGF) is one of the factors that probably play an important role in the process of tissue remodeling. This study was a comprehensive investigation of the effects of VEGF on remodeling of glioma tissue in vivo. C6 cells with expression vectors containing sense (C6/VEGF+) or antisense (C6/VEGF-) VEGF(164) complementary DNA (cDNA) or an empty vector (C6/vec) were implanted into athymic mice, which served as an in vivo model with different levels of VEGF expression. VEGF expression, water content, and morphological characteristics of these tumor tissues were assayed. Expression of VEGF and water content in C6/VEGF- glioma (C6/VEGF-G) tissues were lower than in C6, C6/VEGF+, and C6/vec glioma (C6G, C6/VEGF+G, C6/vecG) (P < 0.01, P < 0.05); water content correlated with VEGF expression (r = 0.791, P = 0.000). In C6/VEGF-G, tumor cells were not tightly adhered to vascular walls, and the basal lamina surrounded by collagen fibers was monolayer and not continuous. However, in C6G, C6/VEGF+G, and C6/vecG, tumor cells were very close to the vascular walls, with some extending their processes to the wall. Generally, loose basal laminae surrounded by small amounts of collagen fibers were multilayer, integrated, and continuous. Vesicular vacuolar organelle (VVO) structures were observed in plasma of vascular endothelial cells (VECs), and significant correlation was found between VEGF expression level and VVO density (Spearman's r = 0.642, P = 0.007). No correlation was found between VEGF expression and fenestrae formation in VECs (Pearson's correlation r = -0.053, P = 0.846), and fenestrae in neither VECs nor intercellular clefts correlated with water content of tumor tissue (Pearson's correlation r = 0.018, P = 0.947). These results demonstrate that VEGF can aggravate edema in tumor tissues by increasing VVOs and plays critical roles in the stickiness of tumor cells to vessel wall and in the integrity and continuity of the basal lamina of vessels. Our data indicate a possible mechanism of remodeling of glioma tissue and suggest that blocking VEGF might contribute to a therapeutic strategy for glioma.

Characterization of two novel ADP ribosylation factors from the shrimp Marsupenaeus japonicus

ADP-ribosylation factors (Arfs) that play an essential role in intracellular trafficking and organelle structure are small GTP-binding proteins, which have been identified recently to be involved in virus infection. However, little is known about the Arfs and their relationships with viral infection in the economically important crustaceans to date. In the present study, two novel members of the Arf family, designated as MjArf1 and MjArfn respectively, were cloned from the shrimp Marsupenaeus japonicus. Sequence and phylogenetic analysis showed that MjArf1 belongs to Class I Arf, which has very high homology in sequence to the known Arf 79F of insects and Arf1 of other animals (96–99%), whereas MjArfn is an unidentified Arf, which has only 62–66% identity to other known Arfs. In High Five cells, the distribution of MjArf1 was dependent on its GDP/GTP binding state but the distribution of MjArfn was not affected by that. Both Arfs were ubiquitously expressed in examined tissues. Further investigation with real-time quantitative PCR revealed that MjArf1 and MjArfn were significantly up-regulated after WSSV challenge. In virus-resistant shrimps, however, no distinct fluctuation of MjArf1 expression was found and MjArfn was even found to be notably repressed. These results suggested that MjArf1 and MjArfn might be involved in the shrimp innate immune response in WSSV infection and MjArfn might play a role in WSSV invasion. These studies may contribute to a better understanding of host defense and/or virus invasion interaction and for the control of marine crustacean diseases.

Comparative physiological, ultrastructural and proteomic analyses reveal sexual differences in the responses of Populus cathayana under drought stress

Drought is a major abiotic stress, limiting the survival and growth of young plants. However, little is known about sex-dependent responses to drought at the proteome level. In this study, we carried out investigations on comparative proteomics, combined with physiological and organelle structure analyses, in males and females of Populus cathayana Rehd. Three-month-old poplar cuttings were treated at 30% of field capacity and at 100% field capacity as a control in a greenhouse for 40 days. Drought greatly inhibited plant growth, damaged the photosynthetic system and destructed the structures of chloroplasts, mitochondria and cellular membranes. However, males suffered less from the adverse effects of drought than did females. Using 2-DE, 563 spots were detected, of which 64 spots displayed significant drought effect and 44 spots displayed a significant sex by drought interaction effect. The results suggest that the different responses to drought stress detected between the sexes have a close relationship to the changes in the expression of sex-dependent proteins, including, e.g. photosynthesis-related proteins, homeostasis-related proteins and stress response proteins. These proteins could contribute to a physiological advantage under drought, giving potential clues for understanding sexual differences in the performance of plants in different environments.

Quantitative 3D imaging of whole, unstained cells by using X-ray diffraction microscopy

Microscopy has greatly advanced our understanding of biology. Although significant progress has recently been made in optical microscopy to break the diffraction-limit barrier, reliance of such techniques on fluorescent labeling technologies prohibits quantitative 3D imaging of the entire contents of cells. Cryoelectron microscopy can image pleomorphic structures at a resolution of 3-5 nm, but is only applicable to thin or sectioned specimens. Here, we report quantitative 3D imaging of a whole, unstained cell at a resolution of 50-60 nm by X-ray diffraction microscopy. We identified the 3D morphology and structure of cellular organelles including cell wall, vacuole, endoplasmic reticulum, mitochondria, granules, nucleus, and nucleolus inside a yeast spore cell. Furthermore, we observed a 3D structure protruding from the reconstructed yeast spore, suggesting the spore germination process. Using cryogenic technologies, a 3D resolution of 5-10 nm should be achievable by X-ray diffraction microscopy. This work hence paves a way for quantitative 3D imaging of a wide range of biological specimens at nanometer-scale resolutions that are too thick for electron microscopy.

Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living cells. The proposed instrument extends the sensitivity of label-free optical microscopy to nanoscale changes in organelle size and shape and can be used to accelerate the study of the structure-function relationship pertaining to organelle dynamics underlying fundamental biological processes, such as programmed cell death or cellular differentiation. The microscope can be easily implemented on existing microscopy platforms, and can therefore be disseminated to individual laboratories, where scientists can implement and use the proposed methods with unrestricted access. The proposed technique is able to characterize subcellular structure by observing the cell through two-dimensional optical Gabor filters. These filters can be tuned to sense with nanoscale (10's of nm) sensitivity, specific morphological attributes pertaining to the size and orientation of non-spherical subcellular organelles. While based on contrast generated by elastic scattering, the technique does not rely on a detailed inverse scattering model or on Mie theory to extract morphometric measurements. This technique is therefore applicable to non-spherical organelles for which a precise theoretical scatter description is not easily given, and provides distinctive morphometric parameters that can be obtained within unstained living cells to assess their function. The technique is advantageous compared with digital image processing in that it operates directly on the object's field transform rather than the discretized object's intensity. It does not rely on high image sampling rates and can therefore be used to rapidly screen morphological activity within hundreds of cells at a time, thus greatly facilitating the study of organelle structure beyond individual organelle segmentation and reconstruction by fluorescence confocal microscopy of highly magnified digital images of limited fields of view. In this demonstration we show data from a marine diatom to illustrate the methodology. We also show preliminary data collected from living cells to give an idea of how the method may be applied in a relevant biological context.

The strategy of “Useful Sun” improves physical endurance and adaptation of cardiac morphology

The action of solar light transformed by special screens has been studied on CD-1 male mice. In the active control group, mice were irradiated through screens absorbing the UV-component. In the experimental group, screens transforming the UV-component into the orange-red light were used. In the active control, changes in the swimming activity, as compared to the same parameter before irradiation, were manifested much less than in animals of the experimental group. A morphological analysis showed changes in the structure of all cardiomyocyte organelles studied: the relative area of mitochondria in the experimental mice increased by more than 20% compared to intact animals (p < 0.05). A significant increase in the area of the sarcoplasmic reticulum, by 23.4% (p < 0.05), and in the volume of the myofibrillar apparatus, by 19.4% (p < 0.05), was detected. The results of our experiment show that the irradiation with using an additional orangered component improves the physical endurance 1.5 times and initiates morphogenetic processes in cardiac muscle cells.

Morphological Heterogeneity of Endothelium

Vascular endothelium lines the entire cardiovascular system where it performs a series of vital functions by its control of microvascular permeability, vessel wall tone, coagulation and anticoagulation cascades, lipid homeostasis, inflammation, angiogenesis, and vasculogenesis. The vertebrate endothelial cells display a remarkable heterogeneity in terms of morphology, molecular makeup, and functional output. This heterogeneity was documented very early by electron microscopy studies that established morphologically recognizable endothelial phenotypes in vascular beds of different organs and, moreover, within the different vascular segments of each organ. This review discusses endothelial heterogeneity from a morphological standpoint and the latest developments in our understanding of the components, structure, and function of the endothelial specific organelles that form the hallmark of these phenotypes.

Transmission electron microscopic observation on ultrastructural alterations in Schistosoma japonicum caused by mefloquine

The purpose of the study was to explore the ultrastructural alterations of adult Schistosoma japonicum induced by mefloquine. Eight out of ten mice infected with 60-80 S. japonuicum cercariae for 35 days were treated orally with mefloquine at a single dose of 400 mg/kg. Four groups of two mice were killed at 8 h, 24 h, 3 days, and 7 days post-treatment, and schistosomes were collected by perfusion technique, fixed, and examined under a transmission electron microscope. Schistosomes obtained from the remaining two mice served as control. Eight hours after mefloquine 400 mg/kg was administered to the infected mice, various alterations in the tegument and subtegument tissues of both male and female worms were seen, which included focal lysis of tegmental matrix resulted in vacuole formation, decrease in rod-like and discoid-like secretary bodies, light swelling or focal lysis of musculature, extensive lysis of internal structure of sensory organelles, and appearance of vacuole or myelin-like structure in perinuclear cytoplasm of syncytium and epithelial cells. In vitelline cells of female worms, the most significant alteration was extensive lysis or fusion of vitelline balls in vitelline droplets and decrease in granular endoplasmic reticulum Twenty-four hours post-treatment, damage to the tegument and subtegument tissues had increased in severity. In male worms, the most prominent alterations were emergence of large vacuoles in the tegument, detachment of cytoplasmic process from the tegumental surface, focal collapse of internal structure of sensory organelle, and loss of definition of syncytium and gut epithelial cell. In female worms, focal lysis in tegumental matrix, musculature, and parenchymal tissues resulted in emergence of vacuole or myelin-like structure, reduction of nucleoli, fusion of partial nuclear membrane together with cytoplasm in epithelial cell, and lysis of interstitial tissues among the vitelline cells which were universal. Three and 7 days post-treatment, besides the aforementioned alterations, the significant damage to the male worms were disrupted outer plasma membrane detached from the cytoplasmic process, swelling of individual cytoplasmic process, extensive swelling and focal lysis in the musculature, parenchymal tissues and perinuclear cytoplasm of syncytium, accompanied by emergence of swollen mitochondria, vacuoles, and myelin-like structure, and severe damage to gut epithelial cell. In female worms, apart from disruption of outer plasmic membrane in cytoplasmic process, severe swelling of tegumental matrix accompanied by emergence of vacuoles, swollen mitochondria and myelin-like structure, focal lysis of heterochromatin and nucleoli, disappearance of microvilli in gut epithelial cells, and emergence of myelin-like structures in vitelline cells were observed. The results indicate that administration of mefloquine to mice infected with adult S. japonicum exhibits an extensive damage to the ultrastructure in tegument and subtegument tissues including syncytium, gut epithelial cells, parenchymal tissues, and vitelline cells of schistosomes.

Saturated fatty acid metabolism is key link between cell division, cancer, and senescence in cellular and whole organism aging

Cellular senescence is an in vivo and in vitro phenomenon, accompanied by physiological changes including cessation of division and disturbances of organelle structure and function. Review of the literature was undertaken to determine whether there is evidence that whole organism aging and cell senescence share a common initiation pathway. In vivo aged cells of different lineages, including aged T lymphocytes, show high expression of the INK4A-p16 gene. In cell culture when telomeres are shortened past a key length or state, the Arf/Ink gene system (p16/p14 humans, p16/p19 mice) switches on and activates p53, which suppresses further cell division. The p53 gene is a key tumor suppressor and its deletion or mutation allows cancerous growth. The switching on of p53 also causes changes in fatty acid metabolism, especially down-regulation of both fatty acid synthase and stearoyl-CoA (delta-9) desaturase. The co-suppression of these genes together with enhanced uptake of extracellular fatty acids, leads to raised levels of cellular palmitate and induction of either apoptosis or senescence. In senescent cells, the fatty acid composition of the cellular membranes alters and leads to changes in both structure and function of organelles, especially mitochondria. Animal models of accelerated aging exhibit repression of stearoyl-CoA desaturase activity while anti-aging calorie restriction stimulates the same enzyme system. It is concluded that aging in cells and whole organisms share a common initiation pathway and that cellular senescence is protective against cancer. Healthy longevity is likely to be most enhanced by factors that actively suppress excessive cell division.

Microbes in Earth’s aqueous environments

Microbes in Earth’s aqueous environments

Mitochondria of human Leydig cells as seen by high resolution scanning electron microscopy

The three-dimensional ultrastructure of over 1000 mitochondria in human Leydig cells (from twelve sexually mature patients) was examined by high resolution scanning electron microscopy (HRSEM) of osmium-macerated specimens, as well as by transmission electron microscopy of conventional ultrathin sections. The stereo-pair imaging of the osmium-macerated specimens by HRSEM is also very useful for investigating the three-dimensional structure of cytoplasmic membranous organelles with great clarity. The mitochondria, which mainly are elongated (although some are ovate), possess cristae that are almost exclusively tubular and that occasionally display constrictions and terminal bulbules. Lamelliform cristae are quite rare. Occasionally, the tubular cristae are joined together to form a simple network. Classic crista junctions could not be identified with certainty, although the base of the tubular cristae might correspond functionally to such junctions. As a whole, in line with the identical and common embryological origin of adrenal cortex and gonads, mitochondria of human Leydig cell closely resemble those of steroidogenic cells of human suprarenal cortex treated by the same maceration method.

Differentiation of E14 Mouse Embryonic Stem Cells into Thyrocytes In Vitro

If methods of differentiating stem cells into thyrocytes can be perfected, they may provide a ready source of normal thyrocytes for basic research and clinical application. We developed a novel culture method capable of differentiating mouse embryonic stem (ES) cells into thyroid follicular cells. E14 mouse ES cells were allowed to differentiate into embryoid bodies and then stimulated with thyroid-stimulating hormone, insulin, and potassium iodide. The resulting differentiated cells were observed for expression of thyrocyte-specific mRNA transcripts with reverse transcriptase (RT)-polymerase chain reaction. To definitively identify thyrocytes, we simultaneously observed the thyrocyte-specific proteins, thyroid transcription factor-1 and PAX-8, with dual-color immunofluorescent labeling. The cells were further characterized by electron microscopy. The ES cells were successfully differentiated into thyrocytes. Differentiated cells expressed PAX-8, thyroid-stimulating hormone receptor, sodium/iodide symporter, thyroperoxidase, and thyroglobulin mRNAs, and coexpressed thyroid transcription factor-1 and PAX-8 proteins. The extent of differentiation was further explored by electron microscopy, which showed that differentiated cells had ultrastructural features similar to adult human thyrocytes, whereas the cells from unstimulated cultures were mostly disintegrated and lacked developed organelle structures. These data show that E14 mouse ES cells can be differentiated into thyrocytes by culturing with thyroid-stimulating hormone, insulin, and potassium iodide. The development of reliable methods to produce thyroid cells from ES cells is important to future research in thyroid biology and medical applications.

Histologic and morphologic effects of valproic acid and oxcarbazepine on rat uterine and ovarian cells

To determine the histologic and morphologic effects of valproic acid (VPA) and oxcarbazepine (OXC) on rat uterine and ovarian cells. Fifty-six female prepubertal Wistar rats (21-24 days old and weighing between 47.5 and 58.1 g) were divided equally into four groups, which were given drinking water (controls), 300 mg/kg/day of VPA, 100 mg/kg/day of OXC or VPA + OXC via gavage, for 90 days. Ovaries and uteri of rats on proestrous and diestrous phases of estrous cycle were extirpated and placed in a fixation solution. The tissue specimens were assessed with apoptosis (TUNEL) staining protocols, eosinophil counting, and electron microscopic techniques. In uteri, apoptosis in stroma, mitochondrial swelling, and cristolysis were observed in the VPA group, and OXC led to negative effects on epithelial cell and intracellular edema. In ovaries, both drugs increased apoptosis and intracytoplasmic edema. Organelle structure disruption was also observed in the OXC group. More conspicuous degenerative modifications were determined in the VPA + OXC group. In uteri, the number of TUNEL-positive luminal epithelial cells was 7.20 +/- 1.32 in controls, and significantly increased to 29.60 +/- 1.58, 34.20 +/- 2.53, and 54.80 +/- 2.04 in VPA, OXC, and VPA + OXC groups, respectively (p < 0.001). The highest number of TUNEL-positive glandular epithelium cells was observed in the VPA + OXC group; however, the number of TUNEL-positive stroma cells was highest in the VPA group. The highest number of eosinophils in stroma was in the VPA group. VPA and OXC trigger apoptotic and degenerative effects on rat uterine and ovarian cells. VPA also prevents implantation of embryo to the uterus and causes abortion via endometrial eosinophil infiltration.

Abstract C101: Subcellular distribution study by means of the inherent fluorescence of the investigational drug Triapine and its zinc(II) complex

Abstract The antineoplastic activity of α-N-heterocyclic thiosemicarbazones was discovered several decades ago. Currently the most promising drug candidate of this class of compounds is Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP), which entered several phase I and II clinical trials as an antitumor agent. We discovered that Triapine possesses intrinsic fluorescence properties. This enabled us to monitor for the first time the uptake and intracellular distribution of an α-N-heterocyclic thiosemicarbazone in living human cancer cells by fluorescence microscopy. In addition we synthesized the first zinc(II) complex [Zn(Triapine)Cl2]•HCl of Triapine, which shows fluorescence as well, compared its cytotoxicity in human cancer cells with that of the parental compound and studied the influence of metal complexation on intracellular distribution. Triapine and its zinc complex were characterized spectroscopically. The UV-Vis spectrum of Triapine in water shows a maximum absorbtion band at 359 nm, whereas the maximum absorbtion of [Zn(Triapine)Cl2]•HCl is slightly shifted to 365 nm. The maxima of the emission spectra of both compounds are at 457 nm when irradiated at ex = 360 nm. Fluorescence microscopy of living SW480 (colon carcinoma) cells treated with Triapine or [Zn(Triapine)Cl2]•HCl showed that both compounds have a striking affinity to the nuclear membrane and to an organelle structure in the cytoplasm, whereas the localization in the nucleus was strongly different. The zinc complex binds to substructures within the nucleus, which could be verified as nucleoli by immunostaining of the nucleolar protein fibrillarin and co-staining with [Zn(Triapine)Cl2]•HCl. On the contrary, a strong affinity of Triapine to the nucleoli was not detected. The cytotoxic potencies of Triapine and [Zn(Triapine)Cl2]•HCl were determined in SW480 (colon carcinoma) and 41M (ovarian carcinoma) cells by means of the colorimetric MTT assay. The IC50 value of Triapine is 0.55±0.2 µM in SW480 cells and 0.45±0.03 µM in the 41M cell line. [Zn(Triapine)Cl2]•HCl showed IC50 values of 0.54±0.02 µM in SW480 and 0.52±0.02 µM in 41M cells. Comparing the two substances, no remarkable differences were observed. Our results demonstrate that Triapine can be visualized with fluorescence microscopy in living cells without any conjugation of fluorophores. Furthermore our findings indicate that metal complexation leads to a different subcellular distribution. Additional studies are ongoing to clearify the influence of metal complexation on the mode of action. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C101.

A Robust and Highly Efficient Immune Cell Reprogramming System

Here we describe a lineage reprogramming system consisting of a B cell line with an estradiol-inducible form of C/EBPalpha where cells can be converted into macrophage-like cells at 100% efficiency within 2 to 3 days. The reprogrammed cells are larger, contain altered organelle and cytoskeletal structures, are phagocytic, and exhibit an inflammatory response. Time-lapse experiments showed that the cells acquire a macrophage morphology and increased migratory activity as early as 10 hr. During induction, thousands of genes become up- or downregulated, including several dozen transcription and chromatin-remodeling factors. Time-limited exposure of cells to the inducer showed that the reprogrammed cells become transgene independent within 1 to 2 days. The reprogramming can be inhibited, at least partially, by perturbation experiments with B cell and macrophage transcription factors. The tightness, robustness, and speed of the system described make it a versatile tool to study biochemical and biological aspects of lineage reprogramming.

Structure and distribution of organelles and cellular location of calcium transporters in Neurospora crassa.

We wanted to examine the cellular locations of four Neurospora crassa proteins that transport calcium. However, the structure and distribution of organelles in live hyphae of N. crassa have not been comprehensively described. Therefore, we made recombinant genes that generate translational fusions of putative organellar marker proteins with green or red fluorescent protein. We observed putative endoplasmic reticulum proteins, encoded by grp-78 and dpm, in the nuclear envelope and associated membranes. Proteins of the vacuolar membrane, encoded by vam-3 and vma-1, were in an interconnected network of small tubules and vesicles near the hyphal tip, while in more distal regions they were in large and small spherical vacuoles. Mitochondria, visualized with tagged ARG-4, were abundant in all regions of the hyphae. Similarly, we tagged the four N. crassa proteins that transport calcium with green or red fluorescent protein to examine their cellular locations. NCA-1 protein, a homolog of the SERCA-type Ca(2+)-ATPase of animal cells, colocalized with the endoplasmic reticulum markers. The NCA-2 and NCA-3 proteins are homologs of Ca(2+)-ATPases in the vacuolar membrane in yeast or in the plasma membrane in animal cells. They colocalized with markers in the vacuolar membrane, and they also occurred in the plasma membrane in regions of the hyphae more than 1 mm from the tip. The cax gene encodes a Ca(2+)/H(+) exchange protein found in vacuoles. As expected, the CAX protein localized to the vacuolar compartment. We observed, approximately 50 to 100 mum from the tip, a few spherical organelles that had high amounts of tagged CAX protein and tagged subunits of the vacuolar ATPase (VMA-1 and VMA-5). We suggest that this organelle, not described previously in N. crassa, may have a role in sequestering calcium.

Developmental changes and organelle biogenesis in the reproductive organs of thermogenic skunk cabbage (Symplocarpus renifolius).

Sex-dependent thermogenesis during reproductive organ development in the inflorescence is a characteristic feature of some of the protogynous arum species. One such plant, skunk cabbage (Symplocarpus renifolius), can produce massive heat during the female stage but not during the subsequent male stage in which the stamen completes development, the anthers dehisce, and pollen is released. Unlike other thermogenic species, skunk cabbage belongs to the bisexual flower group. Although recent studies have identified the spadix as the thermogenic organ, it remains unclear how individual tissues or intracellular structures are involved in thermogenesis. In this study, reproductive organ development and organelle biogenesis were examined during the transition from the female to the male stage. During the female stage, the stamens exhibit extensive structural changes including changes in organelle structure and density. They accumulate high levels of mitochondrial proteins, including possible thermogenic factors, alternative oxidase, and uncoupling protein. By contrast, the petals and pistils do not undergo extensive changes during the female stage. However, they contain a larger number of mitochondria than during the male stage in which they develop large cytoplasmic vacuoles. Comparison between female and male spadices suggests that mitochondrial number rather than their level of activity correlates with thermogenesis. Their spadices, even in the male, contain a larger amount of mitochondria that had greater oxygen consumption, compared with non-thermogenic plants. Taken together, our data suggest that the extensive maturation process in stamens produces massive heat through increased metabolic activities. The possible mechanisms by which petal and pistil metabolism may affect thermogenesis are also discussed.

Lysophosphatidic acid acyltransferase 3 regulates Golgi complex structure and function

Recent studies have suggested that the functional organization of the Golgi complex is dependent on phospholipid remodeling enzymes. Here, we report the identification of an integral membrane lysophosphatidic acid–specific acyltransferase, LPAAT3, which regulates Golgi membrane tubule formation, trafficking, and structure by altering phospholipids and lysophospholipids. Overexpression of LPAAT3 significantly inhibited the formation of Golgi membrane tubules in vivo and in vitro. Anterograde and retrograde protein trafficking was slower in cells overexpressing LPAAT3 and accelerated in cells with reduced expression (by siRNA). Golgi morphology was also dependent on LPAAT3 because its knockdown caused the Golgi to become fragmented. These data are the first to show a direct role for a specific phospholipid acyltransferase in regulating membrane trafficking and organelle structure.

Functional analyses of pilin-like proteins from Francisella tularensis: complementation of type IV pilus phenotypes in Neisseria gonorrhoeae

Accumulating evidence from a number of studies strongly suggests that proteins orthologous to those involved in type IV pili (Tfp) assembly and function are required for Francisella pathogenicity. However, the molecular mechanisms by which the components exert their influence on virulence remain poorly understood. Owing to the conservation and promiscuity of Tfp biogenesis machineries, expression of Tfp pilins in heterologous species has been used successfully to analyse organelle structure-function relationships. In this study we expressed a number of Francisella pilin genes in the Tfp-expressing pathogen Neisseria gonorrhoeae lacking its endogenous pilin subunit. Two gene products, the orthologous PilA proteins from Francisella tularensis subspecies tularensis and novicida, were capable of restoring the expression of Tfp-like appendages that were shown to be dependent upon the neisserial Tfp biogenesis machinery for surface localization. Expression of Francisella PilA pilins also partially restored competence for natural transformation in N. gonorrhoeae. This phenotype was not complemented by expression of the PulG and XcpT proteins, which are equivalent components of the related type II protein secretion system. Taken together, these findings provide compelling, although indirect, evidence of the potential for Francisella PilA proteins to express functional Tfp.

Three-dimensional structure of the cytoskeleton in Trichomonas vaginalis revealed new features

The flagellated protozoan Trichomonas vaginalis has been widely studied owing to its medical significance and unique structure. The complicated three-dimensional (3D) structure of the cellular components of T. vaginalis was reconstructed from serial sections to enable observation of the spatial features of the whole cell. Electron tomography was used to examine the detailed structure of the cellular organelles. Tomographic reconstruction showed the mastigont system and the parabasal filament of T. vaginalis in detail. The last thin filament (Pf3) was located close to the adjacent filament, and the two filaments appeared to be vertically parallel in the cross-sectional view. It is likely that Pf3 cannot be distinguished from the adjacent filament in 2D images obtained from transmission electron microscopy. Our 3D reconstruction of T. vaginalis revealed the presence of an additional striated fiber, and 3D reconstruction by electron tomography showed twisting of the split parabasal filament.