Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes.

Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite Trypanosoma brucei, occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism (i.e. polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.

Molecular composition and ultrastructure of Jurassic paravian feathers.

Feathers are amongst the most complex epidermal structures known, and they have a well-documented evolutionary trajectory across non-avian dinosaurs and basal birds. Moreover, melanosome-like microbodies preserved in association with fossil plumage have been used to reconstruct original colour, behaviour and physiology. However, these putative ancient melanosomes might alternatively represent microorganismal residues, a conflicting interpretation compounded by a lack of unambiguous chemical data. We therefore used sensitive molecular imaging, supported by multiple independent analytical tests, to demonstrate that the filamentous epidermal appendages in a new specimen of the Jurassic paravian Anchiornis comprise remnant eumelanosomes and fibril-like microstructures, preserved as endogenous eumelanin and authigenic calcium phosphate. These results provide novel insights into the early evolution of feathers at the sub-cellular level, and unequivocally determine that melanosomes can be preserved in fossil feathers.

[Dynamic properties of germinal granule ping-body in the testes of Drosophila melanogaster].

Here we investigated dynamic properties of the piNG-body, large perinuclear granule that was discovered previously in spermatocytes of Drosophila. The piNG-body contains ribonucleoprotein complexes involved in piRNA-silencing of genome repeats including transposons in premeiotic spermatocytes with aid of short piRNAs. Confocal microscopy of fixed and native preparations demonstrates that the piNG-body is mobile structure which does not occupy a stationary position near nuclear surface relative to chromosomal territories. FRAP-analysis reveals a high exchange rate of RNA helicase Vasa in the piNG-body and small perinuclear granules with the cytozol Vasa pool. Disruption of microtubule assembly of cytoskeleton does not affect to stability of the piNG-body and small granules. We suppose that the combination of piNG-body mobility and permanent molecular exchange of Vasa protein provides an efficient "scanning" of total volume of the cytoplasm of primary spermatocytes and timely recognition and destruction of unwanted transcripts of the repetitive elements of genome.

The human peroxisome in health and disease: the story of an oddity becoming a vital organelle.

Since the first report by Rhodin in 1954, our knowledge on mammalian microbodies/peroxisomes has known several periods. An initial two decades period (1954-1973) has contributed to the biochemical individualisation of peroxisomes as a new class of subcellular organelles (de Duve, 1965). The corresponding research period failed to define a clear role of mammalian peroxisomes in vital functions and intermediary metabolism, explaining why feeling that peroxisomes might be in the human cell oddities has prevailed during several decades. The period standing from 1973 to nowadays has progressively removed this cell oddity view of peroxisomes by highlighting vital function and metabolic role of peroxisomes in health and disease along with genetic and metabolic regulation of peroxisomal protein content, organelle envelope formation and protein signal targeting mechanisms. Research on peroxisomes and their response to various drugs and metabolites, dietary and physiological conditions has also played a key role in the discovery of peroxisome proliferator activated receptors (PPARs) belonging to the nuclear hormone receptor superfamily and for which impact in science and medicine goes now by far beyond that of the peroxisomes.

Ultrastructural study on dynamics of lipid bodies and plastids during ripening of chili pepper fruits.

Dynamics of lipid bodies and plastids in chili pepper fruits during ripening were investigated by means of transmission electron microscopy. Mesocarp of chili pepper fruits consists of collenchyma, normal parenchyma, and huge celled parenchyma. In mature green fruits, plastids contain numerous thylakoids that are well organized into grana in collenchyma, a strikingly huge amount of starch and irregularly organized thylakoids in normal parenchyma, and simple tubes rather than thylakoids in huge celled parenchyma. These morphological features suggest that plastids are chloroplasts in collenchyma, chloroamyloplasts in normal parenchyma, proplastids in huge celled parenchyma. As fruits ripen to red, plastids in all cell types convert to chromoplasts and, concomitantly, lipid bodies accumulate in both cytoplasm and chromoplasts. Cytosolic lipid bodies are lined up in a regular layer adjacent to plasma membrane. The cytosolic lipid body consists of a core surrounded by a membrane. The core is comprised of a more electron-dense central part enclosed by a slightly less electron-dense peripheral layer. Plastidial lipid bodies in collenchyma, normal parenchyma, and endodermis initiate as plastoglobuli, which in turn convert to rod-like structures. Therefore, plastidial lipid bodies are more dynamic than cytosolic lipid bodies. Both cytosolic and plastidial lipid bodies contain rich unsaturated lipids.

The discovery of GW bodies.

Human autoantibodies were a key to the discovery of GW bodies and their integral protein, GW182. This publication marks the tenth anniversary of the discovery of GW182. As it turns out, the discovery of GW182 was quite timely because it coincided with the elucidation of the RNA interference (RNAi) pathway, which is now known to have a major role in post-transcriptional gene regulation. Following our publication of the essential features of GW182 in 2002, laboratories from around the world began investigations that led to the elucidation of the role of GW182 in RNAi and other pathways of mRNA processing and degradation. This chapter reviews the discovery of GW182 and the description of GWB and some of the observations that followed that still remain to be elucidated.

Glandular trichomes of Ceratotheca triloba (Pedaliaceae): morphology, histochemistry and ultrastructure.

This study was initiated to characterize the distribution, morphology, secretion mode, histochemistry and ultrastructure of the glandular trichomes of Ceratotheca triloba using light and electron microscopy. Its leaves bear two morphologically distinct glandular trichomes. The first type has long trichome with 8-12 basal cells of pedestal, 3-14 stalk cells, a neck cell and a head of four cells in one layer. The second type has short trichome comprising one or two basal epidermal cells, a unicellular or bicellular stalk and a multicellular head of two to eight cells. There is a marked circular area in the upper part of each head cell of the long trichome. This area is provided with micropores to exudate directly the secretory product onto the leaf surface by an eccrine pathway. The secretory product has copious amount of dark microbodies arising from plastids which are positive to Sudan tests and osmium tetroxide for unsaturated lipids. The secretion mode of short trichomes is granulocrine and involves two morphologically and histochemically distinct vesicle types: small Golgi-derived vesicles which are positive to Ruthenium Red test for mucilaginous polysaccharides; the second type is dark large microbodies similar to that of long trichomes with low quantity. These two types are stored in numerous peripheral vacuoles and discharge their contents accompanied by the formation of irregular invaginations of the plasmalemma inside the vacuoles via reverse pinocytosis. These two secretion modes of long and short trichomes are reported for the first time in the family Pedaliaceae. The long trichomes have more unsaturated lipids, while the short trichomes contain more mucilaginous polysaccharides.

Visualization of microbodies in Chlamydomonas reinhardtii.

In Chlorophycean algal cells, these organelles are generally called microbodies because they lack the enzymes found in the peroxisomes of higher plants. Microbodies in some algae contain fewer enzymes than the peroxisomes of higher plants, and some unicellular green algae in Chlorophyceae such as Chlamydomonas reinhardtii do not possess catalase, an enzyme commonly found in peroxisomes. Thus, whether microbodies in Chlorophycean algae are similar to the peroxisomes of higher plants, and whether they use a similar transport mechanism for the peroxisomal targeting signal (PTS), remain unclear. To determine whether the PTS is present in the microbodies of Chlorophycean algae, and to visualize the microbodies in Chlamydomonas cells, we examined the sub-cellular localization of green fluorescent proteins (GFP) fused to several PTS-like sequences. We detected GFP compartments that were spherical with a diameter of 0.3-1.0 µm in transgenic Chlamydomonas. Comparative analysis of the character of GFP-compartments observed by fluorescence microscopy and that of microbodies by electron microscopy indicated that the compartments were one and the same. The result also showed that the microbodies in Chlorophycean cells have a similar transport mechanism to that of peroxisomes of higher plants.

Cytoplasmic ribonucleoprotein (RNP) bodies and their relationship to GW/P bodies.

GW bodies (glycine- and tryptophan-rich cytoplasmic bodies; also known as mammalian processing (P) or Dcp-containing bodies) were described in 2002 when a human autoimmune serum was used to immunoscreen a HeLa expression library. Subsequently, many investigators have focused their attention on elucidating the components and functional relevance of this ribonucleoprotein (RNP)-containing cytoplasmic microdomain to cellular and molecular biology, developmental and pathological processes, and clinical practice. GW/P body components are now known to be involved in the post-transcriptional processing of messenger RNA (mRNA) through the RNA interference pathway, 5'-->3' mRNA degradation as well as mRNA transport and stabilization. It is currently thought that the relevant mRNA silencing and degrading factors are partitioned to these restricted cytoplasmic microdomains thus effecting post-transcriptional regulation and the prevention of accidental degradation of functional mRNA. Although much attention has focused on GW/P bodies, other cytoplasmic RNP bodies, which have highly specialized functions, interact or co-localize with components of GW/P bodies. These include neuronal transport RNP granules, stress granules, RNP-rich cytoplasmic germline granules or chromatoid bodies, sponge bodies, cytoplasmic prion protein-induced RNP granules, U bodies and TAM bodies. This review will focus on the similarities and differences of the various cytoplasmic RNP granules as an approach to understanding their functional relationships to GW/P bodies.

Matching the proteome to the genome: the microbody of penicillin-producing Penicillium chrysogenum cells.

In the filamentous fungus Penicillium chrysogenum, microbodies are essential for penicillin biosynthesis. To better understand the role of these organelles in antibiotics production, we determined the matrix enzyme contents of P. chrysogenum microbodies. Using a novel in silico approach, we first obtained a catalogue of 200 P. chrysogenum proteins with putative microbody targeting signals (PTSs). This included two orthologs of proteins involved in cephalosporin biosynthesis, which we demonstrate to be bona fide microbody matrix constituents. Subsequently, we performed a proteomics based inventory of P. chrysogenum microbody matrix proteins using nano-LC-MS/MS analysis. We identified 89 microbody proteins, 79 with a PTS, including the two known microbody-borne penicillin biosynthesis enzymes, isopenicillin N:acyl CoA acyltransferase and phenylacetyl-CoA ligase. Comparative analysis revealed that 69 out of 79 PTS proteins identified experimentally were in the reference list. A prominent microbody protein was identified as a novel fumarate reductase-cytochrome b5 fusion protein, which contains an internal PTS2 between the two functional domains. We show that this protein indeed localizes to P. chrysogenum microbodies.

Platelet structural pathology in a patient with the X-linked GATA-1, R216Q mutation.

The ultrastructural pathology of GATA-1, V205M and G208S macrothrombocytes was discussed in earlier investigations. This study has used the same technology to evaluate macrothrombocytes from a patient with the GATA-1, R216Q mutation. Some of the pathological features observed in macrothrombocytes from patients with the V205M and G208S variations including hypo- and agranular platelets, tubular inclusions and platelets within platelets, as well as platelets within platelets within platelets were identified. However, tubular membrane sheets in megakaryocytes and platelets of the V205M and G208S types and large groups of platelets attached to platelets to form megathrombocytes were not observed. The unique pathology of the megathrombocytes from this patient was the near absence of dense bodies in his giant cells. Storage Pool Deficiency, together with large platelets, defective adhesion and aggregation of his macrocytes under shear stress to vWF and collagen and defective clot retraction may contribute to the pathogenesis of his bleeding disorder.

Differential expression of glycosomal and mitochondrial proteins in the two major life-cycle stages of Trypanosoma brucei.

Label-free semi-quantitative differential three-dimensional liquid chromatography coupled to mass spectrometry (3D-LC-MS/MS) was used to compare the glycosomal and mitochondrial proteomes of the bloodstream- and insect-form of Trypanosoma brucei. The abundance of glycosomal marker proteins identified in the two life-cycle stages corresponded well with the relative importance of biochemical pathways present in the glycosomes of the two stages and the peptide spectral count ratios of selected enzymes were in good agreement with published data about their enzymatic specific activities. This approach proved extremely useful for the generation of large scale proteomics data for the comparison of different life-cycle stages. Several proteins involved in oxidative stress protection, sugar-nucleotide synthesis, purine salvage, nucleotide-monophosphate formation and purine-nucleotide cycle were identified as glycosomal proteins.

Close association of centrosomes to the distal ends of the microbody during its growth, division and partitioning in the green alga Klebsormidium flaccidum.

Division and partitioning of microbodies (peroxisomes) of the green alga Klebsormidium flaccidum, whose cells contain a single microbody, were investigated by electron microscopy. In interphase, the rod-shaped microbody is present between the nucleus and the single chloroplast, oriented perpendicular to the pole-to-pole direction of the future spindle. A centriole pair associates with one distal end of the microbody. In prophase, the microbody changes not only in shape, from a rodlike to a branched form, but also in orientation, from perpendicular to parallel to the future pole-to-pole direction. Duplicated centriole pairs are localized in close proximity to both distal ends of the microbody. In metaphase, the elongated microbody flanks the open spindle, with both distal ends close to the centriole pair at either spindle pole. The microbody further elongates in telophase and divides after septum formation (cytokinesis) has started. The association between the centrioles and both distal ends of the microbody is maintained throughout mitosis, resulting in the distal ends of the elongated microbody being fixed at the cellular poles. This configuration of the microbody may be favorable for faithful transmission of the organelle during cell division. After cytokinesis is completed, the microbody reverts to the perpendicular orientation by changing its shape. Microtubules radiating from the centrosomes flank the side of the microbody throughout mitosis. The close association of centrosomes and microtubules with the microbody is discussed in respect to the partitioning of the microbody in this alga.

The peroxin PEX14 of Neurospora crassa is essential for the biogenesis of both glyoxysomes and Woronin bodies.

In the filamentous fungus Neurospora crassa, glyoxysomes and Woronin bodies coexist in the same cell. Because several glyoxysomal matrix proteins and also HEX1, the dominant protein of Woronin bodies, possess typical peroxisomal targeting signals, the question arises as to how protein targeting to these distinct yet related types of microbodies is achieved. Here we analyzed the function of the Neurospora ortholog of PEX14, an essential component of the peroxisomal import machinery. PEX14 interacted with both targeting signal receptors and was localized to glyoxysomes but was virtually absent from Woronin bodies. Nonetheless, a pex14Delta mutant not only failed to grow on fatty acids because of a defect in glyoxysomal beta-oxidation but also suffered from cytoplasmic bleeding, indicative of a defect in Woronin body-dependent septal pore plugging. Inspection of pex14Delta mutant hyphae by fluorescence and electron microscopy indeed revealed the absence of Woronin bodies. When these cells were subjected to subcellular fractionation, HEX1 was completely mislocalized to the cytosol. Expression of GFP-HEX1 in wild-type mycelia caused the staining of Woronin bodies and also of glyoxysomes in a targeting signal-dependent manner. Our data support the view that Woronin bodies emerge from glyoxysomes through import of HEX1 and subsequent fission.

Ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-bodies and stress granules.

Tight control of translation is fundamental for eukaryotic cells, and deregulation of proteins implicated contributes to numerous human diseases. The neurodegenerative disorder spinocerebellar ataxia type 2 is caused by a trinucleotide expansion in the SCA2 gene encoding a lengthened polyglutamine stretch in the gene product ataxin-2, which seems to be implicated in cellular RNA-processing pathways and translational regulation. Here, we substantiate a function of ataxin-2 in such pathways by demonstrating that ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6, a component of P-bodies and stress granules, representing cellular structures of mRNA triage. We discovered that altered ataxin-2 levels interfere with the assembly of stress granules and cellular P-body structures. Moreover, ataxin-2 regulates the intracellular concentration of its interaction partner, the poly(A)-binding protein, another stress granule component and a key factor for translational control. Thus, our data imply that the cellular ataxin-2 concentration is important for the assembly of stress granules and P-bodies, which are main compartments for regulating and controlling mRNA degradation, stability, and translation.

Characterization of the role of the receptors PEX5 and PEX7 in the import of proteins into glycosomes of Trypanosoma brucei.

Peroxins 5 and 7 are receptors for protein import into the peroxisomal matrix. We studied the involvement of these peroxins in the biogenesis of glycosomes in the protozoan parasite Trypanosoma brucei. Glycosomes are peroxisome-like organelles in which a major part of the glycolytic pathway is sequestered. We here report the characterization of the T. brucei homologue of PEX7 and provide several data strongly suggesting that it can bind to PEX5. Depletion of PEX5 or PEX7 by RNA interference had a severe effect on the growth of both the bloodstream-form of the parasite, that relies entirely on glycolysis for its ATP supply, and the procyclic form representative of the parasite living in the tsetse-fly midgut and in which also other metabolic pathways play a prominent role. The role of the two receptors in import of glycosomal matrix proteins with different types of peroxisome/glycosome-targeting signals (PTS) was analyzed by immunofluorescence and subcellular fractionation studies. Knocking down the expression of either receptor gene resulted, in procyclic cells, in the mislocalization of proteins with both a type 1 or 2 targeting motif (PTS1, PTS2) located at the C- and N-termini, respectively, and proteins with a sequence-internal signal (I-PTS) to the cytosol. Electron microscopy confirmed the apparent integrity of glycosomes in these procyclic cells. In bloodstream-form trypanosomes, PEX7 depletion seemed to affect only the subcellular distribution of PTS2-proteins. Western blot analysis suggested that, in both life-cycle stages of the trypanosome, the levels of both receptors are controlled in a coordinated fashion, by a mechanism that remains to be determined. The observation that both PEX5 and PEX7 are essential for the viability of the parasite indicates that the respective branches of the glycosome-import pathway in which each receptor acts might be interesting drug targets.

Overproduction of a single protein, Pc-Pex11p, results in 2-fold enhanced penicillin production by Penicillium chrysogenum.

Current industrial production of beta-lactam antibiotics, using the filamentous fungus Penicillium chrysogenum, is the result of many years of strain improvement by classical mutagenesis. More efficient production strains showed significant increases in the number and volume fraction of microbodies in their cells, organelles that harbor key enzymes involved in the biosynthesis of beta-lactam antibiotics. We have isolated the P. chrysogenum cDNA encoding Pc-Pex11p, a peroxin that is involved in microbody abundance. We demonstrate that overproduction of Pc-Pex11p in P. chrysogenum results in massive proliferation of tubular-shaped microbodies and a 2- to 2.5-fold increase in the level of penicillin in the culture medium. Notably, Pc-Pex11p-overproduction did not affect the levels of the enzymes of the penicillin biosynthetic pathway. Our results suggest that the stimulating effect of enhanced organelle numbers may reflect an increase in the fluxes of penicillin and/or its precursors across the now much enlarged microbody membrane.

The glycosome membrane of Trypanosoma cruzi epimastigotes: protein and lipid composition.

Highly purified glycosomes from Trypanosoma cruzi epimastigotes were obtained by differential centrifugation and isopycnic ultracentrifugation. Glycosomal membranes, produced by carbonate treatment of purified glycosomes, exhibited about eight main protein bands and eight minor ones. Essentially the same protein pattern was observed in the detergent-rich fraction of a Triton X-114 fractionation of whole glycosomes, indicating that most of the membrane-bound polypeptides were highly hydrophobic. The orientation of these proteins was studied by in situ labelling followed by limited pronase hydrolysis of intact glycosomes. Three glycosome membrane proteins were characterized as peripheral by comparing the protein bands patterns of membrane fractions obtained by different treatments. Noteworthy membrane polypeptides were: (1) a peripheral 75k Da membrane protein, oriented towards the cytosol, which was the most abundant glycosomal membrane protein in exponentially growing epimastigotes but was essentially absent in stationary phase cells; (2) a pair of integral membrane proteins with molecular masses in the range of 85-100 kDa, which were only present in stationary phase cells; (3) a heme-containing 36k Da protein, strongly associated to the membrane, present in both growth phases; (4) a very immunogenic 41k Da integral membrane polypeptide, oriented towards the cytosol. The lipid composition of the glycosomal membranes was also investigated. The distribution of phospholipid species in glycosomes and glycosomal membranes was very similar to that of whole cells, with phosphatidyl-ethanolamine, phosphatidyl-choline, and phosphatidyl-serine as main components and smaller proportions of sphingomyelin and with phosphatidyl-inositol. On the other hand, glycosomes were enriched in endogenous sterols (ergosterol, 24-ethyl-5,7,22-cholesta-trien-3beta-ol), and precursors, when compared with whole cells, a finding consistent with the proposal that these organelles are involved in the de novo biosynthesis of sterols in trypanosomatids.