Endocytosis by African trypanosomes. I. Three-dimensional structure of the endocytic organelles in Trypanosoma brucei and T. congolense

African trypanosomes multiply rapidly during the course of infection obtaining nutrients from the host blood and other body fluids. The organelles involved in endocytosis were revealed ultrastructurally using horseradish peroxidase (HRP) and colloidal gold coupled to bovine transferrin (Au-Tf) or bovine serum albumin (Au-BSA). At 0 degree C the markers bound to the cell surface and neither entered the flagellar pocket nor were internalized. Upon warming to 37 degrees C, the markers were found in the flagellar pocket and appeared to enter all the intracellular endocytic organelles within 5 min. Serial sectioning of resin-embedded cells was employed to obtain pseudo three-dimensional views of these organelles. The organelles involved were of three types: (1) small vesicles and cisternae (20-25 nm in diameter), (2) large tubular networks (200 nm diameter) similar to endosomes of mammalian cells, and (3) large lysosome-like vesicles. These organelles were located between the flagellar pocket and the nucleus and were also associated with one face of the Golgi apparatus. In pulse-chase experiments HRP was not detected in intracellular organelles after 410 min but Au-Tf was seen in residual bodies. No exocytosis of Au-Tf from the flagellar pocket was observed. The data suggests that the processes of endocytosis in these parasitic protozoa may be similar to the endocytic processes found in mammalian cells.     

Endocytosis of a cytotoxic human high density lipoprotein results in disruption of acidic intracellular vesicles and subsequent killing of African trypanosomes

The host range of Trypanosoma brucei brucei is restricted by the cytolytic effects of human serum high-density lipoprotein (HDL). The lytic activity is caused by a minor subclass of human serum HDL called trypanosome lytic factor (TLF). TLF binds in the flagellar pocket to specific TLF-binding sites. Internalization and localization of TLF to a population of endocytic vesicles, and ultimately large lysosome-like vesicles, precedes lysis of T. b. brucei. The membranes of these large vesicles are disrupted by the accumulation of TLF particles. Inhibitor studies with lysosomotropic amines have shown these large vesicles to be acidic in nature and that prevention of their rupture spares the cells from TLF-mediated lysis. Furthermore, leupeptin inhibition suggests that a thioprotease may be involved in the mechanism of TLF- mediated lysis of T. b. brucei. Based on these results, we propose a lytic mechanism involving cell surface binding, endocytosis and lysosomal targeting. This is followed by lysosomal disruption and subsequent autodigestion of the cell.     

Endocytosis and intracellular processing of BODIPY-sphingomyelin by murine CATH.a neurons

Neuronal sphingolipids (SL) play important roles during axonal extension, neurotrophic receptor signaling and neurotransmitter release. Many of these signaling pathways depend on the presence of specialized membrane microdomains termed lipid rafts. Sphingomyelin (SM), one of the main raft constituents, can be formed de novo or supplied from exogenous sources. The present study aimed to characterize fluorescently-labeled SL turnover in a murine neuronal cell line (CATH.a). Our results demonstrate that at 4 °C exogenously added BODIPY-SM accumulates exclusively at the plasma membrane. Treatment of cells with bacterial sphingomyelinase (SMase) and back-exchange experiments revealed that 55–67% of BODIPY-SM resides in the outer leaflet of the plasma membrane. Endocytosis of BODIPY-SM occurs via caveolae with part of internalized BODIPY-fluorescence ending up in the Golgi and the ER. Following endocytosis BODIPY-SM undergoes hydrolysis, a reaction substantially faster than BODIPY-SM synthesis from BODIPY-ceramide. RNAi demonstrated that both, acid (a)SMase and neutral (n)SMases contribute to BODIPY-SM hydrolysis. Finally, high-density lipoprotein (HDL)-associated BODIPY-SM was efficiently taken up by CATH.a cells. Our findings indicate that endocytosis of exogenous SM occurs almost exclusively via caveolin-dependent pathways, that both, a- and nSMases equally contribute to neuronal SM turnover and that HDL-like particles might represent physiological SM carriers/donors in the brain.     

Flow sorting from organ material by intracellular markers.

BACKGROUND: Fluorescence-activated cell sorting (FACS) is an attractive technique for gene or protein expression studies in rare cell populations. For cell types where specific surface markers are not known, intracellular markers can be used. However, this approach is currently held to be difficult, as the required fixation and permeabilization may cause protein modification and RNA degradation. METHODS AND RESULTS: Using the rat thyroid gland as model, rare (parafollicular) and frequent (follicular) endocrine cell types were sorted based on immunostaining for intracellular calcitonin peptide and thyroglobulin protein expression. The sorted cells were compatible with Western blot analysis of proteins, immunoassay detection of calcitonin peptide hormone and RT-PCR. CONCLUSION: We developed a robust FACS protocol that allows flow sorting of rare cells from dissociated organ material, based on intracellular markers. Our FACS protocol is compatible with downstream analysis of proteins, peptides, and mRNA in the sorted cells.     

Endocytosis and sorting of glycosphingolipids in sphingolipid storage disease

Recent studies on the endocytic itinerary of glycosphingolipids (GSLs) in sphingolipid storage disease (SLSD) fibroblasts have yielded new insights into the mechanisms underlying the endocytosis and intracellular sorting of lipids in normal and disease cells. Here we highlight new data on clathrin-independent endocytosis of GSLs, the involvement of sphingolipid–cholesterol interactions in perturbation of endocytic trafficking, and potential roles for rab proteins in regulation of GSL transport in SLSDs.     

An update on antigenic variation in African trypanosomes.

African trypanosomes can spend a long time in the blood of their mammalian host, where they are exposed to the immune system and are thought to take advantage of it to modulate their own numbers. Their major immunogenic protein is the variant surface glycoprotein (VSG), the gene for which must be in one of the 20--40 specialized telomeric expression sites in order to be transcribed. Trypanosomes escape antibody-mediated destruction through periodic changes of the expressed VSG gene from a repertoire of approximately 1000. How do trypanosomes exclusively express only one VSG and how do they switch between them?     

Uptake and intracellular transport of RNA aptamers in African trypanosomes suggest therapeutic "piggy-back" approach

African trypanosomes are protozoan organisms that multiply as extracellular parasites in the blood of humans and other mammals. The parasites escape destruction by the host immune system by periodically changing their glycoprotein surface coat. This phenomenon is known as antigenic variation and is responsible for the inability of the infected host to clear the infection. Previously we reported the selection of RNA aptamers that bind to a 42 kDa surface protein of Trypanosoma brucei. The polypeptide is localised within a specific substructure on the parasite surface, the so-called flagellar pocket. Here we analyse the fate of the aptamers upon binding to the flagellar pocket. At elevated temperatures, both terminal ends of the RNAs are degraded to form a stable core structure of approximately 50 nucleotides. The RNAs become rapidly internalised by endocytosis and are transported to the lysosome by vesicular transport. The endocytotic process is sequence specific and does not occur with randomised RNA sequences or significantly shortened aptamer fragments. Co-localisation experiments with transferrin suggest a receptor-mediated uptake. The identified internalisation and transport pathway was used to target aptamer-coupled biotin molecules to the lysosome. This demonstrates that the RNAs can be used as 'piggy-back' molecules to target aptamer-coupled compounds/toxins to the lysosomal compartment of the parasite.     

Regulation of protein trafficking by glycosylphosphatidylinositol valence in African trypanosomes

The structure, biosynthesis, and attachment of glycosylphosphatidylinositol (GPI) anchors were all first determined for the variant surface glycoprotein (VSG) of African trypanosomes, and all of the basic aspects of this work have been shown to be universal in eukaryotic organisms. However, the role of GPI anchors in protein trafficking within trypanosomes has lagged behind the more standard eukaryotic model systems such as yeast and polarized epithelial cells. Trypanosomes are also highly polarized cells in which all endocytosis and exocytosis intersect at a discrete domain of the plasma membrane, the flagellar pocket. Within these convergent pathways trafficking of GPI anchored proteins correlates strongly with valence: homodimeric VSG with two GPIs is stably incorporated into the cell surface coat, heterodimeric transferrin receptor with a single GPI is found in the flagellar pocket and is slowly delivered to the lysosome for degradation, and recombinant GPI minus VSG reporters are rapidly degraded in the lysosome. Here we summarize recent data confirming this correlation using a tool kit of recombinant GPI anchored reporters, including a reporter designed to be conditionally modulated between a GPI valence of one and two.     

Surface coats and secretory trafficking in African trypanosomes

Recent advances in transfection technology have been exploited to address fundamental questions relating to secretory trafficking in African trypanosomes. Targeted gene disruptions and ectopic expression of the major stage-specific surface proteins have provided unexpected insights into both the function and assembly of the essential parasite surface coats. A growing list of novel secretory cargo molecules, as well as advances in the characterization of trypanosomal secretory machinery, provide a unique model system for the study of eukaryotic secretory processes.     

Regulation and function of polyamines in African trypanosomes

The polyamine biosynthetic pathway is an important drug target for the treatment of human African trypanosomiasis (HAT), raising interest in understanding polyamine function and their mechanism of regulation. Polyamine levels are tightly controlled in mammalian cells, but similar regulatory mechanisms appear absent in trypanosomes. Instead trypanosomatid S-adenosylmethionine decarboxylase (AdoMetDC), which catalyzes a key step in the biosynthesis of the polyamine spermidine, is activated by dimerization with an inducible protein termed prozyme. Prozyme is an inactive paralog of the active AdoMetDC enzyme that evolved by gene duplication and is found only in the trypanosomatids. In Trypanosoma brucei, AdoMetDC activity appears to be controlled by regulation of prozyme protein levels, potentially at the translational level.     

The biochemical basis of arsenical-diamidine crossresistance in African trypanosomes.

Resistance to currently used drugs is a serious problem in most fields of antimicrobial chemotherapy. Crossresistance between two of the major classes of drug used in the treatment of African trypanosomiasis, the melaminophenyl arsenicals and diamidines is easily selected in the laboratory. Here, Mike Barrett and Alan Fairlamb outline the mechanism underlying this crossresistance, which appears to arise as a result of alterations in an unusual adenosine transporter involved in the uptake of these drugs.     

Microhomology-mediated deletion and gene conversion in African trypanosomes

Antigenic variation in African trypanosomes is induced by DNA double-strand breaks (DSBs). In these protozoan parasites, DSB repair (DSBR) is dominated by homologous recombination (HR) and microhomology-mediated end joining (MMEJ), while non-homologous end joining (NHEJ) has not been reported. To facilitate the analysis of chromosomal end-joining, we established a system whereby inter-allelic repair by HR is lethal due to loss of an essential gene. Analysis of intrachromosomal end joining in individual DSBR survivors exclusively revealed MMEJ-based deletions but no NHEJ. A survey of microhomologies typically revealed sequences of between 5 and 20 bp in length with several mismatches tolerated in longer stretches. Mean deletions were of 54 bp on the side closest to the break and 284 bp in total. Break proximity, microhomology length and GC-content all favored repair and the pattern of MMEJ described above was similar at several different loci across the genome. We also identified interchromosomal gene conversion involving HR and MMEJ at different ends of a duplicated sequence. While MMEJ-based deletions were RAD51-independent, one-sided MMEJ was RAD51 dependent. Thus, we describe the features of MMEJ in Trypanosoma brucei, which is analogous to micro single-strand annealing; and RAD51 dependent, one-sided MMEJ. We discuss the contribution of MMEJ pathways to genome evolution, subtelomere recombination and antigenic variation.     

Endocytosis and intracellular trafficking contribute to necrotic neurodegeneration in C. elegans

Unlike apoptosis, necrotic cell death is characterized by marked loss of plasma membrane integrity. Leakage of cytoplasmic material to the extracellular space contributes to cell demise, and is the cause of acute inflammatory responses, which typically accompany necrosis. The mechanisms underlying plasma membrane damage during necrotic cell death are not well understood. We report that endocytosis is critically required for the execution of necrosis. Depletion of the key endocytic machinery components dynamin, synaptotagmin and endophilin suppresses necrotic neurodegeneration induced by diverse genetic and environmental insults in C. elegans. We used genetically encoded fluorescent markers to monitor the formation and fate of specific types of endosomes during cell death in vivo. Strikingly, we find that the number of early and recycling endosomes increases sharply and transiently upon initiation of necrosis. Endosomes subsequently coalesce around the nucleus and disintegrate during the final stage of necrosis. Interfering with kinesin-mediated endosome trafficking impedes cell death. Endocytosis synergizes with autophagy and lysosomal proteolytic mechanisms to facilitate necrotic neurodegeneration. These findings demonstrate a prominent role for endocytosis in cellular destruction during neurodegeneration, which is likely conserved in metazoans.     

Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells

Platelet-derived microparticles (PMP) bind and modify the phenotype of many cell types including endothelial cells. Recently, we showed that PMP were internalized by human brain endothelial cells (HBEC). Here we intend to better characterize the internalization mechanisms of PMP and their intracellular fate. Confocal microscopy analysis of PKH67-labelled PMP distribution in HBEC showed PMP in early endosome antigen 1 positive endosomes and in LysoTracker-labelled lysosomes, confirming a role for endocytosis in PMP internalization. No fusion of calcein-loaded PMP with HBEC membranes was observed. Quantification of PMP endocytosis using flow cytometry revealed that it was partially inhibited by trypsin digestion of PMP surface proteins and by extracellular Ca(2+) chelation by EDTA, suggesting a partial role for receptor-mediated endocytosis in PMP uptake. This endocytosis was independent of endothelial receptors such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and was not increased by tumour necrosis factor stimulation of HBEC. Platelet-derived microparticle internalization was dramatically increased in the presence of decomplemented serum, suggesting a role for PMP opsonin-dependent phagocytosis. Platelet-derived microparticle uptake was greatly diminished by treatment of HBEC with cytochalasin D, an inhibitor of microfilament formation required for both phagocytosis and macropinocytosis, with methyl-β-cyclodextrin that depletes membrane cholesterol needed for macropinocytosis and with amiloride that inhibits the Na(+)/H(+) exchanger involved in macropinocytosis. In conclusion, PMP are taken up by active endocytosis in HBEC, involving mechanisms consistent with both phagocytosis and macropinocytosis. These findings identify new processes by which PMP could modify endothelial cell phenotype and functions.     

Snapshots of the RNA editing machine in trypanosomes captured at different assembly stages in vivo

Mitochondrial pre‐messenger RNAs in kinetoplastid protozoa are substrates of uridylate‐specific RNA editing. RNA editing converts non‐functional pre‐mRNAs into translatable molecules and can generate protein diversity by alternative editing. Although several editing complexes have been described, their structure and relationship is unknown. Here, we report the isolation of functionally active RNA editing complexes by a multistep purification procedure. We show that the endogenous isolates contain two subpopulations of ～20S and ～35–40S and present the three‐dimensional structures of both complexes by electron microscopy. The ～35–40S complexes consist of a platform density packed against a semispherical element. The ～20S complexes are composed of two subdomains connected by an interface. The two particles are structurally related, and we show that RNA binding is a main determinant for the interconversion of the two complexes. The ～20S editosomes contain an RNA‐binding site, which binds gRNA, pre‐mRNA and gRNA/pre‐mRNA hybrid molecules with nanomolar affinity. Variability analysis indicates that subsets of complexes lack or possess additional domains, suggesting binding sites for components. Together, a picture of the RNA editing machinery is provided.     

Experimental infections of farmed eels with different Trypanosoma granulosum life-cycle stages and investigation of pleomorphism

Trypanosoma granulosum, a flagellate protozoon commonly found in the blood of the European eel Anguilla anguilla, was injected experimentally into uninfected eels purchased from a local farm. In order to investigate the infectivity of different stages in the life cycle, trypanosomes from various sources were used for inoculation. Infectivity was greatly reduced in in vitro culture stages inoculated at 20 C. Isolated bloodstream stages injected into groups of animals held at 12 and 20 C could be detected for over 70 days but did not appear to multiply. Naturally infected Hemiclepsis marginata, a piscivorous leech known to serve as vector, produced detectable, single-peak infections in eels held at 20 C. Infections were characterized by a prepatent stage and a phase of rising parasitemia. Peak infection intensities ranged between 1 and 7 x 10(4) trypanosomes/ml. Trypanosomes in the bloodstream of eels experimentally infected with leeches, divided at a very low rate during the early stages of infection. Small morphs present during the early phase of rising parasitemia were gradually replaced by larger trypanosomes. The overall length frequency distribution of trypanosomes was unimodal.     

Occurrence and biosynthesis of catalase at different stages of seed maturation

It was to be shown whether during the biogenesis of microbodies some of their components were already present in the cell prior to the organelle's assembly. To this end, the occurrence and properties of catalase in soluble and particular fractions of ripening cucumber seeds were examined. Homogenates of seeds from ripening fruits were fractionated by isopycnic density gradient centrifugation, and thus catalase was found in three different fractions: as a soluble enzyme in the gradient supernatant, as a membrane fraction at density d=1.18 kg l^-1, and in association with microbodies. In the early steps of seed formation, catalase was detected at density d=1.18 kg l^-1 and in the gradient supernatant. At a later stage of seed maturation, however, catalase was primarily associated with microbodies which exhibited an equilibrium density of d=1.23 kg l^-1. M _r as well as subunit M _r of catalase were determined, and their close immunological relationship to leaf peroxisomal catalase and glyoxysomal catalase was demonstrated. Biosynthesis of catalase at different stages of seed maturation was investigated by in vivo labeling with l-[³⁵S]methionine, l-[¹⁴C]leucine and -[³H]aminolaevulinic acid. Electrophoretic analysis of de novo synthesized catalase subunits revealed the occurrence of a heavy form (M _r 57,500) in the soluble fraction; this form was preferentially labeled. A light form, M _r 53,500, was detected in microbodies and also in the soluble fraction. The findings lend support to the hypothesis that the rate of catalase synthesis is highest in an early stage of seed formation, when globulins have already been formed, but before de novo synthesis of malate synthase has commenced. Prior to microbody assembling, a cytoplasmic pool of catalase was labeled.Abbreviations EDTA Na₂-ethylenediaminotetraacetate - Hepes 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - M _r molecular weight