The single mitochondrion of the kinetoplastid parasite Crithidia fasciculata is a dynamic network.

Mitochondria are central organelles in cellular metabolism. Their structure is highly dynamic, allowing them to adapt to different energy requirements, to be partitioned during cell division, and to maintain functionality. Mitochondrial dynamics, including membrane fusion and fission reactions, are well studied in yeast and mammals but it is not known if these processes are conserved throughout eukaryotic evolution. Kinetoplastid parasites are some of the earliest-diverging eukaryotes to retain a mitochondrion. Each cell has only a single mitochondrial organelle, making them an interesting model for the role of dynamics in controlling mitochondrial architecture. We have investigated the mitochondrial division cycle in the kinetoplastid Crithidia fasciculata. The majority of mitochondrial biogenesis occurs during the G1 phase of the cell cycle, and the mitochondrion is divided symmetrically in a process coincident with cytokinesis. Live cell imaging revealed that the mitochondrion is highly dynamic, with frequent changes in the topology of the branched network. These remodeling reactions include tubule fission, fusion, and sliding, as well as new tubule formation. We hypothesize that the function of this dynamic remodeling is to homogenize mitochondrial contents and to facilitate rapid transport of mitochondria-encoded gene products from the area containing the mitochondrial nucleoid to other parts of the organelle.

The effect of tunicamycin on the glucose uptake, growth, and cellular adhesion in the protozoan parasite Crithidia fasciculata.

Crithidia fasciculata represents a very interesting model organism to study biochemical, cellular, and genetic processes unique to members of the family of the Trypanosomatidae. Thus, C. fasciculata parasitizes several species of insects and has been widely used to test new therapeutic strategies against parasitic infections. By using tunicamycin, a potent inhibitor of glycosylation in asparaginyl residues of glycoproteins (N-glycosylation), we demonstrate that N-glycosylation in C. fasciculata cells is involved in modulating glucose uptake, dramatically impacting growth, and cell adhesion. C. fasciculata treated with tunicamycin was severely affected in their ability to replicate and to adhere to polystyrene substrates and losing their ability to aggregate into small and large groups. Moreover, under tunicamycin treatment, the parasites were considerably shorter and rounder and displayed alterations in cytoplasmic vesicles formation. Furthermore, glucose uptake was significantly impaired in a tunicamycin dose-dependent manner; however, no cytotoxic effect was observed. Interestingly, this effect was reversible. Thus, when tunicamycin was removed from the culture media, the parasites recovered its growth rate, cell adhesion properties, and glucose uptake. Collectively, these results suggest that changes in the tunicamycin-dependent glycosylation levels can influence glucose uptake, cell growth, and adhesion in the protozoan parasite C. fasciculata.

Detailed interrogation of trypanosome cell biology via differential organelle staining and automated image analysis.

BACKGROUND: Many trypanosomatid protozoa are important human or animal pathogens. The well defined morphology and precisely choreographed division of trypanosomatid cells makes morphological analysis a powerful tool for analyzing the effect of mutations, chemical insults and changes between lifecycle stages. High-throughput image analysis of micrographs has the potential to accelerate collection of quantitative morphological data. Trypanosomatid cells have two large DNA-containing organelles, the kinetoplast (mitochondrial DNA) and nucleus, which provide useful markers for morphometric analysis; however they need to be accurately identified and often lie in close proximity. This presents a technical challenge. Accurate identification and quantitation of the DNA content of these organelles is a central requirement of any automated analysis method. RESULTS: We have developed a technique based on double staining of the DNA with a minor groove binding (4'', 6-diamidino-2-phenylindole (DAPI)) and a base pair intercalating (propidium iodide (PI) or SYBR green) fluorescent stain and color deconvolution. This allows the identification of kinetoplast and nuclear DNA in the micrograph based on whether the organelle has DNA with a more A-T or G-C rich composition. Following unambiguous identification of the kinetoplasts and nuclei the resulting images are amenable to quantitative automated analysis of kinetoplast and nucleus number and DNA content. On this foundation we have developed a demonstrative analysis tool capable of measuring kinetoplast and nucleus DNA content, size and position and cell body shape, length and width automatically. CONCLUSIONS: Our approach to DNA staining and automated quantitative analysis of trypanosomatid morphology accelerated analysis of trypanosomatid protozoa. We have validated this approach using Leishmania mexicana, Crithidia fasciculata and wild-type and mutant Trypanosoma brucei. Automated analysis of T. brucei morphology was of comparable quality to manual analysis while being faster and less susceptible to experimentalist bias. The complete data set from each cell and all analysis parameters used can be recorded ensuring repeatability and allowing complete data archiving and reanalysis.

Flagellar and ciliary beating in trypanosome motility.

The single flagellum of Leishmania and Trypanosoma parasites is becoming an increasingly attractive model for the analysis of flagellar function-driven largely by the abundance of genomic and proteomic information available for the organelle, the genetic manipulability of the organisms and the importance of motility for the parasite lifecycle. However, as yet, there is a paucity of published data on the beating of any genetically malleable trypanosomatid species. Here we undertook an in-depth analysis using high-speed videomicroscopy of the beating of free-swimming Leishmania major cells in comparison to Crithidia species (for which there is some existing literature). In so doing, we describe a simple and generally-applicable technique to facilitate the quantitative analysis of free-swimming cells. Our analysis thoroughly defines the parameters of the expected tip-to-base symmetrical flagellar beat in these species. It also describes beat initiation from points other than the flagellum tip and a completely different, base-to-tip highly-asymmetric beat that represents a ciliary beat of trypanosomatid flagella. Moreover, detailed analysis of parameter interrelationships revealed an unexpected dependency of wavelength on oscillator length that may be the result of reversible constraint of doublet sliding at the tip or resonance of the flagellar beat.

Cell cycle-dependent localization and properties of a second mitochondrial DNA ligase in Crithidia fasciculata.

The mitochondrial DNA in kinetoplastid protozoa is contained in a single highly condensed structure consisting of thousands of minicircles and approximately 25 maxicircles. The disk-shaped structure is termed kinetoplast DNA (kDNA) and is located in the mitochondrial matrix near the basal body. We have previously identified a mitochondrial DNA ligase (LIG kbeta) in the trypanosomatid Crithidia fasciculata that localizes to antipodal sites flanking the kDNA disk where several other replication proteins are localized. We describe here a second mitochondrial DNA ligase (LIG kalpha). LIG kalpha localizes to the kinetoplast primarily in cells that have completed mitosis and contain either a dividing kinetoplast or two newly divided kinetoplasts. Essentially all dividing or newly divided kinetoplasts show localization of LIG kalpha. The ligase is present on both faces of the kDNA disk and at a high level in the kinetoflagellar zone of the mitochondrial matrix. Cells containing a single nucleus show localization of the LIG kalpha to the kDNA but at a much lower frequency. The mRNA level of LIG kalpha varies during the cell cycle out of phase with that of LIG kbeta. LIG kalpha transcript levels are maximal during the phase when cells contain two nuclei, whereas LIG kbeta transcript levels are maximal during S phase. The LIG kalpha protein decays with a half-life of 100 min in the absence of protein synthesis. The periodic expression of the LIG kalpha transcript and the instability of the LIG kalpha protein suggest a possible role of the ligase in regulating minicircle replication.

Presence of a poly(A) binding protein and two proteins with cell cycle-dependent phosphorylation in Crithidia fasciculata mRNA cycling sequence binding protein II.

Crithidia fasciculata cycling sequence binding proteins (CSBP) have been shown to bind with high specificity to sequence elements present in several mRNAs that accumulate periodically during the cell cycle. The first described CSBP has subunits of 35.6 (CSBPA) and 42 kDa (CSBPB). A second distinct binding protein termed CSBP II has been purified from CSBPA null mutant cells, lacking both CSBPA and CSBPB proteins, and contains three major polypeptides with predicted molecular masses of 63, 44.5, and 33 kDa. Polypeptides of identical size were radiolabeled in UV cross-linking assays performed with purified CSBP II and 32P-labeled RNA probes containing six copies of the cycling sequence. The CSBP II binding activity was found to cycle in parallel with target mRNA levels during progression through the cell cycle. We have cloned genes encoding these three CSBP II proteins, termed RBP63, RBP45, and RBP33, and characterized their binding properties. The RBP63 protein is a member of the poly(A) binding protein family. Homologs of RBP45 and RBP33 proteins were found only among the kinetoplastids. Both RBP45 and RBP33 proteins and their homologs have a conserved carboxy-terminal half that contains a PSP1-like domain. All three CSBP II proteins show specificity for binding the wild-type cycling sequence in vitro. RBP45 and RBP33 are phosphoproteins, and RBP45 has been found to bind in vivo specifically to target mRNA containing cycling sequences. The levels of phosphorylation of both RBP45 and RBP33 were found to cycle during the cell cycle.

Mitochondrial histone-like DNA-binding proteins are essential for normal cell growth and mitochondrial function in Crithidia fasciculata.

The Crithidia fasciculata KAP2 and KAP3 proteins are closely related kinetoplast-specific histone-like DNA-binding proteins. The KAP2 and KAP3 genes are 46% identical and are arranged in tandem on the chromosomal DNA. Disruption of both alleles of either gene alone shows no detectable phenotype. However, replacement of both copies of the sequence encoding the entire KAP2 and KAP3 locus increases maxicircle mRNA levels two- to fourfold. These double-knockout cells are viable but grow extremely slowly, have reduced respiration and very abnormal cell morphologies, and accumulate numerous large vacuoles. The extreme phenotype of these mutant cells suggests an important role for the KAP2 and KAP3 proteins in mitochondrial metabolism and cell growth.

Localization of riboproteins in a trypanosomatid mitochondrion.

There is growing evidence in support of mitochondrial translation in trypanosomes but mitoribosomes have never been characterized or localized in these parasites. On RNA-protein blots we identified several proteins from the trypanosomatid Crithidia fasciculata which bound the parasite's 12S and 9S mitochondrial ribosomal RNAs. Two of these proteins had significant amino acid sequence homology to riboproteins S8 and S21 across phyla. Immunoelectron microscopy revealed that antibodies raised against the two proteins react with matrix components in the C. fasciculata mitochondrion. Our data thus provide, we believe for the first time, evidence for the presence of riboproteins within a trypanosomatid mitochondrion, bound, possibly, to the 12S and 9S RNAs. The proteins were immunologically related to two cytosolic riboproteins which were also of identical size, suggesting the interesting possibility that the same set of riboproteins is shared between the cytosol and the mitochondrion in this parasite.

Identification of cis and trans elements involved in the cell cycle regulation of multiple genes in Crithidia fasciculata.

Transcripts of several DNA replication genes, including the RPA1 and TOP2 genes, encoding the large subunit of nuclear replication protein A and the kinetoplast topoisomerase II, accumulate periodically during the cell cycle in the trypanosomatid Crithidia fasciculata. An octamer consensus sequence, CAUAGAAG, present in the 5' untranslated regions (UTR) of these mRNAs is required for periodic accumulation of the TOP2 and RPA1 transcripts and also for binding of a nuclear factor(s) to the 5' UTR RNAs of these genes. We show here that insertion of multiple (six) copies of this octamer sequence (6x octamer) into the 5' UTR of a reporter gene confers periodic accumulation on its transcript. Competition experiments and UV cross-linking studies show that the 6x octamer RNA and TOP2 5' UTR RNA bind to the same nuclear factor(s). Single-nucleotide substitutions in the 6x octamer that abolish the RNA gel shift also prevent cyclic accumulation of the reporter gene transcript. A protein termed cycling element binding protein, purified by affinity chromatography using 6x octamer RNA as a ligand, binds to RNAs containing wild-type octamers and not to those with mutant octamers. These results define a small sequence element in C. fasciculata mRNAs required for their cell cycle regulation and report the identification and purification of a putative regulatory protein that binds specifically to these elements.

A theoretical study of random segregation of minicircles in trypanosomatids.

The kinetoplast (k) DNA network of trypanosomatids is made up of approximately 50 maxicircles and the order of 10(4) minicircles. It has been proposed, based on various observations and experiments, that the minicircles are randomly segregated between daughter cells when the parent cell divides. In this paper, this random segregation hypothesis is theoretically tested in a population dynamics model to see if it can account for the observed phenomena. The hypothesis is shown to successfully explain, in Leishmania tarentolae, the observation that there are a few major and many minor minicircle classes, the fluctuations of minicircle class copy numbers over time, the loss of non-essential minicircle classes, the long survival times of a few of these classes and that these classes are likely to be the major classes within the population. Implications of the model are examined for trypanosomatids in general, leading to several predictions. The model predicts variation in network size within a population, variation in the average network size and large-scale changes in class copy number over long time-scales, an evolutionary pressure towards larger network sizes, the selective advantage of non-random over random segregation, very strong selection for the amplified class in Crithidia fasciculata if its minicircles undergo random segregation and that Trypanosoma brucei may use sexual reproduction to maintain its viability.

Cloning, molecular analysis and differential cell localisation of the p36 RACK analogue antigen from the parasite protozoon Crithidia fasciculata.

The family of the RACK molecules (receptors for activated C kinases) are present in all the species studied so far. In the genus Leishmania, these molecules also induce a strong immune reaction against the infection. We have cloned and characterised the gene that encodes the RACK analogue from the parasite trypanosomatid Crithidia fasciculata (CACK). The molecule seems to be encoded by two genes. The sequence analysis of the cloned open reading frame indicates the existence of a high degree of conservation not only with other members of the Trypanosomatidae but also with mammalians. The study of the protein kinase C phosphorylation sites shows the presence of three of them, shared with the mammalian species, additional to those present in the other protozoa suggesting a certain phylogenetic distance between the protozoon Crithidia fasciculata and the rest of the Trypanosomatidae. The CACK-encoded polypeptide shows an additional sequence of four amino acids at the carboxy-terminal end, which produces a different folding of the fragment with the presence of an alpha-helix instead of the beta-sheet usual in all the other species studied. A similar result is elicited at the amino-terminal end by the change of three amino acid residues. The immunolocalisation experiments show that the CACK displays a pattern with a distribution mainly at the plasma membrane, different from that of the related Leishmania species used as control, that displays a distribution close to the nucleus. Altogether, the data suggest that the existence of the structural differences found may have functional consequences.

Changes in organization of Crithidia fasciculata kinetoplast DNA replication proteins during the cell cycle.

Kinetoplast DNA (kDNA), the mitochondrial DNA in kinetoplastids, is a network containing several thousand topologically interlocked minicircles. We investigated cell cycle-dependent changes in the localization of kDNA replication enzymes by combining immunofluorescence with either hydroxyurea synchronization or incorporation of fluorescein-dUTP into the endogenous gaps of newly replicated minicircles. We found that while both topoisomerase II and DNA polymerase beta colocalize in two antipodal sites flanking the kDNA during replication, they behave differently at other times. Polymerase beta is not detected by immunofluorescence either during cell division or G1, but is abruptly detected in the antipodal sites at the onset of kDNA replication. In contrast, topoisomerase II is localized to sites at the network edge at all cell cycle stages; usually it is found in two antipodal sites, but during cytokinesis each postscission daughter network is associated with only a single site. During the subsequent G1, topoisomerase accumulates in a second localization site, forming the characteristic antipodal pattern. These data suggest that these sites at the network periphery are permanent components of the mitochondrial architecture that function in kDNA replication.

Nuleclear extracts of Crithidia fasciculata contain a factor(s) that binds to the 5'-untranslated regions of TOP2 and RPA1 mRNAs containing sequences required for their cell cycle regulation.

The Crithidia fasciculata replication protein A gene, RPA1, and topoisomerase II gene, TOP2, encode proteins involved in the replication of nuclear and mitochondrial DNA, respectively. Transcripts of both genes accumulate periodically during the cell cycle and attain their maximum levels just before S phase. Octamer consensus sequences within the 5'-untranslated region (UTR) of both genes have been shown to be necessary for cycling of these transcripts. Using a gel retardation assay, we show here that nuclear extracts of C. fasciculata contain a protein factor(s) that binds specifically to RNA from 5'-UTRs of TOP2 and RPA1 genes. In addition, mutations in the consensus octamer sequence abolish binding to the RNA in both cases. Ultraviolet cross-linking using a radiolabeled TOP2 5'-UTR probe identified proteins with apparent molecular masses of 74 and 37 kDa in the RNA-protein complex. Nuclear extracts prepared from synchronized cells show that the binding activity varies during the cell cycle in parallel with TOP2 and RPA1 mRNA levels. These results suggest that the cell cycle regulation of the mRNA levels of trypanosomatid DNA replication genes may be mediated by binding of specific proteins to conserved sequences in the 5'-UTR of their transcripts.

Sequences within the 5' untranslated region regulate the levels of a kinetoplast DNA topoisomerase mRNA during the cell cycle.

Gene expression in trypanosomatids appears to be regulated largely at the posttranscriptional level and involves maturation of mRNA precursors by trans splicing of a 39-nucleotide miniexon sequence to the 5' end of the mRNA and cleavage and polyadenylation at the 3' end of the mRNA. To initiate the identification of sequences involved in the periodic expression of DNA replication genes in trypanosomatids, we have mapped splice acceptor sites in the 5' flanking region of the TOP2 gene, which encodes the kinetoplast DNA topoisomerase, and have carried out deletion analysis of this region on a plasmid-encoded TOP2 gene. Block deletions within the 5' untranslated region (UTR) identified two regions (-608 to -388 and -387 to -186) responsible for periodic accumulation of the mRNA. Deletion of one or the other of these sequences had no effect on periodic expression of the mRNA, while deletion of both regions resulted in constitutive expression of the mRNA throughout the cell cycle. Subcloning of these sequences into the 5' UTR of a construct lacking both regions of the TOP2 5' UTR has shown that an octamer consensus sequence present in the 5' UTR of the TOP2, RPA1, and DHFR-TS mRNAs is required for normal cycling of the TOP2 mRNA. Mutation of the consensus octamer sequence in the TOP2 5' UTR in a plasmid construct containing only a single consensus octamer and that shows normal cycling of the plasmid-encoded TOP2 mRNA resulted in substantial reduction of the cycling of the mRNA level. These results imply a negative regulation of TOP2 mRNA during the cell cycle by a mechanism involving redundant elements containing one or more copies of a conserved octamer sequence within the 5' UTR of TOP2 mRNA.

Changes in network topology during the replication of kinetoplast DNA.

Kinetoplast DNA of Crithidia fasciculata is a network containing several thousand topologically interlocked DNA minicircles. In the prereplicative Form I network, each of the 5000 minicircles is intact and linked to an average of three neighbors (i.e. the minicircle valence is 3). Replication involves the release of minicircles from the interior of the network, the synthesis of nicked or gapped progeny minicircles and the attachment of the progeny to the network periphery. The ultimate result is a Form II network of 10,000 nicked or gapped minicircles. Our measurements of minicircle valence and density, and the network's surface area, revealed striking changes in network topology during replication. During the S phase, the peripheral newly replicated minicircles have a density twice that of minicircles in Form I networks, which suggests that the valence might be as high as 6. Most of the holes in the central region that occur from the removal of intact minicircles are repaired so that the central density and valence remain the same, as in prereplicative networks. When minicircle replication is complete at the end of the S phase, the isolated network has the surface area of a prereplicative network, despite having twice the number of minicircles. During the G2 phase, the Form II network undergoes a remodeling in which the area doubles and the valence is reduced to 3. Finally, the interruptions in the minicircles are repaired and the double-sized network splits in two.

Evidence for close in vivo association of protein with mitochondrial ribosomal RNA in a trypanosome.

Riboprotein particles containing the ribosomal RNA-like 9S and 12S RNAs in the trypanosome mitochondrion have never been isolated. Using the formaldehyde cross-linking procedure I show here that one or more of three proteins, q2 (16.5 kDa), r (15 kDa) and s (13 kDa), are closely associated in vivo with the 9S and 12S RNAs of the trypanosomatid parasite, Crithidia fasciculata. These proteins were also found to be associated with the parasite's cytosolic ribosomal RNA and to have strong immunological cross reactivity with riboprotein S11 of the bacterium Escherichia coli. These data provide the first evidence for the association of riboprotein-like proteins with the 9S and 12S mitochondrial RNA in a trypanosome, possibly as components of a mitochondrial ribosome.