Morphological investigations of the Euglena gracilis isolated paraflagellar body

By means of scanning electron microscopy we have investigated the morphology of the photoreceptive-locomotory apparatus of the flagellate Euglena gracilis. As can be seen from the micrographs, there is a strong connection between the paraflagellar rod and the paraflagellar body; structural and functional details are discussed, and a simple model of the photoreceptive apparatus is given.     

Cryptic paraflagellar rod in endosymbiont-containing kinetoplastid protozoa

Cilia and flagella are central to many biological processes in a diverse range of organisms. The kinetoplastid protozoa are very appealing models for the study of flagellar function, particularly in the light of the availability of extensive trypanosomatid genome information. In addition to the highly conserved 9 + 2 axoneme, the kinetoplastid flagellum contains a characteristic paraflagellar rod structure (PFR). The PFR is necessary for full motility and provides support for metabolic regulators that may influence flagellar beating. However, there is an intriguing puzzle: one clade of endosymbiont-containing kinetoplastids apparently lack a PFR yet are as motile as species that possess a PFR and are able to attach to the invertebrate host epithelia. We investigated how these organisms are able to locomote despite the apparent lack of PFR. Here we have identified a PFR1 gene in the endosymbiont-bearing trypanosome Crithidia deanei. This gene is expressed in C. deanei and is able to partially complement a pfr1 null mutation in Leishmania mexicana cells, demonstrating that the encoded protein is functional. Careful reexamination of C. deanei flagellar ultrastructure revealed a greatly reduced PFR missed by many previous analyses. This affirms the PFR as a canonical organelle of kinetoplastids. Moreover, although PFR proteins have been conserved in evolution, primary sequence differences contribute to particular PFR morphotypes characteristic of different kinetoplastid species.     

NAD+ Kinase Activities in Euglena Gracilis and Phaseolus Vulgaris

After an electrophoretic separation of proteins from Euglena gracilis and dry seeds of Phaseolus vulgaris in native conditions in polyacrylamide gels, gels were incubated in mixtures containing NAD+, Mg-ATP2-, glucose 6-phosphate, G6P dehydrogenase, and either phenazine ethosulfate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (PES/MTT) or phenazine methosulfate and nitro blue tetrazolium (PMS/NBT) as coupled redox system for NAD+ kinase activity detection. In the presence of PES/MTT, 4 bands were revealed for E. gracilis, among which two corresponded to NAD+ kinase activity, the other corresponding to a NAD+ reductase activity due to alcohol dehydrogenase (ADH). In the presence of PMS/NBT, only the bands of NAD+ kinase activity were revealed. With Phaseolus vulgaris, 3 bands of ADH were always revealed in both mixtures, and only the use of PMS/NBT allowed the detection of NAD+ kinase as a fourth band. With both materials, NAD+ reductase staining in gels was intensifed in the presence of GTP or ATP and even further with ADP or GDP. The results demonstrate that: 1) the NAD+ kinase and NAD+ reductase are two distinct enzymes; 2) the NAD+ reductase corresponds to ADH.     

Homologies between paraflagellar rod proteins from trypanosomes and euglenoids revealed by a monoclonal antibody

A Nonidet P 40 insoluble fraction was isolated from Trypanosoma brucei and was used to raise a monoclonal antibody (5E9). The antigen was localized by indirect immunofluorescence in the flagellum of T. brucei and of two species of euglenoids, Euglena gracilis and Distigma proteus. In immunoblot analysis, 5E9 appeared to bind to paraflagellar rod proteins PFR1 and PFR2 of T. brucei (72000 and 75000 mol. wt.) and of E. gracilis (67000 and 76000 mol. wt.). The presence of a common epitope in paraflagellar rod proteins from species of trypanosomes and euglenoids shows that despite distinct structures of the rods some identical domain exists in the proteins that could be involved in their supramolecular assembly into a similar organelle. The antigenic determinant defined by 5E9 was also shown to be present in a 87000 molecular weight polypeptide located in the proximal part of the flagellum of Crithidia oncopelti in which a paraflagellar rod is not detectable at the ultrastructural level.     

The paraflagellar rod of kinetoplastida: solved and unsolved questions

The flagellum of almost every member of the Kinetoplastida contains, next to its canonical 'nine-plus-two' axoneme, structure, a unique, complex and highly organized lattice-like structure called the paraflagellar rod or paraxial rod. Here, Philippe Bastin, Keith Matthews and Keith Gull summarize the latest findings on its structure, the nature of its protein components and their corresponding genes. They also consider the possible functions of this intriguing organelle.     

Expression and functional analysis of Euglena Gracilis chloroplast initiation factor 3

A portion of a cDNA predicted to encode the mature form of Euglena gracilis chloroplast translational initiation factor 3 (IF-3_chlM, molecular mass, 46402) and the portion of this factor homologous to bacterial IF-3 (IF-3_chlH, molecular mass 22829) have been cloned and expressed in Escherichia coli as histidine-tagged proteins. The homology domain can be expressed in reasonable levels in E. coli. However, IF-3_chlM is quite toxic and can only be produced in small amounts. Both forms of the chloroplast factor are associated with E. coli ribosomes. Purification procedures have been developed for both IF-3_chlM and IF-3_chlH using Ni-NTA affinity chromatography followed by ion exchange chromatography. IF-3_chlM and IF-3_chlH are active in promoting ribosome dissociation and in promoting the binding of fMet-tRNA to E. coli ribosomes. However, IF-3_chlH has at least 5-fold more activity than either native IF-3_chl or IF-3_chlM in promoting initiation complex formation on chloroplast 30S ribosomal subunits in the presence of a mRNA carrying a natural translational initiation signal. This observation suggests that regions of IF-3_chl lying outside of the homology domain may down-regulate the activity of this factor.This work was supported in part by National Institutes of Health Grant GM24963.     

Flagellar morphogenesis: protein targeting and assembly in the paraflagellar rod of trypanosomes

The paraflagellar rod (PFR) of the African trypanosome Trypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.     

The paraflagellar rod of kinetoplastid parasites: From structure to components and function

The role of the eukaryotic flagellum in cell motility is well established but its importance in many other aspects of cell biology, from cell signalling to developmental regulation, is becoming increasingly apparent. In addition to this diversity of function the core structure of the flagellum, which has been inherited from the earliest ancestor of all eukaryotes, is embellished with a range of extra-axonemal structures in many organisms. One of the best studied of these structures is the paraflagellar rod of kinetoplastid protozoa in which the morphological characteristics have been well defined and some of the major protein constituents have been identified. Here we discuss recent advances in the identification of further molecular components of the paraflagellar rod, how these impact on our understanding of its function and regulation and the implications for therapeutic intervention in a number of devastating human pathologies.     

The relationship between chlorophyll and the carotenoids in the algal flagellate,Euglena

1. We have obtained an action spectrum for chlorophyll formation in Euglena gracilis. This action spectrum is similar to the absorption spectrum of protochlorophyll. However, efforts to isolate and identify this pigment have been unsuccessful. 2. Porphyrins have been extracted from both the normal and dark-adapted Euglena and a chlorophyll-free mutant. 3. The "action" spectra for chlorophyll and carotenoid synthesis have been found to almost coincide, indicating that the same porphyrin-like molecule may influence the synthesis of both pigments. 4. It is indicated that two porphyrin-like systems are in operation simultaneously, one concerned with carotenoid "removal" and another involved in carotenoid and chlorophyll synthesis.     

Interactions of Pseudomonas Aeruginosa Hemagglutinins With Euglena Gracilis, Chlamydomonas Reinhardi, and Tetrahymena Pyriformis

SYNOPSIS The galactosephilic and mannosephilic hemagglutinins of Pseudomonas aeruginosa adsorbed onto Euglena gracilis, Chlamydomonas reinhardi , and Tetrahymena pyriformis . Furthermore, peroxidase binding to the 3 protozoan species was shown to be mediated by these lectins. Binding of Pseudomonas lectins to E. gracilis and C. reinhardi caused their specific agglutination, whereas no agglutination was observed with T. pyriformis , even after treatment by papain or by NaF. Added to the culture medium, the Pseudomonas hemagglutinins stimulated growth of E. gracilis and T. pyriformis due to their binding to these protozoa: this effect was partly inhibited by the specific sugar.     

The relationship between substrate-induced respiration and swelling in Azotobacter vinelandii

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1. When oxidizable substrates are added to a starved suspension of Azotobacter vinelandii osmotically shrunken in 0.2 M KCl, a decrease in absorbance is observed which results from a change in light scattering as the cells increase in volume.     

Flagellar membrane and paraxial rod proteins of Leishmania: characterization employing monoclonal antibodies

Flagella-specific proteins of Leishmania have been identified employing the monoclonal antibody technique. Six monoclonal antibodies recognized 3 different proteins. A doublet of protein of Mr 69,000 and 74,000 Da identified by monoclonal antibodies F-3, F-4 and F-6 is continuously distributed along the flagellum by immunofluorescence. Immunocytochemical electron microscopic studies localize these molecules to the paraxial rod of the flagellum. A single protein of Mr 13,200 Da is recognized by monoclonal antibodies F-1, F-2 and F-5. The distribution of the Mr 13,200 protein appears irregular, occurring in localized patches along the length of the flagellum, especially at the flagellar tip. Immunocytochemical electron microscopic experiments show that the Mr 13,200 molecule is associated with the membrane of the flagellum. Indirect immunofluorescence experiments demonstrated these monoclonal antibodies cross-reacted with members of the Kinetoplastida family (Endotrypanum, trypanosoma, Leishmania) suggesting that these molecules may be evolutionarily conserved.     

Morphological relationship between the cranial and supraorbital regions in Homo sapiens

Although some hypotheses that attempt to explain the variation in supraorbital region morphology in modern humans have been proposed, this issue is still not well understood. In this study, the craniofacial size and spatial models were tested using a sample of modern human crania from geographically diverse populations, and the co‐occurrence of the degrees of glabella (GL) and superciliary arch (ST) expression were analyzed. The two supraorbital structures were examined by visual assessment, and eight quantitative variables were included in the three‐way ANOVA, canonical variates analysis and partial rank correlation. The influences of sex and the region of origin of the cranial samples on the relationships between the examined variables and the degrees of supraorbital structures expression were also considered. The results only partially supported the craniofacial size and spatial models and suggested that GL and ST experienced separate influences during development. In the sample of all crania, the neurocranial size more strongly influenced the morphological variation of the ST than of the GL, and sex influenced both of these structures the most. The results suggest that sex may be the main factor (having an influence independent of the other traits) on the morphological variation of the GL and ST. Am J Phys Anthropol 156:110–124, 2015 © 2014 Wiley Periodicals, Inc.     

A relationship between adenosine 3'-5'-cyclic monophosphate levels and deoxyribonucleic acid synthesis in Euglena

Using the binding protein method we found that cAMP levels in normal, exponentially growing Euglena stay constant on per cell and protein basis. The level rises slightly when cells enter the stationary stage. Cells growing in low vitamin B12 medium show the same pattern during predeficiency growth. Upon becoming vitamin B12 deficient, the cAMP level decreases. Replenishment of these cells with the vitamin causes an immediate drop, followed by a sharp rise in cAMP. This is followed by resumption of DNA synthesis. The cAMP level drops and rises again when DNA duplication is completed and during the G2 period. The level of the cAMP drops again followed by resumption of cell division. the data suggest a relation exists between cAMP level, resumption and completion of DNA synthesis, and cell division.