Dataset Information

High-speed microscopic imaging of flagella motility and swimming in Giardia lamblia trophozoites.

ABSTRACT: We report, in this paper, several findings about the swimming and attachment mechanisms of Giardia lamblia trophozoites. These data were collected using a combination of a high-contrast CytoViva imaging system and a particle image velocimetry camera, which can capture images at speeds greater than 800 frames/s. Using this system, we discovered that, during rapid swimming of Giardia trophozoites, undulations of the caudal region contributed to forward propulsion combined with the beating of the flagella pairs. It was also discovered, in contrast to previous studies with 10 times slower image sampling technique, that the anterior and posterolateral flagella beat with a clearly defined power stroke and not symmetrical undulations. During the transition from free swimming to attachment, trophozoites modified their swimming behavior from a rapid rotating motion to a more stable planar swimming. While using this planar swimming motion, the trophozoites used the flagella for propulsion and directional control. In addition to examination of the posterolateral and anterior flagella, a model to describe the motion of the ventral flagella was derived, indicating that the ventral flagella beat in an expanding sine wave. In addition, the structure of the ventrocaudal groove creates boundary conditions that determine the form of beating of the ventral flagella. The results from this study indicate that Giardia is able to simultaneously generate both ciliary beating and typical eukaryotic flagellar beating using different pairs of flagella.

SUBMITTER: Lenaghan SC

PROVIDER: S-EPMC3161553 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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High-speed microscopic imaging of flagella motility and swimming in Giardia lamblia trophozoites.

Lenaghan Scott C SC Davis Corinne A CA Henson William R WR Zhang Zhili Z Zhang Mingjun M

Proceedings of the National Academy of Sciences of the United States of America 20110801 34

We report, in this paper, several findings about the swimming and attachment mechanisms of Giardia lamblia trophozoites. These data were collected using a combination of a high-contrast CytoViva imaging system and a particle image velocimetry camera, which can capture images at speeds greater than 800 frames/s. Using this system, we discovered that, during rapid swimming of Giardia trophozoites, undulations of the caudal region contributed to forward propulsion combined with the beating of the f ...[more]

PMID: 21808023

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Project description:The cell membrane of a Giardia lamblia trophozoite is covered with a single species of variant-specific surface protein (VSP) that is replaced by another VSP every 6 to 13 generations of cell growth, possibly for an evasion of host immunity. Experimentally, only six VSP species have been verified to localize to the cell membrane thus far. By assuming that VSP contains multiple CXXC motifs, 219 vsp genes were annotated in GiardiaDB of the WB isolate. By further assuming that VSP possesses both CXXC motifs and a CRGKA tail at the C terminus, Adam et al. (BMC Genomics 11:424, 2010) identified a total of 303 potential vsp genes in Giardia WB. The discrepancies between these two assumed VSP identities have caused some confusion. Here, we used experimental approaches to further verify what is required of the structures of a VSP to localize to the surface of cell membrane. The data led to the following conclusions. (i) The C-terminal CRGKA sequence is not essential for localizing VSPs to the cell membrane. (ii) A "motif 1" of 45 residues, consisting of two CXXCs separated by 12 to 15 amino acid residues, located close to the C terminus and a hydrophobic "motif 2" of 38 residues at the C terminus are both essential and sufficient for localizing the protein to the cell membrane. (ii) An N-terminal sequence upstream from motif 1 is not required for targeting VSPs to the cell membrane. By these criteria, we are able to identify 73 open reading frames as the putative vsp genes in Giardia. IMPORTANCE The intestinal pathogen Giardia lamblia expresses only one variant-specific surface protein (VSP) on the cell membrane surface at a given time, but it changes spontaneously every 6 to 13 generations of growth, presumably for evading the host immunity. Only 6 VSPs have been empirically shown to localize to the cell membrane surface thus far. Here, we used mutations of VSPs and methods of identifying their locations in Giardia cells and found that a "motif 1" of 45 residues, consisting of two CXXCs separated by 12 to 15 amino acid residues, located close to the C terminus and a hydrophobic "motif 2" of 38 residues at the C terminus are the only essential and sufficient structural requirements for localizing a protein to the cell membrane. By these criteria, 73 genes are identified in the Giardia WB strain genome database as the putative repertoire of VSPs.

Project description:BackgroundPolo-like kinases (PLKs) are conserved serine/threonine kinases that regulate the cell cycle. To date, the role of Giardia lamblia PLK (GlPLK) in cells has not been studied. Here, we report our investigation on the function of GlPLK to provide insight into the role of this PKL in Giardia cell division, especially during cytokinesis and flagella formation.MethodsTo assess the function of GIPLK, Giardia trophozoites were treated with the PLK-specific inhibitor GW843286X (GW). Using a putative open reading frame for the PLK identified in the Giardia genomic database, we generated a transgenic Giardia expressing hemagglutinin (HA)-tagged GlPLK and used this transgenic for immunofluorescence assays (IFAs). GlPLK expression was knocked down using an anti-glplk morpholino to observe its effect on the number of nuclei number and length of flagella. Giardia cells ectopically expressing truncated GlPLKs, kinase domain + linker (GlPLK-KDL) or polo-box domains (GlPLK-PBD) were constructed for IFAs. Mutant GlPLKs at Lys51, Thr179 and Thr183 were generated by site-directed mutagenesis and then used for the kinase assay. To elucidate the role of phosphorylated GlPLK, the phosphorylation residues were mutated and expressed in Giardia trophozoites RESULTS: After incubating trophozoites with 5 μM GW, the percentage of cells with > 4 nuclei and longer caudal and anterior flagella increased. IFAs indicated that GlPLK was localized to basal bodies and flagella and was present at mitotic spindles in dividing cells. Morpholino-mediated GlPLK knockdown resulted in the same phenotypes as those observed in GW-treated cells. In contrast to Giardia expressing GlPLK-PBD, Giardia expressing GlPLK-KDL was defective in terms of GIPLK localization to mitotic spindles and had altered localization of the basal bodies in dividing cells. Kinase assays using mutant recombinant GlPLKs indicated that mutation at Lys51 or at both Thr179 and Thr183 resulted in loss of kinase activity. Giardia expressing these mutant GlPLKs also demonstrated defects in cell growth, cytokinesis and flagella formation.ConclusionsThese data indicate that GlPLK plays a role in Giardia cell division, especially during cytokinesis, and that it is also involved in flagella formation.

Dataset Information

High-speed microscopic imaging of flagella motility and swimming in Giardia lamblia trophozoites.

Publications

High-speed microscopic imaging of flagella motility and swimming in Giardia lamblia trophozoites.

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