We developed a series of plasmids that allow C-terminal tagging of any gene in its endogenous locus in Giardia intestinalis, with different epitope tags (triple hemagglutinin [3HA] and triple Myc [3Myc]) and selection markers (puromycin, neomycin, and a newly developed marker, blasticidin). Using these vectors, cyclin B and aurora kinase were tagged, expressed, and localized.The protozoan Giardia intestinalis is a human parasite responsible for considerable morbidity both in humans and in cattle (1,9,15). Due to its high divergence from other eukaryotes, many antibodies useful for cell biology will not recognize Giardia proteins. The process of developing gene-specific antibodies is long and laborious, typically requiring several months. A faster approach is to express a version of the gene tagged with a well-characterized epitope by integrating a construct into the chromosome. Integration of constructs into the Giardia intestinalis genome has been achieved previously (6, 7, 11) but not for the purpose of tagging a gene. Our goal was to facilitate integration of a tagged Giardia gene into the genome and to keep expression as close to endogenous levels as possible.A construct was designed for knocking in a tagged version of cyclin B (GiardiaDB identification [ID] number GL50803_ 3977) into the Giardia genome. This strategy is derived from the gene tagging originally developed with Schizosaccharomyces pombe (2, 4) and since then adapted to other organisms, such as Trypanosoma brucei (10). The C-terminal part of the gene of interest is cloned in frame to an epitope tag in a vector also containing a selectable marker cassette. This region should contain a restriction enzyme site, preferably naturally occurring, that will be unique in the resulting vector; additionally, this site should be located at least 90 bp from either the 5Ј or 3Ј cloning site. The resulting plasmid is then linearized using this restriction enzyme and introduced into cells, and transfected cells are selected using the selectable marker. Single-site homologous recombination and integration of this construct should yield two copies of the gene (Fig. 1A shows more details): one under the control of the original promoter but now with a C-terminal epitope, the other missing any promoters and its entire N terminus and thus presumably not expressed.To tag Giardia cyclin B, an XhoI site was engineered by PCR to eliminate the cyclin B stop codon (see the supplemental material for oligonucleotide details). A StyI/XhoI cyclin B fragment was cloned in frame to a C-terminal triple-hemagglutinin (HA) epitope tag (3HA) (Fig. 1A) and appended to the NotI/KpnI fragment from the plasmid p␥G-GFP (11) containing the puromycin resistance cassette to yield the plasmid pc-cycB-3HA-PAC. The plasmid was then linearized using an original NruI site, ethanol precipitated, and resuspended in distilled water, and 10 g of linear DNA was introduced into Giardia strain WB-C6 by electroporation as previously described (11). Transfected cells were selected using puromycin (10 g/...

The Tousled-like kinases are an evolutionarily conserved family of proteins implicated in DNA repair, DNA replication and mitosis in metazoans and plants. Their absence from the yeasts and other eukaryotic `microbes' suggests a specific role for them in the development of multicellular organisms. In this study, two closely related Tousled-like kinase homologs, TLK1 and TLK2, were identified in Trypanosoma brucei, a unicellular protozoan parasite. Only TLK1 plays an essential role in cell growth, and a deficiency in TLK1 led to an enrichment of S-phase cells, defective spindle formation and aberrant chromosome segregation. Although both TLK proteins localize to the nucleus, only TLK1 also concentrates in the spindle poles during mitosis. Both TLK proteins are phosphorylated by the Aurora kinase (AUK1), and both can autophosphorylate and phosphorylate histone H3 and the chromatin assembly factors Asf1A and Asf1B in vitro, but only TLK1 is autophosphorylated and capable of oligomerizing and interacting with AUK1, Asf1A and Asf1B in vivo. These discrepancies between the two TLK proteins can be attributed to minor differences between their N- and C-terminal sequences. In summary, TLK1 cooperates with Aurora kinase to regulate spindle assembly and chromosome segregation, and it performs a role in DNA replication probably by regulating histone modification in trypanosomes.

Giardia is a single-celled parasite causing significant diarrheal disease in several hundred million people worldwide. Due to limited access to the site of infection in the gastrointestinal tract, our understanding of the dynamics of Giardia infections in the host has remained limited and largely inferred from laboratory culture. To better understand Giardia physiology and colonization in the host, we developed imaging methods to quantify Giardia expressing bioluminescent physiological reporters in two relevant animal models. We discovered that parasites primarily colonize and encyst in the proximal small intestine in discrete, high-density foci. We also show that high parasite density contributes to encystation initiation.

SummaryThe protozoan parasite Giardia intestinalis (also known as Giardia lamblia) is a major waterborne pathogen. During its life cycle, Giardia alternates between the actively growing trophozoite, which has two diploid nuclei with low levels of allelic heterozygosity, and the infectious cyst, which has four nuclei and a tough outer wall. Although the formation of the cyst wall has been studied extensively, we still lack basic knowledge about many fundamental aspects of the cyst, including the sources of the four nuclei and their distribution during the transformation from cyst into trophozoite. In this study, we tracked the identities of the nuclei in the trophozoite and cyst using integrated nuclear markers and immunofluorescence staining. We demonstrate that the cyst is formed from a single trophozoite by a mitotic division without cytokinesis and not by the fusion of two trophozoites. During excystation, the cell completes cytokinesis to form two daughter trophozoites. The non-identical nuclear pairs derived from the parent trophozoite remain associated in the cyst and are distributed to daughter cells during excystation as pairs. Thus, nuclear sorting (such that each daughter cell receives a pair of identical nuclei) does not appear to be a mechanism by which Giardia reduces heterozygosity between its nuclei. Rather, we show that the cyst nuclei exchange chromosomal genetic material, perhaps as a way to reduce heterozygosity in the absence of meiosis and sex, which have not been described in Giardia. These results shed light on fundamental aspects of the Giardia life cycle and have implications for our understanding of the population genetics and cell biology of this binucleate parasite.

SummaryMost cell cycle regulation research has been conducted in model organisms representing a very small part of the eukaryotic domain. The highly divergent human pathogen Giardia intestinalis is ideal for studying the conservation of eukaryotic pathways. Although Giardia has many cell cycle regulatory components, its genome lacks all anaphase-promoting complex (APC) components. In the present study, we show that a single mitotic cyclin in Giardia is essential for progression into mitosis. Strikingly, Giardia cyclin B lacks the conserved N-terminal motif required for timely degradation mediated by the APC and ubiquitin conjugation. Expression of Giardia cyclin B in fission yeast is toxic, leading to a prophase arrest, and this toxicity is suppressed by the addition of a fission yeast degradation motif. Cyclin B is degraded during mitosis in Giardia cells, but this degradation appears to be independent of the ubiquitination pathway. Other putative APC substrates, aurora and polo-like kinases, also show no evidence of ubiquitination. This is the first example of mitosis not regulated by the APC and might reflect an evolutionary ancient form of cell cycle regulation.