The genome of Naegleria gruberi illuminates early eukaryotic versatility.

Genome sequences of diverse free-living protists are essential for understanding eukaryotic evolution and molecular and cell biology. The free-living amoeboflagellate Naegleria gruberi belongs to a varied and ubiquitous protist clade (Heterolobosea) that diverged from other eukaryotic lineages over a billion years ago. Analysis of the 15,727 protein-coding genes encoded by Naegleria's 41 Mb nuclear genome indicates a capacity for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production, with fundamental implications for the evolution of organelle metabolism. The Naegleria genome facilitates substantially broader phylogenomic comparisons of free-living eukaryotes than previously possible, allowing us to identify thousands of genes likely present in the pan-eukaryotic ancestor, with 40% likely eukaryotic inventions. Moreover, we construct a comprehensive catalog of amoeboid-motility genes. The Naegleria genome, analyzed in the context of other protists, reveals a remarkably complex ancestral eukaryote with a rich repertoire of cytoskeletal, sexual, signaling, and metabolic modules.

Disc-associated proteins mediate the unusual hyperstability of the ventral disc in Giardia lamblia.

Giardia lamblia, a widespread parasitic protozoan, attaches to the host gastrointestinal epithelium by using the ventral disc, a complex microtubule (MT) organelle. The 'cup-like' disc is formed by a spiral MT array that scaffolds numerous disc-associated proteins (DAPs) and higher-order protein complexes. In interphase, the disc is hyperstable and has limited MT dynamics; however, it remains unclear how DAPs confer these properties. To investigate mechanisms of hyperstability, we confirmed the disc-specific localization of over 50 new DAPs identified by using both a disc proteome and an ongoing GFP localization screen. DAPs localize to specific disc regions and many lack similarity to known proteins. By screening 14 CRISPRi-mediated DAP knockdown (KD) strains for defects in hyperstability and MT dynamics, we identified two strains - DAP5188KD and DAP6751KD -with discs that dissociate following high-salt fractionation. Discs in the DAP5188KD strain were also sensitive to treatment with the MT-polymerization inhibitor nocodazole. Thus, we confirm here that at least two of the 87 known DAPs confer hyperstable properties to the disc MTs, and we anticipate that other DAPs contribute to disc MT stability, nucleation and assembly.

A detailed look at the cytoskeletal architecture of the Giardia lamblia ventral disc.

Giardia lamblia is a protistan parasite that infects and colonizes the small intestine of mammals. It is widespread and particularly endemic in the developing world. Here we present a detailed structural study by 3-D negative staining and cryo-electron tomography of a unique Giardia organelle, the ventral disc. The disc is composed of a regular array of microtubules and associated sheets, called microribbons that form a large spiral, held together by a myriad of mostly unknown associated proteins. In a previous study we analyzed by cryo-electron tomography the central microtubule portion (here called disc body) of the ventral disc and found a large portion of microtubule associated inner (MIPs) and outer proteins (MAPs) that render these microtubules hyper-stable. With this follow-up study we expanded our 3-D analysis to different parts of the disc such as the ventral and dorsal areas of the overlap zone, as well as the outer disc margin. There are intrinsic location-specific characteristics in the composition of microtubule-associated proteins between these regions, as well as large differences between the overall architecture of microtubules and microribbons. The lateral packing of microtubule-microribbon complexes varies substantially, and closer packing often comes with contracted lateral tethers that seem to hold the disc together. It appears that the marginal microtubule-microribbon complexes function as outer, laterally contractible lids that may help the cell to clamp onto the intestinal microvilli. Furthermore, we analyzed length, quantity, curvature and distribution between different zones of the disc, which we found to differ from previous publications.

The Food Web of Boiling Springs Lake Appears Dominated by the Heterolobosean Tetramitus thermacidophilus Strain BSL.

We studied the protist grazers of Boiling Springs Lake (BSL), an acid geothermal feature in Lassen Volcanic National Park, using a combination of culture and genetic approaches. The major predator in BSL is a vahlkampfiid ameba closely related (95% 18S+ITS rRNA identity) to Tetramitus thermacidophilus, a heterolobose ameboflagellate recently isolated from volcanic geothermal acidic sites in Europe and Russia, as well as an uncultured heterolobosean from the nearby Iron Mountain acid mine drainage site. Tetramitus thermacidophilus strain BSL is capable of surviving the physical extremes of BSL, with optimal growth at 38-50 °C and pH 2-5. This bacterivore also ingested conidiospores of the ascomycete Phialophora sp., but ultrastructural observations reveal the latter may not be readily digested, and conidia were not separable from the ameoboflagellate culture, suggesting a possible symbiosis. DGGE fingerprint transects studies showed the organism is restricted to near-lake environs, and we detected an average of ~500 viable cysts/cm(3) sediment on the shoreline. Other grazing protists were isolated from lakeshore environments, including the lobose amebae Acanthamoeba sp. and Hartmannella sp., and the kinetoplastid flagellate Bodo sp., but none could tolerate both low pH and high temperature. These appear to be restricted to cooler near lake geothermal features, which also contain other potential grazer morphotypes observed but not successfully cultured, including ciliates, euglenids, testate amebae, and possible cercozoans. We compare the food web of BSL with other acidic or geothermal sites, and discuss the impact of protists in this unique environment.

An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins.

Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host.

Harnessing the power of new genetic tools to illuminate Giardia biology and pathogenesis.

Giardia is a prevalent single-celled microaerophilic intestinal parasite causing diarrheal disease and significantly impacting global health. Double diploid (essentially tetraploid) Giardia trophozoites have presented a formidable challenge to the development of molecular genetic tools to interrogate gene function. High sequence divergence and the high percentage of hypothetical proteins lacking homology to proteins in other eukaryotes have limited our understanding of Giardia protein function, slowing drug target validation and development. For more than 25 years, Giardia A and B assemblages have been readily amenable to transfection with plasmids or linear DNA templates. Here, we highlight the utility and power of genetic approaches developed to assess protein function in Giardia, with particular emphasis on the more recent clustered regularly interspaced palindromic repeats/Cas9-based methods for knockdowns and knockouts. Robust and reliable molecular genetic approaches are fundamental toward the interrogation of Giardia protein function and evaluation of druggable targets. New genetic approaches tailored for the double diploid Giardia are imperative for understanding Giardia's unique biology and pathogenesis.

Characterization of a unique attachment organelle: Single-cell force spectroscopy of Giardia duodenalis trophozoites.

The unicellular parasite Giardia duodenalis is the causative agent of giardiasis, a gastrointestinal disease with global spread. In its trophozoite form, G. duodenalis can adhere to the human intestinal epithelium and a variety of other, artificial surfaces. Its attachment is facilitated by a unique microtubule-based attachment organelle, the so-called ventral disc. The mechanical function of the ventral disc, however, is still debated. Earlier studies postulated that a dynamic negative pressure under the ventral disc, generated by persistently beating flagella, mediates the attachment. Later studies suggested a suction model based on structural changes of the ventral discs, substrate clutching or grasping, or unspecific contact forces. In this study, we aim to contribute to the understanding of G. duodenalis attachment by investigating detachment characteristics and determining adhesion forces of single trophozoites on a smooth glass surface (RMS = 1.1 ± 0.2 nm) by fluidic force microscopy (FluidFM)-based single-cell force spectroscopy (SCFS). Briefly, viable adherent trophozoites were approached with a FluidFM micropipette, immobilized to the micropipette aperture by negative pressure, and detached from the surface by micropipette retraction while retract force curves were recorded. These force curves displayed novel and so far undescribed characteristics for a microorganism, namely, gradual force increase on the pulled trophozoite, with localization of adhesion force shortly before cell detachment length. Respective adhesion forces reached 7.7 ± 4.2 nN at 1 µm s-1 pulling speed. Importantly, this unique force pattern was different from that of other eukaryotic cells such as Candida albicans or oral keratinocytes, considered for comparison in this study. The latter both displayed a force pattern with force peaks of different values or force plateaus (for keratinocytes) indicative of breakage of molecular bonds of cell-anchored classes of adhesion molecules or membrane components. Furthermore, the attachment mode of G. duodenalis trophozoites was mechanically resilient to tensile forces, when the pulling speeds were raised up to 10 µm s-1 and adhesion forces increased to 28.7 ± 10.5 nN. Taken together, comparative SCSF revealed novel and unique retract force curve characteristics for attached G. duodenalis, suggesting a ligand-independent suction mechanism, that differ from those of other well described eukaryotes.

Giardia flagellar motility is not directly required to maintain attachment to surfaces.

Giardia trophozoites attach to the intestinal microvilli (or inert surfaces) using an undefined "suction-based" mechanism, and remain attached during cell division to avoid peristalsis. Flagellar motility is a key factor in Giardia's pathogenesis and colonization of the host small intestine. Specifically, the beating of the ventral flagella, one of four pairs of motile flagella, has been proposed to generate a hydrodynamic force that results in suction-based attachment via the adjacent ventral disc. We aimed to test this prevailing "hydrodynamic model" of attachment mediated by flagellar motility. We defined four distinct stages of attachment by assessing surface contacts of the trophozoite with the substrate during attachment using TIRF microscopy (TIRFM). The lateral crest of the ventral disc forms a continuous perimeter seal with the substrate, a cytological indication that trophozoites are fully attached. Using trophozoites with two types of molecularly engineered defects in flagellar beating, we determined that neither ventral flagellar beating, nor any flagellar beating, is necessary for the maintenance of attachment. Following a morpholino-based knockdown of PF16, a central pair protein, both the beating and morphology of flagella were defective, but trophozoites could still initiate proper surface contacts as seen using TIRFM and could maintain attachment in several biophysical assays. Trophozoites with impaired motility were able to attach as well as motile cells. We also generated a strain with defects in the ventral flagellar waveform by overexpressing a dominant negative form of alpha2-annexin::GFP (D122A, D275A). This dominant negative alpha2-annexin strain could initiate attachment and had only a slight decrease in the ability to withstand normal and shear forces. The time needed for attachment did increase in trophozoites with overall defective flagellar beating, however. Thus while not directly required for attachment, flagellar motility is important for positioning and orienting trophozoites prior to attachment. Drugs affecting flagellar motility may result in lower levels of attachment by indirectly limiting the number of parasites that can position the ventral disc properly against a surface and against peristaltic flow.

The Giardia median body protein is a ventral disc protein that is critical for maintaining a domed disc conformation during attachment.

Giardia has unique microtubule structures, including the ventral disc, the primary organelle of attachment to the host, and the median body, a structure of undefined function. During attachment, the ventral disc has a domed conformation and enables Giardia to attach to the host intestinal epithelia within seconds. The mechanism of attachment via the ventral disc and the overall structure, function, and assembly of the ventral disc are not well understood. Our recent proteomic analysis of the ventral disc indicated that the median body protein (MBP), previously reported to localize exclusively to the median body, was primarily localized to the ventral disc. Using high-resolution light and electron microscopy, we confirm that the median body protein localizes primarily to the overlap zone of the ventral disc. The MBP also occasionally localized to the median body during prophase. To define the contribution of MBP to the ventral disc structure, we depleted MBP using an anti-MBP morpholino. We found that the ventral disc was no longer able to form properly and that the disc structure often had an aberrant nondomed or flattened horseshoe conformation. The ability of attached anti-MBP morpholino-treated trophozoites to withstand shear forces and normal forces was significantly decreased. Most notably, the plasma membrane contacts with the surface, including those of the bare area, were defective after the anti-MBP knockdown. To our knowledge, this is the first ventral disc protein whose depletion directly alters ventral disc structure, confirming that the domed ventral disc conformation is important for robust attachment.

The domed architecture of Giardias ventral disc is necessary for attachment and host pathogenesis.

After ingestion of dormant cysts, the widespread protozoan parasite Giardia lamblia colonizes the host gastrointestinal tract via direct and reversible attachment using a novel microtubule organelle, the ventral disc. Extracellular attachment to the host allows the parasite to resist peristaltic flow, facilitates colonization and is proposed to cause damage to the microvilli of host enterocytes as well as disrupt host barrier integrity. The 9 um in diameter ventral disc is defined by a highly complex architecture of unique protein complexes scaffolded onto a spiral microtubule (MT) array of one hundred parallel, uniformly spaced MT polymers that bend approximately one and a quarter turns to form a domed structure. To investigate the role of disc-mediated attachment in causing epithelial cell damage, we used a new approach to rapidly create a stable quadruple knockout of Giardia of an essential ventral disc protein, MBP, using a new method of CRISPR-mediated gene disruption with multiple positive selectable markers. MBP quadruple KO mutant discs lack the characteristic domed architecture and possess a flattened crescent or horseshoe-shaped conformation that lacks the overlapping region, with severe defects in the microribbon-crossbridge (MR-CB) complex structure. MBP KO mutants are also unable to resist fluid flow required for attachment to inert surfaces. Importantly, MBP KO mutants have 100% penetrance off positive selection, which is essential for quantification of in vivo impacts of disc and attachment mutants with host cells. Using a new gastrointestinal organoid model of pathogenesis, we found that MBP KO infections had a significantly reduced ability to cause the barrier breakdown characteristic of wild-type infections. Overall, this work provides direct evidence of the role of MBP in creating the domed disc, as well as the first direct evidence that parasite attachment is necessary for host pathology, specifically epithelial barrier breakdown.

Affinity-purified Plasmodium tubulin provides a key reagent for antimalarial drug development.

Hirst et al. used a TOG-domain-based affinity-purification approach to reconstitute and define the in vitro dynamics of blood-stage Plasmodium falciparum αß-tubulin. This provides a key reagent for defining parasite microtubule (MT) dynamics and for evaluating the efficacy of anti-MT drugs throughout the complex parasite life cycle.

Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis.

CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.

An insider's guide to the microtubule cytoskeleton of Giardia.

Giardia intestinalis is a zoonotic, parasitic protist with a complex microtubule cytoskeleton critical for motility, attachment, intracellular transport, cell division and transitioning between its two life cycle stages - the cyst and the trophozoite. This review focuses on the structures of the primary elements of the microtubule cytoskeleton and cytoskeletal dynamics throughout this complex giardial life cycle. The giardial cytoskeleton has both highly dynamic elements and more stable MT structures, including several novel structures like the ventral disc that change conformation via unknown mechanisms. While our knowledge of the giardial cytoskeleton is primarily cytological, the completed Giardia genome and recently developed reverse genetic tools affords an opportunity to uncover the mechanisms of Giardia's cytoskeletal dynamics. Fundamental areas of giardial cytoskeletal biology remain to be explored, including high resolution imaging and compositional characterization of cytoskeletal structures required for elucidating the molecular mechanisms of cytoskeletal functioning.

Draft Genome Sequence of the Free-Living, Iridescent Bacterium Tenacibaculum mesophilum Strain ECR.

Here, we report the genome sequence of Tenacibaculum mesophilum strain ECR, which was isolated from the river/ocean interface at Trunk River in Falmouth, Massachusetts. The isolation and sequencing were performed as part of the 2016 and 2018 Microbial Diversity courses at the Marine Biological Laboratory in Woods Hole, Massachusetts.

Microtubule organelles in Giardia.

Giardia lamblia is a widespread parasitic protist with a complex MT cytoskeleton that is critical for motility, attachment, mitosis and cell division, and transitions between its two life cycle stages-the infectious cyst and flagellated trophozoite. Giardia trophozoites have both highly dynamic and highly stable MT organelles, including the ventral disc, eight flagella, the median body and the funis. The ventral disc, an elaborate MT organelle, is essential for the parasite's attachment to the intestinal villi to avoid peristalsis. Giardia's four flagellar pairs enable swimming motility and may also promote attachment. They are maintained at different equilibrium lengths and are distinguished by their long cytoplasmic regions and novel extra-axonemal structures. The functions of the median body and funis, MT organelles unique to Giardia, remain less understood. In addition to conserved MT-associated proteins, the genome is enriched in ankyrins, NEKs, and novel hypothetical proteins that also associate with the MT cytoskeleton. High-resolution ultrastructural imaging and a current inventory of more than 300 proteins associated with Giardia's MT cytoskeleton lay the groundwork for future mechanistic analyses of parasite attachment to the host, motility, cell division, and encystation/excystation. Giardia's unique MT organelles exemplify the capacity of MT polymers to generate intricate structures that are diverse in both form and function. Thus, beyond its relevance to pathogenesis, the study of Giardia's MT cytoskeleton informs basic cytoskeletal biology and cellular evolution. With the availability of new molecular genetic tools to disrupt gene function, we anticipate a new era of cytoskeletal discovery in Giardia.

Microbial community dynamics and coexistence in a sulfide-driven phototrophic bloom.

BACKGROUND: Lagoons are common along coastlines worldwide and are important for biogeochemical element cycling, coastal biodiversity, coastal erosion protection and blue carbon sequestration. These ecosystems are frequently disturbed by weather, tides, and human activities. Here, we investigated a shallow lagoon in New England. The brackish ecosystem releases hydrogen sulfide particularly upon physical disturbance, causing blooms of anoxygenic sulfur-oxidizing phototrophs. To study the habitat, microbial community structure, assembly and function we carried out in situ experiments investigating the bloom dynamics over time. RESULTS: Phototrophic microbial mats and permanently or seasonally stratified water columns commonly contain multiple phototrophic lineages that coexist based on their light, oxygen and nutrient preferences. We describe similar coexistence patterns and ecological niches in estuarine planktonic blooms of phototrophs. The water column showed steep gradients of oxygen, pH, sulfate, sulfide, and salinity. The upper part of the bloom was dominated by aerobic phototrophic Cyanobacteria, the middle and lower parts by anoxygenic purple sulfur bacteria (Chromatiales) and green sulfur bacteria (Chlorobiales), respectively. We show stable coexistence of phototrophic lineages from five bacterial phyla and present metagenome-assembled genomes (MAGs) of two uncultured Chlorobaculum and Prosthecochloris species. In addition to genes involved in sulfur oxidation and photopigment biosynthesis the MAGs contained complete operons encoding for terminal oxidases. The metagenomes also contained numerous contigs affiliating with Microviridae viruses, potentially affecting Chlorobi. Our data suggest a short sulfur cycle within the bloom in which elemental sulfur produced by sulfide-oxidizing phototrophs is most likely reduced back to sulfide by Desulfuromonas sp. CONCLUSIONS: The release of sulfide creates a habitat selecting for anoxygenic sulfur-oxidizing phototrophs, which in turn create a niche for sulfur reducers. Strong syntrophism between these guilds apparently drives a short sulfur cycle that may explain the rapid development of the bloom. The fast growth and high biomass yield of Chlorobi-affiliated organisms implies that the studied lineages of green sulfur bacteria can thrive in hypoxic habitats. This oxygen tolerance is corroborated by oxidases found in MAGs of uncultured Chlorobi. The findings improve our understanding of the ecology and ecophysiology of anoxygenic phototrophs and their impact on the coupled biogeochemical cycles of sulfur and carbon.

Recent advances in functional research in Giardia intestinalis.

This review considers current advances in tools to investigate the functional biology of Giardia, it's coding and non-coding genes, features and cellular and molecular biology. We consider major gaps in current knowledge of the parasite and discuss the present state-of-the-art in its in vivo and in vitro cultivation. Advances in in silico tools, including for the modelling non-coding RNAs and genomic elements, as well as detailed exploration of coding genes through inferred homology to model organisms, have provided significant, primary level insight. Improved methods to model the three-dimensional structure of proteins offer new insights into their function, and binding interactions with ligands, other proteins or precursor drugs, and offer substantial opportunities to prioritise proteins for further study and experimentation. These approaches can be supplemented by the growing and highly accessible arsenal of systems-based methods now being applied to Giardia, led by genomic, transcriptomic and proteomic methods, but rapidly incorporating advanced tools for detection of real-time transcription, evaluation of chromatin states and direct measurement of macromolecular complexes. Methods to directly interrogate and perturb gene function have made major leaps in recent years, with CRISPr-interference now available. These approaches, coupled with protein over-expression, fluorescent labelling and in vitro and in vivo imaging, are set to revolutionize the field and herald an exciting time during which the field may finally realise Giardia's long proposed potential as a model parasite and eukaryote.

The Tail of Kinesin-14a in Giardia Is a Dual Regulator of Motility.

Kinesin-14s are microtubule-based motor proteins that play important roles in mitotic spindle assembly [1]. Ncd-type kinesin-14s are a subset of kinesin-14 motors that exist as homodimers with an N-terminal microtubule-binding tail, a coiled-coil central stalk (central stalk), a neck, and two identical C-terminal motor domains. To date, no Ncd-type kinesin-14 has been found to naturally exhibit long-distance minus-end-directed processive motility on single microtubules as individual homodimers. Here, we show that GiKIN14a from Giardia intestinalis [2] is an unconventional Ncd-type kinesin-14 that uses its N-terminal microtubule-binding tail to achieve minus-end-directed processivity on single microtubules over micrometer distances as a homodimer. We further find that although truncation of the N-terminal tail greatly reduces GiKIN14a processivity, the resulting tailless construct GiKIN14a-Δtail is still a minimally processive motor and moves its center of mass via discrete 8-nm steps on the microtubule. In addition, full-length GiKIN14a has significantly higher stepping and ATP hydrolysis rates than does GiKIN14a-Δtail. Inserting a flexible polypeptide linker into the central stalk of full-length GiKIN14a nearly reduces its ATP hydrolysis rate to that of GiKIN14a-Δtail. Collectively, our results reveal that the N-terminal tail of GiKIN14a is a de facto dual regulator of motility and reinforce the notion of the central stalk as a key mechanical determinant of kinesin-14 motility [3].

A standardized kinesin nomenclature.

In recent years the kinesin superfamily has become so large that several different naming schemes have emerged, leading to confusion and miscommunication. Here, we set forth a standardized kinesin nomenclature based on 14 family designations. The scheme unifies all previous phylogenies and nomenclature proposals, while allowing individual sequence names to remain the same, and for expansion to occur as new sequences are discovered.

Cell cycle synchrony in Giardia intestinalis cultures achieved by using nocodazole and aphidicolin.

Giardia intestinalis is a ubiquitous intestinal protozoan parasite and has been proposed to represent the earliest diverging lineage of extant eukaryotes. Despite the importance of Giardia as a model organism, research on Giardia has been hampered by an inability to achieve cell cycle synchrony for in vitro cultures. This report details successful methods for attaining cell cycle synchrony in Giardia cultures. The research presented here demonstrates reversible cell cycle arrest in G(1)/S and G(2)/M with aphidicolin and nocodazole, respectively. Following synchronization, cells were able to recover completely from drug treatment and remained viable and maintained synchronous growth for 6 h. These techniques were used to synchronize Giardia cultures to increase the percentages of mitotic spindles in the cultures. This method of synchronization will enhance our ability to study cell cycle-dependent processes in G. intestinalis.