With eight flagella of four different lengths, the parasitic protist
is an ideal model to evaluate flagellar assembly and length regulation. To determine how four different flagellar lengths are ...maintained, we used live-cell quantitative imaging and mathematical modeling of conserved components of intraflagellar transport (IFT)-mediated assembly and kinesin-13-mediated disassembly in different flagellar pairs. Each axoneme has a long cytoplasmic region extending from the basal body, and transitions to a canonical membrane-bound flagellum at the 'flagellar pore'. We determined that each flagellar pore is the site of IFT accumulation and injection, defining a diffusion barrier functionally analogous to the transition zone. IFT-mediated assembly is length-independent, as train size, speed, and injection frequencies are similar for all flagella. We demonstrate that kinesin-13 localization to the flagellar tips is inversely correlated to flagellar length. Therefore, we propose a model where a length-dependent disassembly mechanism controls multiple flagellar lengths within the same cell.
The evolution of the nucleus, the defining feature of eukaryotic cells, was long shrouded in speculation and mystery. There is now strong evidence that nuclear pore complexes (NPCs) and nuclear ...membranes coevolved with the endomembrane system, and that the last eukaryotic common ancestor (LECA) had fully functional NPCs. Recent studies have identified many components of the nuclear envelope in living Opisthokonts, the eukaryotic supergroup that includes fungi and metazoan animals. These components include diverse chromatin-binding membrane proteins, and membrane proteins with adhesive lumenal domains that may have contributed to the evolution of nuclear membrane architecture. Further discoveries about the nucleoskeleton suggest that the evolution of nuclear structure was tightly coupled to genome partitioning during mitosis.
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.
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Naegleria's genome sequence suggests a versatile and complex ancestral eukaryote ► The genome encodes both aerobic and anaerobic hydrogen-producing metabolism ► We predict novel evolutionarily conserved genes associated with amoeboid motility ► Genomic comparisons identify 4133 protein families likely ancestral to all eukaryotes
Hirst et al. used a TOG-domain-based affinity-purification approach to reconstitute and define the in vitro dynamics of blood-stage Plasmodium falciparum αβ-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.
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.
Giardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of nonhomologous end joining and ...markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia. Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disk MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than 5 d longer than defects associated with transient morpholino-based knockdown. By expressing two guide RNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disk defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems.
, 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.
Giardia lamblia is a flagellated, unicellular parasite of mammals infecting over one billion people worldwide. Giardia's two-stage life cycle includes a motile trophozoite stage that colonizes the ...host small intestine and an infectious cyst form that can persist in the environment. Similar to many eukaryotic cells, Giardia contains several complex microtubule arrays that are involved in motility, chromosome segregation, organelle transport, maintenance of cell shape and transformation between the two life cycle stages. Giardia trophozoites also possess a unique spiral microtubule array, the ventral disc, made of approximately 50 parallel microtubules and associated microribbons, as well as a variety of associated proteins. The ventral disc maintains trophozoite attachment to the host intestinal epithelium. With the help of a combined SEM/microtome based slice and view method called 3View® (Gatan Inc., Pleasanton, CA), we present an entire trophozoite cell reconstruction and describe the arrangement of the major cytoskeletal elements. To aid in future analyses of disc-mediated attachment, we used electron-tomography of freeze-substituted, plastic-embedded trophozoites to explore the detailed architecture of ventral disc microtubules and their associated components. Lastly, we examined the disc microtubule array in three dimensions in unprecedented detail using cryo-electron tomography combined with internal sub-tomogram volume averaging of repetitive domains. We discovered details of protein complexes stabilizing microtubules by attachment to their inner and outer wall. A unique tri-laminar microribbon structure is attached vertically to the disc microtubules and is connected to neighboring microribbons via crossbridges. This work provides novel insight into the structure of the ventral disc microtubules, microribbons and associated proteins. Knowledge of the components comprising these structures and their three-dimensional organization is crucial toward understanding how attachment via the ventral disc occurs in vivo.
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.
Molecular evolutionary studies of eukaryotes have relied on a sparse collection of gene sequences that do not represent the full range of eukaryotic diversity in nature. Anaerobic microbes, ...particularly, have had little representation in phylogenetic studies. Such organisms are the least known of eukaryotes and probably are the most phylogenetically diverse. To provide fresh perspective on the natural diversity of eukaryotes in anoxic environments and also to discover novel sequences for evolutionary studies, we conducted a cultivation-independent, molecular phylogenetic survey of three anoxic sediments, including both freshwater and marine samples. Many previously unrecognized eukaryotes were identified, including representatives of seven lineages that are not specifically related to any known organisms at the kingdom-level and branch below the eukaryotic "crown" radiation of animals, plants, fungi, stramenopiles, etc. The survey additionally identified new sequences characteristic of known ecologically important eukaryotic groups with anaerobic members. Phylogenetic analyses with the new sequences enhance our understanding of the diversity and pattern of eukaryotic evolution.
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