Eight unique basal bodies in the multi-flagellated diplomonad Giardia lamblia

Giardia lamblia is an intestinal parasitic protist that causes significant acute and chronic diarrheal disease worldwide. Giardia belongs to the diplomonads, a group of protists in the supergroup Excavata. Diplomonads are characterized by eight motile flagella organized into four bilaterally symmetric pairs. Each of the eight Giardia axonemes has a long cytoplasmic region that extends from the centrally located basal body before exiting the cell body as a membrane-bound flagellum. Each basal body is thus unique in its cytological position and its association with different cytoskeletal features, including the ventral disc, axonemes, and extra-axonemal structures. Inheritance of these unique and complex cytoskeletal elements is maintained through basal body migration, duplication, maturation, and their subsequent association with specific spindle poles during cell division. Due to the complex composition and inheritance of specific basal bodies and their associated structures, Giardia may require novel basal body-associated proteins. Thus, protists such as Giardia may represent an undiscovered source of novel basal body-associated proteins. The development of new tools that make Giardia genetically tractable will enable the composition, structure, and function of the eight basal bodies to be more thoroughly explored.


Background
Giardia lamblia is a single-celled protistan parasite that causes acute and chronic diarrheal disease, primarily in developing countries with inadequate sanitation and water treatment [1,2]. The life cycle of Giardia includes two stages: the proliferative pathogenic trophozoite and the dormant infective cyst. Giardia belongs to the diplomonads, a group of protists in the supergroup Excavata whose defining cytological characteristics include eight motile flagella and two nuclei [3]. The discovery of Giardia is attributed to Antonie van Leewenhoek, [4] who in 1681 observed teardrop-shaped flagellates in his own stool. More than 300 years later, our understanding of Giardia cytoskeletal biology remains rudimentary. This deficit is primarily due to a lack of tools for genetic manipulation; however, improved cytological descriptions and increasing numbers of genomes of Giardia species and other related diplomonads are aiding comparisons of the cytoskeletal biology of Giardia to other diverse flagellated protists [5].
A recent classification scheme categorized all known eukaryotes into six primary lineages or supergroups: Opisthokonts (e.g., animals, fungi), Amoebozoa, Archaeplastida (e.g., plants and green algae), Rhizaria, Chromalveolata, and the Excavata [6,7]. Excavate protists have been proposed to be a basal lineage of eukaryotes, closest to the common ancestor of all extant eukaryotes [8,9]. Yet, the evolutionary diversity within the Excavata represents genetic distances greater than those between plants, animals, and fungi [6]. Molecular phylogenetic support for the monophyly of this group is controversial [10]. All known excavates have flagellated life cycle stages and, as a group, excavates are defined by the presence of posteriorly directed flagella and flagellar root structures associated with the basal bodies [11]. However, excavate biology is quite varied, and diversity within this group encompasses free-living, commensal, and parasitic forms of the following types of protists: Fornicata (diplomonads, oxymonads, and retortamonads), Parabasalia, Euglenozoa (both euglenids and kinetoplastids), Heterolobosea, Jakobida, and Preaxostyla.
The swimming form of Giardia, or the "trophozoite, " has eight flagella that retain the canonical "9 + 2" structure of a motile flagellum [12]. Each flagellum also has radial spokes, dynein arms, and outer doublet and central pair microtubules [13,14]. The eight flagella are organized into four bilaterally symmetrical pairs: the anterior, the caudal, the posteriolateral, and the ventral (Fig. 1). The basal bodies for all flagella are located in the anterior of the cell between the two nuclei. Each flagellar pair differs in its cytological position within the trophozoite and in its association with ancillary structures. The coordinated beating of Giardia's eight motile flagella results in complex movements essential for motility and cell division, and may aid in parasite attachment to the host gut epithelium [15,16]; however, not all flagellar pairs have characteristic flagellar waveforms [15].
In general, eukaryotic flagella extend from a basal body or centriole and are surrounded by a specialized flagellar membrane after they project from the cell surface. In contrast to other flagellated protists, each of the eight Giardia axonemes has a long cytoplasmic region that extends from a centrally located basal body before exiting the cell body as a membrane-bound flagellum ( Fig. 1 and see [16]). The ratio of the length of the cytoplasmic region to the membrane-bound portion varies between each flagellar pair (e.g., over two-thirds of the length of the caudal axonemes is in the cytoplasmic region, whereas only a third of the anterior axoneme is cytoplasmic). The anterior axonemes cross over the ventral disc spiral before exiting on the right and left sides of the anterior region of the cell. The distance from the exit point from the cell body to the flagellar tip is about 12 µm. Running longitudinally along the anterior-posterior axis of the cell, the two caudal axonemes exit the cell body and extend about 7 µm at the posterior end. The ventral axonemes exit and extend about 14 µm on the ventral side in the "lateral shield" region posterior to the disc. The posteriolateral axonemes angle outward at the lower third of the cell body, extending about 8 µm from the cell body. Electrondense "ciliary pockets" are found at the regions where each flagellum exits the cell body [17]. This review presents detailed findings concerning the structure, duplication, and migration of the eight unique Giardia basal bodies during the parasite life cycle.

Structure and positions of the eight basal bodies
The eight flagellar basal bodies that nucleate the axonemes are positioned between the two nuclei in the cell interior (Fig. 1). The long cytoplasmic regions of the axonemes are not extended transition zones; basal body transition zones are restricted to small regions proximal to the basal bodies rather than to the entire cytoplasmic axoneme [17]. The anterior basal bodies are located toward the anterior ends of the two nuclei and oriented toward the anterior end of the cell. Basal bodies that nucleate the ventral, caudal, and posteriolateral axonemes are positioned posteriorly below the two anterior basal bodies and are oriented toward the posterior of the cell. Interphase trophozoites lack both barren and probasal bodies [18].
Flagellar and basal body proteomics in Giardia have contributed to our overall understanding of flagellar structure and evolution; however, the selective isolation of axonemes or basal bodies from the extensive cytoskeleton in Giardia has proved to be challenging [19]. Canonical basal body-associated proteins (e.g., centrin, delta-tubulin and epsilon tubulin) and some components of the BBSome are present in the Giardia genome (Table 1). Centrin localizes to two distinct clusters adjacent to the two nuclei during interphase, colocalizing with the flagellar basal bodies [20]. Consistent with observations in other flagellated cells, gamma-tubulin also localizes to flagellar basal bodies during interphase; however, gamma-tubulin localization is restricted only to flagella that are newly produced during cell division [18].
Notably, more than 1000 hypothetical proteins (e.g., those lacking significant similarity to proteins in other organisms) have been identified from the Giardia genome, and this genetic novelty is reflected in the analyses of basal body [19] and cytoskeletal proteomes [21]. Proteins localizing to basal bodies may be structural components or may merely dock at basal bodies before they are transported to other parts of the cell. Many known basal body proteins are confirmed in the Giardia genome by homology or from localization studies (see centrin (GL50803_6744) and caltractin (GL50803_104685) in Table 1 and imaged in Fig. 2). Other proteins identified as basal body proteins by comparative proteomics lack basal body localization in Giardia (e.g., FAP52 (GL50803_15956) and PACRG1 (GL50803_15455), see Table 1), or localize to other cytoskeletal structures in addition to the basal bodies (e.g., GL50803_8557 and GL50803_29796, see Table 1, and imaged in Fig. 2). Furthermore, Giardia has proteins that localize to some or all basal bodies, but lack homology to known basal body proteins (e.g., GL50803_15193 and GL50803_6254, see Table 1 and imaged in Fig. 2). In total, there are 49 proteins that localize to some or all Giardia basal bodies. Five components of the BBSome are present in Giardia, although localization of these to basal bodies has not been confirmed (Table 1).

Additional basal body structures or accessories
Additional basal body structures or accessories have not been identified in Giardia. In trophozoites, however, complex axoneme-associated structures are associated with each flagellar pair ( [22] and Fig. 1). These extraaxonemal structures confer a unique structural identity to each flagellar pair; thus, each pair has a unique functional role in parasite motility [15]. Giardia axonemeassociated structures include the "marginal plate" that is associated with the anterior axonemes [22]; the fin structures on the ventral axonemes [23]; the electron dense material on the posteriolateral axonemes, and the "caudal complex" or "funis" microtubules that surround the caudal axonemes. Undiscovered basal body structures may further distinguish and define each axoneme.
The ventral disc microtubules nucleate from the caudal basal bodies and extend to form the right-handed spiral array that mediates attachment to the host intestine during infection. Over 50 proteins associate with the ventral disc (e.g., disc-associated proteins or "DAPS") as seen in proteomic and localization analyses [21].

Origins of the eight basal bodies
The eight axonemal basal bodies are inherited by each daughter cell during a mitotic division in trophozoites that includes two spindles and four spindle poles [20]. In some flagellates, such as Chlamydomonas, flagella are resorbed at the onset of mitosis and the basal bodies (as centrioles) are recruited to function as part of the mitotic spindle poles [24]. Unlike Chlamydomonas, both centrin localization [25,26] and ultrastructural studies [20] indicate that all eight flagella are retained during mitosis, and the flagella and their associated basal bodies migrate to the four spindle poles. Two of the eight flagellar basal bodies associate with each of the four spindle poles during the division of the two nuclei [20]. One basal body at each spindle pole acts as the central structural component of the MTOC, while a second basal body is observed at the periphery of the spindle pole region with an associated axoneme [20]. This peripheral basal body may play an indirect role in spindle nucleation, and its association with the spindle pole may ensure proper segregation to the daughter cells.
During the onset of prophase and spindle assembly, the number of centrin foci increases from two to four due the duplication or separation of the basal body tetrads [18,20]. Spindle microtubule assembly begins with the appearance of microtubules near the duplicated basal bodies; these microtubules extend around each nucleus and continue to elongate as the nuclei migrate  Evidence for candidate basal body proteins is based on homology to known basal body proteins in other organisms (HOM) [9,41,42], enrichments of Giardia basal body proteins in proteomic analyses (PRO) [19,41] and/or subcellular localization using either heterologous antibodies (IFA) [25-27, 43, 44] or epitope-tagging (EPI) [19,41]. Candidate basal body proteins identified with an ongoing, publically available, Giardia C-terminal GFP-tagging project for EuPATHDB [45] in our laboratory are also presented (GFP) to the cell midline. Centrin foci are found at the sites of spindle nucleation during nuclear migration and move to the periphery of the nuclei as the spindle microtubules elongate. Importantly, centrin localizes to only the four basal bodies associated with the spindle poles [18]. Spindle microtubule elongation ceases by the end of prophase, when each spindle surrounds each nucleus and kinetochore microtubules of the spindle capture chromosomes through polar openings [20]. Upon completion of nuclear migration in prophase [27], the microtubules surrounding each nucleus form two independent bipolar spindles that are stacked in the dorsal-ventral plane in metaphase. At this time, centrin is localized to the four spindle poles, where it will remain throughout anaphase A and anaphase B. Gamma-tubulin staining returns during anaphase, but is limited to four of the eight basal bodies and was not observed at the spindle poles [27]. During telophase, the centrin foci at each spindle pole move  Fig. 1). Most notably, the anterior flagella move along the cell periphery from one side of the cell to the other, which is in accordance with the migration of the associate basal body to the spindle poles [18]. Gamma-tubulin immunostaining of basal bodies is reported to disappear early in prophase and reappears during later mitotic stages. This behavior is unique among flagellated protists and resembles what is seen during the reassembly of functional centrosomes in animal cells. Furthermore, these observations suggest a possible association of gamma-tubulin and the kinetochore complex of Giardia [27].

Basal body behavior during encystation and excystation
Giardia cysts are ingested from contaminated water sources and the parasite completes its life cycle in the small intestine of the host. After ingestion, the cyst transforms into a flagellated trophozoite that attaches to the intestinal villi and subsequently colonizes the small intestine. Attachment allows trophozoites to resist peristaltic flow in the gut [16] and is mediated by an elaborate microtubule structure termed the ventral disc [28,29]. Flagellated trophozoites later develop into infectious cysts that are excreted and persist in the environment, disseminating the infection to other hosts [28,30].
The cyst stage contains internalized flagella and is characterized by a thick cyst wall that enables resistance to environmental stresses [31]. During encystation the two nuclei divide without cytokinesis to form tetra-nucleated cysts with 8N ploidy [32]. A subsequent round of DNA replication increases the ploidy of mature cysts to 16N. Throughout the process of encystation, the arrangement of the flagellar apparatus in the majority of cysts is the same as what is observed in the interphase cell. Only a single flagellar apparatus, comprised of four flagellar pairs with associated basal bodies, is present and there is no duplication of either the flagellar apparatus or basal bodies. Ultrastructural analysis of mature cysts shows that basal body tetrads are arranged and localized between the pairs of daughter nuclei, with one nucleus from each pair associated with a basal body tetrad [31].
After a suitable host ingests the Giardia cyst, the parasite undergoes excystation in the small intestine to differentiate into the flagellated "excyzoite. " During excystation, the six flagella directed toward the posterior of the cell, namely the caudal, posteriolateral, and ventral flagella, protrude through the cyst wall allowing the cell to squeeze through this opening. The newly emerged excyzoite contains a single flagellar apparatus with basal bodies positioned similarly to the interphase trophozoite. While few molecular details are known about excystation, it is thought that the excyzoite undergoes two rapid, consecutive cell divisions to produce four trophozoites [31]. In the first division, the basal body tetrads segregate and localize between the pairs of nuclei, and the excyzoite undergoes cytokinesis but not nuclear division. The four intact nuclei resulting from this division are then segregated so that each daughter cell receives two nuclei with previously replicated DNA. Each daughter cell enters into mitotic division without DNA replication to form two trophozoites with two nuclei and two basal body tetrads that nucleate the eight flagella.

Notable findings
Giardia's eight basal bodies have a unique inheritance pattern in daughter cells. In the interphase trophozoite, eight basal bodies are arranged into two tetrads and each basal body pair is associated with a distinct flagellar pair. When the trophozoite is viewed dorsally, the left tetrad consists of anterior/ventral and caudal/posteriolateral basal bodies, while the right tetrad consists of caudal/ ventral and anterior/posteriolateral basal bodies (Fig. 1). The polarity of each daughter cell is thought to be determined through the association of axonemal basal bodies with the dividing nuclei [20]. During division, eight parent flagella persist and are inherited in a semi-conservative manner, with each progeny receiving four flagella from the parent cell while four complementary flagella are assembled de novo in each cell. Importantly, these de novo flagella lack polyglycylated tubulin, which makes this post-translational modification a convenient marker of the parent (inherited) flagella. Thus, the organization of basal body pairs in Giardia informs our understanding of the mitotic distribution of the eight flagella to two daughter cells.
Due to the inheritance and de novo assembly of specific flagella in daughter cells, a multigenerational division cycle has been proposed wherein the relative age of a flagellar axoneme is different based on its anatomical position in the trophozoite [18]. The flagella of some other protists are known to undergo a similar maturation process that takes more than one cell cycle [33], mirroring the behavior of centrioles in metazoans (reviewed in [34]). Based on immunostaining with a polyglycylated tubulin antibody to visualize parental axonemes and an acetylated tubulin antibody to visualize daughter axonemes, eight parental (old) flagella are retained and eight new flagella are synthesized each cell division cycle [18]. Before mitosis is completed, flagellar and basal body duplication occurs [18,20]. Flagellar regeneration begins in anaphase with short flagella (presumably the new ventral and posteriolateral pairs) emerging from the spindle poles [18,20]. While specific molecular markers have not been used to track each flagellar pair to confirm their identity during division [18], the full length parental anterior axonemes are proposed to become the right caudal axonemes in the new daughter cells. Parental right caudal axonemes are then proposed to become the left caudal axonemes. Thus each daughter cell inherits a full complement of eight axonemes and associated basal bodies-four parental (old), and four newly duplicated each generation [18,20].
The division of the caudal axonemes and their associated basal bodies also has notable implications for the de novo nucleation and assembly of the daughter ventral discs. After the daughter nuclei are partitioned and the caudal flagellar basal bodies have been repositioned between the two nuclei [18], two new dorsal daughter ventral discs are assembled during telophase. The parental ventral disc is not disassembled until later in the cell cycle. Thus, the caudal basal bodies nucleate the caudal axonemes and also determine the site of ventral disc assembly, establishing the polarity of the new daughter cells. The left caudal flagellum alone has been proposed to nucleate the spiral MT arrays that form the basis of the ventral disc [22]; however, recent work shows that both caudal basal bodies nucleate the ventral disc MTs (see Fig. 1). Live imaging is required to confirm flagellar migration during cell division and ultimately characterize the forces and mechanisms involved in flagellar maturation and daughter disc nucleation. The timing and mechanism by which the extra-axonemal-associated structures (e.g., marginal plate, caudal complex or funis) are assembled during cell division also remains unclear [17].

Conclusions
While some cellular functions of the cytoskeleton are conserved across eukaryotes (e.g., mitosis and cytokinesis), the molecular components and pathways underlying these processes have extensive variation in less well-studied eukaryotic groups [35]. Based on their evolutionary distances and the complex composition of their diverse cytoskeletal structures, excavate protists such as Giardia may represent an undiscovered reservoir of novel basal body-associated proteins.
The composition, fundamental structure, and functional properties of the eight Giardia basal bodies remain to be explored. We currently lack high-resolution images that distinguish the differences between each individual basal body. We also lack molecular characterizations of any basal body-associated structures during interphase, flagellar duplication, or mitotic division. Giardia basal bodies have unique identities and spatial positions in the cell, and are likely of differing ages due to the mode of basal body inheritance. We have no structural detail that might provide clues linking position or age to particular basal bodies. In addition to trophozoite mitosis, both encystation and excystation are two life cycle transitions that could illuminate basal body duplication, migration, and partitioning into daughter cells. Initial cytological and ultrastructural analyses of the basal bodies and flagellar structures should be updated and revisited at higher resolution using current state of the art fixation techniques and imaging methods, as have been recently used in the analysis of the ventral disc.
Due to our current inability to genetically manipulate Giardia, studying giardial protein function is challenging [29,36]. A complete genome and reverse genetic tools to generate dominant negative mutants [37,38] or antisense [39] and morpholino-based knockdowns [40] have permitted the identification and characterization of novel structural components and the mechanisms underlying Giardia's cytoskeletal dynamics [40]. Current or future genetic methods could be applied to the study of basal body proteins, and the availability of a more complete inventory of basal body-localizing proteins in Giardia will facilitate these efforts.