Open Access

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

Cilia20165:21

https://doi.org/10.1186/s13630-016-0042-4

Received: 30 January 2016

Accepted: 20 April 2016

Published: 4 July 2016

Abstract

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.

Keywords

Giardia lamblia Basal bodyAxonemesFlagella

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].
Fig. 1

Giardia microtubule cytoskeleton emphasizing interphase basal body positions and migration during mitosis. A schematic representation of the characteristic teardrop shape of Giardia and the cytoskeletal features of the cell is shown in panel (a), including: the basal bodies (bb), four pairs of flagella (afl = anterior, cfl = caudal, pfl = posteriolateral, vfl = ventral), median body (mb), and ventral disc (vd). Anti-tubulin immunostaining reveals the cytoplasmic lengths of all eight flagella, which begin at the basal bodies located between the two nuclei labeled with DAPI (b). Panel c shows a schematic of the basal body tetrads arrangements and their association with specific flagellar axonemes (A/A’ = anterior, C/C’ = caudal, P/P’ = posteriolateral, V/V’ = ventral, N = nuclei). A transmission electron micrograph (TEM) of the anterior region of the cell in panel d shows the organization the basal bodies and their associated flagellar axonemes (Abb/A’bb = anterior basal body, Cbb/C’bb = caudal basal body, Pbb = posteriolateral basal body, and AAX = anterior axonemes). The ventral disc is also nucleated from the caudal basal bodies. A TEM cross section of a mitotic cell in panel e shows the migration of basal bodies from their interphase position between the two nuclei (panel c) to the spindle poles, where they are associated with the spindle microtubules and flagellar axonemes (i.e., one anterior axoneme (AAX) is visible)

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. Electron-dense “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].
Table 1

Known and candidate Giardia basal body proteins including supporting evidence

GiardiaDB

Protein name

Protein family

PFAM

Basal body and/or other subcellular localization

Evidence

GL50803_6744

Centrin

Centrin

PF13499; PF13833

All basal bodies

HOM, IFA

GL50803_104685

Caltractin

Caltractin

PF13499

All basal bodies

HOM, IFA, GFP

GL50803_114218

Gamma-tubulin

Tubulin

PF00091; PF03953

All basal bodies, cytoplasm

HOM, PRO, IFA, GFP

GL50803_5462

Delta-tubulin

Tubulin

PF00091

Cytoplasm

HOM, GFP

GL50803_6336

Epsilon-tubulin

Tubulin

PF00091

Cytoplasm

HOM, GFP

GL50803_17429

GCP2

Spc97 Spc98

PF04130

n.d.

HOM

GL50803_12057

GCP3

Spc97_Spc98

PF04130

All basal bodies, median body, cytoplasmic caudal, posteriolateral, and anterior axonemes

HOM, GFP

GL50803_104150

Kinase, PLK

Ser/Thr kinase domain, POLO box domain

PF00069, PF00659

All basal bodies

EPI

GL50803_4689

Hypothetical protein

None

None

All basal bodies

PRO, EPI

GL50803_4692

Hypothetical protein

None

None

All basal bodies

PRO, EPI

GL50803_41512

Flagella associated protein Rib72

DUF1126 domain of unknown function

PF06565

All basal bodies, cytoplasm

PRO, EPI

GL50803_114546

Hypothetical protein

None

None

All basal bodies, all cytoplasmic axonemes

GFP

GL50803_3582

DUF390 domain containing protein

DUF390 domain of unknown function

PF04094

All basal bodies, all cytoplasmic axonemes

GFP

GL50803_8974

Hypothetical protein

None

None

All basal bodies, all cytoplasmic axonemes, median body, ciliary pocket

GFP

GL50803_16935

Hypothetical protein

None

None

All basal bodies, all flagella, ventral disc lateral crest, median body

GFP

GL50803_15218

WD-40 repeat protein

WD40 repeats

PF00400

All basal bodies, all flagella, ventral disc lateral crest, median body

PRO, IFA, GFP

GL50803_9117

CAMP-dependent protein kinase regulatory chain

Cyclic nucleotide binding domain

PF00027

All basal bodies, anterior axonemes, caudal axonemes

EPI

GL50803_11214

Kinase, AGC PKA

Kinase, AGC PKA

PF00069

All basal bodies, anterior axonemes, caudal axonemes

EPI

GL50803_16202

Axoneme central apparatus protein PF16/SPAG6

Central pair associated protein

PF00514

All basal bodies, anterior axonemes, posteriorlateral axonemes, caudal axonemes, median body

PRO, IFA, EPI

GL50803_11867

RIB43A

RIB43A

PF05914

All basal bodies, anterior axonemes, posteriorlateral axonemes, median body

EPI

GL50803_7351

Hypothetical protein

None

None

All basal bodies, ciliary pocket, cytoplasmic ventral, caudal, posteriolateral, and anterior axonemes

GFP

GL50803_24412

Ankyrin-repeat domain containing protein

Ankyrin-repeat domains

PF12796

All basal bodies, cytoplasmic anterior and posteriolateral axonemes

GFP

GL50803_10460

Hypothetical protein

None

None

All basal bodies, cytoplasmic anterior axonemes, median body

GFP

GL50803_17586

Ankyrin-repeat domain containing protein

Ankyrin-repeat domains

PF12796

All basal bodies, cytoplasmic anterior, posteriolateral and ventral axonemes

GFP

GL50803_7192

FWWh domain containing protein

FWWh domain of unknown function

PF14922

All basal bodies, cytoplasmic caudal axonemes, posteriolateral and anterior axonemes

GFP

GL50803_8557

Hypothetical protein

None

None

All basal bodies, cytoplasmic caudal axonemes, posteriolateral and anterior axonemes

GFP

GL50803_102455

GiKIN6a

Kinesin-6

PF00225

All basal bodies, cytoplasmic posteriolateral and anterior axonemes, median body, ventral disc

GFP

GL50803_11775

Kinase, ankyrin-repeat domain containing protein

Ser/Thr kinase domain, ankyrin-repeat domains

PF00069, PF1279

All basal bodies, cytoplasmic posteriolateral and caudal axonemes

GFP

GL50803_15219

Macoilin domain containing protein

Macoilin

PF09726

All basal bodies, cytoplasmic posteriolateral axonemes, cytoplasm

GFP

GL50803_15446

Hypothetical protein

None

None

All basal bodies, cytoplasmic posteriolateral axonemes, median body

GFP

GL50803_6709

Hypothetical protein

None

None

All basal bodies, median body, all flagella, ventral disc lateral crest

GFP

GL50803_92498

Kinase, NEK

Kinase, NEK

PF00069

All basal bodies, median body, anterior axonemes, posteriorlateral axonemes, caudal axonemes, ventral disc

EPI

GL50803_17154

RRP7 domain containing protein

Ribosomal RNA-processing protein 7 (RRP7) domain

PF12923

All basal bodies, median body, cytoplasmic anterior axonemes, caudal axonemes, posteriolateral axonemes

GFP

GL50803_4624

DUF4490 domain containing protein

DUF4490

PF14892

All basal bodies, median body, cytoplasmic caudal, ventral, posteriolateral and anterior axonemes

GFP

GL50803_13352

T-complex protein-10/Sas-4/CENPJ

T complex protein 10 family C terminal domain

PF07202

All basal bodies, median body, cytoplasmic posteriolateral axonemes, ciliary pocket

PRO, IFA, GFP

GL50803_10232

LRR repeat domain containing protein

CENP-F_leu_zip leucine-rich repeats

PF10473

All basal bodies, median body, ventral disc

GFP

GL50803_31671

RPB7 SHS2 domain containing protein

N terminal SHS2 domain of RNA pol II subunit RPB7

PF03876

All basal bodies, nuclei, cytoplasmic posteriolateral and anterior axonemes

GFP

GL50803_16220

Ankyrin-repeat domain containing protein

Ankyrin-repeat domains

PF12796

All basal bodies, plasma membrane, cytoplasm

GFP

GL50803_16973

Hypothetical protein

None

None

All basal bodies, posteriorlateral axonemes, median body

PRO, IFA

GL50803_17096

Ankyrin-repeat domain containing protein

Ankyrin-repeat domains

PF12796

All basal bodies, ventral disc lateral crest

GFP

GL50803_16424

MiflIP domain containing protein

Mlf1IP

PF10248

All basal bodies, ventral disc lateral crest, cytoplasmic anterior axonemes

GFP

GL50803_17097

Ankyrin-repeat domain containing protein

Ankyrin-repeat domains

PF12796

All basal bodies, ventral disc lateral crest, cytoplasmic anterior axonemes, median body

GFP

GL50803_101326

Hypothetical protein

None

None

All basal bodies, ventral disc lateral crest, cytoplasmic caudal axonemes

GFP

GL50803_15499

DUF390 domain containing protein

DUF390 domain of unknown function

PF04094

All basal bodies, ventral disc lateral crest, cytoplasmic posteriolateral axonemes

GFP

GL50803_5568

DUF866 domain containing protein

DUF866

PF05907

All basal bodies, ventral disc lateral crest, ventral axonemes

GFP

GL50803_5010

Ser/Thr phosphatase PP2A-2 catalytic subunit

Metallo-dependent phosphatases

PF00149

All basal bodies, ventral disc, anterior axonemes, posteriorlateral axonemes, caudal axonemes

PRO, IFA

GL50803_17563

Kinase, CMGC MAPK

Kinase, CMGC MAPK

PF00069

All basal bodies, ventral disc, caudal axonemes, median body

EPI

GL50803_16279

Kinase, NEK

Kinase, NEK

PF00069

All basal bodies, ventral disc, caudal axonemes, posteriorlateral axonemes

EPI

GL50803_5358

Aurora kinase

Aurora kinase

PF00069

All basal bodies, ventral disc, nuclei, anterior axonemes, median body

EPI

GL50803_15193

Hypothetical protein

None

None

Some basal bodies

PRO, IFA, GFP

GL50803_29796

Hypothetical protein

None

None

Some basal bodies, anterior flagella, ventral flagella, median body

GFP

GL50803_6254

Hypothetical protein

None

None

Some basal bodies, cytoplasmic posteriolateral axonomes

GFP

GL50803_15455

PACRG1

Parkin coregulated protein

PF10274

Cytoplasm, nuclei

HOM, GFP

GL50803_14048

EB1

EBl-like C terminal motif

PF03271

Nuclear membrane, median body

GFP

GL50803_15956

FAP52

WD40 repeats

PF00400

Plasma membrane, all axonemes

HOM, GFP

GL50803_5333

Calmodulin

EF-hand domain

PF13499

n.d.

HOM

GL50803_33762

POC1

WD repeat protein

PF00400

n.d.

HOM

GL50803_32375

POC18

None

None

n.d.

HOM

GL50803_13372

FAP45

TPH domain

PF13868

n.d.

HOM

GL50803_5167

VFL3

None

None

n.d.

HOM

GL50803_15248

Bub2

TBC domain

PF00566

n.d.

HOM

GL50803_8738

BBS1

BBS1

PF14779

n.d.

HOM

GL50803_23934

BBS2

None

None

n.d.

HOM

GL50803_10529

BBS4

TPR_1 Tetratricopeptide repeat

PF01515

n.d.

HOM

GL50803_8146

BBS5

DUF1448 domain of unknown function

PF07289

n.d.

HOM

GL50803_8508

BBS8

TPR_1 Tetratricopeptide repeat

PF01515

n.d.

HOM

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) [2527, 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)

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).
Fig. 2

Representative GFP-tagged basal body proteins in Giardia. Many known basal body proteins, including caltractin (a) and gamma-tubulin complex 3 (b), have been identified in the Giardia genome by homology and confirmed by GFP tagging to localize to the basal bodies. Giardia also has proteins that localize to some basal bodies (c, d) or all basal bodies (e), but lack homology to known basal body proteins. Further, proteins that localize to basal bodies and other structures, including the cell membrane (f), the cytoplasmic axonemes (g), and the median body (h), have also been identified

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 extra-axonemal structures confer a unique structural identity to each flagellar pair; thus, each pair has a unique functional role in parasite motility [15]. Giardia axoneme-associated 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 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 from their anaphase position near the cell periphery to their interphase position between each pair of nuclei.

Migration of basal bodies and nuclei may be coordinated events facilitated by centrin-dependent attachment of basal bodies to the nuclear envelope. Migration of the nuclei to the center of the cell during prophase displaces the flagellar basal bodies causing a dramatic rearrangement of the flagella ([18] and 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.

Abbreviation

MTOC: 

microtubule organizing center

Declarations

Authors’ contributions

SGM and SCD both conceived and wrote the manuscript. SGM prepared figures. Both authors read and approved the final manuscript.

Acknowledgements

This work was supported by NIH R01AI077571 to SCD, and SGM was supported by Training Grant Number T32-GM007377 from NIH-NIGMS. The authors thank Joel Mancuso and W. Z. Cande for providing unpublished TEM images of basal bodies. The authors thank Kari Hagen and Christopher Nosala for reading draft manuscripts and providing valuable comments.

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Microbiology and Molecular Genetics, University of California Davis

References

  1. Savioli L, Smith H, Thompson A. Giardia and Cryptosporidium join the ‘neglected diseases initiative’. Trends Parasitol. 2006;22(5):203–8.View ArticlePubMedGoogle Scholar
  2. Troeger H, et al. Effect of chronic Giardia lamblia infection on epithelial transport and barrier function in human duodenum. Gut. 2007;56(3):328–35.View ArticlePubMedPubMed CentralGoogle Scholar
  3. Feely D, Hoberton DV, Erlandsen SL. The biology of Giardia. In: Meyer EA, editor. Giardiasis, human parasitic diseases, vol. 3. Amsterdam: Elsevier; 1990. p. 11–50.Google Scholar
  4. Dobell C. Antony van Leeuwenhoek and his “Little animals” being some account of the father of protozoology and bacteriology and his multifarious discoveries in these disciplines; collected, translated, and edited, from his printed works, unpublished manuscripts, and contemporary records. London: J. Bale sons & Danielsson Ltd; 1932. p. vii,** *.Google Scholar
  5. Scholey JM. Intraflagellar transport motors in cilia: moving along the cell’s antenna. J Cell Biol. 2008;180(1):23–9.View ArticlePubMedPubMed CentralGoogle Scholar
  6. Hampl V, et al. Phylogenomic analyses support the monophyly of Excavata and resolve relationships among eukaryotic “supergroups”. Proc Natl Acad Sci USA. 2009;106(10):3859–64.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Adl SM, et al. The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J Eukaryot Microbiol. 2005;52(5):399–451.View ArticlePubMedGoogle Scholar
  8. Baldauf SL. The deep roots of eukaryotes. Science. 2003;300(5626):1703–6.View ArticlePubMedGoogle Scholar
  9. Morrison HG, et al. Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science. 2007;317(5846):1921–6.View ArticlePubMedGoogle Scholar
  10. Parfrey LW, et al. Broadly sampled multigene analyses yield a well-resolved eukaryotic tree of life. Syst Biol. 2010;59(5):518–33.View ArticlePubMedPubMed CentralGoogle Scholar
  11. Simpson AGB, Patterson DJ. The ultrastructure of Carpediemonas membranifera (Eukaryota) with reference to the “excavate hypothesis”. Eur J Protistol. 1999;35(4):353–70.View ArticleGoogle Scholar
  12. Manton IaC B. An electron microscope study of the spermatozoid of Sphagnum. J Exp Bot. 1952;3:265–75.View ArticleGoogle Scholar
  13. Clark JT, Holberton DV. Triton-labile antigens in flagella isolated from Giardia lamblia. Parasitol Res. 1988;74(5):415–23.View ArticlePubMedGoogle Scholar
  14. Carvalho KP, Monteiro-Leal LH. The caudal complex of Giardia lamblia and its relation to motility. Exp Parasitol. 2004;108(3–4):154–62.View ArticlePubMedGoogle Scholar
  15. Campanati L, et al. Video-microscopy observations of fast dynamic processes in the protozoon Giardia lamblia. Cell Motil Cytoskeleton. 2002;51(4):213–24.View ArticlePubMedGoogle Scholar
  16. Dawson SC, House SA. Life with eight flagella: flagellar assembly and division in Giardia. Curr Opin Microbiol. 2010;13(4):480–90.View ArticlePubMedGoogle Scholar
  17. Hoeng JC, et al. High-resolution crystal structure and in vivo function of a kinesin-2 homologue in Giardia intestinalis. Mol Biol Cell. 2008;19(7):3124–37.View ArticlePubMedPubMed CentralGoogle Scholar
  18. Nohynkova E, Tumova P, Kulda J. Cell division of Giardia intestinalis: flagellar developmental cycle involves transformation and exchange of flagella between mastigonts of a diplomonad cell. Eukaryot Cell. 2006;5(4):753–61.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Lauwaet T, et al. Mining the Giardia genome and proteome for conserved and unique basal body proteins. Int J Parasitol. 2011;41(10):1079–92.View ArticlePubMedPubMed CentralGoogle Scholar
  20. Sagolla MS, Dawson SC, Mancuso JJ, Cande WZ. Three-dimensional analysis of mitosis and cytokinesis in the binucleate parasite Giardia intestinalis. J Cell Sci. 2006;119(Pt 23):4889–900.View ArticlePubMedGoogle Scholar
  21. Hagen KD, et al. Novel structural components of the ventral disc and lateral crest in Giardia intestinalis. PLoS Negl Trop Dis. 2011;5(12):e1442.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Friend DS. The fine structure of Giardia muris. J Cell Biol. 1966;29(2):317–32.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Kulda J, Nohynkova E. Giardia in humans and animals. In: Kreier JP, editor. Parasitic protozoa, vol. 10. San Diego: Academic Press Inc; 1995. p. 225–423.View ArticleGoogle Scholar
  24. Ou Y, Rattner JB. The centrosome in higher organisms: structure, composition, and duplication. Int Rev Cytol. 2004;238:119–82.View ArticlePubMedGoogle Scholar
  25. Meng T-C, et al. Immunolocalization and sequence of caltractin/centrin from the early branching eukaryote Giardia lamblia. Mol Biochem Parasitol. 1996;79(1):103–8.View ArticlePubMedGoogle Scholar
  26. Correa G, Morgado-Diaz JA, Benchimol M. Centrin in Giardia lamblia—ultrastructural localization. FEMS Microbiol Lett. 2004;233(1):91–6.View ArticlePubMedGoogle Scholar
  27. Nohynkova E, Draber P, Reischig J, Kulda J. Localization of gamma-tubulin in interphase and mitotic cells of a unicellular eukaryote, Giardia intestinalis. Eur J Cell Biol. 2000;79(6):438–45.View ArticlePubMedGoogle Scholar
  28. Adam RD. Biology of Giardia lamblia. Clin Microbiol Rev. 2001;14(3):447–75.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Elmendorf HG, Dawson SC, McCaffery JM. The cytoskeleton of Giardia lamblia. Int J Parasitol. 2003;33(1):3–28.View ArticlePubMedGoogle Scholar
  30. Gillin FD, Reiner DS, McCaffery JM. Cell biology of the primitive eukaryote Giardia lamblia. Annu Rev Microbiol. 1996;50:679–705.View ArticlePubMedGoogle Scholar
  31. Jirakova K, Kulda J, Nohynkova E. How nuclei of Giardia pass through cell differentiation: semi-open mitosis followed by nuclear interconnection. Protist. 2012;163(3):465–79.View ArticlePubMedGoogle Scholar
  32. Bernander R, Palm JE, Svard SG. Genome ploidy in different stages of the Giardia lamblia life cycle. Cell Microbiol. 2001;3(1):55–62.View ArticlePubMedGoogle Scholar
  33. Beech PL, Heimann K, Melkonian M. Development of the flagellar apparatus during the cell cycle in unicellular algae. Protoplasma. 1991;164(1–3):23–37.View ArticleGoogle Scholar
  34. Beisson J, Wright M. Basal body/centriole assembly and continuity. Curr Opin Cell Biol. 2003;15(1):96–104.View ArticlePubMedGoogle Scholar
  35. Paredez AR, et al. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins. Proc Natl Acad Sci USA. 2011;108(15):6151–6.View ArticlePubMedPubMed CentralGoogle Scholar
  36. Davis-Hayman SR, Nash TE. Genetic manipulation of Giardia lamblia. Mol Biochem Parasitol. 2002;122(1):1–7.View ArticlePubMedGoogle Scholar
  37. Dawson SC, et al. Kinesin-13 regulates flagellar, interphase, and mitotic microtubule dynamics in Giardia intestinalis. Eukaryot Cell. 2007;6(12):2354–64.View ArticlePubMedPubMed CentralGoogle Scholar
  38. Gaechter V, Schraner E, Wild P, Hehl AB. The single dynamin family protein in the primitive protozoan Giardia lamblia is essential for stage conversion and endocytic transport. Traffic. 2008;9(1):57–71.View ArticlePubMedGoogle Scholar
  39. Touz MC, Conrad JT, Nash TE. A novel palmitoyl acyl transferase controls surface protein palmitoylation and cytotoxicity in Giardia lamblia. Mol Microbiol. 2005;58(4):999–1011.View ArticlePubMedGoogle Scholar
  40. Carpenter ML, Cande WZ. Using morpholinos for gene knockdown in Giardia intestinalis. Eukaryot Cell. 2009;8(6):916–9.View ArticlePubMedPubMed CentralGoogle Scholar
  41. Manning G, et al. The minimal kinome of Giardia lamblia illuminates early kinase evolution and unique parasite biology. Genome Biol. 2011;12(7):R66.View ArticlePubMedPubMed CentralGoogle Scholar
  42. Keller LC, Marshall WF. Isolation and proteomic analysis of Chlamydomonas centrioles. Methods Mol Biol. 2008;432:289–300.View ArticlePubMedGoogle Scholar
  43. Belhadri A. Presence of centrin in the human parasite Giardia: a further indication of its ubiquity in eukaryotes. Biochem Biophys Res Commun. 1995;214(2):597–601.View ArticlePubMedGoogle Scholar
  44. House S, Richter D, Pham JK, Dawson SC. Giardia flagellar motility is not directly required to maintain attachment to surfaces. PLoS Pathog. 2011;7:e1002167.View ArticlePubMedPubMed CentralGoogle Scholar
  45. Harb OS, Roos DS. The Eukaryotic Pathogen Databases: a functional genomic resource integrating data from human and veterinary parasites. Methods Mol Biol. 2015;1201:1–18.View ArticlePubMedGoogle Scholar

Copyright

© McInally and Dawson. 2016