© 2012. Published by The Company of Biologists Ltd.
JCS ePress online publication date 24 February 2012
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Nuclear Inheritance and Genetic Exchange without Meiosis in the Binucleate Parasite
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Giardia intestinalis
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Meredith L. Carpenter1, 2, Zoe June Assaf1, 2, Stephane Gourguechon1, W. Zacheus Cande1
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Journal of Cell Science
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1. Department of Molecular and Cell Biology, University of California, Berkeley, CA
94720
2. Current affiliation: Department of Genetics, Stanford University School of Medicine,
Stanford, CA, 94305
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Author for correspondence: W. Z. Cande, [email protected], 510-642-1669
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Running title: Nuclear inheritance in Giardia
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Keywords: Giardia, encystation, excystation, meiosis, homologous recombination
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Summary
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The protozoan parasite Giardia intestinalis is a major waterborne pathogen. During its life cycle,
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Giardia alternates between the actively growing trophozoite, which has two diploid nuclei with
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low levels of allelic heterozygosity, and the infectious cyst, which has four nuclei and a tough
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outer wall. Although the formation of the cyst wall has been studied extensively, we still lack
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basic knowledge about many fundamental aspects of the cyst, including the sources of the four
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nuclei and their distribution during the transformation from cyst to trophozoite. In this study, we
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tracked the identities of the nuclei in the trophozoite and cyst using integrated nuclear markers
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and immunofluorescence staining. We demonstrate that the cyst is formed from a single
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trophozoite by a mitotic division without cytokinesis, and not by the fusion of two trophozoites.
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During excystation, the cell completes cytokinesis to form two daughter trophozoites. The non-
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identical nuclear pairs derived from the parent trophozoite remain associated in the cyst and are
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distributed as pairs to daughter cells during excystation. Thus, nuclear sorting (such that each
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daughter cell receives a pair of identical nuclei) does not appear to be a mechanism by which
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Giardia reduces heterozygosity between its nuclei. Rather, we show that the cyst nuclei
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exchange chromosomal genetic material, perhaps as a way to reduce heterozygosity in the
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absence of meiosis and sex, which have not been described in Giardia. These results shed light
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on fundamental aspects of the Giardia life cycle and have implications for our understanding of
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the population genetics and cell biology of this binucleate parasite.
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Abbreviations used: cyst wall protein (CWP), 4',6-diamidino-2-phenylindole
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(DAPI), encystation-specific vesicles (ESVs), fluorescence in situ hybridization (FISH),
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transmission electron microscopy (TEM)
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Journal of Cell Science
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Introduction
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The divergent eukaryotic parasite Giardia intestinalis (syn. Giardia lamblia, Giardia
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duodenalis) is a major cause of diarrheal disease throughout the world. Additionally, as a
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diplomonad, it is a member of one of the earliest-diverging eukaryotic lineages based on single-
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and multi-gene phylogenies (Baldauf et al., 2000; Baldauf, 2003; Best et al., 2004; Arisue et al.,
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2005; Ciccarelli et al., 2006; Morrison et al., 2007). The Giardia life cycle consists of two
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stages: a flagellated trophozoite with two nuclei and an infectious cyst with four nuclei. The two
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diploid (2N = 10) nuclei in the trophozoite contain complete copies of the genome and are both
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transcriptionally active (Kabnick and Peattie, 1990b; Bernander et al., 2001; Yu et al., 2002).
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They also remain independent in the trophozoite, dividing with separate spindles by a semi-open
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mitosis, and one copy of each parental nucleus is inherited by each daughter cell (Yu et al., 2002;
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Sagolla et al., 2006).
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The differentiation from trophozoite to cyst occurs when a trophozoite, which is usually
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found attached to the wall of the small intestine, is swept towards the large intestine. Changes in
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pH and cholesterol availability prompt the trophozoite to encyst, forming a cyst with four nuclei
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and a thick outer wall (Gillin et al., 1989; Lujan et al., 1996; Lujan et al., 1997). The formation
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of the cyst wall, which involves the regulated secretion of cyst wall protein components, has
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been a topic of intense study over the past few decades (see (Lauwaet et al., 2007a; Faso and
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Hehl, 2011) and references therein). The cyst is water resistant and can persist for weeks in the
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environment until it is ingested by a new host. After passing through the stomach, the cyst
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undergoes excystation, releasing an “excyzoite” that rapidly divides to produce two daughter
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trophozoites (Buchel et al., 1987; Bernander et al., 2001).
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While many advances have been made in our understanding of cyst wall formation,
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particularly with regard to ultrastructural aspects of the transition and to cyst wall morphology
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(Sheffield and Bjorvat, 1977; Luchtel et al., 1980; Buchel et al., 1987; Erlandsen et al., 1989;
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Hetsko et al., 1998; Lanfredi-Rangel et al., 2003; Palm et al., 2005; Chavez-Munguia et al.,
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2007; Midlej and Benchimol, 2009; Bittencourt-Silvestre et al., 2010; Faso and Hehl, 2011), the
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cytoskeletal changes underlying both encystation and excystation have received less attention.
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The trophozoite microtubule cytoskeleton consists of four pairs of flagella, with eight basal
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bodies located between the two nuclei, as well as a ventral adhesive disc (used to attach to the
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intestinal epithelium) and a prominent bundle of microtubules of unknown function called the
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median body (Figure 1) (Elmendorf et al., 2003; Dawson and House, 2010). These structures are
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reorganized and/or disassembled during cyst formation, with only internalized flagella and disc
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fragments thought to remain in the mature cyst (Elmendorf et al., 2003).
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It has been assumed that the cyst is formed via an incomplete mitotic division (as
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opposed to the fusion of two trophozoites) based on morphological observations (reviewed in
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(Adam, 2001; Lauwaet et al., 2007a)), flow cytometric analysis of ploidy (Bernander et al.,
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2001), and the finding that an encystation restriction point exists at the G2 phase of the cell cycle
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(Reiner et al., 2008). However, to our knowledge, the absence of cell fusion has never been
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directly tested (Figure 1A and B), and mitotic spindles in encysting cells have never been
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observed. Addressing this question using solely morphological observations is difficult because
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an encysting cell resulting from cell fusion would likely resemble one resulting from incomplete
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mitosis: i.e., it would contain four nuclei, two discs (or disc fragments), and up to 16 flagella
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(fewer if some were disassembled upon fusion). Similarly, flow cytometry analysis alone cannot
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conclusively distinguish between mitosis and fusion because both would produce cysts with the
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same final DNA content.
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Related to the question of how the quadrinucleate cyst is formed are the identities of the
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nuclei in the cyst and during excystation (Figure 1C and D). The genomes of most Giardia
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isolates contain very low levels of heterozygosity (Morrison et al., 2007; Teodorovic et al., 2007;
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Lasek-Nesselquist et al., 2009; Jerlstrom-Hultqvist et al., 2010), suggesting that the two nuclear
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genomes are able to exchange genetic information during the Giardia life cycle. The Giardia
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genome also contains seven homologs of meiosis-specific genes: Spo11, Hop1, Dmc1a, Dmc1b,
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Hop2, Mnd1, and Mer3 (Ramesh et al., 2005; Malik et al., 2008), which in most organisms are
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expressed only during meiosis and are specifically involved in meiotic homologous
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recombination. However, neither meiosis nor a sexual stage has been identified in the Giardia
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life cycle. We have previously shown that nuclear fusion and the exchange of episomal plasmids
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can occur between nuclei in the Giardia cyst, a process we have termed “diplomixis” (Poxleitner
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et al., 2008). At the same time, we showed that Spo11, Hop1, and Dmc1a are expressed only
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during encystation. Thus, we hypothesized that diplomixis could involve homologous
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recombination between the nuclear genomes. If accompanied by recombination and/or gene
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conversion within nuclei in the trophozoite, this process could help reduce heterozygosity
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between the two nuclei.
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However, another way to maintain low heterozygosity could be the sorting of nuclei
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during excystation such that each daughter cell receives two identical nuclei (rather than one of
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each original nucleus from the parent trophozoite)(Figure 1C and D). Therefore, the goals of the
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present study were twofold: 1) To track the identities of the nuclei during encystation and
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excystation, and 2) To determine whether diplomixis involves the exchange of chromosomal
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genetic material between nuclei. The results of these studies have implications for our
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understanding of both the basic biology and the population genetics of this parasite and could
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also shed light on the evolution of meiosis.
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Results
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Cysts are formed by an incomplete mitotic division
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In vitro, encystation can be prompted by exposing cells to basic media containing high
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levels of bile (which prevents cholesterol absorption) for 24-48 hours (Gillin et al., 1987; Gillin
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et al., 1988; Gillin et al., 1989; Kane et al., 1991; Lujan et al., 1996). Similarly, excystation can
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be triggered by a two-stage procedure in which cells are first exposed to acidic conditions for 30
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minutes, mimicking passage through the stomach, followed by a higher-pH solution containing
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proteases for an additional 30 minutes, which helps break open the cyst wall (Bingham and
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Meyer, 1979; Rice and Schaefer, 1981; Schaefer et al., 1984; Buchel et al., 1987; Hautus et al.,
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1988; Boucher and Gillin, 1990).
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To determine whether cysts can be derived from the fusion of two trophozoites, we first
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mixed two strains with different epitope-tagged markers (histone 2A-3HA and median body
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protein-3myc) and encysted them in vitro. None of the resulting cysts contained both markers
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(Table S1). This result indicates that, at least in vitro, cysts are generally derived from a single
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trophozoite. Following water treatment (to destroy any unencysted cells), excystation, and
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growth to confluency of these mixed cultures, we found that trophozoites always had only one of
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the two markers (Table S1), suggesting that cell fusion also does not occur during excystation.
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Additionally, in a larger-scale experiment performed with 5 x 107 live cysts (500-mL cultures),
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encystation and excystation of a mixture of two strains with different integrated drug resistance
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markers (neomycin and puromycin) failed to produce any trophozoites with resistance to both
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drugs (data not shown). Because we typically observe an in vitro excystation efficiency on the
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order of ~1%, the production of recombinant cells (which would require cell fusion followed by
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chromosomal recombination or nuclear sorting), if it occurs, would have to happen in fewer than
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1 in 500,000 cells.
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Next, we sought to characterize the incomplete mitotic division during encystation by
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staining cells with tubulin, centrin (a basal body and spindle pole marker), and cyst wall protein
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(CWP) antibodies after 24 hours of encystation (Figure 2). Prior to the division, the trophozoites
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produce encystation-specific vesicles (ESVs) full of CWP, and two clusters of four centrin spots
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(marking the basal bodies) are visible between the nuclei (Figure 2A). Anaphase (Figure 2B) and
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telophase (Figure 2C) generally resemble the mitotic division in trophozoites, with two parallel
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spindles and the basal bodies/centrioles at the spindle poles. However, unlike in the trophozoite,
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the encysting cells are concurrently in the process of exporting ESVs to the periphery of the cell
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for deposition. At the end of telophase of the encystation mitotic division (Figure 2D), the
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process diverges further from its vegetative counterpart: cytokinesis does not occur, the cell does
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not grow new flagella, and its existing flagella appear to shorten, perhaps as a result of
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internalization.
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At this stage, the two pairs of nuclei are found at opposite poles of the cell
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(approximately at the previous location of the spindle poles) and each pair remains associated
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with a bundle of internal axonemes, which appear to be derived from the axonemes that remain
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between the nuclei during mitosis (Nohynkova et al., 2006) (Figure 2E). It is at this time that the
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cyst wall is rapidly deposited; by the time the pairs of nuclei have migrated to the same pole of
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the cyst, the cyst wall has been completely formed. The two nuclei within each pair are closely
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associated and the pairs remain motile for some time in the cyst (Poxleitner et al., 2008),
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possibly due to their association with the internal axonemes; this is also presumably the
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mechanism by which they migrate together. The mature cyst contains four nuclei, two bundles of
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internal axonemes with eight centrin loci, and several ventral disc fragments (Figure 2F).
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It has been reported that the mature cyst contains four fragments of the parental disc that
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are reassembled during excystation (Elmendorf et al., 2003; Palm et al., 2005). However, based
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on staining of cells with antibodies to label the tubulin cytoskeleton and the disc (delta-giardin
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antibody), we observed what appear to be the initial stages of two new daughter discs being
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formed during the later stages of the encystation mitotic division (Figure S1A). At the same time,
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the parental disc was being disassembled, as has been described during cytokinesis in the
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trophozoite (Tumova et al., 2007). We most often saw two disc fragments in the mature cyst and
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early excyzoites; however, in some cases, up to four fragments were visible, although they were
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still usually found in two clusters (Figure S1B and C). In the excyzoite, the nascent disc
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fragments appeared to move to the correct position and orientation to complete their
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development into two new discs (Figure S1D).
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Reconstruction of the cytoskeleton during excystation
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Next, we stained the cytoskeleton of excysting cells to monitor the transition from
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excyzoite to trophozoite (Figure 3). As previously reported, the excyzoite emerges from a single
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pole of the cyst, which is weakened by the exposure to acidic conditions in the stomach and
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intestinal proteases (Coggins and Schaefer, 1986; Buchel et al., 1987; Boucher and Gillin, 1990;
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Hetsko et al., 1998) (Figure 3A). The flagella emerge first, followed by the body of the cell
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(Figure 3B). At this stage, the cytoskeleton of the excyzoite still resembles that of the cyst, with
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internal bundles of axonemes, two pairs of nuclei, and eight centrin spots (Figure 3A-C). These
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cells sometimes display one or several structures that resemble ESVs (arrow in Figure 3C) and
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contain CWP; these could result from the residual translation of cyst wall protein transcripts,
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which are extremely abundant during encystation (Hetsko et al., 1998), or could represent
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leftover vesicles from encystation or excystation that have not yet been eliminated.
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The axonemes in the excyzoite are clustered into two prominent bundles, each associated
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with a nuclear pair (Figure 3C). Although this configuration of axonemes is different from that of
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a trophozoite, the cell is still capable of swimming (Buchel et al., 1987; Hautus et al., 1988).
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After additional rearrangements, the excyzoite begins to resemble a heart-shaped (pre-
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cytokinesis) cell that is essentially indistinguishable from that of a dividing trophozoite, with the
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exception that each nucleus is thought to contain a 4C (replicated diploid) DNA content (Figure
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3D-E)(Bernander et al., 2001). By this stage, the number of basal bodies and flagella has
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increased from 8 to 16, and the cell rapidly completes the construction of two ventral discs
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(Figure 3E). Following cytokinesis, the resulting 8C trophozoites presumably divide immediately
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to produce two 4C trophozoites (Bernander et al., 2001).
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Nuclear identity and genetic exchange during differentiation
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The association of pairs of nuclei in the cyst and in the excyzoite suggests that the nuclear
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pairs from the parent trophozoite are preserved throughout differentiation, meaning that a
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mechanism other than nuclear sorting must exist to reduce allelic heterozygosity in Giardia
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populations (compare Figure 1C and Figure 1D). To test this hypothesis, we integrated a plasmid
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containing a 10-kb array of LacO repeats into the chromosomes of a population of trophozoites.
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To ensure the presence of only a single integrated construct, we then made clonal populations
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and tested for the absence of episomes by PCR (Figure S2). We then performed fluorescence in
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situ hybridization (FISH) using a fluorescent probe to label the LacO repeat. Like episomes (Yu
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et al., 2002), integrated plasmids were always found in only one of the two nuclei in the
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trophozoite (Figure 4A). Most cells contained a single spot, but occasionally we observed two
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spots very close together in a single nucleus; we interpret these to represent either replicated
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chromatids that became separated (most trophozoites rest in G2, but few cells contained two
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spots) or cases in which the homolog gained the marker via mitotic recombination/gene
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conversion. In the pre-encystation population, 76% of cells (n = 100) contained one spot (or two
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very close together) in a single nucleus, while 24% of cells contained no spot (Figure 4A).
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Because this population was clonal and the marker was integrated. and therefore stable even in
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the absence of drug selection, we interpret this result as a common artifact of FISH; however, it
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also possible that at least some cells lost the repeat cassette via recombination. After encystation,
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excystation, and growth of excysted trophozoites to confluency, all in the absence of drug
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selection, we observed that 49 ± 2% of cells (average of three biological replicates, each n = 100)
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had one spot, 31 ± 2% had no spots, and 20 ± 2% had two spots (one in each nucleus) (Figure
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4A). Thus, the production of non-parental patterns is common and consistent.
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The observed pattern could be generated in two ways: 1) genetic exchange between
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nuclei, or 2) nuclear sorting such that during excystation, each daughter cell received two
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identical nuclei. However, because only 20% of cells contained two spots, nuclear sorting would
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have had to occur in a minority of excysting cells (excyzoites). To begin to distinguish between
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these possibilities, we examined a strain that contained the same LacO repeat construct, but also
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contained a TetO repeat construct integrated into a different chromosome in the same nucleus
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(Figure 4B). As with the LacO construct, the absence of episomal plasmid in this clonal line was
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confirmed by PCR (data not shown). In this strain, 71% of pre-encysting trophozoites (n = 100)
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contained both markers in a single nucleus; the remaining 29% were missing one or both
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markers, likely due to artifact and/or recombination, as noted above. However, in 25% of
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excysted trophozoites (n = 100), we observed cells with all possible non-parental arrangements
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of markers (i.e., 14% with one in each nucleus, 10% with two in one nucleus and one in the
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other, and 1% with two in both nuclei), further suggesting that chromosomal genetic exchange
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occurs between nuclei in the cyst and/or early excyzoite (Figure 4B). The remaining 75% of
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excysted trophozoites contained either the parental arrangement or were missing one or both
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markers. In theory, all of the observed non-parental arrangements could also be compatible with
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nuclear sorting combined with signal loss; however, our level of signal loss is about 30% overall
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(based on our observations of pre-encysting cells), so if nuclear sorting were solely responsible
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for these patterns, we would expect to see higher levels of cells with both markers in both nuclei.
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Thus, while we cannot completely exclude rare nuclear sorting based on these data, we can
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conclude that it is not responsible for all of the observed non-parental arrangements of markers.
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To further rule out nuclear sorting, we also performed FISH on cysts and excyzoites
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(Figure 4C-F). As expected, the nuclei of most cysts contained two clustered pairs of nuclei, each
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with one labeled nucleus (Figure 4C). This pattern was recapitulated in most excyzoites (Figure
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4D), again supporting the idea that the non-identical nuclear pairs remain associated throughout
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encystation and excystation. While we were able to observe an occasional cyst with three labeled
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nuclei (Figure 4E), as would be expected based on our observation of two spots in the nuclei of
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excysted trophozoites, this was rare; however, we did notice that the spots in the cyst were often
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positioned between the masses of DAPI-stained DNA (Figure 4C), suggesting that the nuclei
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might remain fused in the cyst and only separate during excystation. Consistently, we were able
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to identify an early excyzoite with three labeled nuclei (Figure 4F): i.e., one nuclear pair
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underwent genetic exchange involving movement of the integrated marker, but the other did not.
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Therefore, our results indicate that the predominant mode of nuclear inheritance for excysting
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trophozoites involves the inheritance of one copy of each parental nucleus, and chromosomal
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genetic exchange occurs between nuclear pairs in the cyst.
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Based on our previous observations of the expression patterns of the Giardia meiotic gene
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homologs, we predict that this genetic exchange involves homologous recombination. However,
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because the previous studies were performed using tagged genes on multi-copy episomes (albeit
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under the native promoter), it is possible that the observed patterns may have been the product of
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overexpression. Thus, in the present study, we also used plasmid integration (Gourguechon and
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Cande, 2011) to endogenously tag the seven meiotic gene homologs with 3HA epitope tags.
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Because the genes were tagged at their endogenous locus, the constructs were under the control
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of the native promoter. We then examined the expression and localization of these proteins in
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both trophozoites and cysts. Mer3, which in yeast is a meiosis-specific DNA helicase (Nakagawa
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and Kolodner, 2002), did not localize to the nuclei of trophozoites or cysts (data not shown),
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suggesting that it may not, in fact, be a Mer3 ortholog; consistently, it is annotated as an RNA
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helicase in the Giardia genome database (ORF GL50803_87022 at giardiadb.org). As shown in
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Figure S3 and consistent with our previous findings, only Dmc1b was expressed at high levels in
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the trophozoite; Mnd1 and Hop2 (not previously studied) were present at low levels, and Spo11,
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Dmc1a, and Hop1 were absent. In contrast, Dmc1b was absent in cysts, while Spo11, Dmc1a,
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and Hop1 were upregulated, appearing in encysting trophozoites and remaining in fully encysted
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cells [Figure S3 and (Poxleitner et al., 2008)]. Mnd1 and Hop2 remained at low (but detectable)
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levels in cysts. In all cases and as expected, the proteins were localized to the nuclei in a punctate
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pattern. We also attempted to knock down Dmc1a and Spo11 by electroporation with
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morpholinos (Carpenter and Cande, 2009) immediately prior to encystation to assess their effects
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on exchange of the LacO marker, but we were unable to attain measurable levels of knockdown,
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possibly due to the morpholinos being diluted during the encystation process (data not shown).
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Finally, because we never observe spindles in fully formed cysts (i.e., there is no
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organized separation of chromosomes such as that seen in a meiotic division), the cell must have
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some way in which to anchor its chromosomes to prevent widespread aneuploidy following
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diplomixis. Based on the telomere bouquets observed in meiosis in many organisms, we
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predicted that telomere anchoring might be involved in Giardia as well. Thus, we performed
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FISH on trophozoites and cysts using a probe to detect the Giardia telomeric repeat sequence
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TAGGG (Figure 5). Interestingly, the telomeres are clustered, usually in two spots per nucleus,
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in both trophozoites (Figure 5A) and cysts (Figure 5B). A preliminary BLAST search for
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telomere-binding proteins in the Giardia genome revealed only telomerase (GiardiaDB accession
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GL50803_16225), suggesting that the proteins involved in tethering the chromosomes to the
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nuclear envelope in Giardia are either highly divergent from those in yeast and humans and/or
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are completely novel. This clustering may provide a mechanism by which the nuclei are able to
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undergo recombination without drastically changing the ploidy of each nucleus. However, it
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should be noted that some aneuploidy between nuclei, usually involving the addition/subtraction
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of one chromosome, has been observed in Giardia trophozoites (Tumova et al., 2006);
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diplomixis could be the mechanism by which this aneuploidy was initially created.
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Discussion
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As an organism with two nuclei, Giardia faces unique challenges in coordinating nuclear
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inheritance during its life cycle. Although the functional equivalence of the nuclei in Giardia and
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their independent distribution during mitosis in the trophozoite are relatively well established
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(Kabnick and Peattie, 1990a; Yu et al., 2002), their fates during encystation and excystation have
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only been studied with respect to genome ploidy (Bernander et al., 2001), despite the
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significance of this knowledge to our understanding of patterns of allelic heterozygosity and
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genetic diversity in Giardia populations. Therefore, the goal of the present study was to
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determine how the four nuclei are derived in the cyst and how these nuclei are distributed during
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excystation.
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Cysts are produced by mitosis, not cell fusion
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Based on strain mixing experiments (Table S1) and immunofluorescence staining of
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encysting cells (Figure 2), we have demonstrated that the quadrinucleate cyst is produced via an
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incomplete mitotic division that occurs rapidly during the final stages of cyst wall deposition. In
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addition, we have provided a detailed description of the temporal organization of the microtubule
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cytoskeleton during encystation and excystation. While the fusion of two trophozoites to form a
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cyst—which, if followed by nuclear fusion and genetic exchange, would resemble a parasexual
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cycle with outcrossing—is still theoretically possible, this event would have to be rare and/or
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happen only under certain conditions, such as in the host. It is also possible that fusion only
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happens between different strains or between cells with different variant surface proteins (Lasek-
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Nesselquist et al., 2009). However, given that encystation takes place during transit through the
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small and large intestine, we speculate that a strict requirement for two cells to find each other
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and fuse in order to form a cyst might be disadvantageous; thus, encystation from a single parent
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cell appears to be the predominant (if not sole) mode of cyst formation.
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Nuclei are inherited in non-identical pairs
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Our observations of the cytoskeletal components of encysting and excysting cells both
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synthesize and build upon the results of previous ultrastructural and TEM studies (Sheffield and
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Bjorvat, 1977; Luchtel et al., 1980; Buchel et al., 1987; Hetsko et al., 1998; Bernander et al.,
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2001; Palm et al., 2005; Midlej and Benchimol, 2009; Bittencourt-Silvestre et al., 2010), which
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have primarily focused on mature cysts. We found that the encystation division resembles the
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vegetative trophozoite mitotic division until approximately telophase. At that point, instead of
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continuing on into cytokinesis, the cell internalizes its flagella as the cyst wall rapidly forms. At
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the same time, cyst wall components are being produced and transported to the periphery of the
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cell, a process that appears to be independent from, albeit simultaneous with, the nuclear division
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(Stefanic et al., 2009). In addition, although trophozoites in the early stages of encystation
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display larger, rounded ESVs, by the time the nuclear division takes place, the cyst wall
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components have been transported to the periphery of the cell and exist in a more diffuse
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network, as previously reported (Konrad et al., 2010).
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Based on the patterns of centrin and tubulin staining in our study, the bundles of
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axonemes associated with each nuclear pair in the cyst appear to be derived from the axonemes
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found associated with the nuclei during mitosis (Nohynkova et al., 2006). According to that
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study, each daughter cell (or pair of nuclei, in this case) receives one anterolateral flagellum, one
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caudal flagellum, and either the posterolateral or ventral flagellar pair. These results are
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consistent with those of a TEM-based study that reported two bundles of three internal axonemes
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associated with each nuclear pair in the cyst, as well as two additional flagella found in the
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periphery of the cell (Sheffield and Bjorvat, 1977). It remains unclear whether the flagella are
333
actively depolymerized during encystation or whether they are simply internalized.
334
Based on our observations of the growth of two nascent daughter discs during the
335
encystation mitotic division and the overall morphological similarity of these nascent discs to the
336
fragments in the cyst, we hypothesize that disc growth is paused during encystation and then
337
resumed during excystation. This scenario would be more consistent with a what is observed
338
during cytokinesis in the trophozoite, in which two new ventral discs grow on the opposite side
339
of the cell from the parental ventral disc (Tumova et al., 2007). However, because we cannot
340
distinguish between old and new discs, it remains possible that the disc fragments in the cyst are
11
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Journal of Cell Science
341
derived from the parental disc and are retained as raw material for the construction of new discs
342
during excystation.
343
This is the first study, to our knowledge, to use immunofluorescence staining to examine
344
the Giardia cytoskeleton in depth during excystation, although previous studies have looked at
345
the localizaton of two proteins, PP2A and beta-giardin, in excyzoites (Palm et al., 2005; Lauwaet
346
et al., 2007b). As reported previously (Coggins and Schaefer, 1984; Coggins and Schaefer, 1986;
347
Buchel et al., 1987; Erlandsen et al., 1989), we observed that the excyzoite exits the cyst at one
348
pole, starting with its flagella. The early excyzoite has a similar cytoskeletal organization to the
349
cyst, with each pair of nuclei remaining associated with a bundle of internal axonemes; however,
350
it rapidly re-molds its cytoskeleton to resemble a cytokinetic trophozoite, which is the stage
351
where it paused after the mitotic division during encystation. Its nuclei become decondensed and
352
the distance between nuclei in each pair increases, presumably bringing an end to any nuclear
353
fusion that has occurred. At this stage, each nascent trophozoite is thought to have an 8C genetic
354
content (based on a 16C total in the cyst) (Bernander et al., 2001); thus, after cytokinesis is
355
completed, the cell is primed to complete another round of mitosis without an additional S phase,
356
as the chromosomes were already duplicated in the S phase during encystation. Because the
357
number of centrin spots increases from 8 in the excyzoite to 16 in the heart-shaped cells, we
358
conclude that basal body duplication is also occurring during this time. Taken together, our
359
results suggest that the cytoskeletal changes that happen during encystation, including disc
360
assembly, basal body duplication, and flagellar development, are very similar on the molecular
361
level to their counterparts in the trophozoite (Nohynkova et al., 2006; Tumova et al., 2007;
362
Dawson and House, 2010); the primary difference is the timing of these events in relation to
363
spindle formation.
364
Importantly, we have determined that during excystation, the daughter trophozoites
365
receive one copy of each parental nucleus. To date, the genomes of three Giardia assemblages
366
(genotypes) have been sequenced. Two of these, assemblages A and E, contain extremely low
367
levels of allelic heterozygosity—less than 0.01% (Morrison et al., 2007; Jerlstrom-Hultqvist et
368
al., 2010). These results are surprising because, in a presumed asexual organism with two
369
independent nuclei, the nuclear genomes—and perhaps even homologous chromosomes—would
370
be expected to diverge over time due to genetic drift (Birky, 2010). Fusion and genetic exchange
371
(diplomixis) between the nuclei in the cyst, if accompanied by recombination and gene
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372
conversion within nuclei in the trophozoite, could potentially explain these low levels of
373
heterozygosity (Poxleitner et al., 2008; Andersson, 2011). However, the distribution of identical
374
nuclei to daughter cells during excystation would also explain, independent of diplomixis, the
375
low levels of heterozygosity observed in Giardia populations (see Figure 1). Thus, our
376
observation that nuclei are distributed in non-identical pairs during excystation is significant and
377
implies that there are other mechanisms in place, including but perhaps not limited to diplomixis,
378
to reduce genomic heterozygosity in Giardia. One of these could be highly efficient DNA
379
damage repair in the trophozoite, which rests in the G2 phase of the cell cycle (Bernander et al.,
380
2001); indeed, it is tempting to speculate that this unusual characteristic (most eukaryotic cells
381
rest in G1) arose to allow ample time for repair off the sister chromatid. The expression of
382
Dmc1b in the trophozoite suggests that this Dmc1 homolog could mediate homologous
383
recombination at this stage, and its similarity to Dmc1 (Ramesh et al., 2005) could point to an
384
ability to mediate repair off the homolog. This ability would also be required to maintain the low
385
levels of heterozygosity observed in laboratory strains that have been passaged for long periods
386
of time (Morrison et al., 2007; Teodorovic et al., 2007), as well as for heterozygosity to be
387
reduced within nuclei following diplomixis (Andersson, 2011).
388
389
Chromosomal genetic exchange and meiotic gene expression patterns are consistent with
390
homologous recombination
391
We were surprised to find consistently high rates of chromosomal genetic exchange
392
(~20%) between nuclei in the cyst. This is likely an underestimate of the amount of exchange
393
occurring, as our assay can only detect events involving transfer and preservation of the marker.
394
This finding is consistent with diplomixis being used as a mechanism to reduce nuclear
395
heterozygosity: to be effective, genetic exchange would be expected to be relatively common and
396
involve large portions of the genome. We currently cannot distinguish between homologous
397
recombination and whole-chromosome exchange. One way to directly detect recombination
398
would be to place two integrated markers far apart on the same chromosome and observe their
399
separation; however, this would be technically difficult (if not impossible) in Giardia, both
400
because it is tetraploid and because there would be no clear way to confirm that the markers are
401
in the same chromosome. Nevertheless, we predict that diplomixis involves homologous
402
recombination for two reasons: first, three of the meiotic gene homologs (Dmc1a, Spo11, and
13
Accepted manuscript
Journal of Cell Science
403
Hop1) are expressed specifically at this stage in the life cycle, and second, it is difficult to
404
envision a mechanism for whole-chromosome exchange that would not result in widespread
405
aneuploidy. Therefore, we predict that the meiotic gene homologs are involved in mediating
406
homologous recombination between the nuclei during diplomixis. If this turns out to be the case,
407
it would be one of the first examples of these proteins functioning in a non-meiotic context.
408
Spo11 has been found to mediate homologous recombination during the parasexual cycle of the
409
fungus Candida albicans (Forche et al., 2008). However, the lack of a sexual cycle in this
410
organism is clearly a case of loss, whereas it is unclear whether Giardia or its relatives ever had
411
a true meiotic cycle. Future studies of the functions of the meiotic gene homologs during
412
diplomixis in Giardia could shed light on the evolution of the meiotic homologous
413
recombination machinery.
414
Based on the observations from the present study, we envision a model whereby tethered
415
chromosomes are able to interact and recombine with chromosomes from a neighboring nucleus
416
(Figure 6A-D) at some point either in the cyst or early excyzoite. In this way, widespread
417
crossing-over (facilitated by the meiotic gene homologs) between chromosomes from different
418
nuclei could produce the observed patterns, and subsequent recombination and gene conversion
419
could then act to homogenize the homologous chromosomes within each nucleus. Consistent
420
with this hypothesis, we occasionally observed two paired spots rather than one in some cells
421
(see fourth panel in Figure 4B). These spots could represent replicated chromosomes that became
422
separated; however, these patterns (as well as the increase in unlabeled nuclei following
423
excystation [Figure 4A]) could also result from recombination and gene conversion between
424
homologous chromosomes in the same nucleus.
425
In conclusion, we have demonstrated that, at least in vitro, Giardia cysts are derived from
426
a single cell via an incomplete mitotic division, and daughter trophozoites inherit one copy of
427
each parental nucleus during excystation. Furthermore, as hypothesized based on the results of
428
our previous study (Poxleitner et al., 2008), we have directly demonstrated that chromosomal
429
genetic exchange occurs during diplomixis. This exchange happens at the same time that several
430
homologs of meiosis-specific genes are induced, suggesting that homologous recombination in
431
occurring, presumably as a way to reduce allelic heterozygosity between nuclei. However, while
432
diplomixis could explain the maintenance of low heterozygosity between nuclei, it is also
433
possible that rare meiotic events occur in the wild, as suggested by several recent population
14
Accepted manuscript
Journal of Cell Science
434
genetics studies (Cooper et al., 2007; Lasek-Nesselquist et al., 2009). These studies found
435
evidence for recombination within and between assemblages, which cannot be explained by
436
diplomixis within a single cell, but could potentially be explained by a combination of rare cell
437
fusion followed by diplomixis. Furthermore, in contrast to the low heterozygosity in assemblages
438
A and E, the genome of assemblage B strain GS has 0.5% allelic heterozygosity (Franzen et al.,
439
2009), which has been suggested to be evidence for recent genetic exchange in the lineage
440
(Andersson, 2011).
441
Future studies should aim to test for fusion and diplomixis or a true meiotic event in vivo,
442
similar to what has been done to detect sexual stages in parasites like Trypanosomes and
443
Leishmania (Akopyants et al., 2009; Peacock et al., 2011). Identifying this process may be
444
difficult, particularly if it is rare or occurs only under specific conditions. Nevertheless, a more
445
complete understanding of the Giardia life cycle will not only give us insight into the basic
446
biology of this important parasite but may also shed light on the spread of drug resistance in
447
Giardia populations.
448
449
Materials and Methods
450
Culture conditions
451
Giardia intestinalis trophozoites, WB clone C6 (ATCC 50803), were grown in modified TYI-S-
452
33 medium (Keister, 1983) with bovine bile (Sigma-Aldrich, St. Louis, MO). Cultures were
453
incubated in 16-mL plastic screw top tubes (Fisher Scientific, Pittsburgh, PA) at 37°C and
454
passaged every three days.
455
456
In vitro encystation
457
Encystation was performed essentially as described (Kane et al., 1991). Briefly, trophozoites
458
were seeded into 30- or 50-mL tissue culture flasks (BD Biosciences, Bedford, MA) and grown
459
to 80%-100% confluency. The medium was then decanted and replaced with prewarmed
460
encystation medium (10 mg/mL bovine/ovine bile, pH 7.8). After 24 hours at 37°C, cells were
461
either fixed for observation or incubated on ice for 15 min, pelleted at 500 xg, and resuspended
462
in regular growth medium (0.5 mg/mL bile, pH 7.1). After an additional 24 hours of incubation
463
at 37°C, the cells were again chilled on ice, pelleted, and resuspended in 50 mL ice-cold, sterile
15
464
deionized water to destroy all unencysted cells. Cysts were stored at 4°C for 1-3 days, then
465
excysted as described below.
Journal of Cell Science
Accepted manuscript
466
467
In vitro excystation
468
Excystation was performed essentially as described (Boucher and Gillin, 1990). Briefly, water-
469
treated cysts were pelleted in a 50-mL conical tube, then resuspended in 10 mL prewarmed stage
470
I solution (1X Hank’s Balanced Salt Solution [Invitrogen, Carlsbad, CA], 57 mM L-cysteine
471
HCl, 32.5 mM reduced glutathione, and 25 mM NaHC03; the pH was adjusted to 4.0 with 0.01 N
472
HCl). The tube was vortexed briefly, then incubated at 37°C for 30 minutes. After another brief
473
vortexing, cells were pelleted at 500 xg for 5 minutes. The supernatant was then decanted and
474
replaced with 10 mL prewarmed stage II solution (Tyrode’s buffer [Sigma-Aldrich, St. Louis,
475
MO] containing 1 mg/mL Trypsin II-S [Sigma]; the pH was adjusted to 8.0 with 1 M NaHC03).
476
The tube was vortexed briefly, incubated at 37°C for 30 minutes, then vortexed again and
477
pelleted. The supernatant was decanted and replaced with prewarmed growth medium (see
478
Culture conditions), and cells were moved to 16-mL culture tubes and incubated at 37°C. Cells
479
were then either fixed 1-3 hours later for immunofluorescence staining or grown to confluency
480
(approximately 5-7 days).
481
482
Creation of constructs for endogenous tagging
483
For creating integration constructs for endogenous tagging, the appropriate ORF was amplified
484
from WBC6 genomic DNA and inserted into either the vector pKS-3HA-PAC or pKS-3myc-
485
NEO (Gourguechon and Cande, 2011). The amplified ORFs, primers, and restriction sites used
486
for digestion and linearization are shown Table S2. Transformation and selection of Giardia
487
trophozoites was done as previously described (Gourguechon and Cande, 2011).
488
489
Creation of clonal strains containing integrated LacO and TetO constructs
490
For the LacO array integration construct, a SacI/PvuII fragment containing part of the Spo11
491
coding sequence (GiardiaDB accession GL50803_15279; located on chromosome 4 (Upcroft et
492
al., 2010)) and a puromycin resistance cassette (from the vector pKS-Spo11-3HA-pac) was
493
inserted into the LacO array-containing plasmid SR7 ((Rohner et al., 2008), a gift from S.
494
Gasser) cut with restriction enzymes SacI and EcoRV (Fig. 4.3). For integration, the plasmid
16
Accepted manuscript
Journal of Cell Science
495
was linearized with MfeI, which cuts within the sequence for Spo11. After the selection of stable
496
transformants, single-cell clones were obtained by limiting dilution as previously described
497
(Baum et al., 1988). These clones were screened for the presence of the integrated plasmid and
498
absence of episomal plasmid by PCR as shown in Figure S2, using the primers P1 (5’-
499
CATGATTTTCTTGTTTTAGGG -3’) and P2 (5’- TAATACGACTCACTATAGGG-3’) or P1
500
and P3 (5’- AGACTCTACAGGATACTTTCAG-3’). PCRs were performed by boiling a
501
concentrated culture (200 µl) of approximately 1.3 x 107 cells for 10 minutes at 95°C. One
502
microliter (~65,000 cells) was then used for PCR amplification.
503
504
For the TetO array construct, an EcoRI/XbaI fragment containing the median body protein
505
(MBP; GiardiaDB accession GL50803_16343, located on chromosome 3 (Upcroft et al., 2010))
506
coding sequence and a neomycin resistance cassette (from the vector pKS-MBP-neo) was
507
inserted into the TetO array-containing plasmid SR8 ((Rohner et al., 2008), a gift from S.
508
Gasser) cut with the restriction enzymes EcoRI and NheI. For integration, the plasmid was
509
linearized with MfeI, which cuts within the sequence for MBP. After the selection of stable
510
transformants, clonal lines were created and confirmed as described above, except using primer
511
P4 (5’-TTCCGAATTTTGAGACACCTC-3’) instead of primer P3 listed above.
512
513
Fluorescence in situ hybridization
514
FISH was performed essentially as described (Poxleitner et al., 2008). However, instead of using
515
nick translation to create the probe, the following probes were used at a concentration of 5 ng/µl:
516
a
517
CCACAAATTGTTATCCGCTCACAATTCCACATGTGG-3’); a FITC-labeled TetO probe (5’-
518
TCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAG-3’); and a FITC-labeled
519
telomere probe (5’-TAGGGTAGGGTAGGGTAGGGTAGGG-3’).
Cy3-labeled
LacO
oligonucleotide
probe
(5’-
520
521
Immunostaining
522
Fixation and immunostaining were performed essentially as described (Carpenter and Cande,
523
2009). Briefly, cells were fixed in 1X PEM buffer (100 mM PIPES, 1 mM EGTA, 0.1 mM
524
MgSO4) containing 2% paraformaldehyde. After fixation for 30 minutes at 37°C, cells were
525
washed once with PEM buffer, then resuspended in PEM buffer and attached to poly-L-lysine-
17
Accepted manuscript
Journal of Cell Science
526
coated coverslips for 30 minutes. Alternatively, for delta-giardin antibody staining, cells were
527
fixed for 3 minutes in ice-cold methanol, then washed twice in PBS before being attached to
528
poly-L-lysine coverslips. Fixed cells were permeabilized with 0.1% Triton-X-100 for 15
529
minutes, washed three times with PEM, and blocked in PEMBALG (PEM plus 1% BSA, 0.1%
530
NaN3, 100 mM lysine and 0.5% gelatin from cold water fish skin [Sigma]) for 2 hours. Primary
531
antibody incubations were conducted at room temperature overnight, and antibodies were diluted
532
in PEMBALG as follows: mouse anti-trypanosome alpha-tubulin antibody TAT1, 1:50 ((Woods
533
et al., 1989), a gift from K. Gull); 20H5 mouse anti-centrin antibody, 1:100 (Salisbury et al.,
534
1988); rabbit anti-sea urchin tubulin antibody 1:25 (Fujiwara and Pollard, 1978); rat anti-HA
535
antibody 1:100 (Roche, Basel, Switzerland); mouse anti-HA antibody 1:100 (Sigma); rabbit anti-
536
c-myc antibody 1:100 (Cell Signaling Technology, Danvers, MA); rabbit anti-delta-giardin
537
antibody 1:100 ((Jenkins et al., 2009), a gift from M. Jenkins). After washing with PEMBALG,
538
cells were incubated with secondary antibodies (goat anti-mouse 555 and goat anti-rabbit 488
539
[Invitrogen]) at room temperature for two hours. For labeling with the anti-cyst wall protein
540
antibody, cells were then washed three times with PEMBALG, fixed for 10 minutes in 1%
541
paraformaldehyde, washed three times with PEM, and blocked for an additional 30 minutes in
542
PEMBALG. The cells were then incubated with a Cy5-labeled mouse anti-cyst wall protein
543
antibody (Waterborne, Inc., New Orleans, LA) for two hours. After a final three washes each in
544
PEM and PEMBALG, coverslips were mounted onto slides with Prolong Gold Antifade Solution
545
with DAPI (Invitrogen).
546
547
Fluorescence deconvolution microscopy
548
Images were collected with SoftWorX image acquisition software (Applied Precision, Issaquah,
549
WA) on an Olympus IX70 wide-field inverted fluorescence microscope with an Olympus
550
UPlanApo 100x, NA 1.35, oil-immersion objective and Photometrics CCD CH350 camera
551
cooled to -35°C (Roper Scientific, Tuscon, AZ). Serial sections were acquired at 0.2 µm
552
intervals, and data stacks were deconvolved using the SoftWorX deconvolution software. For
553
presentation purposes, two-dimensional maximum intensity projections were created from the 3D
554
data sets using the DeltaVision image analysis software (Applied Precision).
555
556
Acknowledgements
18
557
We thank current and former members of the Cande Lab, especially L. K. Fritz-Laylin and A. R.
558
Paredez, for helpful discussion. We also thank K. Gull, M. Jenkins, and S. Gasser for reagents.
559
560
Financial disclosure
561
We gratefully acknowledge funding from the NIH (www.nih.gov; grant A1054693 to W.Z.C.)
562
and the NSF (www.nsf.gov; predoctoral fellowship to M.L.C.). The funders had no role in study
563
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Journal of Cell Science
Accepted manuscript
564
565
Competing interests
566
The authors declare no competing interests.
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Palm, D., Weiland, M., McArthur, A. G., Winiecka-Krusnell, J., Cipriano, M. J.,
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Yu, L. Z., Birky, C. W., Jr. and Adam, R. D. (2002). The two nuclei of Giardia each have
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complete copies of the genome and are partitioned equationally at cytokinesis. Eukaryot Cell 1,
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191-199.
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Journal of Cell Science
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Figure legends
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Figure 1: Overview of alternative hypothesis for cyst formation and nuclear distribution
751
during excystation. Shown on the left is a diagram of a Giardia trophozoite, with the major
752
components of the microtubule cytoskeleton labeled: the ventral adhesive disc (vd), used for
753
attachment to the intestinal epithelium; four sets of flagella (afl = anterolateral; pfl =
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posterolateral; vfl = ventral; cfl = caudal); the median body (mb), a microtubule array of
755
unknown function; and the two nuclei (n). While it has often been assumed that the Giardia cyst
756
is formed via an incomplete mitotic division (A), the alternative route of cell fusion between two
757
trophozoites (B) has never been excluded. In addition, during excystation, two possible routes for
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the partitioning of nuclei to daughter cells would have dramatically different consequences for
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Giardia populations. If each daughter cell receives one copy of each parental nucleus (C), any
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nuclear heterozygosity is maintained throughout the life cycle. In contrast, if the nuclei were
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distributed such that each daughter cell received two identical nuclei (D), heterozygosity would
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be eliminated.
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Figure 2: The Giardia cyst is formed by an incomplete mitotic division. Encysting cells were
765
stained with DAPI to label DNA (blue) and antibodies to label tubulin (green), centrin (red), and
766
cyst wall protein (CWP) (grey). (A) An encysting trophozoite, demonstrating typical centrin and
767
tubulin staining and the presence of two nuclei. The median body, a cluster of microtubules of
768
unknown function, is indicated (arrow). (B) An encysting cell in anaphase. The two spindles
769
(arrowheads) and median body (arrow) are indicated. (C and D) Encysting cells in early and late
27
770
stages of telophase. (E) Early cyst in which the nuclear pairs are still at opposite ends of the cell.
771
The arrow indicates what appears to be a nascent disc fragment associated with one pair of
772
nuclei. (F) Mature cyst in which the nuclear pairs have migrated to one pole of the cell. Scale bar
773
= 5 µm.
Journal of Cell Science
Accepted manuscript
774
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Figure 3: Reassembly of the microtubule cytoskeleton during excystation. Excysting cells
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were stained with DAPI to label DNA (blue) and antibodies to label tubulin (green), centrin
777
(red), and cyst wall protein (CWP) (grey). (A) Cyst in the early stages of excystation (note the
778
rupture in cyst wall at the bottom of the image). (B) An excyzoite emerging from a cyst. (C)
779
Completely excysted cell. A residual encystation-specific vesicle is indicated (arrow). (D and E)
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Later-stage excyzoites in which the typical heart-shaped cytokinetic morphology is restored,
781
including two complete discs and 16 flagella. Scale bar = 5 µm.
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Figure 4: Chromosomal exchange occurs between nuclei during differentiation. (A) A
784
clonal population of cells containing an integrated LacO array in one nucleus were subjected to
785
fluorescence in situ hybridization (FISH) to track the marker in the starting population (pre-
786
encystation trophozoites) and in excysted trophozoites. DAPI-stained DNA is shown in blue and
787
the LacO probe is shown in red. Percentages of cells with 0, 1, and 2 spots are shown below each
788
image. (B) Patterns observed after excystation of a clonal population containing two markers,
789
LacO (red) and TetO (green), integrated in the same nucleus (first panel). The number of spots in
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each nucleus is indicated below each image (e.g., N1:0; N2:RG = 0 spots in nucleus 1; one red
791
spot and one green spot in nucleus 2). (C-F) Cysts and excyzoites from the experiment described
792
in (A). DAPI-stained DNA is shown in blue, and the LacO probe is shown in red. Cyst wall
793
protein (CWP) staining is shown in green in (C) and (E), and tubulin staining is shown in green
794
in (D) and (F). Panels C and D show a cyst and an excyzoite, respectively, with one spot in each
795
nuclear pair; panels E and F show a cyst and an excyzoite, respectively, in which one nuclear
796
pair contains two spots. All scale bars = 5 µm.
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798
Figure 5: Telomere clustering in trophozoites and cysts. Fluorescence in situ hybridization
799
was performed on trophozoites (A) and cysts (B), using a probe to label the telomeric repeat
28
800
sequence. DAPI-stained DNA is shown in blue, the telomere probe is shown in green, and cyst
801
wall protein antibody staining is shown in red. Scale bars = 5 µm.
Journal of Cell Science
Accepted manuscript
802
803
Figure 6: Hypothetical model of homologous recombination during diplomixis. (A) Shown
804
is a simplified cell containing an integrated marker (red) in one of its four homologous
805
chromosomes (H1 and H2, shown in orange, in the left nucleus, and H1’ and H2’, shown in blue,
806
in the right nucleus). Throughout the figure, the presence of the integrated marker is indicated by
807
a red outline around the nucleus in which it is found. Because trophozoites typically rest in G2 of
808
the cell cycle, this trophozoite is shown with its chromosomes already duplicated (8C genome
809
content; 4C in each nucleus). (B) During encystation, the meiotic gene homologs Spo11, Dmc1a,
810
and Hop1 are induced. Both nuclei divide and then go through an S phase, producing a 16C cyst.
811
At some point, the nuclei fuse and exchange genetic material, presumably via homologous
812
recombination. The chromosomes remain tethered to the nuclear envelope by their telomeres,
813
which may prevent the development of aneuploidy following diplomixis. (C) When the cell
814
excysts and completes cytokinesis, two trophozoites are formed, one from each nuclear pair
815
found in the cyst. (D) If diplomixis has occurred (left trophozoite in (C)), the first mitotic
816
division produces two possible combinations of daughter trophozoites, depending on the
817
orientation of the chromosomes on the spindle. One of these combinations would produce one
818
cell with markers in both nuclei and one cell with no marker, while the other would produce the
819
parental arrangement (marker in one nucleus). If diplomixis has not occurred (right trophozoite
820
in (C)), only the parental arrangement (marker in one nucleus) is produced.
821
822
Supporting information
823
Figure S1: Development of the ventral adhesive disc during encystation and excystation.
824
Encysting and excysting cells were stained with DAPI to label DNA (blue) and antibodies to
825
label cyst wall protein (red) and either tubulin (A) or delta-giardin (B-D) (green). (A) An
826
encysting cell in telophase showing the growth of a new daughter disc (arrowhead) and the
827
disassembly of the parental disc (arrow). (B) A mature cyst containing two disc fragments. (C)
828
An excysting cell showing that each disc fragment appears to be composed of two pieces. (D) An
829
excyzoite in which the disc fragments are again associated with the nuclear pairs and are in the
29
830
correct orientation (curved arrows) to resume their development into complete discs. Scale bar =
831
5 µm.
Journal of Cell Science
Accepted manuscript
832
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Figure S2: Scheme for the creation and confirmation of a clonal strain with an integrated
834
marker. (A) Map of the plasmid used for integration, which contains an array of 256 LacO
835
repeats. The binding sites for primers P1 and P2 are shown, as is the restriction site (MfeI) that
836
was used to linearize the plasmid for integration. (B) Schematic of the linearized plasmid,
837
showing the binding sites for primers P1, P2, and P3. Primers P3 (which binds upstream of
838
where the plasmid is integrated in the genome) and P1 were used confirm integration of the
839
construct, and primers P1 and P2 (which should not produce a product in the absence of circular
840
plasmid) were used to check for the absence of the circular plasmid. (C) Results of PCR testing
841
of the wild-type (WT), parental (non-clonal) transformed strain, and clonal strain used in
842
subsequent experiments. The presence of a plasmid-specific product in the parental strain
843
suggests that either residual episome was present or that some cells integrated the plasmid in
844
tandem arrays, which would also produce a product with primers P1 and P2.
845
846
Figure S3: Expression of endogenously tagged meiosis-specific gene homologs in
847
trophozoites and cysts. Endogenous tagging constructs were integrated into the endogenous loci
848
of the six giardial meiotic gene homologs, creating C-terminally 3HA-tagged proteins under their
849
native promoters. Control (wild-type) and tagged strains before and after encystation were
850
labeled by immunofluorescence staining using an anti-HA antibody (green) and stained with
851
DAPI to label nuclei (blue).
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Journal of Cell Science
Accepted manuscript