Research Article
2523
Nuclear inheritance and genetic exchange without
meiosis in the binucleate parasite Giardia intestinalis
Meredith L. Carpenter*, Zoe June Assaf*, Stéphane Gourguechon and W. Zacheus Cande`
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
*Present address: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
`
Author for correspondence ([email protected])
Journal of Cell Science
Accepted 23 January 2012
Journal of Cell Science 125, 2523–2532
2012. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.103879
Summary
The protozoan parasite Giardia intestinalis (also known as Giardia lamblia) is a major waterborne pathogen. During its life cycle,
Giardia alternates between the actively growing trophozoite, which has two diploid nuclei with low levels of allelic heterozygosity, and
the infectious cyst, which has four nuclei and a tough outer wall. Although the formation of the cyst wall has been studied extensively,
we still lack basic knowledge about many fundamental aspects of the cyst, including the sources of the four nuclei and their distribution
during the transformation from cyst into trophozoite. In this study, we tracked the identities of the nuclei in the trophozoite and cyst
using integrated nuclear markers and immunofluorescence staining. We demonstrate that the cyst is formed from a single trophozoite by
a mitotic division without cytokinesis and not by the fusion of two trophozoites. During excystation, the cell completes cytokinesis to
form two daughter trophozoites. The non-identical nuclear pairs derived from the parent trophozoite remain associated in the cyst and
are distributed to daughter cells during excystation as pairs. Thus, nuclear sorting (such that each daughter cell receives a pair of
identical nuclei) does not appear to be a mechanism by which Giardia reduces heterozygosity between its nuclei. Rather, we show that
the cyst nuclei exchange chromosomal genetic material, perhaps as a way to reduce heterozygosity in the absence of meiosis and sex,
which have not been described in Giardia. These results shed light on fundamental aspects of the Giardia life cycle and have
implications for our understanding of the population genetics and cell biology of this binucleate parasite.
Key words: Cytoskeleton, Giardia, Encystation, Excystation, Meiosis, Homologous recombination
Introduction
The divergent eukaryotic parasite Giardia intestinalis (syn.
Giardia lamblia, Giardia duodenalis) is a major cause of
diarrheal disease throughout the world. Additionally, as a
diplomonad, it is a member of one of the earliest-diverging
eukaryotic lineages based on single- and multi-gene phylogenies
(Baldauf et al., 2000; Baldauf, 2003; Best et al., 2004; Arisue
et al., 2005; Ciccarelli et al., 2006; Morrison et al., 2007). The
Giardia life cycle consists of two stages: a flagellated trophozoite
with two nuclei and an infectious cyst with four nuclei. The two
diploid (2N510) nuclei in the trophozoite contain complete
copies of the genome and are both transcriptionally active
(Kabnick and Peattie, 1990; Bernander et al., 2001; Yu et al.,
2002). They also remain independent in the trophozoite, dividing
with separate spindles by a semi-open mitosis, and one copy of
each parental nucleus is inherited by each daughter cell (Yu et al.,
2002; Sagolla et al., 2006).
The differentiation from trophozoite into cyst occurs when a
trophozoite, which is usually found attached to the wall of the
small intestine, is swept towards the large intestine. Changes in
pH and cholesterol availability prompt the trophozoite to encyst,
forming a cyst with four nuclei and a thick outer wall (Gillin
et al., 1989; Luján et al., 1996; Luján et al., 1997). The formation
of the cyst wall, which involves the regulated secretion of cyst
wall protein components, has been a topic of intense study over
the past few decades (see Lauwaet et al., 2007a; Faso and Hehl,
2011). The cyst is water resistant and can persist for weeks in the
environment until it is ingested by a new host. After passing
through the stomach, the cyst undergoes excystation, releasing
an ‘excyzoite’ that rapidly divides to produce two daughter
trophozoites (Buchel et al., 1987; Bernander et al., 2001).
Although many advances have been made in our understanding
of cyst wall formation, particularly with regard to ultrastructural
aspects of the transition and to cyst wall morphology (Sheffield
and Bjorvat, 1977; Luchtel et al., 1980; Buchel et al., 1987;
Erlandsen et al., 1989; Hetsko et al., 1998; Lanfredi-Rangel et al.,
2003; Palm et al., 2005; Chávez-Munguı́a et al., 2007; Midlej and
Benchimol, 2009; Bittencourt-Silvestre et al., 2010; Faso and
Hehl, 2011), the cytoskeletal changes underlying both encystation
and excystation have received less attention. The trophozoite
microtubule cytoskeleton comprises four pairs of flagella, with
eight basal bodies located between the two nuclei, as well as a
ventral adhesive disc (used to attach to the intestinal epithelium)
and a prominent bundle of microtubules of unknown function
called the median body (Fig. 1) (Elmendorf et al., 2003; Dawson
and House, 2010). These structures are reorganized and/or
disassembled during cyst formation, with only internalized
flagella and disc fragments thought to remain in the mature cyst
(Elmendorf et al., 2003).
It has been assumed that the cyst is formed after an incomplete
mitotic division (as opposed to the fusion of two trophozoites) on
the basis of morphological observations (reviewed by Adam,
2001; Lauwaet et al., 2007a), flow cytometric analysis of ploidy
(Bernander et al., 2001) and the finding that an encystation
Journal of Cell Science
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Journal of Cell Science 125 (10)
Fig. 1. Overview of alternative hypotheses for cyst formation and nuclear distribution during excystation. Shown on the left is a diagram of a Giardia
trophozoite, with the major components of the microtubule cytoskeleton labeled: the ventral adhesive disc (vd), used for attachment to the intestinal epithelium;
four sets of flagella (afl, anterolateral; pfl, posterolateral; vfl, ventral; cfl, caudal); the median body (mb), a microtubule array of unknown function; and the
two nuclei (N). Although it has often been assumed that the Giardia cyst is formed after an incomplete mitotic division (A), the alternative route of cell fusion
between two trophozoites (B) has never been excluded. In addition, during excystation, two possible routes for the partitioning of nuclei to daughter cells would
have dramatically different consequences for Giardia populations. If each daughter cell receives one copy of each parental nucleus (C), any nuclear heterozygosity
is maintained throughout the life cycle. By contrast, if the nuclei were distributed such that each daughter cell received two identical nuclei (D), heterozygosity
would be eliminated.
restriction point exists at the G2 phase of the cell cycle (Reiner
et al., 2008). However, to our knowledge, the absence of cell
fusion has never been directly tested (Fig. 1A,B), and mitotic
spindles in encysting cells have never been observed. Addressing
this question using solely morphological observations is difficult
because an encysting cell resulting from cell fusion would
probably resemble one resulting from incomplete mitosis [i.e. it
would contain four nuclei, two discs (or disc fragments) and up to
16 flagella (fewer if some were disassembled upon fusion)].
Similarly, flow cytometry analysis alone cannot conclusively
distinguish between mitosis and fusion because both would
produce cysts with the same final DNA content.
Related to the question of how the quadrinucleate cyst is
formed are the identities of the nuclei in the cyst and during
excystation (Fig. 1C,D). The genomes of most Giardia isolates
contain very low levels of heterozygosity (Morrison et al., 2007;
Teodorovic et al., 2007; Lasek-Nesselquist et al., 2009;
Jerlström-Hultqvist et al., 2010), suggesting that the two
nuclear genomes are able to exchange genetic information
during the Giardia life cycle. The Giardia genome also
contains seven homologs of meiosis-specific genes: Spo11,
Hop1, Dmc1a, Dmc1b, Hop2, Mnd1 and Mer3 (Ramesh et al.,
2005; Malik et al., 2008), which in most organisms are expressed
only during meiosis and are specifically involved in meiotic
homologous recombination. However, neither meiosis nor a
sexual stage has been identified in the Giardia life cycle. We
have previously shown that nuclear fusion and the exchange of
episomal plasmids can occur between nuclei in the Giardia cyst,
a process we have termed ‘diplomixis’ (Poxleitner et al., 2008);
we have also shown that Spo11, Hop1 and Dmc1a are expressed
only during encystation. Thus, we hypothesized that diplomixis
could involve homologous recombination between the nuclear
genomes. If accompanied by recombination and/or gene
conversion within nuclei in the trophozoite, this process could
help reduce heterozygosity between the two nuclei.
However, another way to maintain low heterozygosity could
be the sorting of nuclei during excystation such that each
daughter cell receives two identical nuclei (rather than one of
each original nucleus from the parent trophozoite) (Fig. 1C,D).
Therefore, the goals of the present study were twofold: (i) To
track the identities of the nuclei during encystation and
excystation, and (ii) to determine whether diplomixis involves
the exchange of chromosomal genetic material between nuclei.
The results of these studies have implications for our
understanding of both the basic biology and the population
genetics of this parasite and could also shed light on the evolution
of meiosis.
Results
Cysts are formed by an incomplete mitotic division
In vitro, encystation can be prompted by exposing cells to basic
medium containing high levels of bile (which prevents
cholesterol absorption) for 24–48 hours (Gillin et al., 1988;
Gillin et al., 1989; Kane et al., 1991; Luján et al., 1996).
Similarly, excystation can be triggered by a two-stage procedure
in which cells are first exposed to acidic conditions for
30 minutes, mimicking passage through the stomach, followed
by a higher-pH solution containing proteases for an additional
30 minutes, which helps break open the cyst wall (Bingham and
Meyer, 1979; Rice and Schaefer, 1981; Schaefer et al., 1984;
Buchel et al., 1987; Hautus et al., 1988; Boucher and Gillin,
1990).
To determine whether cysts can be derived from the fusion of
two trophozoites, we first mixed two strains with different
epitope-tagged markers [histone 2A tagged with three
hemagglutinin molecules (3HA) and median body protein
tagged with three Myc molecules (3myc)] and encysted them
in vitro. None of the resulting cysts contained both markers
(supplementary material Table S1). This result indicates that,
at least in vitro, cysts are generally derived from a single
Journal of Cell Science
Nuclear inheritance in Giardia
trophozoite. Following water treatment (to destroy any
unencysted cells), excystation and growth to confluency of
these mixed cultures, we found that trophozoites always had only
one of the two markers (supplementary material Table S1),
suggesting that cell fusion also does not occur during excystation.
Additionally, in a larger-scale experiment performed with 56107
live cysts (500-ml cultures), encystation and excystation of a
mixture of two strains with different integrated drug resistance
markers (to neomycin and puromycin) failed to produce any
trophozoites with resistance to both drugs (data not shown).
Because we typically observe an in vitro excystation efficiency
on the order of ,1%, the production of recombinant cells
(which would require cell fusion followed by chromosomal
recombination or nuclear sorting), if it occurs, would have to
happen in fewer than 1 in 500,000 cells.
Next, we sought to characterize the incomplete mitotic division
during encystation by staining cells with antibodies against
tubulin, centrin (a basal body and spindle pole marker) and cyst
wall protein (CWP) after 24 hours of encystation (Fig. 2). Before
the division, the trophozoites produce encystation-specific
vesicles (ESVs) full of CWP, and two clusters of four centrin
spots (marking the basal bodies) are visible between the nuclei
(Fig. 2A). Anaphase (Fig. 2B) and telophase (Fig. 2C) generally
resemble the mitotic division in trophozoites, with two parallel
spindles and the basal bodies (centrioles) at the spindle poles.
However, unlike in the trophozoite, the encysting cells are
concurrently in the process of exporting ESVs to the periphery of
the cell for deposition. At the end of telophase of the encystation
mitotic division (Fig. 2D), the process diverges further from its
vegetative counterpart: cytokinesis does not occur, the cell does
not grow new flagella, and its existing flagella appear to shorten,
perhaps as a result of internalization.
At this stage, the two pairs of nuclei are found at opposite
poles of the cell (approximately at the previous location of the
spindle poles) and each pair remains associated with a bundle of
internal axonemes, which appear to be derived from the
axonemes that remain between the nuclei during mitosis
(Nohynková et al., 2006) (Fig. 2E). It is at this time that the
cyst wall is rapidly deposited; by the time the pairs of nuclei have
migrated to the same pole of the cyst, the cyst wall has
completely formed. The two nuclei within each pair are closely
associated and the pairs remain motile for some time in the cyst
(Poxleitner et al., 2008), possibly owing to their association with
the internal axonemes; this is also presumably the mechanism by
which they migrate together. The mature cyst contains four
nuclei, two bundles of internal axonemes with eight centrin loci
and several ventral disc fragments (Fig. 2F).
It has been reported that the mature cyst contains four
fragments of the parental disc that are reassembled during
excystation (Elmendorf et al., 2003; Palm et al., 2005). However,
on the basis of staining of cells with antibodies to label the
tubulin cytoskeleton and the disc (antibody against delta giardin),
we observed what appeared to be the initial stages of two new
daughter discs being formed during the later stages of the
encystation mitotic division (supplementary material Fig. S1A).
At the same time, the parental disc was being disassembled, as
has been described during cytokinesis in the trophozoite (Tůmová
et al., 2007). We most often saw two disc fragments in the mature
cyst and early excyzoites; however, in some cases, up to four
fragments were visible, although they were still usually found in
two clusters (supplementary material Fig. S1B,C). In the
2525
Fig. 2. The Giardia cyst is formed by an incomplete mitotic division.
Encysting cells were stained with DAPI to label DNA (blue) and antibodies to
label tubulin (green), centrin (red), and cyst wall protein (CWP) (gray).
(A) An encysting trophozoite, demonstrating typical centrin and tubulin
staining and the presence of two nuclei. The median body, a cluster of
microtubules of unknown function, is indicated (arrow). (B) An encysting cell
in anaphase. The two spindles (arrowheads) and median body (arrow) are
indicated. (C,D) Encysting cells in early and late stages of telophase.
(E) Early cyst in which the nuclear pairs are still at opposite ends of the cell.
The arrow indicates what appears to be a nascent disc fragment associated
with one pair of nuclei. (F) Mature cyst in which the nuclear pairs have
migrated to one pole of the cell. Scale bar: 5 mm.
excyzoite, the nascent disc fragments appeared to move to the
correct position and orientation to complete their development
into two new discs (supplementary material Fig. S1D).
Reconstruction of the cytoskeleton during excystation
Next, we stained the cytoskeleton of excysting cells to monitor
the transition from excyzoite into trophozoite (Fig. 3). As
previously reported, the excyzoite emerges from a single pole
of the cyst, which is weakened by the exposure to acidic
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content (Fig. 3D–E) (Bernander et al., 2001). By this stage, the
number of basal bodies and flagella has increased from 8 to 16,
and the cell rapidly completes the construction of two ventral
discs (Fig. 3E). Following cytokinesis, the resulting 8C
trophozoites presumably divide immediately to produce two 4C
trophozoites (Bernander et al., 2001).
Journal of Cell Science
Nuclear identity and genetic exchange during
differentiation
Fig. 3. Reassembly of the microtubule cytoskeleton during excystation.
Excysting cells were stained with DAPI to label DNA (blue) and antibodies to
label tubulin (green), centrin (red), and cyst wall protein (CWP) (gray).
(A) Cyst in the early stages of excystation (note the rupture in the cyst wall at
the bottom of the image). (B) An excyzoite emerging from a cyst.
(C) Completely excysted cell. A residual encystation-specific vesicle is
indicated (arrow). (D,E) Later-stage excyzoites in which the typical heartshaped cytokinetic morphology is restored, including two complete discs and
16 flagella. Scale bar: 5 mm.
conditions in the stomach and intestinal proteases (Coggins and
Schaefer, 1986; Buchel et al., 1987; Boucher and Gillin, 1990;
Hetsko et al., 1998) (Fig. 3A). The flagella emerge first, followed
by the body of the cell (Fig. 3B). At this stage, the cytoskeleton
of the excyzoite still resembles that of the cyst, with internal
bundles of axonemes, two pairs of nuclei and eight centrin spots
(Fig. 3A–C). These cells sometimes display one or several
structures that resemble ESVs (arrow in Fig. 3C) and contain
CWP; these could result from the residual translation of cyst wall
protein transcripts, which are extremely abundant during
encystation (Hetsko et al., 1998), or could represent leftover
vesicles from encystation or excystation that have not yet been
eliminated.
The axonemes in the excyzoite are clustered into two
prominent bundles, each associated with a nuclear pair
(Fig. 3C). Although this configuration of axonemes is different
from that of a trophozoite, the cell is still capable of swimming
(Buchel et al., 1987; Hautus et al., 1988). After additional
rearrangements, the excyzoite begins to resemble a heart-shaped
(pre-cytokinesis) cell that is essentially indistinguishable from
that of a dividing trophozoite, with the exception that each
nucleus is thought to contain a 4C (replicated diploid) DNA
The association of pairs of nuclei in the cyst and in the excyzoite
suggests that the nuclear pairs from the parent trophozoite are
preserved throughout differentiation, meaning that a mechanism
other than nuclear sorting must exist to reduce allelic
heterozygosity in Giardia populations (compare Fig. 1C and
Fig. 1D). To test this hypothesis, we integrated a plasmid
containing a 10-kb array of LacO repeats into the chromosomes
of a population of trophozoites. To ensure the presence of only a
single integrated construct, we then made clonal populations and
tested for the absence of episomes by PCR (supplementary
material Fig. S2). We then performed fluorescence in situ
hybridization (FISH) using a fluorescent probe to label the LacO
repeat. As for episomes (Yu et al., 2002), integrated plasmids
were always found in only one of the two nuclei in the
trophozoite (Fig. 4A). Most cells contained a single spot, but
occasionally we observed two spots very close together in a
single nucleus; we interpret these to represent either replicated
chromatids that became separated (most trophozoites rest in G2,
but few cells contained two spots) or cases in which the homolog
gained the marker through mitotic recombination or gene
conversion. In the pre-encystation population, 76% of cells
(n5100) contained one spot (or two very close together) in a
single nucleus, whereas 24% of cells contained no spot (Fig. 4A).
Because this population was clonal and the marker was
integrated, and therefore stable even in the absence of drug
selection, we interpret this result as a common artifact of FISH;
however, it also possible that at least some cells lose the repeat
cassette through recombination. After encystation, excystation,
and growth of excysted trophozoites to confluency, all in the
absence of drug selection, we observed that 4962% of cells
(6s.d.; average of three biological replicates, each n5100) had
one spot, 3162% had no spots and 2062% had two spots (one in
each nucleus) (Fig. 4A). Thus, the production of non-parental
patterns is common and consistent.
The observed pattern could be generated in two ways: (i)
genetic exchange between nuclei, or (ii) nuclear sorting such that
during excystation each daughter cell received two identical
nuclei. However, because only 20% of cells contained two spots,
nuclear sorting would have had to occur in a minority of
excysting cells (excyzoites). To begin to distinguish between
these possibilities, we examined a strain that contained the same
LacO repeat construct, but also contained a TetO repeat construct
integrated into a different chromosome in the same nucleus
(Fig. 4B). As with the LacO construct, the absence of episomal
plasmid in this clonal line was confirmed by PCR (data not
shown). In this strain, 71% of pre-encysting trophozoites
(n5100) contained both markers in a single nucleus; the
remaining 29% were missing one or both markers, probably
owing to artifacts and/or recombination, as noted above.
However, in 25% of excysted trophozoites (n5100), we
observed cells with all possible non-parental arrangements of
markers (i.e. 14% with one in each nucleus, 10% with two in one
Journal of Cell Science
Nuclear inheritance in Giardia
Fig. 4. Chromosomal exchange occurs between nuclei during
differentiation. (A) A clonal population of cells containing an integrated
LacO array in one nucleus was subjected to FISH to track the marker in the
starting population (pre-encystation trophozoites) and in excysted
trophozoites. DAPI-stained DNA is shown in blue and the LacO probe is
shown in red. Percentages of cells with 0, 1 and 2 spots are shown below each
image. (B) Patterns observed after excystation of a clonal population
containing two markers, LacO (red) and TetO (green), integrated in the same
nucleus (first panel). The number of spots in each nucleus is indicated below
each image (e.g. N1:0; N2:RG50 spots in nucleus 1; one red spot and one
green spot in nucleus 2). (C–F) Cysts and excyzoites from the experiment
described in A. DAPI-stained DNA is shown in blue, and the LacO probe is
shown in red. Cyst wall protein (CWP) staining is shown in green in C and E,
and tubulin staining is shown in green in D and F. Panels C and D show a cyst
and an excyzoite, respectively, with one spot in each nuclear pair; panels E
and F show a cyst and an excyzoite, respectively, in which one nuclear pair
contains two spots. Scale bars: 5 mm.
nucleus and one in the other, and 1% with two in both nuclei),
further suggesting that chromosomal genetic exchange occurs
between nuclei in the cyst and/or early excyzoite (Fig. 4B). The
remaining 75% of excysted trophozoites contained either the
parental arrangement or were missing one or both markers. In
theory, all of the observed non-parental arrangements could also
be compatible with nuclear sorting combined with signal loss;
however, our level of signal loss is about 30% overall (on the
basis of our observations of pre-encysting cells), so if nuclear
sorting were solely responsible for these patterns, we would
expect to see higher levels of cells with both markers in both
nuclei. Thus, although we cannot completely exclude rare nuclear
sorting on the basis of these data, we can conclude that it is not
responsible for all of the observed non-parental arrangements of
markers.
2527
To rule further out nuclear sorting, we also performed FISH on
cysts and excyzoites (Fig. 4C–F). As expected, the nuclei of most
cysts contained two clustered pairs of nuclei, each with one
labeled nucleus (Fig. 4C). This pattern was recapitulated in most
excyzoites (Fig. 4D), again supporting the idea that the nonidentical nuclear pairs remain associated throughout encystation
and excystation. Although we were able to observe an occasional
cyst with three labeled nuclei (Fig. 4E), as would be expected on
the basis of our observation of two spots in the nuclei of excysted
trophozoites, this was rare; however, we did notice that the spots
in the cyst were often positioned between the masses of DAPIstained DNA (Fig. 4C), suggesting that the nuclei might remain
fused in the cyst and only separate during excystation.
Consistently, we were able to identify an early excyzoite with
three labeled nuclei (Fig. 4F) (i.e. where one nuclear pair
underwent genetic exchange involving movement of the
integrated marker, but the other did not). Therefore, our results
indicate that the predominant mode of nuclear inheritance for
excysting trophozoites involves the inheritance of one copy of
each parental nucleus, and chromosomal genetic exchange occurs
between nuclear pairs in the cyst.
On the basis of our previous observations of the expression
patterns of the Giardia meiotic gene homologs, we predict that
this genetic exchange involves homologous recombination.
However, because the previous studies were performed using
tagged genes on multi-copy episomes (albeit under the native
promoter), it is possible that the observed patterns might have
been caused by overexpression. Thus, in the present study, we
used plasmid integration (Gourguechon and Cande, 2011) to
endogenously tag the seven meiotic gene homologs with 3HA
epitope tags. Because the genes were tagged at their endogenous
locus, the constructs were under the control of the native
promoter. We then examined the expression and localization of
these proteins in both trophozoites and cysts. Mer3, which in
yeast is a meiosis-specific DNA helicase (Nakagawa and
Kolodner, 2002), did not localize to the nuclei of trophozoites
or cysts (data not shown), suggesting that it might not, in fact, be
a Mer3 ortholog; consistently, it is annotated as an RNA helicase
in the Giardia genome database (ORF GL50803_87022 at
giardiadb.org). As shown in supplementary material Fig. S3,
and consistent with our previous findings, only Dmc1b was
expressed at high levels in the trophozoite; Mnd1 and Hop2 (not
previously studied) were present at low levels, and Spo11,
Dmc1a and Hop1 were absent. By contrast, Dmc1b was absent in
cysts, whereas Spo11, Dmc1a and Hop1 were upregulated,
appearing in encysting trophozoites and remaining in fully
encysted cells (supplementary material Fig. S3) (Poxleitner et al.,
2008). Mnd1 and Hop2 remained at low (but detectable) levels in
cysts. In all cases, and as expected, the proteins were localized to
the nuclei in a punctate pattern. We also attempted to knockdown
Dmc1a and Spo11 by electroporation with morpholinos
(Carpenter and Cande, 2009) immediately before encystation to
assess their effects on exchange of the LacO marker, but we were
unable to attain measurable levels of knockdown, possibly owing
to the morpholinos being diluted during the encystation process
(data not shown).
Finally, because we never observe spindles in fully formed
cysts (i.e. there is no organized separation of chromosomes such
as that seen in a meiotic division), the cell must have some way in
which to anchor its chromosomes to prevent widespread
aneuploidy following diplomixis. On the basis of the telomere
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Journal of Cell Science 125 (10)
Journal of Cell Science
bouquets observed in meiosis in many organisms, we predicted
that telomere anchoring might be involved in Giardia as well.
Thus, we performed FISH on trophozoites and cysts using a
probe to detect the Giardia telomeric repeat sequence TAGGG
(Fig. 5). Interestingly, the telomeres were clustered, usually in
two spots per nucleus, in both trophozoites (Fig. 5A) and cysts
(Fig. 5B). A preliminary BLAST search for telomere-binding
proteins in the Giardia genome revealed only telomerase
(GiardiaDB accession GL50803_16225), suggesting that the
proteins involved in tethering the chromosomes to the nuclear
envelope in Giardia are either highly divergent from those in
yeast and humans and/or are completely novel. This clustering
might provide a mechanism by which the nuclei are able to
undergo recombination without drastically changing the ploidy of
each nucleus. However, it should be noted that some aneuploidy
between nuclei, usually involving the addition or subtraction of
one chromosome, has been observed in Giardia trophozoites
(Tůmová et al., 2006); diplomixis could be the mechanism by
which this aneuploidy was initially created.
Discussion
As an organism with two nuclei, Giardia faces unique challenges
in coordinating nuclear inheritance during its life cycle. Although
the functional equivalence of the nuclei in Giardia and their
independent distribution during mitosis in the trophozoite are
relatively well established (Kabnick and Peattie, 1990; Yu et al.,
2002), their fates during encystation and excystation have only
been studied with respect to genome ploidy (Bernander et al.,
2001), despite the significance of this knowledge to our
understanding of patterns of allelic heterozygosity and genetic
diversity in Giardia populations. Therefore, the goal of the
present study was to determine how the four nuclei are derived in
the cyst and how these nuclei are distributed during excystation.
Cysts are produced by mitosis and not cell fusion
On the basis of strain mixing experiments (supplementary
material Table S1) and immunofluorescence staining of
encysting cells (Fig. 2), we have demonstrated that the
quadrinucleate cyst is produced through an incomplete mitotic
division that occurs rapidly during the final stages of cyst wall
deposition. In addition, we have provided a detailed description
of the temporal organization of the microtubule cytoskeleton
during encystation and excystation. Although the fusion of two
trophozoites to form a cyst – which, if followed by nuclear fusion
and genetic exchange, would resemble a parasexual cycle with
outcrossing – is still theoretically possible, this event would have
to be rare and/or happen only under certain conditions, such as in
Fig. 5. Telomere clustering in trophozoites and cysts. FISH was performed
on trophozoites (A) and cysts (B), using a probe to label the telomeric repeat
sequence. DAPI-stained DNA is shown in blue, the telomere probe is shown
in green and antibody staining against cyst wall protein is shown in red. Scale
bars: 5 mm.
the host. It is also possible that fusion only happens between
different strains or between cells with different variant surface
proteins (Lasek-Nesselquist et al., 2009). However, given that
encystation takes place during transit through the small and large
intestine, we speculate that a strict requirement for two cells to
find each other and fuse in order to form a cyst might be
disadvantageous; thus, encystation from a single parent cell
appears to be the predominant (if not sole) mode of cyst
formation.
Nuclei are inherited in non-identical pairs
Our observations of the cytoskeletal components of encysting and
excysting cells both synthesize and build upon the results of
previous ultrastructural and transmission electron microscopy
(TEM) studies (Sheffield and Bjorvat, 1977; Luchtel et al., 1980;
Buchel et al., 1987; Hetsko et al., 1998; Bernander et al., 2001;
Palm et al., 2005; Midlej and Benchimol, 2009; BittencourtSilvestre et al., 2010), which have primarily focused on mature
cysts. We found that the encystation division resembles the
vegetative trophozoite mitotic division until approximately
telophase. At that point, instead of continuing on into
cytokinesis, the cell internalizes its flagella as the cyst wall
rapidly forms. At the same time, cyst wall components are being
produced and transported to the periphery of the cell, a process
that appears to be independent from, albeit simultaneous with, the
nuclear division (Stefanic et al., 2009). In addition, although
trophozoites in the early stages of encystation display larger
rounded ESVs, by the time the nuclear division takes place, the
cyst wall components have been transported to the periphery of
the cell and exist in a more diffuse network, as previously
reported (Konrad et al., 2010).
On the basis of the patterns of centrin and tubulin staining in
our study, the bundles of axonemes associated with each nuclear
pair in the cyst appear to be derived from the axonemes found
associated with the nuclei during mitosis (Nohynková et al.,
2006). According to that study, each daughter cell (or pair of
nuclei, in this case) receives one anterolateral flagellum, one
caudal flagellum, and either the posterolateral or ventral flagellar
pair. These results are consistent with those of a TEM-based
study that reported two bundles of three internal axonemes
associated with each nuclear pair in the cyst, as well as two
additional flagella found in the periphery of the cell (Sheffield
and Bjorvat, 1977). It remains unclear whether the flagella are
actively depolymerized during encystation or whether they are
simply internalized.
On the basis of our observations of the growth of two nascent
daughter discs during the encystation mitotic division and the
overall morphological similarity of these nascent discs to the
fragments in the cyst, we hypothesize that disc growth is paused
during encystation and then resumed during excystation. This
scenario would be more consistent with a what is observed during
cytokinesis in the trophozoite, in which two new ventral discs
grow on the opposite side of the cell from the parental ventral
disc (Tůmová et al., 2007). However, because we cannot
distinguish between old and new discs, it remains possible that
the disc fragments in the cyst are derived from the parental disc
and are retained as raw material for the construction of new discs
during excystation.
This is the first study, to our knowledge, to use
immunofluorescence staining to examine the Giardia
cytoskeleton in depth during excystation, although previous
Journal of Cell Science
Nuclear inheritance in Giardia
studies have looked at the localizaton of two proteins, PP2A and
beta-giardin, in excyzoites (Palm et al., 2005; Lauwaet et al.,
2007b). As reported previously (Coggins and Schaefer, 1984;
Coggins and Schaefer, 1986; Buchel et al., 1987; Erlandsen et al.,
1989), we observed that the excyzoite exits the cyst at one pole,
starting with its flagella. The early excyzoite has a similar
cytoskeletal organization to the cyst, with each pair of nuclei
remaining associated with a bundle of internal axonemes;
however, it rapidly re-molds its cytoskeleton to resemble a
cytokinetic trophozoite, which is the stage where it paused after
the mitotic division during encystation. Its nuclei become
decondensed and the distance between nuclei in each pair
increases, presumably bringing an end to any nuclear fusion that
has occurred. At this stage, each nascent trophozoite is thought to
have an 8C genetic content (based on a 16C total in the cyst)
(Bernander et al., 2001); thus, after cytokinesis is completed, the
cell is primed to complete another round of mitosis without an
additional S phase, as the chromosomes were already duplicated
in the S phase during encystation. Because the number of centrin
spots increases from eight in the excyzoite to 16 in the heartshaped cells, we conclude that basal body duplication is also
occurring during this time. Taken together, our results suggest
that the cytoskeletal changes that happen during encystation,
including disc assembly, basal body duplication and flagellar
development, are very similar on the molecular level to their
counterparts in the trophozoite (Nohynková et al., 2006; Tůmová
et al., 2007; Dawson and House, 2010); the primary difference is
the timing of these events in relation to spindle formation.
Importantly, we have determined that during excystation, the
daughter trophozoites receive one copy of each parental nucleus.
To date, the genomes of three Giardia assemblages (genotypes)
have been sequenced. Two of these, assemblages A and E,
contain extremely low levels of allelic heterozygosity – less than
0.01% (Morrison et al., 2007; Jerlström-Hultqvist et al., 2010).
These results are surprising because, in a presumed asexual
organism with two independent nuclei, the nuclear genomes –
and perhaps even homologous chromosomes – would be
expected to diverge over time owing to genetic drift (Birky,
2010). Fusion and genetic exchange (diplomixis) between the
nuclei in the cyst, if accompanied by recombination and gene
conversion within nuclei in the trophozoite, could potentially
explain these low levels of heterozygosity (Poxleitner et al.,
2008; Andersson, 2011). However, the distribution of identical
nuclei to daughter cells during excystation would also explain,
independently of diplomixis, the low levels of heterozygosity
observed in Giardia populations (Fig. 1). Thus, our observation
that nuclei are distributed in non-identical pairs during
excystation is significant and implies that there are other
mechanisms in place, including but perhaps not limited to
diplomixis, to reduce genomic heterozygosity in Giardia. One of
these could be highly efficient DNA damage repair in the
trophozoite, which rests in the G2 phase of the cell cycle
(Bernander et al., 2001); indeed, it is tempting to speculate that
this unusual characteristic (most eukaryotic cells rest in G1) arose
to allow ample time for repair using the sister chromatid as a
template. The expression of Dmc1b in the trophozoite suggests
that this Dmc1 homolog could mediate homologous
recombination at this stage, and its similarity to Dmc1
(Ramesh et al., 2005) could point to an ability to mediate
repair using the homolog as a template. This ability would also be
required to maintain the low levels of heterozygosity observed in
2529
laboratory strains that have been passaged for long periods of
time (Morrison et al., 2007; Teodorovic et al., 2007), as well as
for heterozygosity to be reduced within nuclei following
diplomixis (Andersson, 2011).
Chromosomal genetic exchange and meiotic gene
expression patterns are consistent with homologous
recombination
We were surprised to find consistently high rates of chromosomal
genetic exchange (,20%) between nuclei in the cyst. This is
probably an underestimate of the amount of exchange occurring,
as our assay can only detect events involving transfer and
preservation of the marker. This finding is consistent with
diplomixis being used as a mechanism to reduce nuclear
heterozygosity: to be effective, genetic exchange would be
expected to be relatively common and involve large portions of
the genome. We currently cannot distinguish between homologous
recombination and whole-chromosome exchange. One way to
directly detect recombination would be to place two integrated
markers far apart on the same chromosome and observe their
separation; however, this would be technically difficult (if not
impossible) in Giardia, both because it is tetraploid and because
there would be no clear way to confirm that the markers are in the
same chromosome. Nevertheless, we predict that diplomixis
involves homologous recombination for two reasons: first, three
of the meiotic gene homologs (Dmc1a, Spo11 and Hop1) are
expressed specifically at this stage in the life cycle, and, second, it
is difficult to envision a mechanism for whole-chromosome
exchange that would not result in widespread aneuploidy.
Therefore, we predict that the meiotic gene homologs are
involved in mediating homologous recombination between the
nuclei during diplomixis. If this turns out to be the case, it would be
one of the first examples of these proteins functioning in a nonmeiotic context. Spo11 has been found to mediate homologous
recombination during the parasexual cycle of the fungus Candida
albicans (Forche et al., 2008). However, the lack of a sexual cycle
in this organism is clearly a case of loss, whereas it is unclear
whether Giardia or its relatives ever had a true meiotic cycle.
Future studies of the functions of the meiotic gene homologs
during diplomixis in Giardia could shed light on the evolution of
the meiotic homologous recombination machinery.
On the basis of the observations from the present study, we
envision a model whereby tethered chromosomes are able to
interact and recombine with chromosomes from a neighboring
nucleus (Fig. 6A–D) at some point either in the cyst or early
excyzoite. In this way, widespread crossing over (facilitated by the
meiotic gene homologs) between chromosomes from different
nuclei could produce the observed patterns, and subsequent
recombination and gene conversion could then act to
homogenize the homologous chromosomes within each nucleus.
Consistent with this hypothesis, we occasionally observed two
paired spots rather than one in some cells (see fourth panel in
Fig. 4B). These spots could represent replicated chromosomes that
became separated; however, these patterns, as well as the increase
in unlabeled nuclei following excystation (Fig. 4A), could also
result from recombination and gene conversion between
homologous chromosomes in the same nucleus.
In conclusion, we have demonstrated that, at least in vitro,
Giardia cysts are derived from a single cell through an
incomplete mitotic division and that daughter trophozoites
inherit one copy of each parental nucleus during excystation.
2530
Journal of Cell Science 125 (10)
Journal of Cell Science
Furthermore, as hypothesized on the basis of the results of our
previous study (Poxleitner et al., 2008), we have directly
demonstrated that chromosomal genetic exchange occurs during
diplomixis. This exchange happens at the same time that several
homologs of meiosis-specific genes are induced, suggesting that
homologous recombination in occurring, presumably as a way to
reduce allelic heterozygosity between nuclei. However, although
diplomixis could explain the maintenance of low heterozygosity
between nuclei, it is also possible that rare meiotic events occur
in the wild, as suggested by several recent population genetics
studies (Cooper et al., 2007; Lasek-Nesselquist et al., 2009).
These studies found evidence for recombination within and
between assemblages, which cannot be explained by diplomixis
within a single cell, but could potentially be explained by a
combination of rare cell fusion followed by diplomixis.
Furthermore, in contrast to the low heterozygosity in
assemblages A and E, the genome of assemblage B strain GS
has 0.5% allelic heterozygosity (Franzén et al., 2009), which has
been suggested to be evidence for recent genetic exchange in the
lineage (Andersson, 2011).
Future studies should aim to test for fusion and diplomixis or a
true meiotic event in vivo, similar to what has been performed
to detect sexual stages in parasites like Trypanosomes and
Leishmania (Akopyants et al., 2009; Peacock et al., 2011).
Identifying this process might be difficult, particularly if it is rare
or occurs only under specific conditions. Nevertheless, a more
complete understanding of the Giardia life cycle will not only
give us insight into the basic biology of this important parasite
but might also shed light on the spread of drug resistance in
Giardia populations.
Please note that during the final preparation of this article, a study
describing the mitotic division during encystation and subsequent
nuclear interconnection in the Giardia cyst was published
(Jirakova et al., 2012).
Materials and Methods
Culture conditions
Giardia intestinalis trophozoites, WB clone C6 (ATCC 50803), were grown in
modified TYI-S-33 medium (Keister, 1983) with bovine bile (Sigma-Aldrich).
Cultures were incubated in 16-ml plastic screw top tubes (Fisher Scientific) at 37 ˚C
and passaged every three days.
Fig. 6. Hypothetical model of homologous recombination during
diplomixis. (A) Shown is a simplified cell containing an integrated marker (red)
in one of its four homologous chromosomes (H1 and H2, shown in orange, in the
left nucleus, and H19 and H29, shown in blue, in the right nucleus). Throughout
the figure, the presence of the integrated marker is indicated by a red outline
around the nucleus in which it is found. Because trophozoites typically rest in G2
of the cell cycle, this trophozoite is shown with its chromosomes already
duplicated (8C genome content; 4C in each nucleus). (B) During encystation, the
meiotic gene homologs Spo11, Dmc1a and Hop1 are induced. Both nuclei divide
and then go through an S phase, producing a 16C cyst. At some point, the nuclei
fuse and exchange genetic material, presumably through homologous
recombination. The chromosomes remain tethered to the nuclear envelope by
their telomeres, which might prevent the development of aneuploidy following
diplomixis. (C) When the cell excysts and completes cytokinesis, two
trophozoites are formed, one from each nuclear pair found in the cyst. (D) If
diplomixis has occurred (left trophozoite in C), the first mitotic division
produces two possible combinations of daughter trophozoites, depending on the
orientation of the chromosomes on the spindle. One of these combinations
would produce one cell with markers in both nuclei and one cell with no marker,
whereas the other would produce the parental arrangement (marker in one
nucleus). If diplomixis has not occurred (right trophozoite in C), only the
parental arrangement (marker in one nucleus) is produced.
In vitro encystation
Encystation was performed essentially as described previously (Kane et al., 1991).
Briefly, trophozoites were seeded into 30- or 50-ml tissue culture flasks (BD
Biosciences) and grown to 80–100% confluency. The medium was then decanted
and replaced with prewarmed encystation medium (10 mg/ml bovine-ovine bile,
pH 7.8). After 24 hours at 37 ˚C, cells were either fixed for observation or
incubated on ice for 15 minutes, pelleted at 500 g, and resuspended in regular
growth medium (0.5 mg/ml bile, pH 7.1). After an additional 24 hours of
incubation at 37 ˚C, the cells were again chilled on ice, pelleted, and
resuspended in 50 ml ice-cold, sterile deionized water to destroy all unencysted
cells. Cysts were stored at 4 ˚C for 1–3 days, then excysted as described below.
In vitro excystation
Excystation was performed essentially as described previously (Boucher and
Gillin, 1990). Briefly, water-treated cysts were pelleted in a 50-ml conical tube,
then resuspended in 10 ml prewarmed stage I solution (16 Hank’s balanced salt
solution (Invitrogen), 57 mM L-cysteine HCl, 32.5 mM reduced glutathione and
25 mM NaHCO3; the pH was adjusted to 4.0 with 0.01 M HCl). The tube was
vortexed briefly, then incubated at 37 ˚C for 30 minutes. After another brief
vortexing, cells were pelleted at 500 g for 5 minutes. The supernatant was then
decanted and replaced with 10 ml prewarmed stage II solution [Tyrode’s buffer
(Sigma-Aldrich) containing 1 mg/ml Trypsin II-S (Sigma); the pH was adjusted to
8.0 with 1 M NaHCO3]. The tube was vortexed briefly, incubated at 37 ˚C for
30 minutes, then vortexed again and pelleted. The supernatant was decanted and
replaced with prewarmed growth medium (see the Culture conditions section), and
Nuclear inheritance in Giardia
cells were moved into 16-ml culture tubes and incubated at 37 ˚C. Cells were then
either fixed 1–3 hours later for immunofluorescence staining or grown to
confluency (,5–7 days).
Cy5-labeled mouse anti-CWP antibody (Waterborne, Inc., New Orleans, LA) for 2
hours. After a final three washes each in PEM and PEMBALG, coverslips were
mounted onto slides with Prolong Gold Antifade Solution with DAPI (Invitrogen).
Creation of constructs for endogenous tagging
Fluorescence deconvolution microscopy
For creating integration constructs for endogenous tagging, the appropriate ORF
was amplified from WBC6 genomic DNA and inserted into either the vector pKS3HA-PAC or pKS-3myc-NEO (Gourguechon and Cande, 2011). The amplified
ORFs, primers, and restriction sites used for digestion and linearization are shown
in supplementary material Table S2. Transformation and selection of Giardia
trophozoites was performed as previously described (Gourguechon and Cande,
2011).
Images were collected with SoftWorX image acquisition software (Applied
Precision, Issaquah, WA) on an Olympus IX70 wide-field inverted fluorescence
microscope with an Olympus UPlanApo 1006, NA 1.35, oil-immersion objective
and Photometrics CCD CH350 camera cooled to 235 ˚C (Roper Scientific, Tuscon,
AZ). Serial sections were acquired at 0.2 mm intervals, and data stacks were
deconvolved using the SoftWorX deconvolution software. For presentation
purposes, two-dimensional maximum intensity projections were created from the
three-dimensional data sets using the DeltaVision image analysis software
(Applied Precision).
Creation of clonal strains containing integrated LacO and TetO constructs
Journal of Cell Science
2531
For the LacO array integration construct (supplementary material Fig. S2), a SacIPvuII fragment containing part of the Spo11 coding sequence [GiardiaDB
accession GL50803_15279; located on chromosome 4 (Upcroft et al., 2010)] and a
puromycin resistance cassette (from the vector pKS-Spo11-3HA-pac) was inserted
into the LacO-array-containing plasmid SR7 (Rohner et al., 2008), a gift from
Susan M. Gasser (University of Basel, Basel, Switzerland); cut with restriction
enzymes SacI and EcoRV. For integration, the plasmid was linearized with MfeI,
which cuts within the sequence for Spo11. After the selection of stable
transformants, single-cell clones were obtained by limiting dilution as described
previously (Baum et al., 1988). These clones were screened for the presence of the
integrated plasmid and absence of episomal plasmid by PCR as shown in
supplementary material Fig. S2, using the primers P1 (59-CATGATTTTCTTGTTTTAGGG-39) and P2 (59-TAATACGACTCACTATAGGG-39) or P1 and P3
(59-AGACTCTACAGGATACTTTCAG-39). PCRs were performed by boiling a
concentrated culture (200 ml) of approximately 1.36107 cells for 10 minutes at
95 ˚C. One microliter (,65,000 cells) was then used for PCR amplification.
For the TetO array construct, an EcoRI-XbaI fragment containing the median
body protein [MBP; GiardiaDB accession GL50803_16343, located on
chromosome 3 (Upcroft et al., 2010)] coding sequence and a neomycin
resistance cassette (from the vector pKS-MBP-neo) was inserted into the TetOarray-containing plasmid SR8 (Rohner et al., 2008), a gift from Susan M. Gasser;
cut with the restriction enzymes EcoRI and NheI. For integration, the plasmid was
linearized with MfeI, which cuts within the sequence for MBP. After the selection
of stable transformants, clonal lines were created and confirmed as described
above, except using primer P4 (59-TTCCGAATTTTGAGACACCTC-39) instead
of primer P3 listed above.
Fluorescence in situ hybridization
FISH was performed essentially as described previously (Poxleitner et al., 2008).
However, instead of using nick translation to create the probe, the following probes
were used at a concentration of 5 ng/ml: a Cy3-labeled LacO oligonucleotide probe
(59-CCACAAATTGTTATCCGCTCACAATTCCACATGTGG-39); a FITC-labeled
TetO probe (59-TCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAG-39); and a FITC-labeled telomere probe (59-TAGGGTAGGGTAGGGTAGGGTAGGG-39).
Immunostaining
Fixation and immunostaining were performed essentially as described previously
(Carpenter and Cande, 2009). Briefly, cells were fixed in 16PEM buffer (100 mM
PIPES, 1 mM EGTA, 0.1 mM MgSO4) containing 2% paraformaldehyde. After
fixation for 30 minutes at 37 ˚C, cells were washed once with PEM buffer, then
resuspended in PEM buffer and attached to poly-L-lysine-coated coverslips for
30 minutes. Alternatively, for antibody staining against delta-giardin, cells were
fixed for 3 minutes in ice-cold methanol, then washed twice in PBS before being
attached to poly-L-lysine coverslips. Fixed cells were permeabilized with 0.1%
Triton X-100 for 15 minutes, washed three times with PEM, and blocked in
PEMBALG [PEM plus 1% BSA, 0.1% NaN3, 100 mM lysine and 0.5% gelatin
from cold water fish skin (Sigma)] for 2 hours. Primary antibody incubations were
conducted at room temperature overnight, and antibodies were diluted in
PEMBALG as follows: mouse anti-trypanosome alpha-tubulin antibody TAT1,
1:50 (Woods et al., 1989), a gift from Keith Gull (University of Oxford, Oxford,
England); 20H5 mouse anti-centrin antibody, 1:100 (Salisbury et al., 1988); rabbit
anti-sea urchin tubulin antibody 1:25 (Fujiwara and Pollard, 1978); rat anti-HA
antibody 1:100 (Roche); mouse anti-HA antibody 1:100 (Sigma); rabbit anti-Myc
antibody 1:100 (Cell Signaling Technology); rabbit anti-delta-giardin antibody
1:100 (Jenkins et al., 2009), a gift from Mark C. Jenkins (US Department of
Agriculture, Beltsville, MD, USA). After washing with PEMBALG, cells were
incubated with secondary antibodies (goat anti-mouse antibody conjugated to
Alexa Fluor 555 and goat anti-rabbit antibody conjugated to Alexa Fluor 488
[Invitrogen]) at room temperature for 2 hours. For labeling with the anti-CWP
antibody, cells were then washed three times with PEMBALG, fixed for
10 minutes in 1% paraformaldehyde, washed three times with PEM and blocked
for an additional 30 minutes in PEMBALG. The cells were then incubated with a
Acknowledgements
We thank current and former members of the Cande laboratory,
especially L. K. Fritz-Laylin and A. R. Paredez, for helpful
discussion. We also thank K. Gull, M. Jenkins and S. Gasser for
reagents.
Funding
This work was supported by the National Institutes of Health [grant
number A1054693 to W.Z.C.]; and the National Science Foundation
(predoctoral fellowship to M.L.C.). The funders had no role in
study design, data collection and analysis, decision to publish, or
preparation of the manuscript. Deposited in PMC for release after 12
months.
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.103879/-/DC1
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