Research Article
4889
Three-dimensional analysis of mitosis and cytokinesis
in the binucleate parasite Giardia intestinalis
Meredith S. Sagolla, Scott C. Dawson*, Joel J. Mancuso and W. Zacheus Cande‡
345 LSA, Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA 94720, USA
*Present address: Section of Microbiology, University of California Davis, Davis, CA 95616, USA
‡
Author for correspondence (e-mail: [email protected])
Journal of Cell Science
Accepted 22 September 2006
Journal of Cell Science 119, 4889-4900 Published by The Company of Biologists 2006
doi:10.1242/jcs.03276
Summary
In the binucleate parasite Giardia intestinalis, two diploid
nuclei and essential cytoskeletal structures including eight
flagella are duplicated and partitioned into two daughter
cells during cell division. The mechanisms of mitosis
and cytokinesis in the binucleate parasite Giardia are
poorly resolved, yet have important implications for the
maintenance of genetic heterozygosity. To articulate the
mechanism of mitosis and the plane of cell division, we used
three-dimensional deconvolution microscopy of each stage
of mitosis to monitor the spatial relationships of conserved
cytological markers to the mitotic spindles, the centromeres
and the spindle poles. Using both light- and transmission
electron microscopy, we determined that Giardia has a
semi-open mitosis with two extranuclear spindles that
access chromatin through polar openings in the nuclear
membranes. In prophase, the nuclei migrate to the cell
midline, followed by lateral chromosome segregation in
Introduction
Giardia intestinalis, a widespread zoonotic intestinal parasite,
has two life-cycle stages: a binucleate, double diploid flagellate
or trophozoite form that attaches to the intestinal microvilli,
and an infectious cyst form that persists in the environment
(Adam, 2001; Gillin et al., 1996). Over several million cases
of malabsorptive diarrhea by giardiasis are estimated to occur
annually worldwide, and waterborne outbreaks of giardiasis
are frequent in areas where unfiltered waters are routinely
contaminated. (Savioli et al., 2006). Although Giardia is well
known in terms of disease (Adam, 2001; Savioli et al., 2006)
and has received much recent attention due to evolutionary
controversies (Baldauf, 2003; Baldauf et al., 2000; Best et al.,
2004; Ciccarelli et al., 2006; Dacks et al., 2002; Graczyk, 2005;
Knight, 2004; Sogin et al., 1989), there remains little
knowledge of the assembly and division of the complex
giardial cytoskeletal systems required for the life cycle of the
parasite, including the mechanism of mitosis.
Like all diplomonads, Giardia has two diploid nuclei
(2n=10), which are both transcriptionally active and identical
in DNA content (Adam et al., 1988; Kabnick and Peattie, 1990;
Wiesehahn et al., 1984; Yu et al., 2002). In addition, Giardia
is bilaterally symmetrical and possesses a complex and
distinctive microtubule cytoskeleton that establishes anteriorposterior, left-right and dorsal-ventral polarity and,
importantly, plays a major role in its virulence (Elmendorf et
al., 2003). The microtubule cytoskeleton of the trophozoite, or
anaphase. Taxol treatment results in lagging chromosomes
and half-spindles. Our analysis supports a nuclear
migration model of mitosis with lateral chromosome
segregation in the left-right axis and cytokinesis along the
longitudinal plane (perpendicular to the spindles), ensuring
that each daughter inherits one copy of each parental
nucleus with mirror image symmetry. Fluorescence in situ
hybridization (FISH) to an episomal plasmid confirms that
the nuclei remain separate and are inherited with mirror
image symmetry.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/119/23/4889/DC1
Key words: Giardia intestinalis, Mitosis, Cytokinesis, Spindle, Basal
body, Centromere
intestinal form, is characterized by four main elements: eight
flagellar axonemes and basal bodies, the ventral disc, the funis
and the median body (Fig. 1A,B). Giardia is unique among
diplomonads in that it possesses the ventral disc, a novel
organelle composed of an overlapping spiral lamella of
microtubules that mediates attachment to the intestinal
microvilli (or a laboratory substrate), most likely by a suctionbased mechanism (Hansen et al., 2006).
To complete cell division, the complex giardial cell must
duplicate and partition both diploid nuclei as well as the
multiple cytoskeletal structures. Thus, a detailed analysis of
mitosis in Giardia at both the ultrastructural and molecular
level is needed to resolve two intriguing questions. First, how
does Giardia coordinate the division of two equivalent nuclei
and a complex microtubule cytoskeleton? Second, how
conserved is the mechanism of mitosis in a highly divergent
and putatively early branching eukaryote? Understanding
chromosome segregation in Giardia, however, has been
hampered principally by the lack of cytological descriptions of
intermediate stages of mitosis, including the inability to
identify the mitotic spindle (Solari et al., 2003). Several prior
studies have sought to identify the stages of mitosis using
primarily light microscopy and chromatin staining, yet have
not described a mitotic spindle (Cèrva and Nohynkova, 1992;
Filice, 1952). Furthermore, recent debate concerning the
mechanism of giardial cell division has lead to proposals of
unconventional mechanisms of chromosome segregation,
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Fig. 1. Giardia has two equivalent nuclei and a complex
microtubule cytoskeleton. (A) The microtubule cytoskeleton of
the trophozoite is characterized by eight flagellar axonemes
and basal bodies (afl, anterior; c, caudal; pfl, posterior; vfl,
ventral), the ventral adhesive disc (vd), the funis (fn) and the
median body (mb) (diagramed in A). All basal bodies are
between the nuclei. (B) Anti-tubulin labeling reveals the
microtubule arrays, including the eight flagella, and median
body. The ventral disk is more weakly stained than the other
structures. Although images of Giardia are frequently
presented as two-dimensional projections in dorsal perspective
as in A, describing cell division in Giardia requires analysis of
cell morphology in three dimensions. (C) The cell is shown in
3D from a side angle. The spatial positions of the four nuclei
following nuclear division are shown arrayed along the leftright (L-R) and dorsal-ventral (D-V) axes. Three possible
planes of cytokinesis are represented in C; longitudinal
[anterior-posterior (A-P)], transverse (L-R) and frontal (D-V).
The dorsal-posterior nuclei are marked with asterisks in C,D.
including the role of non-spindle microtubular organelles in
cell division (Benchimol, 2004b; Solari et al., 2003) or of
the presence of multiple or incongruent planes of division
(Benchimol, 2004a; Ghosh et al., 2001; Yu et al., 2002).
Owing to the cytoskeletal complexity of the binucleate
Giardia cell, understanding the mechanism of mitosis and cell
division requires the use of three-dimensional (3D) imaging
and monitoring of crucial aspects of the spindle such as
the spatial orientation of spindle poles and kinetochore
attachments of chromosomes (at the centromere) to the spindle.
Centrin, a ubiquitous protein found in association with basal
bodies and centrioles, is located with the centrioles at the
spindle poles during mitosis in metazoans (Levy et al., 1996;
Salisbury, 1995). To test hypotheses of both the mode and
mechanism of mitosis, we imaged mitotic cells in 3D with
conserved molecular markers of the mitotic spindle (antitubulin), the centromere (cenH3::GFP), and the basal bodies
and the spindle poles (anti-centrin), allowing us to monitor the
behavior of hallmark features of the spindle during mitosis.
This study is the first to observe dual mitotic spindles, to
confirm the role of centrin directly at the spindle poles and to
infer spindle attachment to the chromosomes at the kinetochore
(using the centromere histone variant cenH3). By our detailed
analysis of mitotic stages using 3D deconvolution light
microscopy and transmission electron microscopy (TEM), we
find that mitosis is semi-open, with two extranuclear central
spindles and microtubules that enter the nuclei through polar
openings in the nuclear envelopes. Chromosomes are
segregated along the left-right (L-R) axis, and cytokinesis
occurs along the longitudinal axis – perpendicular to the
spindle – suggesting that daughter cells inherit one copy of
each parent nucleus.
By monitoring these cytological markers at all stages of
mitosis and cytokinesis, we present a model of cell division in
Giardia that: (1) establishes both the spatial orientation and the
timeline of chromosome segregation (mitosis) and cytokinesis;
(2) illustrates the timing of the duplication of cytoskeletal
structures including the median body, the ventral disc and the
eight flagella; and (3) accounts for the fate of each nucleus
during cell division. This model of cell division can explain
prior studies on nuclear partitioning during cytokinesis, albeit
with an alternative mode of cytokinesis than that originally
proposed (Ghosh et al., 2001).
Results
Traditionally, identifying individual mitotic stages in Giardia
has been difficult due to the inability to generate synchronized
cultures. Therefore, we first developed a method for enriching
cultures of Giardia for mitotic cells (supplementary material
Table 1) followed by indirect immunofluorescence of key
cytological markers to visualize changes in the microtubule
cytoskeleton throughout mitosis. To describe mitotic cells we
immunostained mitotic spindles (anti-tubulin) and the spindle
poles (anti-centrin), visualized green fluorescent protein
(GFP)-tagged centromeres (cenH3::GFP) and tracked hallmark
features of the spindle during mitosis using 3D deconvolution
microscopy (Materials and Methods). Between 50-100 cells
were analyzed for each mitotic stage; representative images are
presented in Fig. 2.
These analyses allow the establishment of a timeline of the
crucial events of mitosis and the determination of the plane of
nuclear division and cytokinesis. Although we emphasize the
dynamics of chromosome segregation during mitosis at high
levels of resolution, we also present a timeline of the
duplication and partitioning of major cytoskeletal elements
including the eight flagella, the median body and the ventral
disc, albeit in less detail.
Interphase
Microtubule cytoskeleton: Giardia trophozoites have an
elaborate microtubule cytoskeleton, consisting of four major
structural microtubule arrays: a ventral disc, flagellar
axonemes with basal bodies, the funis and the median body (an
enigmatic microtubular array of unknown function) (see Fig.
1A,B) (see also Elmendorf et al., 2003). In the images
presented here, the ventral disc is weakly stained relative to the
other microtubule organelles.
Chromosomes: in interphase, chromatin is decondensed, as
Mitosis in Giardia
Journal of Cell Science
indicated by the uniform fluorescence of 4,6-diamino-2phenylindole (DAPI)-stained nuclei (Fig. 2A,D). Although
eukaryotic centromeric sequences vary greatly at the
nucleotide level, centromeric nucleosomes contain the
specialized histone H3 variant cenH3 (Malik and Henikoff,
2003). To visualize the centromeres of Giardia chromosomes,
we identified the cenH3 homolog in Giardia and constructed
a GFP fusion protein (S.C.D. and W.Z.C., unpublished
data). In interphase, cenH3::GFP foci were seen throughout
the DAPI-stained nuclei, indicating that the cenH3:GFP
marks discrete centromeric loci on the chromosomes (Fig.
2A).
Basal bodies: to determine the role of basal bodies in
organizing the giardial mitotic spindles, we monitored the
4891
movement of flagellar basal bodies during interphase and
mitosis using anti-centrin immunostaining. The basal bodies of
all eight flagella are located between the two adjacent nuclei
in the anterior region of the cell. In interphase trophozoites, we
observed that centrin localized to two clusters between the two
nuclei, colocalizing with the flagellar basal bodies (Fig. 2D) as
has been shown previously (Belhadri, 1995; Correa et al.,
2004; Meng et al., 1996).
Prophase
Mitotic prophase is characterized by extensive chromatin
condensation and the nucleation of spindle microtubules. As
in most eukaryotes, we found that individual giardial
chromosomes could be resolved following chromatin
condensation in mitotic prophase (Fig. 2B). In prophase,
each chromosome contained a single cenH3::GFP focus,
indicating that each of the ten chromosomes in each
nucleus has a single centromeric locus. Spindle
microtubule nucleation was also initiated during
prophase: microtubules first appeared between the two
nuclei near the flagellar basal bodies and extended
around each nucleus individually (Fig. 2B).
An unanticipated repositioning of the nuclei occurred
throughout prophase. The two nuclei migrated to the cell
midline one nucleus on top of the other along the dorsalventral axis, with the ventral nucleus slightly anterior to
the dorsal nucleus (supplementary material Movie 1).
Three-dimensional reconstructions of these cells showed
the two nuclei remained separate from one another
(supplementary material Movie 1). At the completion of
nuclear migration individual chromosomes could be
distinguished. During nuclear migration the spindle
Fig. 2. During mitosis two autonomous spindles are
responsible for chromosome segregation after nuclei migrate
to the cell center. The centromere-specific histone cenH3
marks centromeres (cenH3:GFP in green, A-C,G-I), and
centrin antibodies mark spindle poles (green, D-F,J-L). TAT1
(anti-tubulin) labels the microtubule cytoskeleton including
the mitotic spindles (all cells, in red) and DAPI labels
chromatin (all cells, in blue). Behavior of left (red) and right
(blue) nuclei is diagramed above each stage. (A) Interphase;
centromeres are discreet foci in each nucleus. (D) Interphase;
centrin stains two foci in association with the flagellar basal
bodies between the two nuclei. (B) Prophase; spindle
microtubules appear between the two nuclei and extend
around each nucleus. Centromeres are one spot on each
condensed chromosome. (E) Prophase; four centrin foci are
associated with the forming spindles. (C) Metaphase;
following nuclear migration the nuclei become stacked along
the dorsal-ventral axis. The microtubules organized into two
bipolar spindles. (F) Metaphase; centrin foci are at the four
spindle poles. (G) Anaphase; chromatin is segregated to
spindle poles along the left-right axis of the cell with
centromeres clustered at the spindle poles. (J) Anaphase;
centrin remains at the spindle poles. (H) Telophase; a linear
microtubule structure replaces the bipolar spindles and
centromeres remain clustered. (K) Telophase; centrin foci are
between the two nuclei of each daughter. (I) Cytokinesis;
chromatin decondenses and the cleavage furrow forms at the
anterior end, creating a heart-shaped cell. (L) Cytokinesis;
centrin stains two foci between the nuclei. Cytokinesis divides
the cell into left and right halves. Bar, 2 m.
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microtubules continued to elongate, encompassing each
nucleus by the end of prophase.
Duplication of the centrin foci also occurred during
prophase. At the onset of prophase and spindle assembly the
number of centrin foci increased from two to four as the result
of either duplication or separation of the basal bodies (Fig. 2E).
During nuclear migration the centrin foci were present at the
sites of microtubule nucleation. These foci moved around the
periphery of the nucleus as the spindle microtubules continued
to elongate and the nuclei migrated to the center. When the two
nuclei were stacked, two centrin foci, each with its own
complement of microtubules, were positioned on opposite
sides of each nucleus.
Metaphase
Upon completion of nuclear migration, the microtubules
surrounding each nucleus formed two independent bipolar
spindles, stacked in the dorsal-ventral plane (Fig. 2C,F). The
opposing poles of each spindle were oriented along the leftright axis of the cell. The chromatin was clustered tightly in
the center of each spindle axis so that individual chromosomes
were not visible (Fig. 2C). We were unable to observe a
canonical metaphase alignment of centromeres along a
metaphase plate. At this time, centrin localized to each of the
four spindle poles (Fig. 2F).
Anaphase A and B
Chromosome segregation in both nuclei occurred in two
stages: chromatid segregation to the spindle poles in anaphase
A, followed by spindle elongation along the left-right axis of
the cell in anaphase B (Fig. 2G). In Giardia, anaphase A
initiates first in one nucleus (data not shown), however
anaphase B occurs simultaneously between the two nuclei. As
a result of both nuclear migration and lateral chromosome
segregation, the sister chromatids from each nucleus were
segregated to opposite sides (L-R) of the cell. At the
completion of anaphase, nuclei of different parental origin
reside on opposite sides of the cell with the daughters of the
dorsal nucleus remaining dorsal and slightly posterior with
respect to the daughter nuclei of the ventral nucleus.
During anaphase A, the cenH3::GFP localized to the leading
edge (near the spindle pole) of the segregating DNA, a
behavior characteristic of kinetochore attachment to
microtubules. The cenH3::GFP foci remained tightly clustered
together at the spindle poles during anaphase B (Fig. 2G). This
distribution pattern implicates centromeres as the site of
microtubule attachment during chromosome segregation in
Giardia as in other eukaryotes. Centrin foci remained at the
spindle poles throughout anaphase A and anaphase B (Fig. 2J).
Telophase and cytokinesis
In telophase, a microtubule bundle with unfocused ends
extended between the nuclei on each side of the cell replacing
the bipolar spindle arrays (Fig. 2H). We interpret this structure
as the remaining spindle microtubules following the loss of
focused spindle poles. We are not able to rule out formation of
this structure from de novo microtubule polymerization;
however, a de novo origin is unlikely given that intermediate
structures were not observed. The single cenH3::GFP foci
observed in the daughter nuclei indicated that the centromeres
remained clustered in telophase nuclei.
During telophase, the two centrin foci on each side of the
nuclei moved from their anaphase position near the cell
periphery to their position between each pair of nuclei as seen
in interphase (Fig. 2K). By the onset of cytokinesis, the DNA
was decondensed, cenH3::GFP foci were seen throughout the
nucleus, the nuclei were adjacent to one another and all
cytoskeletal structures were regenerated (described below).
To complete cytokinesis, a furrow formed at the anterior end
of the cell creating a heart-shaped four nuclear stage cell (Fig.
2I,L). The furrow progressed from anterior to posterior, in the
longitudinal plane, separating the left and right sides of the
heart into the two daughter cells. As a result, each daughter cell
inherits two nuclei each derived from one left and one right
nucleus of the mother cell.
Patterns of nuclear inheritance using FISH of an
episomal plasmid in mitotic nuclei
To determine the pattern of nuclear partitioning and
inheritance, we used fluorescence in situ hybridization (FISH)
to visualize an episomal plasmid in the cenH3::GFP strain,
which is maintained by one nucleus in each Giardia
trophozoite (Ghosh et al., 2001; Singer et al., 1998; Yu et al.,
2002). In each cell, we observed the episomal plasmid FISH
signal in only one nucleus (either the left or the right) as
previously reported (Ghosh et al., 2001). Since a nucleus
contains more than one episome, by using deconvolution
microscopy we are able to detect multiple discrete FISH
signals in the labeled nucleus. In prophase nuclei, during the
late stages of nuclear migration, the FISH signal was observed
only in one nucleus (see dorsal nucleus in Fig. 3B,B’). Given
our previous observation that daughter nuclei from each
Fig. 3. FISH to an episomal
plasmid maintained in one
nucleus shows nuclei are inherited
with mirror image symmetry and
that the nuclei never mix.
(A) Interphase; the FISH probe
hybridizes to the episome
contained in the right nucleus.
(B) In the metaphase nuclei,
stacked on top of each other in the
cell center, FISH signal is
detected in the dorsal nucleus.
(B’) Projection of the first eight z-sections, dorsal nucleus, of the cell seen in B contains the FISH signal. (B’’) Projection of the last eight zsections, ventral nucleus, of the cell shown in B shows the FISH signal is excluded. (C) During cytokinesis, one nucleus in each daughter is
labeled with mirror image symmetry between the two cells. Red, DAPI; green, FISH. Bars, 2 m.
Mitosis in Giardia
Journal of Cell Science
nucleus are segregated laterally, we would expect only one
nucleus in each daughter cell to contain the episome. As
predicted, in heart-shaped cells entering cytokinesis, only one
nucleus in each future daughter cell showed the episomal
plasmid FISH signal (Fig. 3C); the two cells showed mirror
image symmetry of the labeled nuclei as has been previously
reported (Ghosh et al., 2001).
Fig. 4. As shown by TEM, the mitotic spindle is semi-open and basal
bodies with axonemes are present at the four spindle poles. (A) Crosssection of a mitotic nucleus; microtubules form a sheath surrounding the
nucleus. The nuclear envelope is intact except for large openings at the
poles. Bar, 200 nm. (B) High-magnification view of area outlined by box
in A. Cytoplasmic microtubules enter the nucleus through a large opening
in the nuclear membrane. Bar, 50 nm. (B’) Diagram of nuclear membrane
(blue) and microtubules (red) seen in B. (C) Spindle microtubules radiate
from the basal body seen in cross-section, which organizes the spindle
microtubules. Bar, 300 nm. (D) Basal bodies are located between the two
daughter telophase nuclei. One basal body with associated axoneme is
seen in longitudinal section, the others are in cross-section. Bar, 400 nm.
bb, basal body; fl, flagellum; n, nucleus.
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Giardia has a semi-open mitosis
We used TEM to visualize spindle ultrastructure, to assess the
integrity of the nuclear envelope during mitosis and to
determine the identity of the spindle pole. TEM images of both
early and late mitotic nuclei showed that the spindles are
extranuclear and that the nuclear envelope remains intact
throughout mitosis (Fig. 4A). Microtubules originating from
each spindle pole form a sheath around the nucleus
outside of the nuclear envelope. The nuclear envelope
elongates with the spindle in anaphase B, extending
between the spindle poles of fully elongated spindles.
The persistence of the nuclear envelope throughout
mitosis precludes mixing of the chromatin between
nuclei. Furthermore, we did not observe fusion of the two
nuclei at any mitotic stage.
Although the nuclear envelope is present throughout,
this type of mitosis can be classified as semi-open. The
right spindle pole (outlined by the box in Fig. 4A) is
shown at greater magnification in Fig. 4B. Cytoplasmic
microtubules from spindle poles passed through large
openings in the nuclear membrane (Fig. 4B). In the latestage (anaphase B) nuclei, the internal (presumably
kinetochore) microtubules extended a few microns into
the nucleus near the chromatin, seen as an electron-dense
region near the pole (Fig. 4B).
We observed both basal bodies as well as centrin
localization at each of the four spindle poles. Each
spindle pole was associated with at least one axoneme
(Fig. 4A,C). We imaged at least two basal bodies
positioned at right angles to one other at each spindle
pole: one basal body is in cross-section, whereas the
second basal body with associated axoneme – at a more
peripheral location relative to the spindle pole – is seen
in longitudinal section (Fig. 4A,C). The basal body
cross-section revealed a ring of doublet microtubules that
lack a central microtubule pair (Fig. 4C), resembling
the transition zone of a basal body as described in
Chlamydomonas (O’Toole et al., 2003). Spindle
microtubules radiated from one of the basal bodies (seen
in cross-section) in proximity to spindle poles (Fig. 4C).
In telophase the flagellar basal bodies were positioned
between the two daughter nuclei on each side of the cell
(Fig. 4D).
Microtubule stabilization disrupts mitotic spindles
and causes chromosome missegregation
Prior studies have suggested that chromosome
segregation in Giardia occurs through a spindleindependent mechanism (Solari et al., 2003). To
determine conclusively whether dynamic microtubules
are required for chromosome segregation, we treated
mitotic cells with taxol [which binds to the -tubulin
subunit of the tubulin heterodimer in polymerized
microtubules (Parness and Horwitz, 1981; Rao et al.,
1995)] and evaluated segregation defects. Although
giardial tubulin subunits are highly divergent, the tubulin subunit has retained the amino acid sequence
associated with taxol binding (see supplementary
material Fig. S1). Nearly 100% of mitotic cells treated
with either 10 M or 20 M taxol (n>100) showed
dramatic defects in chromosome segregation and
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spindle morphology (Fig. 5). In control cells treated
with dimethyl sulfoxide (DMSO) abnormal cell
divisions were rarely observed. Many cells had
broken spindles with two half-spindles emerging
from opposite spindle poles (Fig. 5C). Some
elongated spindles were bent around the anterior
edge of the cell with the chromatin lying outside the
spindle axis (Fig. 5A,D).
In wild-type, untreated cells, anaphase A is
completed in one nucleus before the other (data not
shown). In cells treated with taxol, anaphase A was
delayed and chromosome segregation occurred in both
nuclei together (Fig. 5B). In some cases chromosome
missegregation occurred in cells in the absence of any
obvious spindle defects (Fig. 5B). In cells in which
anaphase A segregation had taken place, the
chromosomes did not remain tightly clustered at the
poles (data not shown). Chromosome segregation
defects ranged from one or two lagging chromosomes
remaining in the middle of the dividing nucleus (Fig.
5C), to a trail of lagging chromosomes extending
between the two spindle poles (Fig. 5D). Treatment
with nocodazole also resulted in lagging
chromosomes (data not shown).
Flagellar duplication
In some flagellates, such as Chlamydomonas, flagella
Fig. 5. Treatment with the microtubule-stabilizing compound taxol reveals
are resorbed at the onset of mitosis and the basal
that dynamic microtubules are required for spindle integrity and proper
bodies (as centrioles) are recruited to function as part
chromosome segregation. (A) Late-anaphase cell treated with 10 M taxol
of the mitotic spindle poles (Ou and Rattner, 2004).
for 15 minutes. Elongated spindle microtubules are bent towards anterior of
Both centrin localization and TEM data showed that
cell; lagging chromosomes remain in the spindle midzone. (B-D) Cells
while giardial flagellar basal bodies are present at the
treated with 20 M taxol for 1 hour. (B) Anaphase spindle morphology is
spindle poles, all eight flagella were retained during
normal but anaphase A chromosome segregation is delayed. (C) Anaphase
mitosis.
spindles are broken into four half-spindle microtubule arrays. Arrows point
A dramatic rearrangement of the flagella coincided
to distal tips of broken microtubule arrays. (D) Late anaphase; spindle is bent
around anterior edge of cell, with lagging chromosomes along spindle axis.
with nuclear migration in prophase; flagellar
Median bodies persist in mitosis and are marked with asterisks. Red, tubulin
rearrangements were most easily seen with the
(TAT1); blue, DNA (DAPI). Bars, 2 m.
anterior and caudal flagellar pairs. Migration of the
nuclei to the center of the cell displaces the flagellar
basal bodies. By TEM, we observed the central nuclei
in an early mitotic cell separating the caudal flagellar pair (Fig.
separation of spindle poles and the eventual duplication of
6A). Movements of the anterior flagella were assessed by light
other cellular structures prior to cytokinesis. Although we
microscopy. Basal bodies associated with each anterior
were unable to identify an aligned metaphase plate in giardial
flagellum moved to the outer periphery of the nuclei,
mitosis, which could be attributed to the transient nature of
associating with one of the four spindle poles (arrowheads in
this stage or to the small size of the chromosomes, it is clear
Fig. 6C,D). The fluorescence images of the anterior flagella
the chromosomes congress to the spindle midzone during
matched the position of the flagella seen in longitudinal
metaphase. Both spindles are bipolar arrays of microtubules
sections in TEM images of the spindle pole (Fig. 4B). These
with attachments to chromosomes at kinetochores. Thus, the
data confirm and extend a recent report of flagellar
organization and function of the giardial spindle is highly
rearrangements during mitosis in Giardia (Nohynkova et al.,
conserved with these aspects of the metazoan spindle, despite
2006).
the strikingly different morphology (i.e. two nuclei and eight
flagella) and evolutionary distance between Giardia and
Discussion
metazoans.
Recent discourse has suggested that mitosis in Giardia
Distinctive aspects of giardial mitosis (as compared with
proceeds by a unique and unprecedented mechanism
metazoan or fungal mitoses) include: the semi-open structure
(Benchimol, 2004a; Benchimol, 2004b; Solari et al., 2003).
of the nuclear envelope and spindle; the movement of each
We find, however, that the major cytological events of mitosis
nucleus to the cell center (one dorsal and one ventral); and the
in Giardia proceed as defined in many organisms: it begins
extended telophase spindle structure. Further, we infer some
with prophase condensation of chromatin, followed by the
involvement of the flagellar basal bodies in spindle microtubule
alignment of chromosomes in the spindle midzone, the
nucleation and/or spindle positioning (Fig. 4C). The unique
movement of chromosomes to the spindle poles, the
features of Giardia mitosis are highlighted in detail below.
Mitosis in Giardia
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mitotic spindle structure in protists. Owing to the
ubiquity of extranuclear spindles in protists, the
ancestral state of eukaryotic mitosis may be similar to
Giardia and trichomonads, an extranuclear spindle that
interacts with chromatin either through a semi-open
nucleus or across an intact nuclear envelope. In this
scenario, both closed mitosis with intranuclear spindles
(yeasts) and open mitosis (plants and metazoans) are
derived mitotic forms.
Fig. 6. During mitosis the flagella and basal bodies are displaced by the
migrating nuclei and become associated with the spindle poles. (A) TEM
of a metaphase mitotic cell shows that the caudal flagella (cfl) are
displaced following nuclear migration. Bar, 1 m. (B) Interphase; arrow
marks position of basal bodies of anterior flagella. (C) Early prophase;
arrow marks position of basal bodies, located at the periphery of the
migrating nuclei. (D) Metaphase; the anterior flagella is wrapped around
the dorsal nucleus, and the basal body is associated with the right spindle
pole. Median body marked by asterisk. Bar, 2 m.
Spindle structure
Based on ultrastructural analyses, mitotic spindles in protists
have been classified using several cytological features: the
continuity of the nuclear envelope during mitosis (open, semiopen and closed); the position of the spindle relative to the
nuclear envelope (pleuromitosis versus orthomitosis); and the
position of chromosomes relative to the spindle axis (Raikov,
1994). The nuclear envelope sequesters chromatin throughout
most of the cell cycle. In most plants and animals, a complete
nuclear envelope breakdown occurs during prophase, allowing
microtubules direct access to chromatin. In protists, both
kinetoplastids (e.g. trypanosomes) and heteroloboseans (e.g.
Naegleria gruberi) retain an intact nuclear envelope, or closed
mitosis, with an intranuclear spindle, a trait that is shared with
both fission and budding yeast (O’Toole et al., 2003; Ogbadoyi
et al., 2000; Schuster, 1975; Winey et al., 1995). The
trichomonads also have a closed mitosis with an extranuclear
spindle that attaches to kinetochores embedded in the nuclear
envelope (Brugerolle, 1975b; Gomez-Conde et al., 2000).
During giardial mitosis, the nuclear envelope also acts as a
barrier between cytoplasmic microtubule arrays and chromatin.
By TEM, we provide the first evidence that Giardia has a semiopen mitosis in which microtubules from the two extranuclear
spindles penetrate the nucleus through polar openings in the
nuclear envelope (Fig. 4B). This is similar to a description of
semi-open mitosis in a related diplomonad, Hexamita inflata
(Brugerolle, 1975a), and is consistent with the diversity of
Evidence for kinetochore microtubules in
chromosome segregation
The mitotic spindle links the microtubule cytoskeleton
to chromosomes through kinetochore microtubules to
facilitate chromosome segregation. Two experimental
observations strongly support the role of kinetochore
microtubules in chromosome segregation in Giardia: the
cenH3::GFP localization pattern in mitosis and the
presence of intranuclear microtubules seen by TEM.
A histone H3 variant cenH3 is found exclusively at
centromeres, and has been proposed as a universal
marker of the eukaryotic centromere (Sullivan et al.,
2001). Using a GFP-tagged cenH3 strain, we have
shown that cenH3::GFP localizes to a discrete focus on
each prophase chromosome, allowing us to visualize
the giardial centromere and demonstrate that giardial
chromosomes are monocentric (S.C.D. and W.Z.C.,
unpublished data). During prophase in other eukaryotes,
centromeres containing cenH3 are required to build the
mitotic kinetochore by recruiting motors, checkpoint
proteins and additional structural elements (Van Hooser
et al., 2001). In giardial mitosis, the clustering of cenH3marked centromeres at the leading edge of segregating
DNA (Fig. 2G) implies that chromosome segregation is
mediated by microtubule attachments at the kinetochore.
The number of microtubules attached to the kinetochore
ranges from a single kinetochore microtubule per chromosome
in budding yeast to more than 20 per chromosome in some
metazoans (McDonald et al., 1992; Winey et al., 1995).
Although we have not directly observed kinetochoremicrotubule associations, given the number of microtubules
seen in a single TEM section and the number of kinetochores
available for attachment in the nucleus, i.e. ten, we predict
more than one microtubule is attached per kinetochore in
Giardia.
Microtubule-stabilizing compounds generate lagging
chromosomes on the anaphase spindle in Giardia. By
inhibiting microtubule depolymerization using taxol, we were
able to generate many of the same anaphase B spindle defects
as previously observed in metazoans (Amin-Hanjani and
Wadsworth, 1991; De Brabander et al., 1986; Jordan et al.,
1993). Primarily, we found that taxol treatment caused the loss
of interzonal microtubules, resulting in the formation of two
half-spindles. This observation provides further evidence
for the role of kinetochore microtubules in chromosome
segregation and demonstrates the bipolar organization of
spindle microtubules.
A role for the flagellar basal bodies in spindle
organization and positioning
The morphology of the microtubule organizing centers
Journal of Cell Science
4896
Journal of Cell Science 119 (23)
(MTOCs) of mitotic spindles is diverse among eukaryotes. The
centrosomes of metazoan cells consist of a pair of centrioles
surrounded by pericentriolar material containing factors, such
as gamma tubulin, required for microtubule nucleation (Ou and
Rattner, 2004). Yeasts lack centrioles but are able to nucleate
and organize the mitotic spindle using spindle pole bodies
(SPBs) (Jaspersen and Winey, 2004). Flagellated protists such
as Chlamydomonas recruit flagellar basal bodies, structurally
related to centrioles, to function as part of the MTOC at the
spindle poles following resorption of the flagella. These basal
bodies of Chlamydomonas organize the spindle microtubules
in a bipolar array and also appear to play a role in spindle
positioning (Coss, 1974; Ehler et al., 1995). The association of
flagellar basal bodies with the spindle poles may serve to
ensure equal partitioning of basal bodies in each division cycle,
thereby preserving the number of flagella in each daughter cell
(Dutcher, 2003; Marshall and Rosenbaum, 2000; Heath, 1980;
Lechtreck and Grunow, 1999; Wright et al., 1989).
In Giardia, eight basal bodies are inherited by each daughter
cell during a mitotic division that includes two spindles and four
spindle poles. How are the 16 giardial basal bodies accounted
for during division and what roles do they play in spindle
formation, positioning and cell-polarity determination? We
hypothesize that the polarity of each daughter cell is established
through the association of the eight basal bodies with the four
spindle poles during mitosis. This proposal is based on our
analysis of spindle structure using TEM, which demonstrates
the association of multiple basal bodies with spindle poles (Fig.
4). We presume that one basal body at each pole acts as the
central structural component of the MTOC (as indicated by the
spindle microtubules radiating from the basal body seen in
cross-section, see Fig. 4C). We predict that careful analysis of
-tubulin in the intermediate stages of mitosis as outlined in our
study would reveal -tubulin as part of the spindle-pole
complex. In a recent study, -tubulin localization was detected
at basal bodies both before and after nuclear division, although
spindles were not directly observed (Nohynkova et al., 2000).
We found that a second basal body is found at the periphery of
the spindle-pole region, peripheral to the focused spindle
microtubules (Fig. 4C). This basal body, with its associated
flagellum, may play an indirect role in spindle nucleation and
organization; its association with a spindle pole may ensure its
proper segregation to the daughter cells. Our data support the
recent description of movements of flagellar complexes during
cell division (Nohynkova et al., 2006).
Outside of spindle nucleation, the association of flagellar
basal bodies with spindle poles could establish and maintain cell
polarity in each generation. We propose that specific
combinations of two axonemes and associated basal bodies at
each pole confer a unique positional identity to each of the four
spindle poles. This is supported by both centrin immunostaining
(Fig. 2K) and the TEM of telophase cells (Fig. 4D), which
reveal flagellar basal bodies positioned between the two
daughter nuclei on each side of the cell. The specific association
of the eight basal bodies (with different developmental origins)
with the four individual spindle poles can provide spatial cues
for the proper establishment of cell polarity after mitosis.
Nuclear repositioning, cytokinesis and the orientation of
daughter cells
The bilaterally symmetrical Giardia trophozoite is complex,
and one would expect mitosis and cytokinesis to be tightly
controlled and spatially defined processes. Based on our
analysis of mitotic stages, we present a new model of giardial
mitosis, the nuclear migration model, named for the
repositioning of the two nuclei during prophase (Fig. 2C). In
this model, the left and right nuclei are inherited with mirror
image symmetry in the two daughter cells (Fig. 7). Although
this pattern of nuclear inheritance has been predicted by
previous studies (Benchimol, 2004a; Benchimol, 2004b; Cèrva
and Nohynkova, 1992; Filice, 1952; Ghosh et al., 2001; Yu et
al., 2002), the nuclear migration model differs in how the
mirror image symmetry of daughter nuclei is achieved. To
clarify the possible planes of cell division the axes of mitosis
and cytokinesis are defined in Fig. 1C.
Three primary observations provide empirical support for
our proposed model of mitosis: nuclear migration during
prophase, cytokinesis perpendicular to the spindle axes and
mirror image symmetry of nuclei using FISH. To illustrate this,
we find that following prophase nuclear migration, nuclear
division occurs along the left-right axis. The two nuclei remain
separate during this time and thus do not exchange genetic
information. Second, cytokinesis occurs in the longitudinal
axis perpendicular to the axis of nuclear division.
Although it has been proposed that Giardia divides along
multiple division planes depending on whether the cells are
attached or unattached to a substrate (Benchimol, 2004a),
this is not consistent with our observations. We examined
populations of cells that were both attached and unattached at
the time of fixation, and determined that chromosome
segregation only occurs along the left-right axis of the cell with
cytokinesis perpendicular to that plane. In the course of our
experiments hundreds of mitotic cells were imaged and we
never detected cytokinesis occurring in the dorsal-ventral
plane.
Despite differences in terms of the timing and mechanism
of cell division among diverse eukaryotes, the cell division
plane is always perpendicular to the axis of chromosome
segregation as defined by the mitotic spindle (Balasubramanian
et al., 2004). Our model for cytokinesis, occurring in the
longitudinal plane perpendicular to the spindle axis, is
consistent with patterns of cytokinesis in other eukaryotes.
Finally, we show that nuclei are inherited with mirror image
symmetry. In cytokinesis, we observed mirror image symmetry
of nuclei containing the episomal FISH signal, as has been
previously reported (Ghosh et al., 2001). Given that cytokinesis
occurs in the longitudinal plane, the only outcome for nuclear
identity is mirror image symmetry between the daughter cells.
Prior attempts to define the pattern of nuclear inheritance
have also used FISH of an episomal plasmid to track the fate
of either nucleus during cell division, but did not look at
intermediate stages of mitosis, particularly during the prophase
nuclear migration or the later four nuclear pre-cytokinesis stage
(Ghosh et al., 2001; Yu et al., 2002). In addition, these studies
presumed that cytokinesis occurs in the frontal plane or dorsalventral axis. Ghosh et al. reported that 50% of the FISH signal
was present in either the left or right nucleus and in one nucleus
per daughter in heart-shaped cells undergoing cytokinesis
(Ghosh et al., 2001). Based on these observations, Ghosh and
colleagues concluded that frontal cell division occurred,
resulting in daughter cells in a ventral-ventral orientation.
Alternatively, Yu et al. observed that the same nucleus always
Mitosis in Giardia
4897
A
V
R
L
D
P
Journal of Cell Science
*
A-P
D-V
A-P
D-V
†
D-D–V-V
‡
contained the episomal plasmid, concluding that the cells were
in a dorsal-ventral orientation (Yu et al., 2002). The pattern of
FISH we observe supports the observations of Ghosh et al.,
wherein the two nuclei are inherited with mirror image
symmetry. The fact that no cells contained two labeled nuclei
in this or prior studies implies that: (1) there is no nuclear
fusion during giardial mitosis; and (2) daughters inherit one
copy of each parental nucleus in each generation.
Implications and possible mechanisms of the nuclear
migration model for the maintenance of heterozygosity
in the two nuclei
Beyond establishing the plane of cytokinesis, the mode of
Giardia cell division has important implications that bear on
the maintenance of a unique genetic identity for each nucleus.
Based on our observations, the nuclei remain physically and
genetically distinct from one another with daughter cells
inheriting one copy of both nuclei. Thus, if neither nuclear
fusion events nor meiosis occur, asexually dividing
Giardia should theoretically accumulate substantial allelic
heterozygosity between each nucleus in a short time, which
Fig. 7. The nuclear migration model, to the left and shaded
in green, is proposed from this analysis (Figs 2-4). Each
model presents three events that determine the pattern of
nuclear inheritance in each division cycle: the plane of
nuclear division, the plane of cytokinesis and the
orientation of the daughter cells relative to one another.
The schematic depicts the relative positions of the left
(red) and right (blue) nuclei before and after mitosis and
cytokinesis as seen from the dorsal side of the cell (see
also Fig. 1). Following nuclear migration to the cell
midline, the left-right (L-R) axis of nuclear division yields
two daughter nuclei, one from each parental nucleus
located on opposite (L-R) sides of the cell. The
longitudinal plane of cytokinesis results in inheritance of
nuclei with mirror image symmetry in the daughter cells.
Dorsal-ventral (D-V) cytokinesis leads to inheritance of
two nuclei of the same parental origin and is not supported
by our data or by previous FISH studies (Ghosh et al.,
2001; Yu et al., 2002). Models outlined in previous studies
are presented on the right. Nuclear division was assumed
to be in the D-V plane, by an unspecified mechanism,
resulting in nuclei of the same parental origin residing on
one side (L-R) of the cell (Filice, 1952; Ghosh et al., 2001;
Yu et al., 2002; Benchimol, 2004a). Division along the
longitudinal axis results in each daughter cell inheriting
nuclei of the same parental origin. Cytokinesis in the
frontal plane (Ghosh et al., 2001; Yu et al., 2002;
Benchimol, 2004a) (Fig. 1C) results in either identical or
mirror image symmetry of the two nuclei in daughter cells
depending on the orientation of the cells to one another. AP, anterior-posterior; D-D, dorsal-dorsal; D-D–V-V, dorsaldorsal–ventral-ventral. †(Yu et al., 2002; Benchimol,
2004a). ‡(Ghosh et al., 2001; Benchimol, 2004a).
has not been observed (Baruch et al., 1996; Kabnick and
Peattie, 1990; Lu et al., 1998; Yu et al., 2002). The recent
identification of putative meiotic genes in Giardia (Ramesh et
al., 2005) has raised the possibility that rare nuclear fusions
or meiotic events provide a mechanism for chromosomal
recombination and reduction of such heterozygosity. Yet
canonical meiosis, including the presence of characteristic
cytological evidence such as a synaptonemal complex, has not
been directly observed in Giardia trophozoites. One possibility
is that two genetically disparate nuclei could be maintained in
Giardia, if during rare points in the life cycle (i.e. encystation)
the two nuclei could fuse and homogenize genetic material.
Alternatively, the inheritance of genetic material from both
nuclei may somehow be important for cell survival.
What is the mechanism of nuclear migration? Centrin, a
calcium binding protein associated with basal bodies and
centrosomes of eukaryotic cells (Salisbury, 1995), may
represent a link between flagellar segregation and nuclear
migration. In algal cells, centrin is a major component of the
nuclear basal body connector, a fibrous network that physically
links the basal bodies to the nucleus (Brugerolle and Mignot,
4898
Journal of Cell Science 119 (23)
Journal of Cell Science
2003; Marshall and Rosenbaum, 2000; Salisbury et al., 1988;
Wright et al., 1989; Wright et al., 1985). In interphase, we
observed centrin immunolocalization solely at basal bodies
(Fig. 2) in contrast to prior studies (Belhadri, 1995; Meng et
al., 1996). During mitosis, we determined that centrin localized
only to basal bodies associated with the four spindle poles (Fig.
2). Although centrin is not associated with all eight basal
bodies, centrin fibers may connect spindle pole-associated
basal bodies with other basal bodies in preparation for
segregation during mitosis. Thus, basal body migration and
nuclear migration during mitosis may be coordinated events,
facilitated by the centrin-dependent attachment of basal bodies
to the nuclear envelope.
Conclusions
Diplomonads have been proposed to represent the earliest
diverging lineage of extant eukaryotes, based on single rRNA
and single and/or concatenated protein phylogenies using the
best available phylogenetic methods when an archaeal
outgroup is included (Baldauf, 2003; Baldauf et al., 2000;
Ciccarelli et al., 2006; Van de Peer et al., 2000). In any
evolutionary analysis, diplomonads remain monophyletic and
lack any statistically supported affiliation with late-emerging
groups of eukaryotes or with the majority of so-called excavate
taxa (with the possible exception of retortamonads or
trichomonads) (Simpson, 2003; Simpson et al., 2006; Simpson
et al., 2002).
Regardless of phylogenetic position, giardial mitosis shares
common aspects of mitosis with other eukaryotes. In this way,
insights into mitotic function in Giardia are informative for
understanding general questions of spindle organization in
diverse eukaryotes as compared with commonly studied
experimental models. In terms of the evolution of the mitotic
spindle, the observations of spindle organization in
diplomonads such as Giardia with the comparison with other
protists leads to two main conclusions. First, mitosis in both
Giardia and the trichomonads occurs with extranuclear
spindles that interact with chromatin across the nuclear
membrane. Therefore, this may represent a more ancestral state
of mitotic organization than either the open mitosis of plants
and metazoans or closed mitosis of fungi and trypanosomes.
Second, monocentric chromosome structure is probably
ancestral to holocentric chromosome structure.
At a minimum, understanding both the molecular
conservation of structure and function of the dual spindles in
such a highly divergent eukaryote provides a unique
perspective on the mechanism of mitosis and its evolution.
Intriguing prospects remain to be determined; in particular,
how conserved is mitosis at the molecular level in Giardia?
Also, how do we account for the observed homogeneity
between the nuclei in populations of Giardia given our
proposed nuclear migration model? In the long-term, a detailed
understanding of both the mode and mechanism of giardial cell
division will be crucial toward developing anti-giardial
compounds that target the cell division machinery.
Materials and Methods
Strains and culture conditions
Giardia intestinalis trophozoites, strain WBC6, were grown in modified TYI-S-33
medium at 37°C (Keister, 1983). Cultures were incubated in 15 ml or 6 ml plastic
screw-cap tubes (Fisher Scientific). Cell cultures were enriched for mitotic cells by
growing 1-2 days past confluency. Fresh, warmed medium was added to the tube
and cultures were incubated at 37°C for 3-7 hours. Cells were collected at one-hour
intervals to collect the maximum number of mitotic cells. We generally observed
10-20% mitotic cells in each culture (supplementary material Movie 1).
GFP-tagging of essential markers in mitosis
The giardial cenH3 gene (GenBank acc. no. EAA41448), plus approximately 80 bp
of upstream promoter sequence was PCR amplified from genomic DNA and
subcloned into Asc1-Age1 sites of the pMCS-GFP vector. Transformation of Giardia
trophozoites was also as previously described (Singer et al., 1998); however,
electroporation conditions were modified using the GenePulserXL (Bio-Rad) at
375 mV, 1000 F and 700 ohms. Following electroporation, the cells were added
to 12 ml fresh medium and allowed to recover at 37°C overnight and selected with
10 g/ml puromycin (Calbiochem) for 4-7 days. Strains were maintained at a final
concentration of 50 g/ml puromycin.
Immunolocalization of the microtubule cytoskeleton with GFP
fusion proteins
Cells were fixed in the culture tubes with 1% paraformaldehyde, centrifuged,
washed in PEM buffer (100 mM PIPES, 1 mM EGTA, 0.1 mM MgSO4) and
attached to poly-L-lysine-coated coverslips. Cells attached to coverslips include
cells that were both attached and unattached at the time of fixation. Cells were
permeabilized in 0.1% Triton X-100 for 10 minutes. Coverslips were washed PEM
buffer and blocked for 30 minutes in PEMBALG [PEM+ 1% bovine serum albumin
(BSA), 0.1% sodium azide, 100 mM lysine and 0.5% cold-water fish skin gelatin
(Sigma, St Louis, MO)]. We visualized microtubules by incubating coverslips with
the monoclonal -tubulin antibody TAT1 (Woods et al., 1989) diluted 1:75 in
PEMBALG at room temperature overnight. The TAT1 antibody was directly labeled
(1:1) with a Zenon fragment conjugated with an Alexa Fluor 555 (Molecular Probes,
Eugene, OR) at room temperature for 5 minutes before dilution in PEMBALG.
Coverslips were washed in PEMBALG, then PEM before mounting with ProLong
AntiFade with DAPI (Molecular Probes).
Direct fixation of the cenH3::GFP fusion protein was performed to visualize GFP
protein localization in conjunction with immunostaining of the cytoskeleton.
Trophozoites were cultured as described above, and medium was exchanged with
1 HEPES buffered saline (HeBs) for 30 minutes at 37°C, prior to fixation with
1% paraformaldehyde.
Centrin antibody 20H5 (a gift of J. L. Salisbury) was directly labeled with the
Zenon fragment conjugated with the Alexa Fluor 555 (above). Coverslips with
mitotic cells were incubated in a 1:50 dilution in PEMBALG overnight at room
temperature. In double-labeling experiments with -tubulin, the centrin antibody
was directly labeled with an Alexa Fluor 488-conjugated Zenon fragment. Centrin
immunostaining was performed first, followed by a brief fixation, followed by
tubulin staining with TAT1 (Woods et al., 1989).
FISH of the episomal plasmid cenH3::GFP in mitotic cells
FISH probes to the episomally maintained cenH3::GFP plasmid were made by
incorporation of Cy3-labeled dUTPs by nick translation (Roche) of an AscI-AgeI
digest of the cenH3::GFP plasmid (removing the cenH3 sequence). Labeled probe
was precipitated in LiCl, resuspended in 100% formamide and used at
approximately 10 ng/l final concentration in 37°C hybridizations of mitotic
trophozoites as previously described (Yu et al., 2001).
Fluorescence deconvolution microscopy
Images were collected using SoftWorX image acquisition software (Applied
Precision, Issaquah, WA) on an Olympus IX70 wide-field inverted fluorescence
microscope with an Olympus UPlanApo 100, NA 1.35, oil-immersion objective
and Photometrics CCD CH350 camera cooled to –35°C (Roper Scientific, Tuscon,
AZ). Serial sections were acquired at 0.2 m intervals, and data stacks were
deconvolved using the SoftWorX deconvolution software. For presentation
purposes, two-dimensional projections were created from the 3D data sets using the
DeltaVision image analysis software (Applied Precision).
Transmission electron microscopy
Giardia trophozoites were grown on Aclar® tabs and were fixed in 2.0%
glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, overnight at room temperature,
and post-fixed with 1% OsO4 + 0.8% potassium ferricyanide + 5 mM CaCl2 in 0.1
M cacodylate buffer, pH 7.2, for 45 minutes. Aclar tabs with cells were dehydrated
with ethanol and infiltrated with Epon resin. Mitotic trophozoites were embedded
as previously described (Muller-Reichert et al., 2003). Briefly, trophozoites attached
to Aclar tabs were flat embedded on top of coated glass-slides and cured at 60°C
for two days. Aclar tabs were removed and cells of interest were identified using
phase microscopy and then marked, using a diamond scribe. Regions containing
mitotic cells were excised and remounted for sectioning. Ultra-thin sections (50-65
nm) were collected and stained with uranyl acetate and lead citrate and imaged in
a JEOL 1200 transmission electron microscope.
Taxol-treatment of trophozoites
Taxol (pacitaxel; Sigma) was stored as a 5 mg/ml stock in DMSO at –20°C. For
Mitosis in Giardia
experiments determining the contribution of the microtubule cytoskeleton to
chromosome segregation, taxol was diluted directly into culture tubes containing
media to a working concentration of either 10 M or 20 M. An equal concentration
of DMSO was added to control cells.
We would like to acknowledge Heidi Elmendorf and Steve Singer
(Georgetown University, Washington, DC), C.C. Wang and colleagues
(UCSF) and contributors to the Giardia Genome Project, Mitch Sogin,
Hillary Morrison and Andrew MacArthur as well as Keith Gull
(Oxford University, UK) and Jeff Salisbury (Mayo Clinic) for
plasmids, reagents and methodologies. We also thank members of the
Cande laboratory for helpful discussions. This work was supported by
an NIH grant A1054693 to W.Z.C.
Journal of Cell Science
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