The Plant Cell, Vol. 15, 854–862, April 2003, www.plantcell.org © 2003 American Society of Plant Biologists
Asymmetric Division in Fucoid Zygotes Is Positioned by
Telophase Nuclei
Sherryl R. Bisgrove,1 David C. Henderson,2 and Darryl L. Kropf
Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840
The relative contributions of cell polarity and nuclear position in specifying the plane of asymmetric division in fucoid zygotes were investigated. In zygotes developing normally, telophase nuclei were positioned parallel to the polar growth axis,
and the division plane bisected both axes. To assess division plane specification, the colinearity of the nuclear and growth
axes was uncoupled by treatment with pharmacological agents. Spatial correlations between the growth axis, telophase
nuclei, and the division plane were analyzed in the treated zygotes. In all cases, cytokinesis was oriented transverse to the
telophase mitotic array and was less well aligned with the growth axis. Telophase nuclei also played a predominant role in
positioning the division plane in polyspermic zygotes. Microtubules from the telophase nuclei interdigitated throughout the
plane of subsequent cytokinesis, and we speculate that they specify the division plane. Morphological markers of the division plane were not observed before telophase; the earliest division marker detected was a plate of actin that assembled in
the zone of microtubule overlap late in telophase. These findings are consistent with division plane specification at cytoplast boundaries.
INTRODUCTION
Asymmetric cell division is fundamental to development in all
higher eukaryotes because it produces unequal daughter cells
of different fates. Examples include early divisions in Caenorhabditis elegans embryos (White and Strome, 1996), neural
development in Drosophila melanogaster (Ceron et al., 2001),
reproduction in the green alga Volvox carteri (Kirk et al., 1993),
and several formative divisions in higher plants (Gallagher and
Smith, 1997; Scheres and Benfey, 1999; Smith, 1999;
Sylvester, 2000). In most instances, these divisions are positioned precisely with respect to the polarity of the cell or tissue.
Positioning cytokinesis is a matter of choosing a plane of division, and there are two general mechanisms for specifying the
division plane. In the first mechanism, the orientation of the
spindle (or the position of nuclei) determines the plane of division. This mechanism specifies division in metazoan, protist,
and some plant cells (White and Strome, 1996; Field et al.,
1999; Otegui and Staehelin, 2000; Brown and Lemmon, 2001).
To correctly orient asymmetric division, the spindle (or nuclei)
must be positioned in a precise location and orientation within
the cell, presumably in accordance with polarity markers. In
cells that divide centripetally by furrowing (e.g., metazoan
cells), the spindle is thought to signal the cortical division site,
perhaps using astral microtubules (Hales et al., 1999; Glotzer,
2001). A variation on this theme occurs during cellularization in
some plant tissues, including nuclear endosperm and female
1 To
whom correspondence should be addressed. E-mail bisgrove@
biology.utah.edu; fax 801-581-4668.
2 Current address: Department of Botany, University of Georgia, Athens,
GA 30602.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.009415.
gametophytes. During cellularization, radial microtubules from
nuclei define cellular spaces and centrifugally expanding
phragmoplasts deposit cell plates at the boundaries (PickettHeaps et al., 1999; Otegui and Staehelin, 2000; Brown and
Lemmon, 2001).
In the other general mechanism for positioning cytokinesis,
cell polarity directly determines the division plane in accordance with localized cortical cues (Fowler and Quatrano, 1997).
Once the division plane has been specified, the spindle need
only align well enough to ensure that telophase nuclei are partitioned to different daughter cells. This mechanism often is used
by cells that determine the division plane before mitosis, such
as fission yeast, budding yeast, and somatic plant cells (Field et
al., 1999; Smith, 1999; Sylvester, 2000). Asymmetric division in
Azolla root cells (Gunning et al., 1978) and in onion guard
mother cells (Mineyuki and Palevitz, 1990) provides clear examples: a preprophase band of microtubules forms in the cell cortex, marking the plane of future division, and the nucleus migrates to this site before spindle formation. These two general
mechanisms are not mutually exclusive, and in many divisions
in higher plants, both cellular asymmetry and nuclear position
contribute to division plane specification (Gallagher and Smith,
1997; Smith, 1999; Sylvester, 2000; Baluska et al., 2001).
Zygotes of fucoid algae, genera Fucus and Silvetia (formerly
Pelvetia; Serrao et al., 1999), provide an excellent model system for investigating the processes that establish cell polarity
and position asymmetric division (Fowler and Quatrano, 1995;
Kropf, 1997; Kropf et al., 1999; Belanger and Quatrano, 2000b;
Brownlee et al., 2001). Symmetry is broken at fertilization, and
the sperm entry site defines the rhizoid pole of a growth axis
(Hable and Kropf, 2000). This initial axis can be overridden by
vectorial cues in the environment until germination, when
growth becomes localized to the rhizoid pole. Rhizoid elongation gives the zygote an asymmetric, pear-shaped morphology.
Orienting Cytokinesis in Fucoid Zygotes
Before mitosis, the nucleus rotates, bringing the axis defined by
the centrosomes into crude alignment with the growth axis
(Bisgrove and Kropf, 1998). Because centrosomes become
spindle poles in mitosis, the metaphase spindle is aligned approximately with the growth axis. During spindle elongation at
anaphase and telophase, the mitotic apparatus comes into perfect registry with the growth axis, and cytokinesis bisects both
the growth axis and the axis defined by telophase nuclei
(Bisgrove and Kropf, 2001). Rhizoid and thallus cells resulting
from this division differ in morphology, cellular composition,
and developmental fate; the rhizoid cell is the progenitor of the
holdfast, whereas the thallus cell gives rise to much of the stipe
and frond tissues. The orientation of the first asymmetric division is important for subsequent embryogenesis; treatments that
misorient this division result in developmental arrest (Bisgrove
and Kropf, 1998).
Because the growth axis and telophase mitotic array are
colinear at the onset of cytokinesis, it is difficult to assess the
relative contributions of the two axes in determining the division plane. We addressed this issue experimentally by perturbing the orientation of the mitotic apparatus, effectively uncoupling the two axes, and measuring the spatial alignment of
division. The results indicate that the orientation of telophase
nuclei is the primary determinant of the division plane. We conclude that cell polarity is used to position telophase nuclei,
which in turn specify the division plane, probably via interdigitating microtubules.
RESULTS
Division Plane Markers
Because the mitotic apparatus is not positioned transverse to
the subsequent cytokinetic plane until telophase (Bisgrove and
Kropf, 2001), identification of markers at the division site before
telophase would indicate that cell polarity is primarily responsible for specifying the plane of the first asymmetric division.
Mitosis and cytokinesis last for several hours in Silvetia compressa zygotes, making it possible to conduct detailed temporal and spatial analyses of putative markers. The plane of future division is marked by unique cytoskeletal arrays in many
eukaryotic cells (Field et al., 1999; Wasteneys, 2002), so microtubule and actin arrays were examined. Microtubules were labeled with antibodies throughout G2 and mitosis, and thousands of images were examined. In no case was a conspicuous
microtubule array found in the cortex at the division site. A preprophase band of microtubules, which marks the division plane
in somatic plant cells (Otegui and Staehelin, 2000; Brown and
Lemmon, 2001; Kost and Chua, 2002), was not present. Instead, microtubules radiated from two centrosomes into the
cortex of premitotic cells (Figure 1A). These microtubules are
thought to be captured at the cortex and to participate in nuclear rotation (Bisgrove and Kropf, 2001). In metaphase (Figure
1B) and anaphase (Figure 1C), the crudely aligned spindle nucleated short astral microtubules that did not extend to the cortex. A vast cytoplasmic array reformed at telophase, and microtubules from daughter nuclei interacted with the cell cortex and
855
Figure 1. Microtubule Arrays of the First Zygotic Cell Cycle.
G2 (A), metaphase (B), anaphase (C), and telophase (D) of the first zygotic cell cycle are shown. Predicted cortical division sites reside within
the white boxes shown in (A). Each image is a projection of confocal images representing 5 to 15 m near the center of the zygote. Bar in (D) 10 m for all panels.
interdigitated in the midzone of the disassembling spindle (Figure 1D).
Actin filaments were present throughout the cortex in interphase (Kropf et al., 1992), metaphase, and anaphase but
showed no preferential localization or orientation with respect
to the division site (data not shown). The first actin structure
that indicated the division plane was a plate of F-actin that assembled in the cytokinetic plane; the actin plate formed after
the zygotes had entered telophase but several hours before
partition membrane deposition (Figures 2A, 2C, and 2D). At approximately the same time, the plane of subsequent division
became optically more translucent than adjacent cytoplasm
(Figures 2B and 2D). The clear zone likely resulted from the exclusion of pigmented organelles and granules. Actin plates and
clear zones persisted at fully mature cross-walls.
Cellular organization also was examined by transmission
electron microscopy, but structural markers of the division
plane were not apparent before telophase (data not shown).
Because indicators of the division plane were not detected until
zygotes had entered telophase, these data do not discriminate
cell polarity from nuclear position as the primary determinant of
the division plane.
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Uncoupling Nuclear Position from Cell Polarity
In a second approach to assess the relative contributions of
cell polarity and the division plane, the orientation of division
was examined in zygotes in which telophase nuclei were not
colinear with the growth axis. In any population of untreated zygotes, a few cells fail to complete nuclear alignment, causing
the nuclear axis to be skewed from the growth axis (Figures 3A
and 3B). We examined 4732 cells and found 63 zygotes (1.3%)
with skewed relative positions. The division plane was measured with respect to the growth axis and the axis defined by
the telophase nuclei in these zygotes. In every cell examined,
the division plane was more transverse to the nuclear axis than
to the growth axis. On average, the division plane was oriented
84.3 4.3 to the nuclear axis and 66.1 12.7 to the growth
axis (Figure 3C).
The colinearity of the growth axis and the telophase mitotic
apparatus was perturbed experimentally by disrupting nuclear
rotation. Nuclear rotation occurs before mitosis and is driven by
microtubules that interact with the cell cortex (Bisgrove and
Kropf, 2001). Agents that perturb the cell cortex prevent rotation, resulting in mispositioned telophase nuclei. The cell cortex
Figure 3. Division Plane Alignment in Untreated Zygotes.
(A) Fluorescein diacetate labeling to visualize nuclei and the division
plane. In the vast majority of embryos (4669 of 4732), telophase nuclei
were aligned with the growth axis and division bisected this axis.
(B) Nuclear and growth axes were not colinear in 63 zygotes. Bar 25 m
for (A) and (B).
(C) Angles between the division plane and the nuclear axis (white bar)
and the division plane and the growth axis (hatched bar) in the 63 zygotes with misaligned axes. Error bars indicate SD.
Figure 2. Markers of the Division Plane.
(A) to (C) Formation of an actin plate ([A]; projection of confocal images
representing 2 m near the median plane of the zygote) and a clear
zone ([B]; bright field) preceded the deposition of the partition membrane ([C]; FM4-64 labeling). Bar in (C) 25 m for (A) to (C).
(D) Time course of formation of structures associated with mitosis and
cytokinesis. The y axis indicates the percentage of zygotes that are in,
or have completed, each stage. The data shown are from one population of cells; similar results were obtained when the experiment was repeated except that the rate of development varied between populations.
was disrupted in two ways, by plasmolysis and by treatment
with latrunculin B, which depolymerizes cortical actin arrays
(Alessa and Kropf, 1999; Bisgrove and Kropf, 2001). Although
cytokinesis was blocked in latrunculin B–treated zygotes, a
clear zone formed between daughter nuclei, indicating that a
division plane had been chosen. Two treatment regimes were
used to analyze the spatial relationships between the clear
zone, cell polarity, and the nuclear position at first telophase.
First, zygotes were treated with latrunculin B before germination (6 to 9 h after fertilization [AF]), and angles between the
clear zone and the growth axis, or the clear zone and the mitotic axis, were measured on randomly selected zygotes. The
clear zone formed transverse to the axis of the daughter nuclei
but was not well aligned with respect to the growth axis (Figure
4A). In the second protocol, zygotes were allowed to germinate
before treatment (latrunculin B added at 13 h AF), and only zygotes with severely misaligned nuclei were analyzed. In controls, telophase nuclei were in register with the growth axis, and
the clear zone formed transverse to both axes (Figure 4B). In
treated cells with severely misaligned nuclei (Figure 4C), the
Orienting Cytokinesis in Fucoid Zygotes
857
clear zone also formed transverse to the nuclear axis but was
skewed with respect to the growth axis (Figures 4C and 4D).
Plasmolysis also was used to disrupt the cortex and misposition telophase nuclei (Henry et al., 1996; Bisgrove and Kropf,
2001). Zygotes in artificial sea water (see Methods) were transferred to artificial sea water containing 0.6 M sucrose at 12 to
13 h AF, and relevant angles were measured at 28 to 30 h AF.
Cell plates formed between telophase nuclei without regard to
the orientation of the growth axis (Figure 5A).
Treatment with oryzalin depolymerizes microtubules and inhibits rotational alignment, presumably by preventing interaction between the nucleus and the cortex (Bisgrove and Kropf,
2001). Because chronic microtubule depolymerization blocks
the cell cycle and prevents mitosis (Corellou et al., 2000), 1 M
oryzalin was applied in a pulse treatment. Zygotes were exposed to oryzalin during the period of rotational alignment,
from 15 to 19 h AF, and then rinsed back into artificial sea water. Not all zygotes recovered from the treatment, and those
that did developed slowly, so measurements were made at 39 h
AF, when many of the surviving zygotes had completed the first
division. The division plane was oriented nearly randomly with
respect to the growth axis (alignment angle of 45.0 19.7) but
was transverse to the nuclear axis (Figure 5B).
Polyspermy
Polyspermy severely alters the stereotypical spatial relation between nuclei and cell polarity. Centrioles are inherited paternally in fucoid algae (Motomura, 1994), and polyspermy induced
by fertilization in low-Na seawater introduces supernumerary
centrioles (Brawley, 1987; Nagasato et al., 1999). Labeling of
cytoplasm extruded from squashed, polyspermic zygotes has
shown that the supernumerary centrosomes nucleate a multipolar spindle at first mitosis (Nagasato et al., 1999). To investigate the spatial aspects of division in polyspermic zygotes, we
labeled microtubules, actin, and partition membrane in intact
cells. At telophase, each spindle pole organized a radial microtubule array and actin plates formed approximately equidistant
between poles (Figure 6). Subsequently, partition membranes
and cell plates formed in the zones containing actin plates (data
not shown).
Positioning of division planes between daughter nuclei in untreated, polyspermic, and treated zygotes strongly indicates that
nuclear position is the primary factor in determining the division
plane.
Figure 4. Analysis of the Division Plane in Zygotes Treated with Latrunculin B.
(A) Zygotes treated chronically with 30 nM latrunculin B beginning at 6
to 9 h AF. Black bar, angle between the growth axis and the clear zone
in solvent-treated control zygotes; white bar, angle between the nuclear
axis and the clear zone in latrunculin B–treated zygotes; hatched bar,
angle between the growth axis and the clear zone in latrunculin B–treated
zygotes. Error bars indicate SD.
(B) and (C) Treatments begun at 13 h AF. Solvent-treated control (B)
and latrunculin B–treated (C) zygotes were labeled with fluorescein diacetate at 26 h AF to visualize clear zones (arrowheads) and nuclear positions. Images are overlays with bright-field images. Bar in (C) 25 m
for (B) and (C).
Microtubules and Division Plane Specification
How might telophase nuclei position the division plane? In
green algae and cellularizing plant tissues, nuclei specify
the division plane by defining a cytoplast (also termed a nuclear
(D) Angles between the clear zone and the nuclear axis (white bar) and
the clear zone and the growth axis (hatched bar) in 94 latrunculin B–treated
zygotes. Error bars indicate SD.
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The Plant Cell
sis. At later stages of telophase, microtubules parallel to the cytokinetic plane (transverse to the radial array) became prevalent
in the region of microtubule overlap (Figures 7D and 7E). Microtubules residing in, and parallel to, the cleavage plane are
present in various groups of green algae and collectively form a
structure termed a phycoplast, which is thought to form a barrier
that keeps daughter nuclei apart during cytokinesis (PickettHeaps, 1975). Finally, an actin plate formed in the cytokinetic
plane, where both interdigitating microtubules and phycoplast
microtubules were present (Figure 7F).
DISCUSSION
Although previous work has shown that the division plane can
be skewed from the growth axis (Shaw and Quatrano, 1996;
Bisgrove and Kropf, 2001), in this report we have investigated
the spatial relation between the division plane and telophase
nuclei. Analysis of cell polarity, nuclear position, and cytokinesis in individual cells demonstrates that nuclear position is the
primary determinant of the division plane. This conclusion is
based on the observation that whenever the nuclear axis is uncoupled from the growth axis, either naturally or experimentally,
cytokinesis bisects the nuclear axis. Cellular polarity is fundamental in positioning the telophase nuclei; therefore, it plays an
important, albeit indirect, role in division plane specification.
Figure 5. Uncoupling Growth and Nuclear Axes by Plasmolysis or Microtubule Depolymerization.
(A) Plasmolysis.
(B) Microtubule depolymerization.
Division plane alignment with telophase nuclei (white bars) and with the
growth axis (hatched bars) on individual zygotes. See text for details.
Error bars indicate SD.
cytoplasmic domain [Brown and Lemmon, 2001]) using radial
microtubules, with cytokinesis occurring in the zone of microtubule overlap (Pickett-Heaps et al., 1999; Baluska et al., 2001;
Brown and Lemmon, 2001). The organization of microtubule arrays in polyspermic S. compressa zygotes is consistent with
this mechanism; radial microtubule arrays extending outward
from nuclei interdigitate in the plane of subsequent division
(Figure 6). Treatments with pharmacological agents that alter
microtubule arrays were not informative in investigating the role
of microtubules in specifying the division plane because the
treatments invariably blocked cell cycle progression (Corellou
et al., 2000; S.R. Bisgrove, unpublished observations). Instead,
microtubule arrays in untreated zygotes were examined in detail throughout telophase. Early in telophase, microtubules nucleated at centrosomes associated with the reforming nuclei interdigitated in the midzone between the two nuclei (Figures 1D
and 7A). As telophase progressed, the region of interdigitating
microtubules expanded outward to the cortex (Figures 7B and
7C). Interdigitating microtubules persisted throughout cytokine-
Figure 6. Double Labeling of Microtubules (Green Channel) and Actin
(Red Channel) in a Polyspermic Zygote at Telophase.
Some microtubules appear yellow as a result of bleeding from the red
channel. Actin plates formed equidistant between each of four nuclei.
See Methods for details. Projection of images representing 18 m. Bar 20 m.
Orienting Cytokinesis in Fucoid Zygotes
859
Figure 7. Microtubule and Actin Arrays during Telophase.
Microtubules initially interdigitated in the midzone between telophase nuclei (A) and later throughout the division plane (cortical section [B] and median section [C]). Before cytokinesis, microtubules in the plane of division were observed (arrows in [D] and [E]). (F) shows double labeling of microtubules and actin in the cytokinetic plane of the apical cell in the rhizoid of a multicellular embryo. Projections of confocal images are as follows: total
section thickness is 2 to 5 m ([B], [D], and [E]), 8 m (F), 15 m (A), and 20 m (C). Images were taken with a 63 objective except in (E), in which
a 100 objective was used. Bars 10 m in (C), (D), and (F) and 5 m in (E). (A) to (C) are the same scale.
Cellular polarity provides cortical spatial cues that guide both
phases of nuclear alignment. Early in development, the paternally inherited centrosomes migrate to opposite sides of the zygotic nuclear envelope; the axis defined by the centrosomes
initially is oriented randomly with respect to the growth axis
(Bisgrove and Kropf, 1998). Before entry into metaphase, microtubules from the perinuclear centrosomes extend into the
cellular cortex preferentially at the rhizoid pole, although some
microtubules clearly associate with the thallus cortex. Microtubules are thought to be captured at specific sites in the cell cortex and to exert force; because most capture sites are in the
rhizoid cortex, the centrosome is effectively pulled toward the
rhizoid (Kropf, 1997). One centrosome, presumably that with
the most anchored microtubules, wins this tug-of-war and is
translocated toward the rhizoid; the other centrosome moves in
the direction of the thallus. Because the centrosomes are embedded in the nuclear envelope, the result is a rotation of the
entire nucleus. Although the nature of the cortical capture sites
is uncertain, they apparently involve transmembrane adhesions. Membrane wall adhesions from cortical actin to extracellular protein are concentrated in the rhizoid (Henry et al., 1996),
and treatments that disrupt these cortical adhesions prevent
nuclear rotation (Bisgrove and Kropf, 2001). Available evidence
suggests that nuclear rotation in S. compressa is mechanistically similar to spindle alignment processes associated with
asymmetric division in yeast and C. elegans embryos (White
and Strome, 1996; Adames and Cooper, 2000; Tirnauer and
Bierer, 2000).
Nuclear rotation crudely aligns the centrosomal axis with the
growth axis, and at metaphase, a spindle with short astral microtubules is assembled. The spindle is brought into perfect registry with the growth axis as the spindle elongates during anaphase and telophase. Interestingly, dynamic microtubules interact
with the cortex during this phase of alignment, but cortical adhesions are not required (Bisgrove and Kropf, 2001). Although
the mechanism of postmetaphase alignment is unclear, nuclear
centering mechanisms may participate. Centering involves dynamic centrosomal microtubules that either push or pull on the
cell cortex (Hill and Kirschner, 1982; Bjerknes, 1986; Holy et al.,
1997; Reinsch and Gonczy, 1998). In the asymmetrically shaped
S. compressa zygote, centrosomal centering forces that act on
the separating spindle poles could align the spindle with the long
axis of the cell (Bjerknes, 1986). There is ample evidence in
plant cells that cell morphology can affect spindle position (Oud
and Nanninga, 1992; Palevitz, 1993; de Ruijter et al., 1997).
The current findings indicate that once positioned along the
growth axis, the telophase nuclei specify the division plane.
Specification of the cytokinetic plane must occur late in telophase, because only then do the daughter nuclei attain a position that is transverse to the subsequent cell plate (Bisgrove
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and Kropf, 2001). This conclusion is supported by the absence
of division plane markers at earlier times. The late specification
of the division plane is in contrast with that in many algae,
yeast, and somatic plant cells, which establish a division plane
before mitosis (Field et al., 1999; Pickett-Heaps et al., 1999). Instead, the timing of division plane specification in S. compressa
is more similar to that in metazoan cells, in which the anaphase
spindle orients subsequent division by a mechanism involving
unknown signals from the spindle midzone to the cell cortex
(White and Strome, 1996; Hales et al., 1999; Glotzer, 2001). In
S. compressa, the mechanism of cytokinesis is not well understood; if it occurs centrifugally, as recent evidence suggests
(Belanger and Quatrano, 2000a), the telophase mitotic apparatus need not signal the cell cortex. Instead, the expanding cell
plate may fuse with the parental wall wherever contact is made.
The telophase array may specify the division plane via interdigitating microtubules that define cytoplast boundaries. A cytoplast is a tensegral unit in which compression-resistant struts
integrate with tension elements in the cell periphery (Ingber,
1993). In the most evolutionarily conserved arrangement, the
struts are microtubules emanating from the nuclear region and
a network of cortical actin constitutes the tension elements
(Ingber, 1993; Pickett-Heaps et al., 1999; Baluska et al., 2000).
At cytokinesis, microtubules from daughter nuclei overlap in
the region of the remnant spindle midzone, thereby defining a
new cytoplast border, and the cytokinetic machinery assembles there.
The first asymmetric division in S. compressa zygotes is consistent with the cytoplast concept. Throughout interphase, radial microtubules extend into the cellular cortex, which contains
a meshwork of actin filaments. In telophase, remnant spindle
microtubules interdigitate in the midzone between nuclei, and
with time this structure expands centrifugally such that microtubules eventually interdigitate across the entire cell. We speculate that the interdigitating microtubules specify the cytokinetic plane. An actin plate assembles in the plane of
interdigitating microtubules, ensuring that both thallus and rhizoid cells inherit a complete tensegral unit when cytokinesis is
complete. It should be noted that an actin plate is not required
for division plane specification, because a clear zone forms between telophase nuclei in latrunculin B–treated zygotes. Unfortunately, the requirement for microtubules in division plane
specification is more difficult to assess with pharmacological
agents, because microtubule depolymerization or stabilization
during mitosis blocks cell cycle progression (Corellou et al.,
2000; S.R. Bisgrove, unpublished observations).
Among photosynthetic organisms, division plane specification
at cytoplast boundaries defined by interdigitating radial microtubules occurs in the most ancient taxa, most notably green and
red algae (Pickett-Heaps et al., 1999), and recent findings indicate that it also may be common in brown algae (Karyophyllis
et al., 2000; Nagasato and Motomura, 2002). In the plant lineage, this mechanism has been retained in multinucleate tissues, such as meiocytes, nuclear endosperm, and female gametophytes (Otegui and Staehelin, 2000; Baluska et al., 2001;
Brown and Lemmon, 2001), which cellularize after repeated
rounds of karyokinesis. During cellularization, each nucleus
forms an extensive radial microtubule array, and cell plate de-
position occurs in the zone of microtubule overlap by means of
a centrifugally expanding phragmoplast (or several miniature
phragmoplasts). As in S. compressa zygotes, preprophase bands
generally are not formed, and there is no evidence that cortical
division sites are specified before cytokinesis.
Two defining features of plant evolution are changes in the
organization of the cytoplast and in the mechanism of division
plane specification in vegetative, somatic cells (for an evolutionary perspective on these issues, see Pickett-Heaps et al.,
1999). Microtubules, rather than forming radial struts from the
nucleus, reside predominantly in the cell cortex. The division
plane is specified in G2, before mitosis, and is marked in the
cortex by a preprophase band of microtubules, from which actin becomes excluded (Wasteneys, 2002). The preprophase
band disassembles during mitosis, leaving an actin-depleted
zone to mark the cortical division site. Cell plate deposition
progresses centrifugally by means of a phragmoplast that contains microtubules, actin, and many other proteins, including
motor and signaling molecules (Otegui and Staehelin, 2000; Liu
and Lee, 2001; Wasteneys, 2002). Phragmoplast actin is thought
to guide the expanding cell plate to the predetermined cortical
division site.
Although division plane specification in somatic cells is not
well understood, analyses of several mutations in maize (e.g.,
tangled, discordia, warty, and brick) that affect the positioning
of the cell plate have begun to identify the processes and molecules involved (Smith, 1999; Gallagher and Smith, 2000;
Sylvester, 2000). As might be expected, some of the mutations
reside in genes that encode proteins that interact with the cytoskeleton; TANGLED encodes a microtubule binding protein
(Smith et al., 2001), and the DISCORDIA and BRICK proteins
are thought to regulate actin arrays (Gallagher and Smith, 1999;
Frank and Smith, 2002).
Although superficially similar, the phragmoplast of plant cells is
distinct from the interdigitating microtubule array in S. compressa
in several important respects. Microtubules in the S. compressa
array are attached to perinuclear centrosomes, whereas phragmoplast microtubules are much shorter and have minus ends
that are free in the cortical cytoplasm. In addition, phragmoplast microtubules in the center of the array disassemble as it
expands outward, whereas the interdigitating array in Silvetia
persists throughout the cytokinetic plane. Importantly, the
phragmoplast of somatic cells does not determine the division
plane but instead fuses with cortical sites occupied previously
by the preprophase band (Wasteneys, 2002). Actin also is likely
to have different functions in the two structures. Phragmoplast
actin is thought to guide the expanding phragmoplast to the
cortical division sites, whereas in Silvetia, the actin plate forms
after the interdigitating microtubule array has expanded to the
cell cortex; therefore, it is unlikely to play a role in guidance.
In summary, we suggest the following scenario for specifying
the position of the first asymmetric division in S. compressa zygotes. Cell polarity participates indirectly by providing spatial cues
for nuclear and spindle alignment during G2 and M phases, respectively. In telophase, a centrifugally expanding array of interdigitating microtubules defines a plane in which the cytokinetic
machinery, including actin, assembles. Ongoing studies are focused on identifying the molecules that link the radial microtu-
Orienting Cytokinesis in Fucoid Zygotes
bules to the cortical actin during nuclear alignment and on understanding cytoskeletal function during cell plate deposition.
861
Received November 22, 2002; accepted January 23, 2003.
REFERENCES
METHODS
Sexually mature receptacles of the fucoid alga Silvetia compressa (Serrao
et al., 1999) were collected near Santa Cruz, California, shipped cold,
and stored at 4C until use. To induce the release of zygotes, receptacles
were placed in the light (100 mol·m2·s1) at 16C in artificial sea water
(ASW; 10 mM KCl, 0.45 M NaCl, 9 mM CaCl2, 16 mM MgSO4, and 0.040
mg/mL chloramphenicol, buffered to pH 8.2 with 10 mM Tris base) overnight and then transferred to the dark for 30 to 45 min. The time of fertilization was considered to be the midpoint of the dark period. All zygotes
were grown at 16C in unidirectional light.
The cytoskeleton was imaged by confocal microscopy using a monoclonal anti--tubulin antibody (DM1A; Sigma, St. Louis, MO) to label microtubules and rhodamine phalloidin to label actin filaments, as described
previously (Alessa and Kropf, 1999; Bisgrove and Kropf, 2001). Actin also
was labeled with a monoclonal anti-actin antibody (C4; ICN Biomedicals,
Aurora, OH) after fixation in actin-stabilizing buffer at pH 6.9 (50 mM
Na2-Pipes, 0.75 M sucrose, 1 mM EGTA, 5 mM MgSO4, and 0.05% Triton X-100) with 3% paraformaldehyde, 6.3 M m-maleimidobenzoyln-hydroxysuccinimide ester, and 0.5% glutaraldehyde. The remainder of
the protocol was identical to that used to label microtubules (Bisgrove and
Kropf, 2001). Secondary antibodies were goat anti-mouse IgG conjugated
to Alexa 488 or Alexa 546 (Molecular Probes, Eugene OR). Because both
primary antibodies were raised in mouse, double labeling of microtubules
and actin was performed as previously described for microtubule labeling
(Bisgrove and Kropf, 2001), and antibodies were added sequentially to
avoid cross-reactivity of secondary antibodies; DM1A was followed by Alexa 488 secondary antibody, C4 antibody, and finally Alexa 546 secondary antibody. Images were collected through narrow band-pass emission
filters on a LSM510 (Carl Zeiss, Thornwood, NY) or a MRC-600 (Bio-Rad
Laboratories, Richmond, CA) laser scanning confocal microscope. Confocal images were routinely collected with 63 and 100 objectives.
Partition membranes were visualized with 5 M FM4-64 (Molecular
Probes) as described previously (Belanger and Quatrano, 2000a; Hable
and Kropf, 2000), and the cleavage plane and nucleus were stained with
0.1 g/mL fluorescein diacetate (Bisgrove and Kropf, 1998). These probes
were imaged by confocal and conventional epifluorescence microscopy;
with epifluorescence microscopy, images were captured with a CoolSnap digital camera (Roper Scientific Photometrics, Tucson, AZ).
Polyspermy was induced in ASW in which Na had been reduced to
2.5 mM and substituted with N-methylglucamine (Brawley, 1987; Hable
and Kropf, 2000). Potentiated receptacles in reduced-Na ASW were
placed in the dark for 30 min to induce gamete release, and 1 h later, zygotes were rinsed five times in normal ASW and allowed to develop in
the light.
Actin filaments were depolymerized by chronic treatment with 30 or
100 nM latrunculin B (stock, 5 mM in DMSO), and microtubules were depolymerized by pulse treatment with 1 M oryzalin (stock, 10 mM in
DMSO). Appropriate solvent controls were included in all experiments
and were found to have no effect on development.
Upon request, all novel materials described in this article will be made
available in a timely manner for noncommercial research purposes.
ACKNOWLEDGMENTS
This research was supported by U.S. Department of Agriculture award
99-35304-8230 and National Science Foundation award IBN 0110113 to
D.L.K. and by Natural Sciences and Engineering Research Council postdoctoral award PDF-219981-1999 to S.R.B.
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