Cytokinesis in brown algae: studies of asymmetric division in fucoid zygotes.

The mechanism of cytokinesis was investigated during the first asymmetric division in fucoid zygotes. A plate of actin assembled midway between daughter nuclei where microtubules interdigitated and defined the cytokinetic plane. A membrane was then deposited in islands throughout the cytokinetic plane; the islands eventually fused into a continuous partition membrane and cell plate material was deposited in the intermembrane space. All of these structures matured from the center of the cell outward (centrifugal maturation). Pharmacological agents were used to investigate the roles of microtubules, actin, and secretion in cytokinesis. The findings indicate a mechanism of cytokinesis that may be unique to the brown algae.

Polarity establishment requires dynamic actin in fucoid zygotes.

Previous work has demonstrated that actin plays important roles in axis establishment and polar growth in fucoid zygotes. Distinct actin arrays are associated with fertilization, polarization, growth, and division, and agents that depolymerize actin filaments (cytochalasins, latrunculin B) perturb these stages of the first cell cycle. Rearrangements of actin arrays could be accomplished by transport of intact filaments and/or by actin dynamics involving depolymerization of the old array and polymerization of a new array. To investigate the requirement for dynamic actin during early development, we utilized the actin-stabilizing agent jasplakinolide. Immunofluorescence of actin arrays showed that treatment with 1-10 microM jasplakinolide stabilized existing arrays and induced polymerization of new filaments. In young zygotes, a cortical actin patch at the rhizoid pole was stabilized, and in some cells supernumerary patches were formed. In older zygotes that had initiated tip growth, massive filament assembly occurred in the rhizoid apex, and to a lesser degree in the perinuclear region. Treatment disrupted polarity establishment, polar secretion, tip growth, spindle alignment, and cytokinesis but did not affect the maintenance of an established axis, mitosis, or cell cycle progression. This study suggests that dynamic actin is required for polarization, growth, and division. Rearrangements in actin structures during the first cell cycle are likely mediated by actin depolymerization within old arrays and polymerization of new arrays.

Asymmetric cell division in fucoid algae: a role for cortical adhesions in alignment of the mitotic apparatus.

The first cell division in zygotes of the fucoid brown alga Pelvetia compressa is asymmetric and we are interested in the mechanism controlling the alignment of this division. Since the division plane bisects the mitotic apparatus, we investigated the timing and mechanism of spindle alignments. Centrosomes, which give rise to spindle poles, aligned with the growth axis in two phases--a premetaphase rotation of the nucleus and centrosomes followed by a postmetaphase alignment that coincided with the separation of the mitotic spindle poles during anaphase and telophase. The roles of the cytoskeleton and cell cortex in the two phases of alignment were analyzed by treatment with pharmacological agents. Treatments that disrupted cytoskeleton or perturbed cortical adhesions inhibited pre-metaphase alignment and we propose that this rotational alignment is effected by microtubules anchored at cortical adhesion sites. Postmetaphase alignment was not affected by any of the treatments tested, and may be dependent on asymmetric cell morphology.

Cell wall deposition during morphogenesis in fucoid algae.

Cell was deposition was investigated during morphogenesis in zygotes of Pelvetia compressa (J. Agardh) De Toni. Young zygotes are spherical and wall is deposited uniformly, but at germination (about 10 h after fertilization) wall deposition becomes localized to the apex of the tip-growing rhizoid. Wall deposition was investigated before and after the initiation of tip growth by disrupting cytoskeleton, secretion or cellulose deposition; effects on wall strength and structure were examined. All three were involved in generating wall strength in both spherical and tip-growing zygotes, but their relative importance were different at the two developmental stages. Much of the wall strength in young zygotes was dependent on F-actin, whereas cellulose and a sulfated component, probably a fucan (F2), were most important in tip growing zygotes. Some treatments had contrasting effects at the two developmental stages; for example, disruption of F-actin or inhibition of secretion weakened walls in spherical zygotes but strengthened those in tip-growing zygotes. Transmission electron microscopic analysis showed that most treatments that altered wall strength induced modifications of internal wall structure.

A S/M DNA replication checkpoint prevents nuclear and cytoplasmic events of cell division including centrosomal axis alignment and inhibits activation of cyclin-dependent kinase-like proteins in fucoid zygotes.

S/M checkpoints prevent various aspects of cell division when DNA has not been replicated. Such checkpoints are stringent in yeast and animal somatic cells but are usually partial or not present in animal embryos. Because little is known about S/M checkpoints in plant cells and embryos, we have investigated the effect of aphidicolin, a specific inhibitor of DNA polymerases (alpha) and (delta), on cell division and morphogenesis in Fucus and Pelvetia zygotes. Both DNA replication and cell division were inhibited by aphidicolin, indicating the presence, in fucoid zygotes, of a S/M checkpoint. This checkpoint prevents chromatin condensation, spindle formation, centrosomal alignment with the growth axis and cytokinesis but has no effect on germination or rhizoid elongation. This S/M checkpoint also prevents tyrosine dephosphorylation of cyclin-dependent kinase-like proteins at the onset of mitosis. The kinase activity is restored in extracts upon incubation with cdc25A phosphatase. When added in S phase, olomoucine, a specific inhibitor of cyclin-dependent kinases, has similar effects as aphidicolin on cell division although alignment of the centrosomal axis still occurs. We propose a model involving the inactivation of CDK-like proteins to account for the S/M DNA replication checkpoint in fucoid zygotes and embryos.

Sperm entry induces polarity in fucoid zygotes.

Fucoid zygotes establish a rhizoid-thallus growth axis in response to environmental signals; however, these extrinsic cues are not necessary for polarization, suggesting that zygotes may have inherent polarity. The hypothesis that sperm entry provides a default pathway for polarization of zygotes cultured in the absence of environmental signals was tested, and was supported by several lines of evidence. First, an F-actin patch, a cortical marker of the rhizoid pole, formed at the sperm entry site within minutes of fertilization. Second, the sperm entry site predicted the site of polar adhesive secretion (the first morphological manifestation of the rhizoid pole) and the position of rhizoid outgrowth. Third, when fertilization was restricted to one hemisphere of the egg, rhizoid outgrowth always occurred from that hemisphere. Fourth, delivery of sperm to one location within a population of eggs resulted in polarization of both adhesive secretion and rhizoid outgrowth toward the sperm source. Finally, induction of polyspermy using low sodium seawater increased the frequency of formation of two rhizoids. Sperm entry therefore provides an immediate default axis that can later be overridden by environmental cues.

F-actin marks the rhizoid pole in living Pelvetia compressa zygotes.

Spatial and temporal changes in F-actin during polarity establishment in Pelvetia compressa zygotes were investigated using vital staining with rhodamine phalloidin (RP). F-actin was localized to a patch in the cortex of young zygotes. When unilateral light was applied to induce a growth axis (photopolarization) in a population of zygotes, the cortical F-actin patches localized at the shaded pole (rhizoid pole of growth axis). Treatments that prevented photopolarization prevented localization of F-actin patches to the shaded pole. When the direction of the light treatment was reversed, the previous growth axis was abandoned and a new axis was established in the opposite direction. The F-actin patch repositioned to the new rhizoid pole within minutes of light reversal, indicating that F-actin was an immediate marker of the nascent growth axis. Repositioning probably occurred by disassembly of the initial patch and reassembly of a new one. The patch grew in size as zygotes developed, eventually becoming a ring just prior to rhizoid outgrowth. The rhizoid emerged at the site of the F-actin ring and, following germination, the ring was located in the subapical zone of the elongating tip.

Roles of secretion and the cytoskeleton in cell adhesion and polarity establishment in Pelvetia compressa zygotes.

During the establishment of polarity, fucoid algal zygotes adhere to the substratum and select a growth axis according to environmental cues. Since little is known about the early events leading to axis selection, we investigated the chronology of cell adhesion, adhesive deposition, and axis selection induced by light (photopolarization). The requirements for secretion and the cytoskeleton in these processes and in the process of changing the orientation of an axis in response to new environmental cues (axis realignment) were also tested. Adhesive deposition occurred in two distinct stages: it was deposited uniformally on young zygotes (uniform primary adhesive) and later was deposited asymmetrically (polar secondary adhesive). Uniform primary adhesive deposition, cell adhesion, and photopolarization occurred simultaneously, and shortly thereafter, polar secondary adhesive deposition occurred at the future growth site. Uniform primary adhesive deposition and cell adhesion required secretion, but were independent of filamentous-actin (F-actin) and microtubule function. Photopolarization of young zygotes and polar secondary adhesive deposition required secretion but not microtubules. F-actin served to localize secondary adhesive deposition at the rhizoid pole; its function in polarization was more complex. F-actin was required for axis selection; however, its role in realignment of an axis depended on the light regime. The differing requirements for F-actin during development indicates that the axis is not static, but changes with time. These findings indicate that previous and future work on "axis formation" must be interpreted in the context of the developmental stage of the zygote.

Alignment of centrosomal and growth axes is a late event during polarization of Pelvetia compressa zygotes.

Zygotes and embryos of the fucoid brown alga Pelvetia compressa undergo a series of asymmetric cleavages. We are interested in the developmental role of these cleavages and the mechanism controlling their alignment. To assess the importance of division plane alignment, the orientation of the first asymmetric division was altered and the effects on subsequent embryo elongation rates were analyzed. Although this division is normally oriented transversely (90 degrees) to the growth axis, deviations up to 45 degrees had no significant effects on embryo elongation. However, division planes that were parallel with the growth axis (0-45 degrees) had drastic effects. Embryo elongation was severely inhibited and the wall often bifurcated and avoided the rhizoid tip. The orientation of the division plane is determined by the position of the centrosomes. We therefore investigated centrosomal position and function during the first cell cycle within the three-dimensional context of the cell. We found that, after karyogamy, microtubule organization changed from a radially symmetric circumnuclear array into a bipolar centrosomal array. The reorganization coincided with the migration of the centrosomes around the nucleus. The centrosomes separated slowly and asynchronously until they reached opposite sides of the nuclear envelope. At this time the centrosomal axis, defined by the position of the two centrosomes, was oriented randomly with respect to the cortical growth axis. The centrosomal axis then rotated into alignment parallel with the growth axis late in the first cell cycle. These results indicate that the growth axis and the centrosomal axis develop independently of each other and that the centrosomal axis does not align with the growth axis until just prior to mitosis.

Cytoskeletal control of polar growth in plant cells.

There are two quite different modes of polar cell expansion in plant cells, namely, diffuse growth and tip growth. The direction of diffuse growth is determined by the orientation of cellulose microfibrils in the cell wall, which in turn are aligned by microtubules in the cell cortex. The orientation of the cortical microtubule array changes in response to developmental and environmental signals, and recent evidence indicates that microtubule disassembly/reassembly and microtubule translocation participate in reorientation of the array. Tip growth, in contrast, is governed mainly by F-actin, which has several putative forms and functions in elongating cells. Longitudinal cables are involved in vesicle transport to the expanding apical dome and, in some tip growers, a subapical ring of F-actin may participate in wall-membrane adhesions. The structure and function of F-actin within the apical dome may be variable, ranging from a dense meshwork to sparse single filaments. The presence of multiple F-actin structures in elongating tips suggests extensive regulation of this cytoskeletal array.

Measurement and manipulation of cytosolic pH in polarizing zygotes.

The role of cytosolic pH (pHc) in establishment and expression of developmental polarity was examined in zygotes of the brown alga Pelvetia. pHc was measured and manipulated at specific developmental stages during the first zygotic cell cycle. pHc was measured using pH-sensitive microelectrodes and by confocal ratio imaging of dextran-conjugated SNARF 1 (dc SNARF 1) loaded cells. The two techniques yielded very similar values of pHc in the cellular cortex, but ratio imaging was not effective in measuring endoplasmic pHc values. As zygotes formed a developmental axis, cortical pHc decreased abruptly by approximately 0.1 units, and a small but significant difference in pHc was detected at the thallus and rhizoid poles. The cortical cytosol was relatively acidic at the presumptive rhizoid pole. The magnitude of the pHc difference increased following initiation of rhizoid growth. pHc was manipulated by treating zygotes with membrane-permeant weak acids (propionic and benzoic acid) or bases (methylamine and procaine), which effectively clamp pHc to specific values in a concentration-dependent manner. pHc values in treated zygotes were measured for each concentration of acid or base, and a dose response curve was generated. Zygotes in which pHc had been clamped were examined for their ability to form a developmental axis and to initiate rhizoid outgrowth (germination). Both developmental processes were inhibited by relatively small (0.2-0.3 pH units) perturbations of pHc. The permissive ranges of pHc were slightly different, germination (permissive pHc range-pH 7.0 to 7.7) being more acid tolerant than axis formation (permissive pHc range-7.2 to 7.8).

Cytosolic pH Gradients Associated with Tip Growth.

The presence of a cytosolic pH gradient and its relation to polar tip growth was investigated in rhizoid cells of Pelvetia embryos with the use of pH-sensitive microelectrodes and by ratio imaging. Growing rhizoid cells generated a longitudinal pH gradient in which the apical cytosol was 0.3 to 0.5 units more acidic than the cytosol at the base of the cell. Treatment with a membrane-permeant weak acid, propionic acid, dissipated the cytosolic pH gradient and inhibited growth. The magnitude of the pH gradient correlated well with the rate of tip elongation. The pH gradient spatially superimposed on the cytosolic calcium gradient, and inhibition of calcium fluxes by treatment with lanthanum abolished the pH gradient and inhibited growth.

Cell wall and rhizoid polarity in Pelvetia embryos.

The developmental potentials of the rhizoid and thallus cells of two-celled Pelvetia embryos were investigated. Ablation of either the thallus or the rhizoid cell was accomplished by puncture with a micropipette. Thallus cells continued to divide repeatedly after rhizoid ablation, and in no case did the thallus initiate new rhizoid growth. In the reciprocal experiment, rhizoid cells elongated and divided in the normal transverse orientation after ablation of the thallus cell. The rhizoid did not appear to initiate a new thallus. Similar results were obtained when laser irradiation was applied to arrest division in one of the two cells. Thus, neither thallus nor rhizoid cell compensated for ablation or arrest of its sibling cell. The role of the cell wall-plasma membrane connections in organizing polar growth in the rhizoid cell was investigated by separating the wall from the protoplast. This was accomplished in two ways, by plasmolysis and by enzymatic wall digestion. Wall digestion yielded rhizoid cell protoplasts capable of wall regeneration and division, but the polar growth habit was irreversibly lost. Loss of polar growth correlated with loss of polarity in the microtubule cytoskeleton as visualized by indirect immunofluorescence using confocal microscopy. Transient plasmolysis of rhizoid cells resulted in abandonment of the preexisting apex and the initiation of a new rhizoid tip after rehydration. We suggest that interaction between the cell wall and the plasma membrane is involved in organizing polar growth.

Intracellular pH and its regulation in Pelvetia zygotes.

We have investigated the role of intracellular pH (pHi) in early plant development using ion-selective microelectrodes to record from eggs and embryos of the brown alga Pelvetia. Temporal changes in pHi were investigated by recording from zygotes at all stages of the first cell cycle. pHi was 7.57 +/- 0.09 in recently fertilized eggs, but decreased by approximately 0.2 units a few hours postfertilization. Proton motive force (pmf) was also monitored and found to be less than -50 mV throughout the first cell cycle. Because of the low pmf values, we suggest that secondary active transport is probably not coupled to H+, and instead we propose that solute transport is driven by the Na+ electrochemical potential. Zygotes strictly regulated pHi over a wide range of extracellular pH (pHo); pHi varied by less than 0.2 units over pHo values from 6.2 to 9.2. Inhibitor studies were conducted to investigate the mechanism of regulation. Agents known to inhibit the H(+)-ATPase (antimycin A, KCN, carbonylcyanide m-chlorophenylhydrazone, N,N'-dicyclohexylcarbodiimide, and erythrosin B) caused marked cytoplasmic acidification. Addition of amiloride, an inhibitor of Na+/H+ antiport, also resulted in acidification, as did removal of NaCl from the medium. By contrast, increasing extracellular NaCl concentration caused transient alkalinization of the cytoplasm. Taken together, these results indicate that proton pumping by an H(+)-ATPase and Na+/H+ antiporter contribute to pH regulation.

Pronuclear positioning and migration during fertilization in Pelvetia.

The position and migration of egg and sperm pronuclei were studied in the brown alga Pelvetia. The egg pronucleus was located near the center of the cell before and after fertilization and, unlike pronuclei in animal eggs, did not migrate. Inhibitor studies indicated that anchoring of the egg pronucleus in the cell center was dependent on microtubules and microfilaments. An extensive array of microtubules, many of which extended into the actin-rich egg cortex, was associated with the egg pronucleus. Migration of the sperm pronucleus was investigated quantitatively in both living and fixed zygotes. Migration occurred linearly at rates from 0.11 to 0.29 microns/min and was oriented directly toward the egg pronucleus in the cell center. Sperm penetration was inhibited by cytochalasin D, which disrupts F-actin function, whereas sperm pronuclear migration was sensitive to the microtubule-depolymerizing drug, nocodazole. Microtubules associated with the migrating sperm pronucleus formed a sperm trail that terminated at the egg cortex. As these were the only microtubules associated with the sperm at early stages of migration, we conclude that they provide the force for migration.

Establishment and expression of cellular polarity in fucoid zygotes.

Zygotes of fucoid algae have long been studied as a paradigm for cell polarity. Polarity is established early in the first cell cycle and is then expressed as localized growth and invariant cell division. The fertilized egg is a spherical cell and, by all accounts, bears little or no asymmetry. Polarity is acquired epigenetically a few hours later in the form of a rhizoid/thallus axis. The initial stage of polarization is axis selection, during which zygotes monitor environment gradients to determine the appropriate direction for rhizoid formation. In their natural setting in the intertidal zone, sunlight is probably the most important polarizing vector; rhizoids form away from the light. The mechanism by which zygotes perceive environmental gradients and transduce that information into an intracellular signal is unknown but may involve a phosphatidylinositol cycle. Once positional information has been recorded, the cytoplasm and membrane are reorganized in accordance with the vectorial information. The earliest detectable asymmetries in the polarizing zygote are localized secretion and generation of a transcellular electric current. Vesicle secretion and the inward limb of the current are localized at the presumptive rhizoid. The transcellular current may establish a cytoplasmic Ca2+ gradient constituting a morphogenetic field, but this remains controversial. Localized secretion and establishment of transcellular current are sensitive to treatment with cytochalasins, indicating that cytoplasmic reorganization is dependent on the actin cytoskeleton. The nascent axis at first is labile and susceptible to reorientation by subsequent environmental vectors but soon becomes irreversibly fixed in its orientation. Locking the axis in place requires both cell wall and F-actin and is postulated to involve an indirect transmembrane bridge linking cortical actin to cell wall. This bridge anchors relevant structures at the presumptive rhizoid and thereby stabilizes the axis. Approximately halfway through the first cell cycle, the latent polarity is expressed morphologically in the form of rhizoid growth. Elongation is by tip growth and does not appear to be fundamentally different from tip growth in other organisms. The zygote always divides perpendicular to the growth axis, and this is controlled by the microtubule cytoskeleton. Two microtubule-organizing centers on the nuclear envelope rotate such that they align with the growth axis. They then serve as spindle poles during mitosis. Cytokinesis bisects the axial spindle, resulting in a transverse crosswall. Although the chronology of cellular events associated with polarity is by now rather detailed, causal mechanisms remain obscure.

Ionic requirements for establishment of an embryonic axis in Pelvetia zygotes.

Previous reports have shown that fertilized Pelvetia fastigiata (J. Ag.) DeToni eggs generate a transcellular ionic current which correlates temporally and spatially with establishment of a rhizoid/thallus axis. In order to understand more fully the ionic controls of development, we have determined the ionic requirements for axis formation induced by unilateral light. Formation of the developmental axis was independent of the presence of individual ions in artificial seawater. This finding indicates that transcellular circulation of a particular ion is not obligatory for polarization, and it confirms earlier work showing that calcium circulation is not fundamental to axis establishment in Fucus zygotes. Polarization was not, however, completely independent of ionic conditions; zygotes were unable to form an axis in pure sucrose solutions. Single salts were added to sucrose to determine which ions were sufficient to permit polarization. Salts of impermeant monovalent cations and salts of divalent cations supported polarization weakly or not at all. By contrast, zygotes photopolarized well in KCl, and other alkali metals substituted for K(+) with varying effectiveness (Rb(+)>Na(+)> Cs(+)≫ Li(+)). The anion was unimportant; a variety of different potassium salts all supported polarization equally well. The mechanism by which KCl promotes polarization is not yet understood, but may involve transport through K(+) channels.