lnduction of Polarity in Fucoid Zygotes - NCBI

The Plant Cell, Vol. 9, 1 O1 1-1 020, July 1997 O 1997 American Society of Plant Physiologists

lnduction of Polarity in Fucoid Zygotes

Darryl L. Kropf' Department of Biology, University of Utah, Salt Lake City, Utah 841 12

INTRODUCTION

Early events 'in the vegetative development of higher land plants are difficult to investigate because the young embryo is encased in the ovarian tissue of the previous gametophytic and sporophytic generations. Recently, genetic approaches have succeeded in identifying mutations that alter early devel- opment (Jürgens, 1995; see also Laux and Jürgens, 1997, in this issue), but cellular and physiological analyses of higher plant embryogenesis remain tedious.

Brown algae of the family Fucales (comprising the genera Fucus and Pelvetia) are, however, very tractable for investi- gating early plant embryogenesis, especially the establish- ment of developmental polarity. These marine organisms grow attached to rocks in the intertidal zone and are typi- cally 0.25 to 0.5 m in height at maturity. Zygotes, eggs, and sperm are shed from fronds of mature plants in the labora- tory and can easily be harvested in gram quantities, free of other tissues. Young zygotes attach firmly to the substra- tum, and populations of adhering zygotes transit synchro- nously through early development.

In the Fucales, mature eggs are radially symmetric, and cell polarization does not occur until after fertilization. The primary developmental axis is organized early in the first cell cycle and defines the growth axis of the young embryo. Un- like higher plants, in which positional cues are often conveyed by neighboring cells, fucoid zygotes develop autonomously and therefore rely on environmental signals for spatial infor- mation to orient their nascent axes. In the laboratory, an en- tire population can be induced to establish axes in parallel by the application of externa1 vectors such as unidirectional light (Jaffe, 1968). A few hours after polarization, localized growth occurs at one pole of the nascent axis, as shown in Figure 1.

The first zygotic division is an invariant, asymmetric divi- sion, with the cell plate oriented transverse to the growth axis. The smaller rhizoid cell contains the growing apex and is the precursor to the holdfast of the alga; the larger thallus cell gives rise to the stipe and fronds of the mature plant (Figure 1). After the initial division of the zygote, the rhizoid cell elongates and repeatedly divides transverse to the growth axis, whereas the thallus cell undergoes proliferative divisions, each of which is transverse to the previous division.

E-mail [email protected]; fax 801 -581 -4668.

Developing embryos of many land plants, including Arabidop- sis, undergo a similar pattern of cell divisions (Figure 1; see also Laux and Jürgens, 1997, in this issue).

This review highlights recent progress in understanding the polarization of fucoid zygotes, with an emphasis on early events involved in polarity induction in Pelvetia. Readers are directed to a comprehensive review (Kropf, 1992) and to other more specialized reviews (Goodner and Quatrano, 1993; Kropf, 1594; Berger and Brownlee, 1995; Fowler and Quatrano, 1995; Quatrano and Shaw, 1997) for additional analyses and opinions.

POLARITY INDUCTION

The induction of polarity in fucoid zygotes is a continuum of overlapping events that occur during the first 10 hr after fer- tilization (AF). For convenience, I consider polarity induction to occur in two stages: axis selection, followed by axis am- plification. Events occurring during these stages are consid- ered below, and models describing polarity induction are presented later.

Axis Selection

Axis selection is the process by which the position of the de- velopmental axis is specified. To select an axis, a zygote must perceive a signal, transduce that signal, and mark the developmental polarity.

Fucoid eggs have no demonstrable cellular asymmetries, and axis selection must therefore occur during or after fertil- ization. The egg pronucleus resides in the center of the egg cell, and microtubules nucleate uniformly from the surface of the pronucleus and radiate toward the cortex (Swope and Kropf, 1993). Short rods of F-actin are randomly oriented and uniformly distributed throughout the cortex (Kropf et al., 1992), and organelles are uniformly distributed throughout the cytoplasm (Brawley et al., 1976b). Glycoproteins are dis- tributed in patches over the entire surface of the egg (Stafford et al., 1992).

1012 The Plant Cell

derived organelles persist through much of the first cell cy- cle and have the potential to serve as positional markers. However, preliminary evidence indicates that the nucleus and perinuclear material, as marked by centrosomal posi- tion, do not align with the developmental axis until after growth begins (S.R. Bisgrove, unpublished results); there- fore, they are unlikely to be involved in a i s selection. In- stead, polarity is likely to be perceived and marked in the plasma membrane and cell cortex (see below). Although the mechanisms by which sperm may mark the plasma mem- brane or cortex are unknown, Ca2+ levels increase just be- neath the plasma membrane at the sperm entry site (Roberts et al., 1994), and it is possible that, in turn, this local elevation in Ca2+ activity triggers regional structural changes (see Trewavas and Malhó, 1997, in this issue).

A B

C D

Environmental Cues

The putative sperm-induced axis is weak and can be over- ridden by vectorial information in the environment (Jaffe, 1958; Robinson, 1996b). Approximately 3 hr AF, Pelvetia and Fucus zygotes secrete an adhesive symmetrically over the surface of the zygote and attach firmly to the substratum (Vreeland et al., 1993; Vreeland and Epstein, 1996). Once at-

Figure 1. Embryogenesis in Pelvetia and Arabidopsis.

(A) to (C) Pelvetia embryogenesis. (A) Unfertilized egg. (B) Two- celled embryo displaying localized rhizoid growth and invariant divi- sion. (C) Eight-celled embryo with transverse divisions in descen- dants of the rhizoid cell. (D) Arabidopsis embryo. The young embryo is morphologically simi- lar to the Pelvetia embryo. Arrows indicate the first division plane of the zygote.

The Role of Sperm Entry: An Open Question

The role of sperm entry in inducing polarity in fucoid eggs is unclear (Roberts et al., 1993). Sperm entry is sufficient for polar development; externa1 vectorial information (e.g., light) is not required for localized rhizoid outgrowth, oriented cell division, or subsequent development. These observations are consistent with sperm entry inducing an initial axis, and in a related brown alga, Cystosira, rhizoid growth occurs at the sperm entry site in the absence of vectorial cues (Knapp, 1931). However, the spatial relationship between sperm entry and rhizoid outgrowth has not been investigated in Pelvetia or Fucus, the organisms in which nearly all of the subsequent polarity studies have been conducted.

If sperm entry induces polarity, then the sperm entry site must somehow be marked in the egg cytoplasm. The sperm eyespot, mitochondria (Brawley et al., 1976a), and cen- trosomes (Motomura, 1994) migrate with the sperm pronu- cleus and are deposited on the zygotic nucleus at the site of pronuclear fusion, as shown in Figure 2A. These sperm-

tached, zygotes begin to sense and respond to an impres- sive number of very different environmental cues. Indeed, the developmental axis, and, hence, the position of rhizoid outgrowth, can be controlled by the application of unidirec- tional light, gradients of ions or ionophores, gravity (G. Muday, personal communication), flow of seawater, temperature gra- dients, and many other factors (Jaffe, 1968). Plane polarized light induces the formation of two rhizoids (Jaffe, 1958), indi- cating that vectorial information does not simply result in ròta- tion of the putative sperm-induced axis. Zygotes are sensitive to each gradient for a restricted period, and the windows of sensitivity overlap for many gradients (Bentrup, 1984).

The mechanisms by which environmental gradients are perceived and transduced are almost totally unknown in plants (see Trewavas and Malhó, 1997, in this issue). In- deed, the diversity of stimuli that can elicit developmental axis selection in zygotes of the Fucales indicates that multi- ple, converging signal transduction pathways are involved. However, to date, only the photopolarization pathway has been investigated in detail. Unidirectional ultraviolet or blue light induces rhizoid growth from the shaded hemisphere of the zygote (Hurd, 1920; Robinson, 1996a), and the activa- tion of cortical photoreceptors (Jaffe, 1958) preferentially stimulates plasma membrane redox chains at the presump- tive rhizoid (i.e., shaded) pole (Berger and Brownlee, 1994). Photopolarization is inhibited by acidification or alkaliniza- tion of the cytosol (Kropf et al., 1995) and by disruption of F-actin (Quatrano, 1973), but the roles of redox potential, cytosolic ions, and the cytoskeleton in signal transduction remain a mystery. Likewise, the mechanism by which the rhizoid pole is marked is unknown.

Polarity in Fucoid Zygotes 1013

sperm pronucleusegg and zygotic nucleicentrosomeMtOCmicrotubule

\ F-actinA current flow

3B cell wall'.<- jellyo secretory vesicle

| DHP receptor•:vi-.cytosolic ion gradient* F2; vitronectin-like protein~ actin mRNA

Figure 2. Summary of Structural and Physiological Changes during the First Zygotic Cell Cycle.

(A) One hr AF. Centrosomes are inherited paternally, and organelles, microtubules, and F-actin are uniformly distributed in the zygotic cytoplasm.(B) Four to 7 hr AF. Zygotes secrete local jelly, and electrical current flows through the cell. Inward current and jelly are positioned on the shadedside of the zygote. Centrosomes separate and begin to migrate along the nuclear envelope.(C) Seven to 10 hr AF. F-actin in the cortex and dihydropyridine receptors in the plasma membrane accumulate at the presumptive rhizoid. Se-cretion becomes targeted to this site, depositing the sulfated fucan F2 in the wall and causing local cortical clearing. Gradients of H* and Ca2+

are detectable.(D) Ten to 13 hr AF. The rhizoid begins to elongate, Centrosomes become the major site of microtubule nucleating activity, and microtubules ex-tend preferentially into the rhizoid cortex.(E) Thirteen to 16 hr AF. The nucleus rotates, bringing the Centrosomes into axial alignment; the spindle forms.(F) Twenty to 24 hr AF. At cytokinesis, the cross-wall forms transverse to the growth axis. F-actin and actin mRNA accumulate at the cross-wall.In the next division, only the rhizoid nucleus rotates, so spindles in the two-celled embryo are aligned transverse to one another.DHP, dihydropyridine; MtOC, microtubule organizing center.

1014 The Plant Cell

Axis Amplification

After axis selection, there is a prolonged period, lasting from~4 to 10 hr AF, during which there is a gradual reorganizationof the cytoplasm, plasma membrane, and cell wall in theyoung zygote. These rearrangements, which presumably am-plify and strengthen the weak initial axis, overlap temporallyand are discussed in their approximate chronological order.

Polar Jelly Deposition

One to 2 hr after Pelvetia zygotes adhere to a substratumand begin to sense environmental cues, they deposit a jellymaterial at the presumptive rhizoid pole of the nascent axis,as shown in Figure 3A (Schroter, 1978; Weisenseel, 1979).The jelly, which contains alginate, phenolic compounds, andsulfated fucans (Vreeland and Epstein, 1996), is deposited asan amorphous, translucent layer outside the cell wall. Fucuszygotes are slightly delayed compared with Pelvetia', uniformadhesive is secreted ~4 hr AF, and conspicuous polar jellyis deposited 6 to 8 hr AF (Vreeland et al., 1993). The rapiditywith which jelly deposition occurs probably relates to func-tion; the rhizoid-localized jelly strengthens the attachment ofthis region of the spherical zygote to the substratum, usuallya rock in the intertidal zone (Vreeland and Epstein, 1996).Robust and rapid attachment helps prevent the cell from be-ing washed out to sea in the next tidal cycle.

Jelly continues to be deposited over the surface of Fucusand Pelvetia embryos throughout early development, withgreatest accumulations occurring toward the rhizoid pole(Schroter, 1978). However, jelly deposition is not required forearly embryogenesis. Growth in seawater lacking sulfateprevents sulfation of fucans and thereby interferes with ionicinteractions between polysaccharide molecules. The jellylayer is not deposited, yet zygotes germinate and divide nor-mally (Schroter, 1978).

The jelly is likely deposited via vesicle secretion (Vreelandet al., 1993), but it is not yet clear whether uniformly distrib-uted vesicles fuse with the plasma membrane locally orwhether a subpopulation of secretory vesicles is preferen-tially localized at the presumptive rhizoid. Electron micros-copy studies of Fucus zygotes provide no evidence forasymmetries in Golgi or vesicle distributions at this early stageof development (Quatrano, 1972; Brawley et al., 1976b). Theearly secretion of polar jelly in fucoid zygotes is surprising,because in most polarizing cells, secretion is a late event,occurring only after an axis has been amplified and rein-forced (Drubin and Nelson, 1996).

Despite the lack of obvious structural polarity in the cell atthe time of jelly secretion, there are local physiologicalchanges in the cellular cortex that can be detected by plas-molysis or measurement of electrical current. When 5-hr-oldphotopolarized zygotes are placed in hypertonic seawater,plasmolysis occurs preferentially at the presumptive rhizoidpole (Reed and Whitaker, 1941). These changes in interactions

Figure 3. Jelly Deposition.

(A) Jelly layer surrounding a 4-hr-old Pelvetia zygote outlined withfluorescent microspheres. The jelly (arrowhead) extends >5 (j.mfrom the cell surface (arrow) at the presumptive rhizoid but has nomeasurable thickness at the presumptive thallus. The arrow in theupper right corner indicates direction of light treatment initiated at 1hr AF. Bar = 25 H.ITI.(B) The position of jelly deposition changes during axis realignment.In response to light 1 (L1), jelly is deposited at jelly 1 (J1). When thedirection of the light is reversed to L2, jelly is deposited at J2.The images, kindly provided by Whitney Hable, are 1-(jim opticalsections taken by laser scanning confocal microscopy. Fluorescentorganelles inside the cells are chloroplasts.

between plasma membrane and cell wall at the presumptiverhizoid are detectable within 1 hr of photopolarization.

Ionic Currents

A transcellular ionic current (Figure 2B), which is generatedat the plasma membrane, has been thoughtfully discussedin numerous articles and reviews (Jaffe et al., 1974; Jaffeand Nuccitelli, 1977; Robinson and McCaig, 1980) and is onlydiscussed briefly here. In very young zygotes, unstablepatches of inward and outward current (defined as the flow ofpositive charge) can be detected, but by 6 hr AF, an inwardcurrent stabilizes at the presumptive rhizoid pole (Nuccitelli,1978). Treatment with cytochalasin D prevents localized cur-rent flux (Brawley and Robinson, 1985), indicating a depen-dence on cortical F-actin.

Polarity in Fucoid Zygotes 1 O1 5

The current is thought to result in local differences in cyto- solic ion concentrations at the presumptive rhizoid and thal- lus poles. Indeed, a fraction of the inward current is carried by Caz+ at 5 to 6 hr AF (Robinson and Jaffe, 1975), yet cyto- solic Caz+ gradients have not been detected until 10 hr AF (Berger and Brownlee, 1993; Taylor et al., 1996). By contrast, a small but measurable cytosolic pH gradient (acidic at the presumptive rhizoid) has been detected at 5 hr AF, and the magnitude of the gradient increases throughout the period of amplification (Kropf et al., 1995). However, the difference in pH between rhizoid and thallus poles is <0.1 pH units and may not be intense enough to induce regional specialization.

Late Evenfs

Severa1 cellular rearrangements occur later in axis amplifica- tion, between 7 and 10 hr AF. Dihydropyridine receptors, which may be Ca2+ channels that carry part of the inward current, localize to the plasma membrane at the presump- tive rhizoid soon after the stable current is first detectable (Shaw and Quatrano, 1996a). Shortly thereafter, F-actin (Brawley and Robinson, 1985; Kropf et al., 1989), cytosolic Ca2+ (Berger and Brownlee, 1993), and a clearing between plasma membrane and cell wall (cortical clearing; Peng and Jaffe, 1976; Nuccitelli, 1978) become localized to the cell cortex at the rhizoid pole, and a sulfated fucan (F2; Novotny and Forman, 1974) becomes localized to the rhizoid cell wall (Figure 2C). The precise temporal relationships between these localizations have not been investigated.

Both cortical clearing and polar plasmolysis may be mani- festations of localized secretion, which locally disrupts inter- actions between the plasma membrane and the cell wall (Peng and Jaffe, 1976). Polar secretion also may function to deposit F2 into the wall at the rhizoid pole (Novotny and Forman, 1974). Because these localizations occur late in amplification, they may be important for subsequent events such as axis fixation or rhizoid growth (see below).

though these experiments are reported to investigate axis formation, it seems more likely that they address realign- ment of an axis selected before jelly secretion.

AXlS FlXATlON

Just before germination, polar zygotes become insensitive to external cues, and the site of rhizoid outgrowth becomes fixed in space. Axis fixation occurs 10 to 12 hr AF and is marked by the polar secretion of F2 into the wall. For the first time in zygotic development, the secretory apparatus shows marked polarity; Golgi complexes accumulate in the perinu- clear region on the rhizoid-facing side, and vesicles are trans- ported preferentially toward the site of impending outgrowth (Brawley and Quatrano, 1979; Quatrano and Shaw, 1997). Cytochalasin treatment causes F2-containing vesicles (F gran- ules) to accumulate near the Golgi complexes, indicating that vesicle transpor? is mediated by F-actin (Brawley and Quatrano, 1979). However, actin filaments in the region between the Golgi and the cortex have not been observed, perhaps be- cause F-actin is difficult to preserve. Total mRNA and actin mRNA are excluded from the rhizoid and are abundant at the thallus pole at the time of axis fixation (Bouget et al., 1996), but the significance of these observations is unclear.

Agents that block axis fixation extend the period in which zygotes are sensitive to external stimuli; zygotes germinate in accordance with the most recently perceived light vector when the inhibitor is removed. Secretory inhibition (Shaw and Quatrano, 1996b), disruption of F-actin (Quatrano, 1973), and enzymatic removal of the cell wall (Kropf et al., 1988) all have been shown to prevent axis fixation. lncubation in hy- pertonic seawater of proper osmolarity also prevents axis fixation but not rhizoid outgrowth, demonstrating that the two phenomena can be uncoupled (Robinson, 1996b).

GROWTH

Axis Realignment

The developmental axis is labile and susceptible to realign- ment by applied vectors throughout the period of amplifica- tion (4 to 10 hr AF). Components of the axis become aligned with the most recently perceived vectorial cue, as demon- strated for polar jelly in Figure 38. In addition to jelly (Schroter, 1978), dihydropyridine receptors (Shaw and Quatrano, 1996a), the ionic current, and cortical clearing (Nuccitelli, 1978) can all be repositioned. F2, however, is deposited after the axis can no longer be realigned (Fowler and Quatrano, 1995) and is therefore a marker for axis fixation (see below). Repositioning of F-actin, Ca2+, and H+ has not been investigated.

Traditionally, experiments designed to investigate polarity induction are conducted on well-attached, 6- to 8-hr-old zy- gotes that are then exposed to an environmental vector. Al-

At germination, vesicle secretion intensifies, and a rhizoid, which elongates by tip growth, emerges from the chosen site (Figure 2D). The insertion of secretory vesicles deposits new membrane and wall components, including F2 (Brawley and Quatrano, 1979) and a vitronectin-like protein (Wagner et al., 1992), at the elongating tip. Some of the cellular com- ponents that are localized to the presumptive rhizoid during axis induction and fixation are important for tip elongation. This is the case for F-actin and cytosolic ions, which are thought to play fundamental roles in the elongation of most, if not all, tip growing cells (Steer and Steer, 1989; Heath, 1990; Jackson and Heath, 1993; Hepler, 1997).

The Ca2+ gradient in Fucus zygotes intensifies at germina- tion and is maintained during rhizoid growth (Berger and Brownlee, 1993). This gradient likely results from flux through

1 O1 6 The Plant Cell

stretch-activated channels that are uniformly distributed but preferentially activated in the rhizoid apex (Taylor et al., 1996). In addition, the magnitude of the H+ gradient also increases at germination (Kropf et al., 1995). These ionic gradients appear to function in growth; when they are dissi- pated by treatment with H+ (Kropf et al., 1995) or Ca2+ (Speksnijder et al., 1989) buffers, tip elongation is inhibited. Presumably, the elevated ionic activities localize specific physiological processes to the tip.

Cortical F-actin also has an important role in rhizoid growth. F-actin is localized to the elongating tip, and treatment with cytochalasin results in the immediate cessation of growth (Kropf et al., 1989). However, the functions of F-actin during tip growth are not clear. lnhibitor studies suggest that vesicles are transported along F-actin (Brawley and Quatrano, 1979), but this has not been demonstrated directly. Cortical F-actin may also provide structural support to reinforce the apex against turgor pressure andor may play a more direct role in generating force for rhizoid expansion (Harold et al., 1996).

Recently, cortical F-actin has been shown to be present at adhesion sites near the apex of growing Pelvetia rhizoids (Henry et al., 1996). These wall-membrane adhesions are visible during plasmolysis of germinated zygotes and are lo- calized to the base of the apical dome, where they form a ringlike structure. They contain F-actin (Henry et al., 1996), dihydropyridine receptors (Shaw and Quatrano, 1996a), and Ca2+ (Kropf and Quatrano, 1987) on the cytoplasmic face and are thought to be manifestations of transmembrane complexes (see below). Although they do not appear to be needed for tip elongation, these adhesions may play a role in defining and maintaining the growth site (Kropf et al., 1993; Henry et al., 1996).

DlVlSlON PLANE ALIGNMENT

The first zygotic division is an invariant, asymmetric division with the wall oriented transverse to the growth axis. This ori- entation is determined by spindle alignment, which in turn is determined by the positioning of the centrosomes (Allen and Kropf, 1992). Two centrosomes are inherited paternally (Fig- ure 2A) and separate to opposite sides of the zygotic nu- cleus before growth (Figure 2B; Motomura, 1994). Soon after germination, the nucleus and centrosomes rotate so that the axis defined by the centrosomes aligns with the growth axis, and the spindle subsequently forms in axial (longitudinal) alignment (Figure 2E; Kropf et al., 1990). Cy- tokinesis occurs centripetally by invagination and is sup- ported by vesicle fusion (Brawley et al., 1977). The cleavage bisects the spindle, resulting in a transverse cross-wall (Fig- ure 2F). Subsequent divisions in the rhizoid are also trans- verse (Figure 1 D) and are thought to involve nuclear rotation (Allen and Kropf, 1992). Rotational alignment implies that in- formation regarding the position of the rhizoid is communi- cated to the nucleus.

Division plane alignment can be perturbed by pulsed treatments with cytochalasins, brefeldin A, or microtubule depolymerizing agents (e.g., oryzalin or nocodazole; Allen and Kropf, 1992; Shaw and Quatrano, 1996b). These agents cause abnormal nuclear rotations that result in misaligned spindles and skewed division planes. Embryos with slightly misaligned division planes can develop normally, but more severe misalignment reduces growth rate. Embryos in which the division plane is displaced 90" and bisects the rhizoid tip often develop two rhizoids (Shaw and Quatrano, 1996b; Quatrano and Shaw, 1997), suggesting that rotational align- ment may have evolved as a mechanism to prevent the for- mation of multiple rhizoids on a single embryo.

WORKING MODELS

The working models presented below are intended to provide a framework for interpreting the diverse findings discussed above and to help clarify the most important questions that need to be addressed.

Polarity lnduction

A developmental axis is selected very early in fucoid zy- gotes, perhaps even at sperm entry. lnvestigation of the spatial relationships between the sperm entry site and the sites of jelly secretion and rhizoid outgrowth in the absence of externa1 vectors will resolve the role of sperm entry in po- larization. If sperm entry is found to induce polarity, the next challenge will be to identify the cortical markers of this site. F-actin is a likely candidate because localized cortical do- mains of F-actin mark important sites in other polar cells (Drubin and Nelson, 1996; White and Strome, 1996). Appli- cation of high-pressure freezing techniques will aid in pre- serving cortical F-actin structure, and microinjection of fluorescently tagged actin should provide insight into the dy- namic properties of F-actin during fertilization.

The chain of causality during the early stages of polarity induction is not well understood. In published models, chan- nel localization, transcellular current, and cytosolic Ca2+ ac- cumulation are postulated to be causal to polar secretion (Jaffe et al., 1975; Brawley and Robinson, 1985). Alterna- tively, polar secretion may precede ionic localizations, as depicted in Figure 4A. In this version of polarity induction, axis selection is followed closely by vesicle secretion at the presumptive rhizoid, which inserts ion channels and/or cy- toskeletal anchoring proteins into the presumptive rhizoid membrane (Drubin and Nelson, 1996). These membrane asymmetries then give rise to local current influx and Ca2+ localization. ldentification of the spatial and temporal rela- tionships among dihydropyridine receptor localization, jelly secretion, and cytosolic gradients by double-labeling stud- ies will help distinguish between these two chronologies.

Polarity in Fucoid Zygotes 1 O1 7

A A

C

T cytoskeletal anchoring protein o ion I\ integral meinhrane protein jelly

A cell wall protein 0 secretory vesicle [ ) ion channel

- F-actin

Figure 4. Working Model.

(A) and (B) Axis amplification. (A) Polar secretion at the presumptive rhizoid pole of 4-hr zygotes establishes asymmetries in the plasma membrane by locally inserting membrane proteins, including ion channels and cytoskeletal anchoring proteins. (B) The weak asym- metry is amplified by continued local insertion of channels, recruit- ment of channels already in the plasma membrane, F-actin stabilization, and the establishment of locally elevated ionic activities at the presumptive rhizoid pole. (C) Axis fixation. Transmembrane complexes (right side of drawing) containing F-actin, an integral membrane protein, and a secreted cell wall component fix the developmental axis and are involved in the rotational alignment of the nucleus. For simplicity, putative actin- binding proteins of the complex are not shown.

The initial asymmetry is hypothesized to be amplified and reinforced over the ensuing hours, as depicted in Figure 4B (Brawley and Robinson, 1985). Continued vesicle insertion adds new channels and amplifies the local increase in the activity of inward-flowing cations, particularly H+ and Caz+. F-actin is stabilized locally by elevated H+ and Caz+ and by interaction with cytoskeletal-anchoring proteins. This F-actin in turn anchors and stabilizes ion channels and other mem- brane proteins, which results in further accumulations at the presumptive rhizoid and reinforcement of the vesicle secretion site. Furthermore, F-actin may mediate the movement of addi- tional plasma membrane components, such as dihydropyridine

receptors, to the rhizoid site. This amplification loop increases asymmetries in Caz+, H+, F-actin, and polar secretion.

This amplification model raises severa1 important issues. First, is a Caz+ gradient causal to the initial stages of axis in- duction? Previous experiments investigating the require- ment for Ca2+ in early development produced contradictory and equivoca1 results (Kropf and Quatrano, 1987; Hurst and Kropf, 1991 ; Robinson, 1996b). Manipulating cytosolic Ca2+ in specific cellular domains may help resolve the issue. Cy- tosolic Ca2+ can be locally elevated by microinjection of caged Caz+ followed by local Ca2+ release using laser irradi- ation. Putative interactions between cytosolic ionic activities and the cytoskeleton can be investigated by manipulating ionic activities and examining the effects on cytoskeletal as- sembly and stability. Finally, an investigation of secretory vesicle distribution at the time of jelly secretion should clarify whether vesicles are transported directionally at this early stage of development.

Axis Fixation and Rotational Alignment

Wall-membrane interactions mediated by transmembrane complexes are postulated to be important in axis fixation and rotational alignment of the nucleus. The essence of fixa- tion is thought to be the formation of transmembrane com- plexes that link F-actin in the rhizoid cortex through the plasma membrane to the cell wall (Figure 4C). F-actin accu- mulates in the rhizoid by differential stabilization (see above), and plasma membrane and/or cell wall components are localized by the polar secretion of Golgi-derived material (Fowler and Quatrano, 1995; Shaw and Quatrano, 1996b). Although the components of these proposed complexes have not been identified, proteins that cross-react with anti- bodies raised against mammalian focal adhesion complex components, such as vinculin, p-1 integrin, and vitronectin (Burridge and Chrzanowska-Wodnicka, 1996), have been identified in Fucus (Quatrano et al., 1991; Wagner et al., 1992). However, the vitronectin-like protein and F2 are un- likely to be the cell wall components needed for axis fixation because fixation proceeds even when localization of these molecules is disrupted (Fowler and Quatrano, 1995). It is im- portant to identify the true adhesion components, which may be different from molecules comprising focal adhesions in animal cells. Regardless of their composition, the forma- tion of transmembrane complexes is thought to stabilize the rhizoid cortex and define a target site for vesicle fusion dur- ing ensuing growth.

However, available evidence indicates that there may not be a close association of plasma membrane and cell wall at the time of axis fixation. A clear zone in the cortex at the pre- sumptive rhizoid pole is thought to be caused by displace- ment of the plasma membrane from the cell wall (Figure 2C; Peng and Jaffe, 1976). Moreover, wall-membrane adhesions visualized by plasmolysis are not localized at the presumptive rhizoid at the time of axis fixation (Henry et al., 1996).

1 O1 8 The Plant Cell

Rotational alignment of the nucleus before mitosis is also thought to involve transmembrane complexes. At the time of rotational alignment, wall-membrane adhesions, presum- ably composed of transmembrane complexes, are localized at the elongating rhizoid tip (Henry et al., 1996), and treat- ments that disrupt adhesions, such as the application of cy- tochalasin D, cause improper rotation (Allen and Kropf, 1992). Treatment with brefeldin A also prevents nuclear rota- tion (Shaw and Quatrano, 1996b), perhaps by preventing deposition of the cell wall component of the transmembrane complexes (Quatrano and Shaw, 1997).

The current working model for rotational alignment postu- lates that microtubules anchored at the centrosomes grow into the rhizoid cortex and are captured there (Kropf, 1994). Although the nature of the microtubule-capturing site is un- known, it is postulated to contain the cortical F-actin associ- ated with adhesion sites. Motors located at the centrosome or cortex exert force on the captured microtubules and pull the centrosome apically. Stochastically, one centrosome will be closer to the apex and is therefore likely to have more mi- crotubules captured. This centrosome wins the tug-of-war and rotates apically (Figures 2D and 2E).

Similar mechanisms have been proposed to orient asym- metric divisions in Caenorhabdifis elegans embryos (White and Strome, 1996) and in Saccharomyces cerevisiae (Palmer et al., 1992; Li et al., 1993; Muhua et al., 1994). The emerg- ing picture is that a cortical patch of actin and capping pro- tein serves as a nucleation site for a dynactin complex and dynein, which pull on the astral microtubules (vallee et al., 1995). Whether the same cortical machinery operates in rota- tional alignment in fucoid zygotes remains to be determined.

FUTURE DIRECTIONS

Recent technological advances promise further rapid ad- vances in studies of early development in fucoid algae. Re- porter molecules can be introduced into the cytoplasm of living cells by microinjection, and redistributions can be fol- lowed during polarization (Hepler et al., 1993). lnjection of multiple reporters will permit spatial and temporal relation- ships to be examined. Fluorescent reporters for ionic activity and for cytoskeleton are currently being injected into zy- gotes, and others will soon follow. Application of molecular tools has led to the cloning and spatial characterization of cytoskeletal sequences (Bouget et al., 1996; Coffman and Kropf, 1997), and microinjection of antisense RNA and anti- bodies will permit analyses of gene function.

ACKNOWLEDGMENTS

I thank Tasuku Hishinuma, Whitney Hable, Sherryl Bisgrove, and Mary Slocum for valuable discussions and critical comments on the

manuscript. Part of this work was supported by a grant from the Na- tional Science Foundation (No. IBN-9506094) to D.L.K.

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