THE CYTOPLAST CONCEPT IN DIVIDING PLANT CELLS:

Transcription

1 American Journal of Botany 86(2): INVITED SPECIAL PAPER THE CYTOPLAST CONCEPT IN DIVIDING PLANT CELLS: CYTOPLASMIC DOMAINS AND THE EVOLUTION OF SPATIALLY ORGANIZED CELL DIVISION 1 JEREMY D. PICKETT-HEAPS, 2,5 BRIAN E. S. GUNNING, 3 ROY C. BROWN, 4 BETTY E. LEMMON, 4 AND ANN L. CLEARY 2 2 School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia; 3 Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra, A.C.T. 2601, Australia; and 4 Department of Biology, University of Southwestern Louisiana, Lafayette, Louisiana The unique cytokinetic apparatus of higher plant cells comprises two cytoskeletal systems: a predictive preprophase band of microtubules (MTs), which defines the future division site, and the phragmoplast, which mediates crosswall formation after mitosis. We review features of plant cell division in an evolutionary context and from the viewpoint that the cell is a domain of cytoplasm (cytoplast) organized around the nucleus by a cytoskeleton consisting of a single tensegral unit. The term tensegrity is a contraction of tensional integrity and the concept proposes that the whole cell is organized by an integrated cytoskeleton of tension elements (e.g., actin fibers) extended over compression-resistant elements (e.g., MTs). During cell division, a primary role of the spindle is seen as generating two cytoplasts from one with separation of chromosomes a later, derived function. The telophase spindle separates the newly forming cytoplasts and the overlap between half spindles (the shared edge of two new domains) dictates the position at which cytokinesis occurs. Wall MTs of higher plant cells, like the MT cytoskeleton in animal and protistan cells, spatially define the interphase cytoplast. Redeployment of actin and MTs into the preprophase band (PPB) is the overt signal that the boundary between two nascent cytoplasts has been delineated. The actin-depleted zone that marks the site of the PPB throughout mitosis may be a more persistent manifestation of this delineation of two domains of cortical actin. The growth of the phragmoplast is controlled by these domains, not just by the spindle. These domains play a major role in controlling the path of phragmoplast expansion. Primitive land plants show different morphological changes that reveal that the plane of division, with or without the PPB, has been determined well in advance of mitosis. The green alga Spirogyra suggests how the phragmoplast system might have evolved: cytokinesis starts with cleavage and then actin-related determinants stimulate and positionally control cell-plate formation in a phragmoplast arising from interzonal MTs from the spindle. Actin in the PPB of higher plants may be assembling into a potential furrow, imprinting a cleavage site whose persistent determinants (perhaps actin) align the outgrowing edge of the phragmoplast, as in Spirogyra. Cytochalasin spatially disrupts polarized mitosis and positioning of the phragmoplast. Thus, the tensegral interaction of actin with MTs (at the spindle pole and in the phragmoplast) is critical to morphogenesis, just as they seem to be during division of animal cells. In advanced green plants, intercalary expansion driven by turgor is controlled by MTs, which in conjunction with actin, may act as stress detectors, thereby affecting the plane of division (a response clearly evident after wounding of tissue). The PPB might be one manifestation of this strain detection apparatus. actin; cytoplast; cytoskeleton; microtubule; phragmoplast; polarization; preprophase band; Spirogyra; ten- Key words: segrity. THE CYTOPLAST CONCEPT In 1858, Virchow enunciated the great truism in biology that all cells arise from pre-existing cells. Within 20 years, Flemming had stained chromosomes. Soon, he and others, notably Van Beneden, Boveri, and Strasburger, discovered mitosis. These great discoveries provided the structural basis for the expanding science of genetics, re- 1 Manuscript received 11 May 1998; revision accepted 19 October J.P-H. gratefully thanks the Australian Research Council for the grants (numbers A and A ) that enabled this work to be carried out. R.C.B. and B.E.L. thank the NSF (USA) for continued support, most recently from grant MCB Author for correspondence. 153 vealing the essential mechanistic basis of information transfer from the pre-existing cell to each of the two cells arising from it by division. In this paper, we return to Virchow s truism and explore aspects of the spatial and cytoplasmic information inherent in the structure of the cell and the means by which that information is passed from one living cell to its progeny. We take the view, like Trewavas and Malho (1997), that every cell contains two information systems: the genetic system by which information in DNA is translated into protein and an epigenetic system in which interacting genes and gene products are structurally and functionally integrated into networks that transduce internal and external signals. In this scenario, structural, particularly biophysical, information can play major roles in governing the behavior of living cells and tissues. Accordingly, Virchow s truism then in-

2 154 AMERICAN JOURNAL OF BOTANY [Vol. 86 cludes the possibility that structural and perhaps dynamic information (as well as genetic information) is passed directly from one living cell to the next. Porter and colleagues (e.g., Porter and McNiven, 1982; Porter, Beckerle and McNiven, 1983) propose that the ground substance of the animal cell is highly structured; they visualize the cytoplasm in toto as a cytoplast in which this matrix is permeated by cytoskeletal elements such as microtubules (MTs), actin, and intermediate filaments. Ingber and colleagues (e.g., Ingber, 1993, 1998; Ingber et al., 1994) have proposed a related concept, arguing convincingly that the interphase cell is a single tensegral (a term derived from tensional integrity ) unit whose cytoskeleton consists of rigid struts (MTs) supporting both tension and compression created over them by tensile elements, a lattice of actin filaments, and other components. Ingber states: Tensegrity-based force interactions between MTs, MFs (microfilaments) and ECM (extracellular matrix) also provide an efficient mechanism for local regulation of CSK (cytoskeleton) filament polymerization. Thus, structure, function, dynamics and control are interdependent in the living cytoplast. Can the cytoplast concept be applied to plant as well as animal cells? Plant cells share many features with animal cells, but also possess unique attributes, mostly associated with their mode of life within a cell wall. This wall is intimately related to many of the special features of the higher plant cell cytoskeleton. First, cortical ( wall ) MTs line the plasma membrane and they are part of a cell surface apparatus that governs the geometry in which cellulose fibers are deposited in the wall and, consequently, the shape the cell assumes when it expands under turgor pressure. Second, the mechanical support offered by walls acting in concert with turgor pressure allows the development of large, vacuolated cells in which the volume of cytoplasm can be small in relation to the volume of the cell. With this dispersion of cytoplasm came the need for an internal cytoskeletal system to power intracellular motion so that substrates, enzymes, solutes, macromolecules, vesicles, and organelles, are all delivered to the farthest reaches of the cytoplasm. Cytoplasmic streaming generated primarily by an acto-myosin system fulfilled this requirement. Third, cytokinesis in the virtually immobile cells of plants is radically different to that in animal cells, which can migrate during development. The plant cytokinetic apparatus comprises two spatially and temporally distinct components: the preprophase band (PPB) of MTs and the phragmoplast (Gunning, 1982). (The acronym PPB is imprecise since the discovery that PPBs contain actin as well as MTs. Traditionally, PPB refers to the microtubular array, a connotation retained for this paper.) The PPB arises as wall MTs gather into a specific site in the cell that precisely predicts the plane of subsequent cytokinesis; it then disappears completely as the cell moves into prophase. The phragmoplast is a very complex array of actin, other cytoskeletal elements, and membranes, which typically arises at the end of mitosis between daughter nuclei and forms the new crosswall (the cell plate ) in exactly the position predicted by the PPB. There is ample evidence that MTs and actin-based systems work together in these as in other situations, in the form of tensegral structures. In spite of these and other distinctively different botanical features, we think that the cytoplast concept is applicable to plant as well as animal cells. The cytoplast concept may provide new insights into the processes that prepare plant cells for division and later control cytokinesis. For 30 years plant biologists have speculated on the relationship between the phragmoplast and the PPB without achieving any satisfying explanation. Our current approach in rethinking this seminal issue is evolutionary. We commence our discourse with a review of salient features of cell division in algae and lower land plants in which we see strong evidence that cytoplasts are delineated, i.e., the plane of division(s) has been determined, well before division starts. The PPB and phragmoplast are advanced, derived structures arising in ancestors that probably shared some of the features evident in lower plants and algae. In comparing cell division in these primitive and more advanced cells, our objective is to better understand the significance of the PPB and how precisely determined cell divisions might be set up and controlled. THE SPINDLE: DELINEATION AND REPRODUCTION OF THE CYTOPLAST IN ANIMAL CELLS Let us now view the interphase cell as a unit cytoplast (single cytoplasmic domain) whose limits and form are defined by its cytoskeleton. Animal cells generally contain a centrosome, an amorphous body located near the nucleus that often contains a pair of centrioles. During interphase, it is the focus of the MT cytoskeleton radially extending to the cell margin. The MTs, intimately connected to the array of intermediate and actin filaments, create a unified tensegral structure as defined by Ingber and his colleagues (Ingber, 1993, 1998; Ingber et al., 1994). It follows that one requirement of cell division is to create two cytoplasts from the original cell sensu Virchow. Before prophase in animal cells, the centrosome replicates. During prophase, the interphase MT cytoskeleton emanating from the two centrosomes breaks down. Then asters, star-shaped arrays of spindle MTS, grow from the centrosomes. Some MTs from one centrosome interact laterally with MTs from the other so as to create continuous fibers that define the spindle axis while further separating the centrosomes. In our interpretation, the centrosomes are already starting to define two new cytoplasts. The interaction of MTs from each aster creates the central region of MT overlap at the middle of the spindle. This coincides with the interface of the nascent cytoplasts. As the spindle grows and then separates the chromosomes during anaphase, the overlap necessarily defines the plane of cytokinesis. As the cell cleaves, the overlap is compressed into the midbody. During late telophase, the new cytoplasts become fully delineated by their reforming cytoskeleton, particularly the MTs growing from the centrosome. An immediate consequence of this rationale is that the spindle represents the cell s mechanism of creating two similar cytoplasts from one (Pickett-Heaps, Forer, and Spurck, 1997). We proposed that attachment of chromosomes to the spindle arose later during evolution of the eucaryotic cell.

3 February 1999] PICKETT-HEAPS ET AL. CYTOPLASMIC DOMAINS 155 Porter (1976) has described the metaphase stage of mitosis as two sets of chromosomes becoming arrayed between the edges of two nascent cytoplasts. This description accurately reflects the preceding argument since metaphase chromosomes, by lining up halfway between the asters, are coincident with the spindle overlap region. To physically separate the new cytoplasts, the primitive cytokinetic apparatus, cleavage, must coincide positionally with the overlap of MTs of opposite polarity in the interzone of the spindle: this must be one of the most ancient features of the eucaryotic cell. As Rappaport (1986,1996) showed, cleavages occur between asters, even in abnormal situations, for example, between nondaughter nuclei in multinucleate cells. The latter phenomenon has long been known in higher plants where phragmoplasts form between nondaughter nuclei in multinucleate cells (discussed later). A few examples are given to support this scenario. Zhang and Nicklas (1996) demonstrated that a spindle can proceed into and through anaphase even when all of its chromosomes have been mechanically removed. Preprogrammed series of mitoses and attempted cytokineses may continue in the absence of any nuclei (Sluder, Miller, and Rieder, 1986) or alternatively, cleavage can be induced around half spindles (single cytoplasts) in certain circumstances (discussed later). Leslie (1992) has demonstrated that chromosomes become arrayed around the periphery of a domain defined by a supernumerary, monopolar spindle. The existence of cytoplasts or cytoplasmic domains is strongly supported, for example, during embryogenesis in Drosophila (Warn, 1986; Miller and Kiehart, 1995). Initial rounds of mitosis are not followed by cytokinesis, and the embryo is at first multinucleate. Then incipient cleavage furrows isolate nuclei into cytoplasmic domains during rounds of mitoses, but the furrows regress (Rappaport, 1996, p. 312, summarizes these events). At the next round of nuclear division, cleavages between nuclei continue deeper, finally partitioning the cytoplasm to effect cellularization. Thus, while mitosis and cytokinesis are at first uncoupled, spatial cues for cellularization remain in the cytoplasm and/or cell membrane. When DNA synthesis and nuclear division are blocked by aphidicolin, the centriole (i.e., centrosome, in our context) cycle is unaltered. Leaving their nuclei behind in the center of the egg, replicating centrioles migrate to the periphery and develop asters (radial MTs). Then, cleavage around monoasters creates pole cells devoid of nuclei (Raff and Glover, 1989); Alberts et al. (1994, p. 914) state:... it is clear that the division cycle of the cell as a whole depends on and is at least in part organized by the microtubule aster... MTs and actin cooperatively generate domains (Karr and Alberts, 1986), with actin forming a cap over each nucleus, just under the plasma membrane. Precellular cap domains are predicted and probably imprinted by a pattern of spectrin (a protein that anchors actin to membranes) decorating the cytoplasmic side of the plasma membrane, after which actin is involved in cellularization (Pesacreta et al., 1989) via contractile rings (Warn, 1986). Spectrin-like proteins isolated from higher plants might function similarly in localizing actin-dependent cytokinetic structures (cleavage and/or the phragmoplast), but their role is unknown (Michaud et al., 1986; Faraday and Spanswick, 1993). Cytoplasts can remain spatially defined even when cytokinesis fails or is interrupted in some way. We were reminded of this possibility by unexpected observations on cleaving eggs of the starfish Coscinasterias muricata (Pickett-Heaps, unpublished data). For the first 3 6 sets of divisions, synchronous mitoses are followed by cleavages, which progress deeply inwards from the egg surface but which then relax completely until the egg recovers its spherical shape (Fig. 1A D); this unusual behavior appears normal for these cells. At some point during the third and seventh sets of cleavages, the furrows that had formed and then relaxed during all the previous divisions now are completed, creating a multicellular embryo, which thereafter develops normally (Fig. 1E, F). Thus, cytoplasts are defined and re-expressed successively around each set of divided nuclei. THE CYTOPLAST IN DIVIDING PLANT CELLS Increasingly of late, some of us (Brown and Lemmon, 1992b, 1997) have stressed the cytoplasmic domain (references below) in connection with plant cell morphogenesis. The idea of a nuclear-cytoplasmic domain (NCD), a unit of cytoplasm administered by a nucleus, can be traced back to the early cytologists Strasburger and Hertwig (Mazia, 1961). The modern connotation includes cytoskeletal organization into tensegral units equivalent to cytoplasts sensu Porter (Porter and Mc- Niven, 1982; Porter, Beckerle, and McNiven, 1983). Nuclear-cytoplasmic domains and cytokinesis Spatial apportionment of the cytoplasm and control over cytokinesis in multinucleate cells are dependent upon nuclear-based radial (i.e., aster-like) systems of MTs which define NCDs (Brown and Lemmon, 1988a, 1992b). In land plants, phragmoplasts are stimulated to form at the interface of opposing arrays of MTs radiating from adjacent nuclei and new walls are deposited at the edges of NCDs (see below). Certain animal and fungal cells develop cleavages around radial MTs originating from a centrosome or polar body close to each nucleus. These NCDs may be discernible during interphase; in red algae, nuclei are hexagonally spaced and the size of the domain around each reflects its ploidy (Goff and Coleman, 1987). The importance of NCDs becomes obvious during spatially complex cleavages. In Phytophthora, MTs establish NCDs around nuclei in multinucleate sporangia while actin guides the orderly growth of numerous cleavage furrows (Hyde et al., 1991; Hyde and Hardham, 1992). NCDs are established by phycoplast and other MTs and/ or actin systems in multinucleate chlorococcalean (Pickett-Heaps, 1975a) and other green algae (e.g., Menzel, 1986; Menzel, Jonitz, and Elsner-Menzel, 1992). Cytoplast division and reformation after cytokinesis in algae The architecture of the cytoskeleton of many algae and other protists is precisely defined by an array of cytoskeletal MTs (sometimes called rootlet MTs) emanating from MT-organizing regions (MTOCs) which include the flagellar bases. Before cell division, the MT cytoskeleton is usually disassembled and in Ochromonas

4 156 AMERICAN JOURNAL OF BOTANY [Vol. 86 Fig. 1. Cleaving egg, starfish Coscinasterias muricata (fertilization at T 0 h). The first cleavage furrow, (A) T 2.7 h, always relaxes completely (B) T 2.9 h. Following the next rounds of mitosis, previously formed furrows reform along with new furrows; they again relax to varying extents in different cells. (C) T 3.3 h; (D) T 3.78 h. By the third-fifth round of mitoses, all cleavages continue to completion and a normal embryo develops. (E) T 4.67 h; (F) T 5.47 h. The egg measures 195 m across in (A).

5 February 1999] PICKETT-HEAPS ET AL. CYTOPLASMIC DOMAINS 157 (Bouck and Brown, 1972), for example, the duplicated MTOCs (equivalent to centrosomes) associate with the spindle poles and reform the cytoskeleton in daughter cells. In algae with closed spindles, the MTOCs are also usually located near the poles; in certain cases, the flagellar bases generate a mini-spindle alongside, but separate from the mitotic spindle (Seegar, Gerritsen, and De Bakker, 1989). This system ensures correct segregation of each MTOC accompanying its daughter nucleus. This paper also suggests how the phycoplast of green algae arose: the two mini-half spindles fold inwards during cleavage and as their MTs become coplanar, they define the plane of cytokinesis, becoming the phycoplast of parallel MTs coplanar with cleavage. In the primitive charophycean alga Chlorokybus (Lokhorst, Sluiman, and Star, 1988) nuclear and cytoplasmic divisions are remarkably coordinated. Division site selection starts with the annular cleavage of chloroplast and pyrenoids, as in other green algae (e.g., Coleochaete, below; Stichococcus and Klebsormidium [Pickett-Heaps, 1972, 1974a]). The nucleus is drawn into the constriction, now coplanar with a precocious furrow opposite the chloroplast. The centrioles (flagellar bases) migrate into this plane between the furrow and nucleus. Astral MTs from the flagellar bases extend to the furrow, envelop the premitotic nucleus, and pass between the two newly divided chloroplasts. The flagellar bases move to the spindle poles, becoming closely associated with the plastid tips. The astral MTs emanating from the flagellar bases form the half spindles and the overlap region is thereby aligned with the previously determined plane of cytokinesis (Figs. 6, 24B in Lokhorst, Sluiman, and Star, 1988). Cleavage follows mitosis with the cell having established all spatial parameters for cytokinesis before mitosis (see next section for similar phenomena in bryophyte spore mother cells). NCDs and the phragmoplast Cytokinesis in plant cells is brought about within the phragmoplast, a complex structure that arises between telophase daughter nuclei. In living cells, this area is initially relatively clear of larger organelles and is faintly striated by very fine fibers parallel with the axis of the spindle. In most cases, the phragmoplast appears to arise from remnant spindle fibers, and in living cells, it displays intense activity that soon results in the appearence of an initially fragile, flexible cross wall, the cell plate. The fibrous texture of the phragmoplast becomes more evident as it grows from the center outwards to the side walls, carrying with it the expanding cell plate, which soon attaches to the walls and consolidates. Electron microscopy confirms that the fibrous nature of the phragmoplast is due to its complement of MTs, one set from each half of the spindle, overlapping in the middle. Among the MTs are actin filaments, numerous larger and smaller vesicles, and membranous components (endoplasmic reticulum); the vesicles probably come from Golgi bodies and are transported into the overlap region where they fuse to form the cell plate. The phragmoplast is without counterpart in animal cells. The midbody is quite different, although similar in consisting of overlapping MTs. Unlike phragmoplast MTs, midbody MTs are very stable, and the midbody is essentially inert, being pinched by the cleavage furrow and persisting long after mitosis is over (Byers and Abramson, 1968). Also the midbody does not have mitotic cyclins that associate with cyclindependent kinases to effect changes in MT organization earlier in mitosis. In contrast, the phragmoplast of dividing maize cells carries cyclins II and III in a postspindle association that may reflect kinase-regulated control of MT dynamics (Mews et al., 1997). In most higher plant cells, mitosis is so quickly followed by cytokinesis that cytokinetic systems appear to be dependent upon the prior existence of the mitotic spindle, suggesting that... developmentally, the phragmoplast MTs appear to arise from remnants of the mitotic spindle apparatus... (Staehelin and Hepler, 1996). MTs radiate from both telophase nuclei, perhaps participating in delineation of new cytoplasts, but those MTs that remain from the spindle are special in that they already have interacted with each other at the midplane. It is among this set of MTs that the phragmoplast first appears. There are, however, alternative situations. New walls form between nonsister nuclei during cellularization of plant coenocytes, e.g., following meiosis in bryophytes (Brown and Lemmon, 1988a) or flowering plants (Brown and Lemmon, 1991), and in endosperm (Bajer, 1968; Morrison and O Brien, 1976; Van Lammeren, 1988; Brown, Lemmon and Olsen, 1994; Olsen, Brown, and Lemmon, 1995). MTs radiating from sister and nonsister nuclei interact to form bipolar arrays in which phragmoplasts develop. Thus, the telophase spindle is not essential as a precursor of a phragmoplast. Moreover, a few plant cells show phragmoplast organization and, thus, evidence of daughter domains, even before prophase (next section), while elsewhere, NCDs can remain established after telophase in situations where a crosswall is not formed (e.g., Brown and Lemmon, 1992a, b). Uncoupling of phragmoplast formation from wall deposition is common in male and female reproductive cell lineages, from mosses (e.g., Brown and Lemmon, 1987a; Busby and Gunning, 1988a) to angiosperms (e.g. Hogan, 1987; Webb and Gunning, 1991). The appearance, disappearance, and reappearance of phragmoplasts, sometimes in association with partial cleavage furrows, argue strongly that long-lived cytoplasmic domains have been set up. Phragmoplasts form after the first division in spore mother cells of mosses, but then disappear; later, after the second division, phragmoplasts appear between all nuclei and cell plate formation proceeds simultaneously (Busby and Gunning, 1988a; Brown and Lemmon, 1991). Wall ingrowths (perhaps sometimes cleavage furrows) predict the arrangement of the spore tetrad before division in sporocytes of almost all bryophytes (Brown and Lemmon, 1993). In flowering plants, similar ingrowths may occur after nuclear divisions. During pollen morphogenesis in Magnolia (Brown and Lemmon, 1992a), a phragmoplast appears after the first meiosis but no cell plate or crosswall is formed. Instead, the cell develops deep wall ingrowths. After the second meiotic division, phragmoplasts appear between all nuclei (not just sister nuclei). Only when the phragmoplasts grow out to the walls does new cross-wall deposition restart. The phragmoplasts are positioned at the interface of MTs radiating from the nuclei (Brown and Lemmon, 1992b). The close spatial relationship of cleavage/wall ingrowths and the phragmo-

6 158 AMERICAN JOURNAL OF BOTANY [Vol. 86 Fig. 2. Endosperm, young seed of Ranunculus scleratus. During cellularization, phragmoplasts develop simultaneously at edges of all Nuclear- Cytoplasmic Domains. Wall deposition creates alveolate endosperm with each nucleus enclosed by walls. Confocal Laser Scanning Microscope (CLSM); MTs labelled by indirect immunofluoresence. plast in these situations suggests evolutionary conservation as described earlier. In early stages of nuclear endosperm development, mitosis and phragmoplast formation are uncoupled from cytokinesis entirely (not unlike Drosophila embryogeny discussed earlier). Waves of mitosis create multinucleate cytoplasm; each mitosis initiates a phragmoplast, which soon disappears, having formed no wall (Brown, Lemmon, and Olsen, 1994). The entire multinucleate cytoplasm is organized into a single layer of NCDs, and anticlinal walls are initiated at their boundaries. Subsequent growth of the anticlinal walls is associated with adventitious phragmoplasts that form at the interface of MTs radiating from adjacent nuclei. The anticlinal walls compartmentalize the cytoplasm into open-ended alveoli (Fig. 2), each containing a nucleus. Mitosis in the alveoli is followed by the appearance of typical interzonal phragmoplasts. Thereafter, repeated cycles of adventitious phragmoplasts guiding the growth of anticlinal walls between nonsister nuclei during interphase alternate with interzonal phragmoplasts guiding periclinal walls after mitosis. Monoplastidy and cytoplasmic domains in early land plants Cells in many lower land plants are monoplastidic; provision is made during cell division for the single plastid to divide and the two halves to be correctly segregated into daughter cells (reviewed by Brown and Lemmon, 1984, 1990a, 1997; Cleary, Brown, and Lemmon, 1992a, b). To achieve this partitioning, the chloroplast is precisely positioned so that its cleavage is integrated with the future cytoplasmic division site and the plastid serves as an MTOC for bipolar MT arrays that mark the cytokinetic plane and also transform into the spindle (as for meiosis). As in other land plants, cells of hornworts lack centrosomes except in the final division leading to differentiation of flagellated spermatozoids where centrioles appear de novo. All dividing cells of hornworts are monoplastidic in both gametophytic and sporophytic generations, and the spindle poles are intimately associated with the plastid. As in Coleochaete and Chlorokybus, the dividing plastid becomes positioned with its isthmus at the division site. An axial MT system extends from the plastid isthmus to the plastid tips, becoming bipolar. The division site at the interface of the two half spindles is further marked by a cortical PPB encircling this axial array of MTs (Fig. 3). The bipolar axial MT array encloses the nucleus from the chloroplast side and is transformed into the spindle. The phragmoplast arises in overlap region of spindle MTs, from newly generated MTs emanating from proximal faces of reforming nuclei and from the proximal surfaces of daughter plastids. Thus, the cytoplast is precisely organized into daughter domains before karyoki-

7 February 1999] PICKETT-HEAPS ET AL. CYTOPLASMIC DOMAINS 159 Fig. 3. Monoplastidic mitosis, hornwort; two planes of focus. Bipolar axial MT system lying parallel to long axis of plastid. Region of MT overlap coincides with plastid isthmus and future plane of division indicated by the encircling PPB. CLSM; MTs labelled by indirect immunofluoresence. Fig. 4. Quadrilobed sporocyte; the moss Entodon seductrix. During sporogenesis, the quadripolar microtubular system (QMS) defines four spore domains and is later transformed into a bipolar spindle. CLSM of MTs labelled by indirect immunofluoresence. nesis starts and the eventual plane of cytokinesis coincides with plastid division, the region of overlap in the axial MT array, and the plane of the PPB. Establishment of daughter domains indicated by phragmoplast-like structures before prophase In certain cell types, daughter domains are established prior to nuclear division by precocious bipolar arrays of MTs defining future cleavage planes. In bryophyte sporogenesis, quadripolar MT systems (QMSs) emanating from plastids in the four future spore domains establish the pattern of spore cleavage before prophase of meiosis (Brown and Lemmon, 1987a, b; Busby and Gunning, 1988b: reviewed by Brown and Lemmon, 1997). These tetrahedrally arranged daughter domains (Fig. 4) do not receive a nucleus until telophase of the second meiosis, at which time the four spores are cleaved simultaneously. The cytoplasm is usually deeply lobed with each of the cleavage furrows precisely aligned with the overlap of the bipolar arrays of the QMS. The cytokinetic planes may be partially or deeply marked by dense material like that in the phragmoplast (Busby and Gunning, 1988b). The QMS is converted into a functional bipolar spindle with poles located in the plane of opposite cleavage furrows; this re-

8 160 AMERICAN JOURNAL OF BOTANY [Vol. 86 sults in placement of daughter nuclei in proper position to be distributed into the four spore domains after second meiosis. In meiotic prophase of the fern ally Selaginella, a procytokinetic plate marks the first division plane. This phragmoplast-like array on either side of the prophase nucleus marks the midzone of the bipolar MTs that merge to form the meiotic spindle (Brown and Lemmon, 1985). All these observations indicate that morphogenetic cues for cytokinesis, a process we equate with definition of daughter cytoplasts, are set up well in advance of nuclear division. CLEAVAGE AND EVOLUTION OF THE PHRAGMOPLAST Cytokinesis by cleavage, common to all procaryotic cells, is mediated by a specific set of proteins, notably FtsZ (Bramhill, 1997). This protein can exist as monomers and in filaments and has a sequence similarity to eucaryotic tubulins (Erickson, 1997). In most eucaryotic animal cells, cleavage is generated by a ring of actin filaments assembled on the plasmamembrane at a site determined during early mitosis by the spindle, particularly the asters (reviewed by Strome, 1993). Later, the furrow constricts onto the midbody, the region of MT overlap in the non-kinetochore spindle fibers. Force is generated by acto-myosin-ii interactions (Fujiwara and Pollard, 1976) with modulating and membrane attachment proteins concentrated in the furrow (Satterwhite and Pollard, 1992; Yonemura et al., 1993). Cleavage is the common mechanism of cytokinesis in eucaryotic algae but whether this is invariably an actinbased phenomenon is unproven. In the simplest colonial algae of many major groups (greens, reds, chrysophytes), cells are held together loosely in gelatinous colonies. A major morphological advance was the evolution of the filament, which required, first, that cells tightly adhere after division, and, second, that the plane of division is consistently perpendicular to the axis of the filament. To achieve the latter, the division apparatus has to be sensitive to the direction of cell growth, perhaps via cytoskeletal components, which concurrently detect strain and control organization of wall microfibrils. (For a discussion on the origin of the filamentous condition, see Pickett-Heaps, 1975a.) Simple green algae cleave, while the phragmoplast is found in a few advanced forms (the Charales, Coleochaete) considered to be related to the ancestors of the land plants. Spirogyra: an intermediate in the evolution of the phragmoplast from a cleavage apparatus That the land plants arose from charophycean algae (e.g., Pickett- Heaps, 1975a; Graham and Kaneko, 1991) is supported by biochemical, molecular, and comparative morphological evidence (reviewed by Graham, 1996). Ancestors of land plants had a persistent telophase spindle; the nuclei remained widely separated during cytokinesis by cleavage (Pickett-Heaps, 1975a). In Spirogyra (and the related Mougeotia: Pickett-Heaps and Wetherbee, 1987; Galway and Hardham, 1991), cytokinesis is initiated by an ingrowing furrow lined with actin (Goto and Ueda, 1988; Sawitzky and Grolig, 1995). Once this furrow impinges on the persistent overlapping spindle MTs, a small phragmoplast develops at the region of contact (McAlister, 1931; Fowke and Pickett-Heaps, 1969a, b); it contains dense material, aggregations of vesicles, and proliferating MTs. If the telophase spindle becomes tilted as it elongates, it straightens once contact is made (Pickett-Heaps, unpublished in vivo observations); thus, the furrow affects spindle orientation. Spirogyra suggests that the phragmoplast could originally have evolved from a specialisation of the cytoplasm at the edge of the cleavage furrow. There is a similar tight association of the cleavage furrow with overlapping MTs in the animal midbody (Wheatley and Yang, 1996); destruction of these MTs causes regression of the cleavage furrow. Spirogyra: experiments with anti-mt and anti-actin drugs The functional relationship between these two successive modes of cytokinesis is revealed by simple drug experiments (McIntosh, Pickett-Heaps, and Gunning, 1995). Both cleavage and the phragmoplast are required for full cytokinesis. Cleavage alone cannot complete cytokinesis; thus, when the anti-mt drug oryzalin breaks down the phragmoplast at late cytokinesis, cells finish up with a central perforation in the cross wall, i.e., infurrowing is incomplete (see also Sawitzky and Grolig, 1995). Cleavage can be inhibited by the drug cytochalasin D (CD) and two different outcomes can be observed. Figure 5A C shows the result of inhibition of cytokinesis with CD, after the cleavage furrow has contacted the interzonal spindle: the phragmoplast is now able to complete cytokinesis. Figure 5C F shows another result from the same experiment, but in another cell in which cleavage was stopped early, before the ingrowing furrow impinged upon the interzonal spindle; a phragmoplast did form and it even succeeded in generating a long-lived (several hours) cell plate. However, the latter could not consolidate into a cross wall; after some hours, this phragmoplast and the cell plate dispersed and the cell remained binucleate (Fig. 5F). These experiments show that physical interaction of the cleavage apparatus with this phragmoplast is vital for a successful transition from one cytokinetic system to the other and, therefore, for successful cytokinesis. In living Spirogyra, this interaction becomes evident as soon as the interzonal spindle contacts the furrow. Interaction not only triggers assembly of the cell plate into a cross wall, but it also locates the edge of cell plate/overlap of the interzonal MTs, in precisely the right position with respect to the previously formed furrow. Such an ancient juxtaposition, the same as that in the animal cells midbody, ensures that daughter nuclei are separated into daughter cells. This scenario suggests how the phragmoplast might have evolved, from an initial stimulation of wall synthesis occasioned by the presence of spindle fibers into a cytoskeletal system that became predominant over cleavage. It poses the important question: why did the phragmoplast become the dominant cytokinetic structure in higher plants? Coleochaete: a later step in the evolution of organized divisions? Some Coleochaete (particularly C. scutata and C. orbicularis) display a disc-like vegetative thallus

9 February 1999] PICKETT-HEAPS ET AL. CYTOPLASMIC DOMAINS 161 Fig. 5. Cytokinesis in Spirogyra treated with CD (20 g/ml). In (A C) the cleavage furrow had reached the cell plate when the CD was applied. While cleavage stopped, a normal cell plate finished cytokinesis. In a second cell in the same filament, cleavage had stopped before the furrow reached the cell plate. A phragmoplast containing a cell plate lasted many hours in the cell (D, E) but no cross wall was formed. (F) shows the same cell 12 h later, confirming that the cross wall was absent.

10 162 AMERICAN JOURNAL OF BOTANY [Vol. 86 Fig. 6. Divisions are confined to marginal cells of the discoid green alga Coleochaete obicularis. Migration of centrosomes and orientation of half spindles predict division polarity. The cell at the left is preparing for a circumferential (periclinal) division, the cell on the right will divide radially (anticlinal division). CLSM; MTs labelled by indirect immunofluoresence. that arises from coordinated divisions confined to the marginal cells. Division occurs in either of two planes (Fig. 6): radial or circumferential (i.e., periclinal), and a phragmoplast is present in both types of division. The circumferential division adds cells to preexisting files of cells, and it is the primitive type, equivalent to transverse division of an elongating filament. This division somewhat resembles cytokinesis in Spirogyra. An ingrowing cleavage furrow impinges on the interzonal spindle (Marchant and Pickett-Heaps, 1973) and a collarlike phragmoplast grows outwards until it contacts the outer wall, but no cell plate is laid down. Instead, cytokinesis occurs centripetally as an ill-defined cleavage impinging upon a persistent column of MTs in the center of the cell, whereupon the final separation of cells appears to be an irregular twisting (Brown, Lemmon, and Graham, 1994). Immunofluorescence studies suggest that the ingrowing furrow and the expanding phragmoplast fail to coincide, a positional discrepancy possibly related to two unusual features of circumferential division. The division is unequal; the spindle is tilted and the outer cell is considerably larger and will continue to divide whereas the smaller inner cell will normally cease to divide. The latter feature of this division makes it difficult to make live observations, but this cytokinesis begs reinvestigation since it may hold clues to the requirements for evolution of successful cytokinesis by the phragmoplast/cell plate. We interpret the radial division as the more derived since it adds new files of cells to create the monostromic thallus. Well before prophase, the impending site of this radial cytokinesis is indicated by a deep cleavage in the chloroplast (see earlier) and a cytoplasmic furrow that precisely aligns with the future division site. During this division, the phragmoplast and the cell plate within it grow from the center of the spindle outwards, and the cell plate fuses with peg-like ingrowths from the wall (Marchant and Pickett-Heaps, 1973; Brown, Lemmon, and Graham, 1994). This cytokinesis is quite different from that in Spirogyra, being mostly accomplished by the phragmoplast with cleavage relegated to a minor role although the wall pegs might result from slow cleavage. (The terms cleavage and wall ingrowths may be descriptions of the same process.) Thus this radial cytokinesis resembles that in higher plants. We return now to the question of why the phragmoplast became the dominant cytokinetic system in green plants. To evolve from a one-dimensional, filamentous type of simple division into a two- or three-dimensional pattern of division was an absolute requirement for the evolution of complex plant bodies. Perhaps the phragmoplast represents the cell s mechanism for escaping from the primitive restraint of transverse cleavage. Spirogyra and Coleochaete exemplify two key stages in the origin of the phragmoplast: originally it might have been an alternative way of achieving transverse division and later exploited so that the plane of division could be varied. Apices of Chara and Nitella exemplify still more complex situations, also making use of phragmoplasts (Pickett-Heaps, 1975a; Cook et al., 1997). THE CYTOPLAST CONCEPT AND DIVISION IN HIGHER PLANT CELLS Higher plant cells do not have an interphase MT array even remotely resembling that of animal cells; nor do they display centrosomes (except, briefly, in those cell lineages leading to flagellated sperm). The periphery of the cytoplast is defined by the transversely oriented wall MTs, which profoundly affect the properties and morphogenesis of the wall. Higher plants also acquired another, enigmatic structure. When they prepare to divide,

11 February 1999] PICKETT-HEAPS ET AL. CYTOPLASMIC DOMAINS 163 they redeploy their cytoskeleton to form the PPB of MTs. As stated by Brown and Lemmon (1990a: p. 561):... Introduction of the preprophase band into the cytokinetic apparatus is a topic of extreme interest in the evolution of land plants. The preprophase band (PPB) of microtubules Since its discovery over 30 years ago (Pickett-Heaps and Northcote, 1966a, b), the PPB of MTs has proved a ubiquitous feature of premitotic, vegetative, noncoenocytic cells in land plants. During the G 2 phase of the cell cycle, wall MTs steadily collect into a narrowly defined band girdling the cell. The location of this PPB predicts the site and plane of the subsequent cytokinesis in those divisions in which a new wall is inserted into a preexisting pattern of parental walls (reviewed by Pickett-Heaps, 1974b; Gunning, Hardham, and Hughes, 1978; Gunning, 1982; Lloyd, 1989; Gunning and Wick, 1985; Gunning and Sammut, 1990; Wick, 1991a, b). The PPB disappears as the cell goes into prophase and its role (if any) in determining the position of the cell plate invariably predicted with exquisite accuracy has remained enigmatic. Actin and other interesting antigens (e.g., p34 cdc2 : Mineyuki, Marc, and Palevitz, 1991; Colasanti et al., 1993; mitotic cyclins: Mews et al., 1997) have recently been found to co-localize with MTs in PPBs (see below). There may be additional unrecognized, positive markers (i.e., determinants) at the PPB site as well. The distribution of the PPB in the plant kingdom The PPB is characteristic of dividing cells in land plants but not algae, including Coleochaete and Chara, which display precisely controlled symmetric and asymmetric divisions (Pickett-Heaps, 1975a; Brown and Lemmon, 1990a; Brown, Lemmon, and Graham, 1994). The PPB of bryophytes is sometimes diffuse or asymmetric (reviewed in Brown and Lemmon, 1988b, 1993). Since bryophytes could be the earliest divergent extant embryophytes (Graham, 1996), they may display primitive features of land plant division. Even in the pteridophyte Azolla PPBs are frequently incomplete (Gunning, Hardham, and Hughes, 1978). The PPB is absent during tip growth and initiation of branches in filamentous moss protonema (Doonan et al., 1987; Doonan and Duckett, 1988; Doonan, 1991), but is present once multidimensional tissues are developed in both gametophytic and sporophytic generations. Hepatics such as Reboulia may show a transitional stage in PPB evolution. The spindle arises from astral MTs emanating from discrete polar organizers that develop de novo in preprophase. The PPB forms at the boundary of the two domains defined by astral MTs, the two sets of MTs displaying considerable interaction (Brown and Lemmon, 1990b). This differs from the situation in higher plants where the PPB precedes assembly of visible spindle MTs. Throughout the land plants, the PPB is absent in cells of reproductive lineages: (a) archesporial cells in bryophytes (Busby and Gunning, 1988a, b; Gambardella and Alfano, 1990; Brown and Lemmon, 1992a); (b) meiotic microsporogenesis and pollen mitosis (Hogan, 1987; Brown and Lemmon, 1991; Palevitz, 1993); (c) megasporogenesis and megagametogenesis (Webb and Gunning, 1990, 1991,1994; Huang and Sheridan, 1994); and d) endosperm development (Van Lammeren, 1988; Brown, Lemmon, and Olsen, 1994). The PPB is absent from cells entering a developmental pathway where they are no longer part of an organized tissue. For example, compare the development of the embryo and endosperm in flowering plants. Both originate from fertilization, yet the zygote displays a PPB in the first division (Webb and Gunning, 1991); in contrast, endosperm develops as a coenocyte (at least in cereals and Arabidopsis thaliana) before becoming cellular and does not exhibit a PPB until later when divisions occur in the aleurone layer (Brown, Lemmon, and Olsen, 1994). Thus, the PPB appears to be a land plant (embryophyte) autapomorphy. It appears in cells inserting spatially predetermined new walls within the pattern of neighboring cells during histogenesis of the multicellular plant. The PPB clearly shows that the division plane has been established before mitosis just as do the various examples of NCDs in algae and bryophytes, reviewed above. Actin and MTs in the PPB and phragmoplast Factin is frequently, and perhaps ubiquitously, co-localized with MTs at the PPB site (Kakimoto and Shibaoka, 1987; Palevitz, 1987; Traas et al., 1987; Lloyd and Traas, 1988; Katsuta, Hashiguchi, and Shibaoka, 1990; McCurdy and Gunning, 1990; Mineyuki and Palevitz, 1990; Ding, Turgeon, and Parthasarathy, 1991; Eleftheriou and Palevitz, 1992; Liu and Palevitz, 1992; Panteris, Apostolakos, and Galatis, 1992; Hepler et al., 1993; Cleary et al. 1992c; Cleary, 1995; Cleary and Mathesius, 1996). The behavior of the actin and MTs is very different between determination of the division site in preprophase and fulfilment at cytokinesis. PPB MTs disappear during prophase. Cortical actin becomes largely excluded from the PPB site, and so narrow actin-depleted zones (Liu and Palevitz, 1992; Cleary et al., 1992c; Cleary and Mathesius, 1996) mark the division site throughout mitosis in a negative sense. Both actin and MTs appear to be redeployed cooperatively in establishing the limits of new cytoplasts, and perhaps the actin-depleted zone is the most precise spatial indication of this determination. One possibility is that the PPB must prepare the division site, whereupon it is disassembled (see John, 1996, for mechanisms whereby this step could be integrated into the molecular controls of the plant cell cycle) and the parental cell has initiated cytoplast division via redistribution of cortical actin. An alternative hypothesis is that the persistent actin flanking the depleted zone is a positive marker delimiting the future cytoplasts; the zone may represent a gap between the cytoskeletal systems of the two new cytoplasts into which the phragmoplast directs deposition of the new wall. The structure of the actin cytoskeleton during cytokinesis is complex. It is absent (or perhaps masked) where the phragmoplast MTs are densest, at the mid-plane (Cleary et al., 1992c; Zhang, Wadsworth, and Hepler, 1993; Hepler et al., 1993; Cleary, 1995; Cleary and Mathesius, 1996; Valster and Hepler, 1997). Thus, it appears as two opposing arrays between daughter nuclei, separated by a narrow gap; this arrangement persists after the nearby MTs disappear, but it too, finally disperses. The gap matches the width of the actin-depleted zone towards

12 164 AMERICAN JOURNAL OF BOTANY [Vol. 86 which it progresses as the phragmoplast grows. Currently available images show this actin as finely textured, somewhat contrasting with and also merging into the coarser actin bundles nearby which extend between the daughter nuclei (Kakimoto and Shibaoka, 1987; Seagull, Falconer and Weerdenburg, 1987; Traas et al., 1987; Lloyd and Traas, 1988; Cho and Wick, 1990; Wick, 1991b; Valster and Hepler, 1997). An interpretation of this morphology is that assembly of the fine actin defines the extent of the new cytoplasts, with the phragmoplast becoming integrated via the coarser bundles so as to build the wall in the intervening gap. The phenomena involved exhibit typical tensegral properties: for example, the internal stiffness of the phragmoplast (and spindle pole) contrasting with the elastic tensile forces acting on it. THE DIVISION SITE AND PHRAGMOPLAST IN THE CONTEXT OF THE DIVIDING CYTOPLAST Preparations for cytoplast division In symmetrical divisions of highly vacuolated cells, the nucleus becomes centrally located and a sheet or strands of cytoplasm, the phragmosome, indicates the plane of incipient cell division (Sinnott and Bloch, 1941; Sinnott, 1960) and connects the nucleus with the site of the PPB (Venverloo et al., 1980). If division is asymmetrical, the nucleus migrates into position prior to division, either before (e.g., Pickett-Heaps, 1969a) or after the PPB has appeared. The actin in the phragmosome (Traas et al., 1987; Katsuta, Hashiguchi, and Shibaoka, 1990) remains, perhaps to constitute a memory (Lloyd and Traas, 1988) of the division plane. The PPB may prepare the division site for subsequent insertion of the new wall into the parental wall (Mineyuki and Gunning, 1990), but how this function is actually achieved remains unknown. Pickett-Heaps (1969b) and Gimenaez-Abiam et al. (1998) used caffeine treatment to show that the signal, whatever its nature, can persist at the PPB site through a division cycle. Numerous observations show that wall stubs can be formed at the PPB site, for example, after disruption of cell plates with caffeine (references in Mineyuki and Gunning, 1990). The stubs are similar to the wall ingrowths that mark the division site in spore mother cells, particularly in bryophytes (see next subsection), although the latter arise without the participation of PPBs. If the actin-depleted zone represents a gap between the nascent cytoplasts, wall ingrowths may be a consequence of lessened cytoskeletal support of the plasma membrane or of altered local control over wall deposition, permitting wall material to intrude inwards. The fact that actin is localized among PPB MTs raises the interesting possibility that higher plant cells retain a derivative of an ancient cleavage furrow at the division site. O Brien (1983) suggested that the PPB is a device to block cleavage; however, one would then expect it to persist throughout mitosis. When wall-less plant protoplasts divide, they appear to cleave (Meyer and Abel, 1975) even after a phragmoplast is initiated (Meyer and Herth, 1978; Sonobe, 1990). Meristematic plant cells plasmolyzed during cytokinesis (Cleary, unpublished data) undergo slow indenting where the edge of the cell plate approaches the margin of the protoplast (Sonobe, 1990). This phenomenon differs from true cleavage (as in animal cells), being sensitive to anti-mt drugs (Cleary, unpublished data) and stimulated by CB (Sonobe, 1990). It may be that when turgor is reduced, the inbuilt tensegrity of the cytoskeletal elements of the daughter cytoplasts can induce a constriction between them, i.e. at the interface between the new NCDs that were set up before division, first at the actin-depletion zone and then at the spindle overlap zone. If so, such deformation may not be powerful enough to be manifest in walled cells dividing under full turgor. That internal, tensegral, forces exist within walled cells is apparent from various studies. Hahne and Hoffman (1984) show that freshly isolated protoplasts have a dimpled surface, the dimples occurring at the outer extremity of transvacuolar strands; when the strands are cut by laser microsurgery, the dimples relax. Goodbody, Venverloo, and Lloyd (1991) and Grolig (1998) have analyzed internal tension in the cytoplasmic strands that position (and reposition) the nucleus. The role of the wall in providing anchoring points for the internal tensegral system is highlighted by the smoothing out of surface dimples when protoplasts regenerate walls (Katsuta and Shibaoka, 1989). Currently, trans-plasma membrane molecular linkages that can let the cytoskeleton exploit the mechanical strength of the wall are under intense investigations. There are a few observations that suggest that higher plant cells retain and can on occasion use cleavage instead of the phragmoplast for cytokinesis. Cell division in stress-induced pollen embryoids are of two different forms, characteristic of two types of cells: densely cytoplasmic and vacuolate (Huang, 1986). Vacuolate cells cleave while dense cells use the phragmoplast/cell-plate system. While the mechanisms by which both the formation of wall stubs and these cleavages occur are undetermined, the phenomena point to an alteration in the physical (i.e., tensegral) properties of the parental cytoplasm at the division site, where the actin-depleted zones delineate the future cytoplasts. Phragmoplast initiation and expansion The division site predicted by the PPB marks only a surface band where the future cross wall will intersect the parental cell surface. In many divisions, the surface thus defined is a simple plane, but it can be a complex curved surface, symmetrically or asymmetrically placed. In all cases, the initial position and orientation of the mitotic apparatus do not determine final positioning of the new cross wall. Centrifugal growth of the phragmoplast from the middle of the spindle is subject to accurate guidance processes toward the previously prepared division site. For example, the mitotic spindle in guard mother cells (GMCs) is often tilted but invariably undergoes an actin-based reorientation (Palevitz and Helper, 1975; Palevitz, 1987). Likewise, spindles and the initial phragmoplasts in developing epidermis of the maize leaf are frequently tilted but later become aligned with the division site (Cleary and Smith, 1998). Tradescantia stamen hairs can be centrifuged to displace the spindle without prejudice to the achievement later of a correctly oriented division (Ota, 1961). What the spindle does establish in these circumstances

13 February 1999] PICKETT-HEAPS ET AL. CYTOPLASMIC DOMAINS 165 Fig. 7. Destruction of cellular polarization by cytochalasin D in two epidermal cells that were dividing to form subsidiary cells next to two guard mother cells (g) in Tradescantia. During asymmetrical mitosis in cell Y, the spindle, at prophase in (A), reverted back to a poorly placed transverse division. In cell X, the future SC nucleus detached from the GMC (B D), while its phragmoplast (arrowhead) wandered off across the cell. (E, F) show the malpositioned new walls arising after the drug treatment. From time-lapse video recording. is the development of the overlap between the half spindles. In the context of the cytoplast model, the spindle has initiated one region of cytoplast demarkation within a much larger cell volume that has already been prepared for division. During later telophase, these two are brought into conjunction, probably by the actin cytoskeleton. When phragmoplasts are formed adventitiously between nonsister nuclei, the required overlap is established at the interface of opposing MT systems much as it is in the inetrzonal region of the spindle, so much so that they have been referred to as secondary spindles. The highly asymmetric divisions required for morphogenesis of stomatal complexes (Stebbins and Jain, 1960; Stebbins and Shah, 1960) are classic polarized divisions, and they provided the first unambiguous evidence that the PPB is related to plane of division (Pickett-Heaps and Northcote, 1966b; Pickett-Heaps, 1969a). Stomatal morphogenesis requires two distinct phenomena: positioning of the spindle and precise spatial control over the path of phragmoplast expansion. Treatment of forming stomatal complexes with cytochalasin B and D (CB, CD) severely interferes with stomatal differentiation (Cho and Wick, 1990; Fig. 7A F), particularly of subsidiary cells (SCs) around the GMC. First, spindle polarization (attachment of the pole and SC daughter nucleus to the GMC) is strong enough to resist centrifugation (Pickett-

14 166 AMERICAN JOURNAL OF BOTANY [Vol. 86 Heaps, 1969c), but under CD treatment, the spindle pole and SC nucleus slowly detach from the wall at the GMC and drift away (Fig. 7A F). Second, the phragmoplast normally curves sharply around the SC nucleus, creating a hemispherical cell plate attaching to the site previously occupied by the PPB. However, when in CD, this phragmoplast completely loses orientation; its growing edge is no longer pulled into the correct position, and instead, it wanders off to generate a randomly situated cross wall (Fig. 7A C; Palevitz and Hepler, 1974; Gunning and Wick, 1985; Mineyuki and Gunning, 1990 and others). Young phragmoplasts are not always aligned in the division plane, but if initially skew, they soon become adjusted as they grow (e.g., Wick, 1991b; see above). Lloyd (1988) poses the important question: Why doesn t the expanding edge of the phragmoplast wander off into another place? and suggests that actin remaining in the phragmosome in vacuolated plant cells normally ensures that such wandering is not allowed. Numerous observations show that adjustment is an active process. The phragmoplast often wavers while becoming aligned, until it comes under a more direct influence of the division site, whereupon it appears to be attracted with great precision to the midline of the former PPB site. A striking example is given in Fig. 8A D, in which the SC phragmoplast was unusually crooked at first (Fig. 8A, B) and the cell plate underwent rocking movement (Fig. 8E). A quite rapid (Fig. 8E) twisting then aligned one edge of the phragmoplast into the correct position (Fig. 8C), and subsequent SC formation was normal (Fig. 8D). Even during normal, symmetrical divisions, the edge of the phragmoplast usually undergoes hunting movements, back and forth near the wall, before becoming firmly attached to it (Mineyuki and Gunning, 1990). In the tangled mutant of maize, skewed spindles fail to adjust correctly and its gene product appears to be necessary to bring the skewed phragmoplast into proper alignment (Cleary and Smith, unpublished data). The positioning of the phragmoplast is the final expression of the mysterious process called polarization. Our experience in interpreting the behavior of living cells in algae as well as higher plants strongly suggests to us that polarized activity (e.g., the movement of cell organelles like the nucleus, spindle, phragmoplast or chloroplasts into specific positions) is generated by strands of cytoplasm that display tensegral properties (rigidity concurrent with tension and force generation); we believe Fig. 8. Cytokinesis forming lateral SC, normal Tradescantia. (A) The spindle had temporarily rotated untill the pole (*) was no longer adjacent to the GMC. (B) Cell plate forming. (C) The spindle had rotated, the cell plate moving into the expected position adjacent to the GMC. (D) Cell plate completed in correct position. (E) Plot of distance of the right-hand edge of the cell plate from its final position vs. time. Between T 0 (A ) and T 5.5 min (B ), the cell plate underwent small iregular movements often toward the upper division site, then rapidly (in 3.5 min) repositioned into the lower division site (C ).

15 February 1999] PICKETT-HEAPS ET AL. CYTOPLASMIC DOMAINS 167 these properties result from the close interactions of MTs with actin. Finally, we point out that such tensegral interactions, so apparent in plant cells, may apply universally. For example, accurate orientation of the spindle in yeast division requires interaction of the poles, via spindle MTs, and cytoskeletal actin (Palmer et al., 1992). Establishment and maintenance of asymmetry in cleaving nematode eggs specifically require actin for rotation of one prophase spindle in the two-celled embryo (Hill and Strome, 1988; Waddle, Cooper, and Waterson, 1994); thereafter the pole is tethered to a cortical site, just as with the GMC. Establishment of cellular polarity in fucoid zygotes similarly requires actin, as does the subsequent reorientation of the centrosomal MTs and the mitotic spindle (reviewed in Kropf, 1992). SPATIAL CUES AND THE PLANT CYTOPLAST Clearly, the determination of the plane of cytokinesis occurs early and is marked by subtle changes in the whole cell, particularly its cytoskeleton. Let us now briefly consider the cues that might guide this determination. While chemical cues and messengers have long been considered paramount in this role, we believe that physical cues may be equally significant, a view recently exciting more interest in animal cells (e.g., Ingber et al., 1994; Chicurel, Chen, and Ingber, 1998). Strain detection: growth in large cylindrical cells Expansion of cells in higher plants cells and some algae (e.g., Spirogyra) takes place along the length of a wall lined by transverse wall MTs. Very fine, transversely oriented filaments of actin (Cleary, unpublished data) lie close to these MTs (Lancelle and Hepler, 1991; Cleary, unpublished data). In cells stripped of their walls, resumption of cylindrical growth coincides with the establishment of wall MTs (Galway and Hardham, 1986). The cytoskeletal system of wall MTs and actin appears responsive to forces induced in the wall by normal growth (Kotenko, 1986; Green, 1987), and so these forces probably provide spatial cues and/or physical determinants that affect cell division. The cytoskeleton in individual cells responds to external cues generated near a wound site (Lintilhac and Vesecky, 1981; Gunning, 1982). That MTs are concurrently involved in controlling elongation while being sensitive to external cues has been demonstrated by Murata, Kadota, and Wada (1997; see also Kropf, Williamson, and Wasteneys, 1997); they irradiated single cells in photosensitive tissue and showed that wall MTs in just the irradiated cell became reoriented within an hour of the stimulus. Strain detection: control of the plane of cell division After plant tissue is wounded, repair is initiated by cell divisions around the wound site, coplanar with the wound surface (Venverloo, 1990). MTs and the actin cytoskeleton respond rapidly by becoming reoriented parallel to the plane of the wound (Goodbody and Lloyd, 1990; Hush, Hawes, and Overall, 1990). Subsequent alignment of the phragmosome, PPB, and plane of cell divisions follow to initiate wound repair. Moreover, this response is not confined to cells within tissues. Lynch and Lintilhac (1997) have demonstrated that shear force applied to dividing, single, cultured plant cells orients the division plane to be either parallel or perpendicular to the direction of that force. We suggest that this response is mediated through a tensegral cytoskeleton, which includes the PPB when the cell is preparing for division. MTs may act as strain detectors: reorientation of F-actin is not needed for reorientation of MTs (Hush and Overall, 1992). The wound response can be invoked to explain the geometry of formation of stomatal complexes: the GMC might mimic a point injury, creating polarization in neighboring cells. Evidence that stomatal differentiation may be a highly evolved adaptation of a wound response polarized around a point injury is given by Kennard and Cleary (1997) who showed that physical pressure applied by a micropipette tip can induce the nucleus of an epidermal cell to migrate toward the pressure point, just as it does naturally toward the GMC during the G 1 phase of its normal division. A similar relationship between osmotically induced stress and the plane of division may apply in vacuolated green algal cells. For example, nuclear polarization and asymmetric division (and ultimately, branching and hair cell differentiation) in Bulbochaete (Pickett-Heaps, 1974c, 1975b) strongly resemble what happens in antheridium formation in a fern (Kotenko, 1986), while in Coleochaete, the switch from a transverse to radial division could be triggered by the increased stress in the outer wall of a cell effectively situated between two diverging files of cells. The same general relationship of force to the plane of cytokinesis seems to apply to animal cells. For example (Mazia, 1961, p. 328), in sea urchin eggs subjected to centrifugal force, cleavage (inducible even without prior mitosis) was always at right angles to the direction of applied force as it seems to be in phragmoplast formation. Such responses suggest a central role of force in determining the geometry of differentiation. The concept of tensegrity (Ingber, 1993) provides an excellent starting point for probing the structural basis for such responses, as an alternative to any theories based on chemistry and physiological gradients. SUMMARY: THE CYTOPLAST AND EVOLUTION OF THE PPB We use the cytoplast/ncd (nuclear-cytoplasmic domain) concept as a basis for exploring morphological and dynamic observations on morphogenesis. A cell is usually a single NCD, but multinucleate cells contain many NCDs, one around each nucleus. The spindle is seen as the cell s mechanism for replicating the cytoplast. During cell division, the margin between the two new cytoplasts is defined by the interdigitated MTs of the interzonal spindle fibers. However, in many plants, the plane of future division is determined well before division and is therefore not just a function of the spindle. Cytokinesis is necessarily coincident with the spindle overlap (the midbody in animal cells, the phragmoplast in plant cells), an association mediated by unidentified actin-related, morphogenetic cues (molecular determinants). Spirogyra exemplifies how the phragmoplast may have arisen; interaction between the spindle and cleavage furrow stimulated proliferation of phragmoplast MTs whose

16 168 AMERICAN JOURNAL OF BOTANY [Vol. 86 transport properties contributed to evolution of the cell plate. As the phragmoplast became established, assembly of actin into a cleavage furrow became less important. Acquisition of the phragmoplast may have enabled cells to divide at right angles to the transverse cytokinesis of the primitive filament. However, this ancient cleavage site may still be expressed during determination of the plane of cytokinesis and certain higher plant cells retain the ability to cleave. During cell division in lower land plants, NCDs are expressed in various ways; they can be set up before preprophase/prophase and they create phragmoplasts in various situations not immediately related to the presence of mitotic spindles. In higher land plants, ancient positional cues that located actin at the site of cleavage are apparently now expressed during preprophase. Establishment of this cleavage site may define the two nascent cytoplasts as manifested by relocation of actin and wall MTs into the PPB. The PPB delineates the perimeter of the interface between the future daughter cytoplasts, and the actin-depleted zone that becomes visible at the cell surface when the PPB disperses is viewed as an indication that the surface cytoskeleton has been rearranged into daughter cytoplasts well before cytokinesis. During telophase, the positional cues previously indicated by the PPB are still related to actin localization and function, even when visible as an actin-depleted zone. The determinant(s) of actin function are activated to draw the expanding edge of the phragmoplast to the primitive cleavage site formerly occupied by the PPB. Polarization evident in asymmetric divisions also requires actin to hold the spindle pole in position. Large plant cells expanding under turgor pressure detect stress in their wall using their MT/actin cytoskeleton, maintaining uniaxial growth via control over orientation of wall microfibrils. This stress-detection system also apparently controls the plane of cell division, ensuring that (in most cases) it is transverse to the axis of growth. Its controlling role is evident during wound response, when altered stress patterns in the wall may provide the physical cues for realigning the plane of cell division around the wound. WHAT NEXT? After 30 years of speculating, we still do not understand the significance of the preprophase band perhaps because we have been overly preoccupied with finding role(s) for its complement of MTs. The concept of tensegrity emphasizes that the major cytoskeletal elements of the cell work together in subtle and interdependent ways, not easily amenable to reductionistic analysis. From this perspective, it is logical that actin is involved in polarized cell division, and not unexpected that it appears to have some subtle involvement in the PPB. Several mutations have recently been uncovered that affect the PPB and subsequent division in various ways. In the tonneau (Traas et al., 1995) and the fass (McClinton and Sung, 1997) mutations, PPBs are absent and in their absence causes abnormally oriented cell divisions during embryogenesis. The recessive tangled mutation (Smith, Hake and Sylvester, 1996; Cleary and Smith, 1998) results in the telophase spindle being unable to reorient correctly into the division site marked previously by the PPB. While tissue is disorganized by these mutations, the morphology of the plant is unaffected. These results confirm that the PPB accompanies highly organized cell divisions that are normal during histogenesis, but they do not yet suggest any particular role for it. Such mutations offer hope that we may be able to identify specific proteins involved in histogenesis, determine where they are located, and whether and how they interact with the cytoskeleton. But Harold (1995) in a provoking and thoughtful review reminds us that morphological mutants in the simple, intensively studied system, yeast, still offer no interpretable correlation between genotype and phenotype. The relationship between genes and morphogenesis will be more complex in the multicelled tissues of plants. The importance of membrane-bound proteins that attach to cytoskeletal components is well established in animal cells and equivalent proteins surely exist in plant cells, a possibility being explored in Fucus embryos (Fowler and Quatrano, 1997) in which the planes of cell division are strictly controlled (Quatrano and Shaw, 1997). One might expect to uncover such proteins at the PPB site and we suspect that they may have attributes more associated with actin (derived from an ancient cleavage apparatus) than MTs. Such spatial determinants presumably exert their influence via force-generating, tensegral systems and the means by which they establish their three-dimensional expression will surely help explain how asymmetric mitosis and the phragmoplast are controlled. The spindle itself may be a tensegral structure (Pickett-Heaps, Forer, and Spurck, 1997), in which event it is one expression of the cell s global cytoskeletal system responsive to physical (e.g., force) as well as chemical cues. We believe that plant cells offer tremendous advantages, ripe for further exploitation, in such investigations into fundamental aspects of cellular and tissue morphogenesis. LITERATURE CITED ALBERTS, B., D. BRAY, J.LEWIS, M.RAFF, K.ROBERTS AND J. D. WAT- SON Molecular biology of the cell. Garland, New York, NY. BAJER, A. S Fine structure studies on phragmoplast and cell plate formation. Chromosoma (Berlin) 24: BOUCK, G. B., AND D. L. BROWN Microtubule biogenesis and cell shape in Ochromonas. I. The distribution of cytoplasmic and mitotic microtubules. Journal of Cell Biology 56: BRAMHILL, D Bacterial cell division. Annual Review of Cell and Developmental Biology 13: BROWN, R. C., AND B. E. LEMMON Plastid apportionment and preprophase microtubule bands in monoplastidic root meristem cells of Isoetes and Selaginella. Protoplasma 123: , AND. 1985a. A cytoskeletal system predicts division plane in meiosis of Selaginella. Protoplasma 127: , AND. 1985b. Preprophasic establishment of division polarity in monoplastidic mitosis of hornworts. Protoplasma 124: , AND. 1987a. Division polarity, development and configuration of microtubule arrays in bryophyte meiosis I. Meiotic prophase to metaphase I. Protoplasma 137: , AND. 1987b. Division polarity, development and configuration of microtubule arrays in bryophyte meiosis II. Anaphase I to the tetrad. Protoplasma 138: 1 10., AND. 1988a. Cytokinesis occurs at boundaries of domains delimited by nuclear-based microtubules in sporocytes of

17 February 1999] PICKETT-HEAPS ET AL. CYTOPLASMIC DOMAINS 169 Conocephalum conicum (Bryophyta). Cell Motility and the Cytoskeleton 11: , AND. 1988b. Preprophasic microtubule systems and development of the mitotic spindle in hornworts (Bryophyta). Protoplasma 143: , AND. 1990a. Monoplastidic cell division in lower land plants. American Journal of Botany 77: , AND. 1990b. Polar organizers mark division axis prior to preprophase band formation in mitosis of the hepatic Reboulia hemispherica (Bryophyta). Protoplasma 156: , AND The cytokinetic apparatus in meiosis: control of division plane in the absence of a preprophase band of microtubules. In C. Lloyd [ed.]. The cytoskeletal basis of plant growth and form, Academic Press, London., AND. 1992a. Control of division plane in normal and griseofulvin-treated microsporocytes of Magnolia. Journal of Cell Science 103: , AND. 1992b. Cytoplasmic domain: a model for spatial control of cytokinesis in reproductive cells of plants. EMSA Bulletin 22: , AND Diversity of cell division in simple land plants holds clues to evolution of the mitotic and cytokinetic apparatus in higher plants. Memoirs of the Torrey Botanical Club 25: , AND The quadripolar microtubule system in lower plants. Journal of Plant Research 110: ,, AND L. E. GRAHAM Morphogenetic plastid migration and microtubule arrays in mitosis and cytokinesis in the green alga Coleochaete orbicularis. American Journal of Botany 81: ,, AND O.-A. OLSEN Endosperm development in barley: microtubule involvement in the morphogenetic pathway. Plant Cell 6: BUSBY, C. H., AND B. E. S. GUNNING. 1988a. Establishment of plastidbased quadripolarity in spore mother cells of the moss Funaria hygrometrica. Journal of Cell Science 91: , AND. 1988b. Development of the quadripolar meiotic cytoskeleton in spore mother cells of the moss Funaria hygrometrica. Journal of Cell Science 91: BYERS, B., AND D. H. ABRAMSON Cytokinesis in HeLa: posttelophase delay and microtubule-associated motility. Protoplasma 66: CHICUREL, M. E., C. S. CHEN, AND D. E. INGBER Cellular control lies in the balance of forces. Current Opinion in Cell Biology 10: CHO, S.-O., AND S. M. WICK Distribution and function of actin in the developing stomatal complex of winter rye (Secale cereale cv. Puma). Protoplasma 157: CLEARY, A. L F-actin redistribution at the division site in living Tradescantia stomatal complexes as revealed by microinjection of rhodamine-phalloidin. Protoplasma 185: , R. C. BROWN, AND B. E. LEMMON. 1992a. Microtubule arrays during mitosis in monoplastidic root tip cells of Isoetes. Protoplasma 167: ,, AND. 1992b. Establishment of division plane and mitosis in monoplastidic guard mother cells of Selaginella. Cell Motility and the Cytoskeleton 23: , B. E. S. GUNNING, G.O.WASTENEYS, AND P. K. HEPLER. 1992c. Microtubule and F-actin dynamics at the division site in living Tradescantia stamen hair cells. Journal of Cell Science 103: , AND U. MATHESIUS Rearrangements of f-actin during stomatogenesis visualised by confocal microscopy in fixed and permeabilized Tradescantia leaf epidermis. Botanical Acta 109: , AND L. G. SMITH The Tangled1 gene is required for spatial control of cytoskeletal arrays associated with cell division during maize leaf development. Plant Cell 10: COLASANTI, J., S.-O. CHO, S. WICK, AND V. SUNDARESAN Localization of the functional p34 cdc2 holmolog of maize in root tip and stomatal complex cells: association with predicted division sites. Plant Cell 5: COOK, M. E., L. E. GRAHAM, C.E.J.BOTHA, AND C. A. LAVIN Comparative ultrastructure of plasmodesmata of Chara and selected bryophytes: toward an elucidation of the evolutionary origin of plant plasmodesmata. American Journal of Botany 84: DING, B., R. TURGEON, AND M. V. PARTHASARATHY Microfilaments in the preprophase band of freeze substituted tobacco root cells. Protoplasma 165: DOONAN, J. H The cytoskeleton and moss morphogenesis. In C. Lloyd [ed.], The cytoskeletal basis of plant growth and form, Academic Press, London., D. J. COVE, F.M.R.CORKE, AND C. W. LLOYD Preprophase bands of microtubules, absent from tip-growing moss filaments, arises in leafy shoots during transition to intercalary growth. Cell Motility and the Cytoskeleton 7: , AND J. G. DUCKETT The bryophyte cytoskeleton: experimental and immunofluoresence studies of morphogenesis. Advances in Bryology 3: ELEFTHERIOU, E. P., AND B. A. PALEVITZ The effects of cytochalasin D on preprophase band organization in root tip cells of Allium. Journal of Cell Science 103: ERICKSON, H. P FtsZ, a tubulin homologue in prokaryotic cell division. Trends in Cell Biology 7: FARADAY, C. D., AND R. M. SPANSWICK Evidence for a membrane skeleton in higher plants: a spectrin-like polypeptide co-isolates with rice root plasma membranes. Federation of European Biochemical Societies 318: FOWKE, L. C., AND J. D. PICKETT-HEAPS. 1969a. Cell division in Spirogyra: I. Mitosis. Journal of Phycology 5: , AND. 1969b. Cell division in Spirogyra: II. Cytokinesis. Journal of Phycology 5: FOWLER, J. E., AND R. S. QUATRANO Plant cell morphogenesis: plasma membrane interactions with the cytoskeleton and cell wall. Annual Review of Cell and Developmental Biology 13: FUJIWARA, K., AND T. D. POLLARD Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow and the mitotic spindle. Journal of Cell Biology 71: GALWAY, M. E., AND A. R. HARDHAM Microtubule reorganization, cell wall synthesis and establishment of the axis of elongation in regenerating protoplasts of the alga Mougeotia. Protoplasma 135: , AND Immunofluorescent localization of microtubules throughout the cell cycle in the green alga Mougeotia (Zygnemataceae). American Journal of Botany 78: GAMBARDELLA, R., AND F. ALFANO Monoplastidic mitosis in the moss Timmiella barbuloides (Bryophyta). Protoplasma 156: GIMENAEZ-ABIAN, M. I., J. L. UTRILLA, G.CANOVAS, M.H.GIMENEZ- MARTIN, M.H.NAVARRETE, AND C. DE LA TORRE The positional control of mitosis and cytokinesis in higher plant cells. Planta 204: GOFF, L. J., AND A. W. COLEMAN The solution to the cytological paradox of isomorphy. Journal of Cell Biology 104: GOODBODY, K. C., AND C. W. LLOYD Actin filaments line up across Tradescantia epidermal cells, anticipating wound-induced division planes. Protoplasma 157: , C. J. VENVERLOO, AND C. W. LLOYD Laser microsurgery demonstrates that cytoplasmic strands anchoring the nucleus across the vacuole of premitotic plant cells are under tension: implications for division plane alignment. Development 113: GOTO, Y., AND K. UEDA Microfilament bundles of F-actin in Spirogyra observed by fluorescence microscopy. Planta 173: GRAHAM, L. E Green algae to land plants: an evolutionary transition. Journal of Plant Research 109: , AND Y. KANEKO Subcellular structures of relevance to the origin of land plants (Embryophytes) from green algae. Critical Reviews in Plant Sciences 10: GREEN, P. B Inheritance of pattern: analysis from phenotype to gene. American Zoologist 27: GROLIG, F Nuclear centering in Spirogyra: force integration by microfilaments along microtubules. Planta 204: GUNNING, B. E. S The cytokinetic apparatus: its development and spatial regulation. In The cytoskeleton in plant growth and development, Academic Press, London.

18 170 AMERICAN JOURNAL OF BOTANY [Vol. 86, A. R. HARDHAM, AND J. E. HUGHES Pre-prophase bands of microtubules in all categories of formative and proliferative cell division in Azolla roots. Planta 143: , AND M. SAMMUT Rearrangements of microtubules involved in establishing cell division planes start immediately after DNA synthesis and are completed just before mitosis. Plant Cell 2: , AND S. M. WICK Preprophase bands, phragmoplasts and spatial control of cytokinesis. Journal of Cell Science Supplement 2: HAHNE, G., AND F. HOFFMAN The effect of laser microsurgery on cytoplasmic streaming in isolated plant protoplasts. European Journal of Cell Biology 33: HAROLD, F. M From genes to morphogenesis. Microbiology 141: HEPLER, P. K., A. L. CLEARY, B. E. S. GUNNING, P. WADSWORTH, G. O. WASTENEYS, AND D. H. ZHANG Cytoskeletal dynamics in living plant cells. Cell Biology International 17: HILL, P., AND S. STROME An analysis of the role of microfilaments in the establishment and maintenance of asymmetry in Caenorhabitis elegans. Developmental Biology 125: HOGAN, C. J Microtubules during meiosis in two higher plant species. Protoplasma 198: HUANG, B Ultrastructural aspects of pollen embryogenesis in Hordeum, Triticum and Paeonia. In H. Xu and H. Yang [eds.]. Haploids of higher plants in vitro, Springer-Verlag, Vienna., AND W. F. SHERIDAN Female gametophyte development in maize: microtubular organization and embryo sac polarity. Plant Cell 6: HUSH, J. M., C. R. HAWES, AND R. L. OVERALL Interphase microtubule re-orientation predicts a new cell polarity in wounded pea roots. Journal of Cell Science 96: , AND R. L. OVERALL Re-orientation of cortical F-actin is not necessary for wound-induced microtubule re-orientation and cell polarity establishment. Protoplasma 169: HYDE, G. J., AND A. R. HARDHAM Confocal microscopy of microtubule arrays in cryosectioned sporangia of Phytophora cinnamomi. Experimental Mycology 16: , S. LANCELLE, P. K. HEPLER, AND A. R. HARDHAM Freeze substitution reveals a new model for sporangial cleavage in Phytophora, a result with implications for cytokinesis in other eucaryotes. Journal of Cell Science 100: INGBER, D. E Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton. Journal of Cell Science 104: The architecture of life. Scientific American 278 (January): , L. DIKE, L. HANSEN, S. KARP, H. LILEY, A. MANIOTIS, H. MCNAMEE, D. MOONEY, G. PLOPPER, J. SIMS, AND N. WANG Cellular tensegrity: exploring how mechanical changes in the cytoskeleton regulate cell growth, migration, and tissue pattern during morphogenesis. International Review of Cytology 150: JOHN, P. C. L The plant cell cycle: conserved and unique features in mitotic control. Progress in Cell Cycle Research 2: KAKIMOTO, T., AND H. SHIBAOKA Actin microfilments and microtubules in the preprophase band and phragmoplast of tobacco cells. Protoplasma 140: KARR, T. L., AND B. M. ALBERTS Organization of the cytoskeleton in early Drosophila embryos. Journal of Cell Biology 102: KATSUTA, J., Y. HASHIGUCHI, AND H. SHIBAOKA The role of the cytoskeleton in positioning of the nucleus in premitotic tobacco BY-2 cells. Journal of Cell Science 95: , AND H. SHIBAOKA The roles of the cytoskeleton and the cell wall in nuclear positioning in tobacco BY-2 cells. Plant Cell Physiology 29: KENNARD, J. L., AND A. CLEARY Pre-mitotic nuclear migration in subsidiary mother cells of Tradescantia occurs in G1 of the cell cycle and requires F-actin. Cell Motility and the Cytoskeleton 36: KOTENKO, J. L Antheridium formation in Onoclea sensibilis L.: cytoplasmic polarity and determination of wall positions. Botanical Gazette 147: KROPF, D. L Establishment and expression of cellular polarity in fucoid zygotes. Microbiological Reviews 56: , R. E. WILLIAMSON, AND G. O. WASTENEYS Microtubule orientation and dynamics in elongating characean internodal cells following cytosolic acidification, induction of ph bands, or prematuire growth arrest. Protoplasma 197: LANCELLE, S. A., AND P. K. HEPLER Association of actin with cortical microtubules revealed by immunogold localization in Nicotiana pollen tubes. Protoplasma 165: LESLIE, R. J Chromosomes attain a metaphase position on halfspindles in the absence of an opposing pole. Journal of Cell Science 103: LINTILHAC, P. M., AND T. B. VESECKY Mechanical stress and cell wall orientation in plants. II. The application of controlled directional stress to growing plants: with a discussion on the nature of the wound reaction. American Journal of Botany 68: LIU, B., AND B. A. PALEVITZ Organization of cortical microfilaments in dividing root cells. Cell Motility and the Cytoskeleton 23: LLOYD, C. W Actin in plants. Journal of Cell Science 90: The plant cytoskeleton. Current Opinion in Cell Biology 1: , AND J. A TRAAS The role of F-actin in determining the division plane of carrot suspension cells: drug studies. Journal of Cell Science 102: LOKHORST, G. M., H. J. A. SLUIMAN, AND W. STAR The ultrastructure of mitosis and cytokinesis in the sarcinoid Chlorokybus atmosphyticus (Chlorophyta, Charophyceae) revealed by rapid freeze fixation and freeze substitution. Journal of Phycology 24: LYNCH, T. M., AND P. M. LINTILHAC Mechanical signals in plant development: a new method for single cell studies. Developmental Biology 181: MARCHANT, H. J., AND J. D. PICKETT-HEAPS Mitosis and cytokinesis in Coleochaete scutata. Journal of Phycology 9: MAZIA, D Mitosis and the physiology of cell division. In J. Brachet and A. E. Mirsky [eds.], The cell, vol. 3, Academic Press, New York, NY. MCALISTER, F The formation of the achromatic figure in Spirogyra. American Journal of Botany 10: MCCLINTON, R. S., AND Z. R. SUNG Organization of cortical microtubules at the plasma membrane in Arabidopsis. Planta 201: MCCURDY, D. W., AND B. E. S. GUNNING Reorganization of cortical actin microfilaments and microtubules at preprophase and mitosis in wheat root-tip cells: a double label immunofluoresence study. Cell Motility and the Cytoskeleton 15: MCINTOSH, K., J. D. PICKETT-HEAPS, AND B. E. S. GUNNING Cytokinesis in Spirogyra: integration of cleavage and cell-plate formation. International Journal of Plant Science 156: 1 8. MENZEL, D Visualization of cytoskeletal changes through the life cycle in Acetabularia. Protoplasma 134: , H. JONITZ, AND C. ELSNER-MENZEL The cytoskeleton in the life cycle of Acetabularia and other related species of dasyclad green algae. In D. Menzel [ed.], The cytoskeleton of the algae, CRC Press, Boca Raton, FL. MEWS, M., F. J. SEK, R. MOORE, D. VOLKMANN, B. E. S. GUNNING, AND P. L. C. JOHN Mitotic cyclin distribution during maize cell division: implications for the sequence diversity and functions of cyclins in plants. Protoplasma, in press. MEYER, Y., AND W. O. ABEL Importance of the wall for cell division and in the activity of the cytoplasm in cultured tobacco protoplasts. Planta 123: , AND W. HERTH Chemical inhibition of cell wall formation and cytokinesis, but not of nuclear division, in protoplasts of Nicotiana tabacum L. cultivated in vitro. Planta 142: MICHAUD, D., G. GUILLET, P. A. ROGERS, AND P. M. CHAREST Identification of a 220kDa membrane-associated plant protein immunologically related to human spectrin. Federation of European Biochemical Society 294:

19 February 1999] PICKETT-HEAPS ET AL. CYTOPLASMIC DOMAINS 171 MILLER, K. G., AND D. P. KIEHART Fly division. Journal of Cell Biology 131: 1 5. MINEYUKI, Y.,AND B. E. S. GUNNING A role for the preprophase band of microtubules in maturation of new cell walls, and a general proposal on the function of preprophase band sites in cell division in higher plants. Journal of Cell Science 97: , J. MARC, AND B. A. PALEVITZ Relationship between the preprophase band, nucleus and spindle in dividing Allium cotyledon cells. Journal of Plant Physiology 138: , AND B. A. PALEVITZ Relationship between preprophase band organization, F-actin and the division site in Allium. Journal of Cell Science 97: MORRISON, I. N., AND T. P. O BRIEN Cytokinesis in the developing wheat grain; division with and without a phragmoplast. Protoplasma 130: MURATA, T., A. KADOTA, AND M. WADA Effects of blue light on cell elongation and microtubule orientation in dark-grown gametophytes of Ceratopteris richardii. Plant Cell Physiology 38: O BRIEN, T. P The preprophase band of microtubules: does it block cleavage? Cytobios 37: OLSEN, O.-A., R. C. BROWN, AND B. E. LEMMON Pattern and process of wall formation in developing endosperm. BioEssays 17: OTA, T The role of cytoplasm in cytokinesis of plant cells. Cytologia 26: PALEVITZ, B. A Actin in the preprophase band of Allium cepa. Journal of Cell Biology 104: Organization of the mitotic apparatus during generative cell division in Nicotiana tabacum. Protoplasma 174: , AND P. K. HEPLER The control of plane of cell division during stomatal differentiation in Allium. II. Drug studies. Chromosoma 47: PALMER, R. E., D. S. SULLIVAN, T. HUFFAKER, AND D. KOSHLAND Role of astral microtubules and actin in spindle orientation and migration in the budding yeast, Saccharomyces cerevisiae. Journal of Cell Biology 119: PANTERIS, E., P. APOSTOLAKOS, AND B. GALATIS The organization of F-actin in root tip cells of Adiantum capillus veneris throughout the cell cycle. A double label fluoresence microscopy study. Protoplasma 170: PESACRETA, T. C., T. J. BYERS, R. DUBREUIL, D. P. KIEHART, AND D. BRANTON Drosophila spectrin: the membrane skeleton during embryogenesis. Journal of Cell Biology 108: PICKETT-HEAPS, J. D. 1969a. Preprophase microtubules and stomatal differentiation in Commelina cyanea. Australian Journal of Biological Sciences 22: b. Preprophase microtubule bands in some abnormal mitotic cells of wheat. Journal of Cell Science 4: c. Proprophase microtubules and stomatal differentiation; some effects of centrifugation on symmetrical and asymmetrical cell division. Journal of Ultrastructural Research 27: Cell division in Klebsormidium subtilissimum (formerly Ulothrix subtilissima), and its possible phylogenetic significance. Cytobios 6: a. Cell division in Stichococcus. British Phycological Journal 9: b. Plant microtubules. In A. W. Robards [ed.], Dynamic aspects of plant ultrastructure, McGraw-Hill, London c. Cell division in Bulbochaete. II. Hair cell formation. Journal of Phycology 10: a. Green algae: structure, reproduction and evolution in selected genera. Sinauer, Stamford, CT b. Cell division and evolution in Bulbochaete. III. Sexual reproduction and evolution of the branched habit. Cytobiologie 12: , A. FORER, AND T. S. SPURCK Traction fibre: toward a tensegral model of the spindle. Cell Motility and the Cytoskeleton 37: 1 6., AND D. H. NORTHCOTE. 1966a. Organisation of microtubules and endoplasmic reticulum during mitosis and cytokinesis in wheat meristems. Journal of Cell Science 1: , AND. 1966b. Cell division in the formation of the stomatal complex in the young leaves of wheat. Journal of Cell Science 1: , AND R. WETHERBEE Spindle function in the green alga Mougeotia: absence of anaphase A correlates with post-mitotic nuclear migration. Cell Motility and the Cytoskeleton 7: PORTER, K. R Motility in cells. In C. R. Goldman, T. Pollard, and J. Rosenbaum [eds.], Cell motility, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., M. BECKERLE, AND M. A. MCNIVEN The cytoplasmic matrix. Modern Cell Biology 2: , AND M. A. MCNIVEN The cytoplast: a unit structure in chromatophores. Cell 29: QUATRANO, R. S., AND S. L. SHAW Role of the cell wall in the determination of cell polarity and the plane of cell division in Fucus embryos. Trends in Plant Science 2: RAFF, J. W., AND D. M. GLOVER Centrosomes, and not nuclei, initiate pole cell formation in Drosophila embryos. Cell 57: RAPPAPORT, R Establishment of the mechanism of cytokinesis in animal cells. International Review of Cytology 105: Cytokinesis in animal cells. Cambridge University Press, Cambridge. SATTERWHITE, L. L., AND T. D. POLLARD Cytokinesis. Current Opinion in Cell Biology 14: SAWITZKY, H., AND F. GROLIG Phragmoplast of the green alga Spirogyra is functionally different from the higher plant phragmoplast. Journal of Cell Biology 130: SEAGULL, R. W., M. M. FALCONER, AND C. A. WEERDENBURG Microfilaments: dynamic arrays in higher plant cells. Journal of Cell Biology 104: SEEGAR, P. J., A. F. GERRITSEN, AND M. A. G. DE BAKKER The cytokinetic apparatus during sporulation in the unicellular green flagellate Gloeomonas kupfferi: the phycoplast as a spatio-temporal differentiation of the cortical microtubule array that organises cytokinesis. Nova Hedwigia 49: SINNOTT, E. W Plant morphogenesis. McGraw-Hill, New York, NY., AND R. BLOCH Division in vacuolate plant cells. American Journal of Botany 28: SLUDER, G., F. J. MILLER, AND C. L. RIEDER The reproduction of centrosomes: nuclear versus cytoplasmic controls. Journal of Cell Biology 103: SMITH, L. G., S. HAKE, AND A. W. SYLVESTER The tangled-1 mutation alters cell division orientations throughout maize leaf development without altering leaf shape. Development 122: SONOBE, S Cytochalasin B enhances cytokinetic cleavage in miniprotoplasts isolated from cultured tobacco cells. Protoplasma 155: STAEHELIN, L.A.,AND P. K. HEPLER Cytokinesis in higher plants. Cell 84: STEBBINS, G. L., AND S. K. JAIN Developmental studies of cell differentiation in the epidermis of monocotyledons. II. Allium, Rhoeo and Commelina. Developmental Biology 2: , AND S. S. SHAH Developmental studies of cell differentiation in the epidermis of monocotyledons. II. Cytological features of stomatal development in the Gramineae. Developmental Biology 2: STROME, S Determination of cleavage planes. Cell 72: 3 6. TRAAS, J. A., C. BELLINI, P. NACRY, J. KRONENBURGER, D. BOUCHEZ, AND M. CABOCHE Normal differentiation patterns in plants lacking microtubular preprophase bands. Nature 375: , J. H. DOONAN, D. J. RAWLINS, P. J. SHAW, J. WATTS, AND C. W. LLOYD An actin network is present in the cytoplasm throughout the cell cycle of carrot cells and associates with the dividing nucleus. Journal of Cell Biology 105: TREWAVAS, A., AND R. MALHO Signal perception and transduction. Plant Cell 9: VALSTER, A. H., AND P. K. HELPER Caffeine inhibition of cytokinesis: effect on the phragmoplast cytoskeleton in living Tradescantia stamen hair cells. Protoplasma 196: VAN LAMMEREN, A. M. M Structure and function of the microtubular cytoskeleton during endosperm development in wheat: an immuno-fluorescent study. Protoplasma 146:

20 172 AMERICAN JOURNAL OF BOTANY [Vol. 86 VENVERLOO, C. J Regulation of the plane of cell division in vacuolated cells. II. Wound-induced changes. Protoplasma 155: , P. H. HOVENKAMP, A. J. WEEDA, AND K. R. LIBBENGA Cell division in Nautilocalyx explants. I. Phragmosome, preprophase band and plane of division. Zeitschrift fur Pflanzenphysiologie 100: WADDLE, J. A., J. A. COOPER, AND R. H. WATERSON Transient localized accumulation of actin in Caenorhabditis elegans with oriented asymmetric divisions. Development 120: WARN, R. M The cytoskeleton of the early Drosophila embryo. Journal of Cell Science Supplement 5: WEBB, M. C., AND B. E. S. GUNNING Embryo sac development in Arabidopsis thaliana. I. Megamorphogenesis, including the microtubular cytoskeleton. Sexual Plant Reproduction 3: , AND The microtubule cytoskeleton during development of the zygote, proembryo and free-nuclear endosperm in Arabidopsis thaliana (L.) Heynh. Planta 184: , AND Embryo sac development in Arabidopsis thaliana. II. Embryo sac development, including the microtubular cytoskeleton. Sexual Plant Reproduction. 7: WHEATLEY, S. P., AND Y.-L. YANG Midzone microtubule bundles are continuously required for cytokinesis in cultured epithelial cells. Journal of Cell Biology 135: WICK, S. M. 1991a. The preprophase band. In C. Lloyd [ed.], The cytoskeletal basis of plant growth and form, Academic Press, London b. Spatial aspects of cytokinesis in plant cells. Current Opinions in Cell Biology 3: YONEMURA, S., A. NAGAFUCHI, N. SATO, AND S. TSUKITA Concentration of an integral membrane protein, CD43 (Leukosialin, Sialophorin), in the cleavage furrow through the interaction of its cytoplasmic domain with actin-based cytoskeletons. Journal of Cell Biology 120: ZHANG, D., AND R. B. NICKLAS Anaphase and cytokinesis in the absence of chromosomes. Nature 382: , P. WADSWORTH, AND P. K. HEPLER Dynamics of microfilaments are similar, but distinct from microtubules during cytokinesis in living, dividing plant cells. Cell Motility and the Cytoskeleton 24: