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2School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia; 3Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra, A.C.T. 2601, Australia; and 4Department of Biology, University of Southwestern Louisiana, Lafayette, Louisiana 70504-2451
Received for publication May 11, 1998. Accepted for publication October 19, 1998.
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ABSTRACT |
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 TOP ABSTRACT THE "CYTOPLAST" CONCEPTTHE SPINDLE: DELINEATION AND...THE CYTOPLAST IN DIVIDING...CLEAVAGE AND EVOLUTION OF...THE CYTOPLAST CONCEPT AND...THE DIVISION SITE AND...SPATIAL CUES AND THE...SUMMARY: THE CYTOPLAST AND...WHAT NEXT?LITERATURE CITED |
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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.
Key Words: actin • cytoplast • cytoskeleton • microtubule • phragmoplast • polarization • preprophase band • Spirogyra • tensegrity
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THE "CYTOPLAST" CONCEPT |
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TOPABSTRACTTHE "CYTOPLAST" CONCEPTTHE SPINDLE: DELINEATION AND...THE CYTOPLAST IN DIVIDING...CLEAVAGE AND EVOLUTION OF...THE CYTOPLAST CONCEPT AND...THE DIVISION SITE AND...SPATIAL CUES AND THE...SUMMARY: THE CYTOPLAST AND...WHAT NEXT?LITERATURE CITED |
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, 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 includes 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.
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THE SPINDLE: DELINEATION AND REPRODUCTION OF THE CYTOPLAST IN ANIMAL CELLS |
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TOPABSTRACTTHE "CYTOPLAST" CONCEPTTHE SPINDLE: DELINEATION AND...THE CYTOPLAST IN DIVIDING...CLEAVAGE AND EVOLUTION OF...THE CYTOPLAST CONCEPT AND...THE DIVISION SITE AND...SPATIAL CUES AND THE...SUMMARY: THE CYTOPLAST AND...WHAT NEXT?LITERATURE CITED |
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, 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.
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 11–13 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.
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THE CYTOPLAST IN DIVIDING PLANT CELLS |
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TOPABSTRACTTHE "CYTOPLAST" CONCEPTTHE SPINDLE: DELINEATION AND...THE CYTOPLAST IN DIVIDING...CLEAVAGE AND EVOLUTION OF...THE CYTOPLAST CONCEPT AND...THE DIVISION SITE AND...SPATIAL CUES AND THE...SUMMARY: THE CYTOPLAST AND...WHAT NEXT?LITERATURE CITED |
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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 McNiven, 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 (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).
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). Also the midbody does not have mitotic cyclins that associate with cyclin-dependent 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 phragmoplast 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.
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, 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 karyokinesis 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.
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, 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 results 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.
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CLEAVAGE AND EVOLUTION OF THE PHRAGMOPLAST |
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TOPABSTRACTTHE "CYTOPLAST" CONCEPTTHE SPINDLE: DELINEATION AND...THE CYTOPLAST IN DIVIDING...CLEAVAGE AND EVOLUTION OF...THE CYTOPLAST CONCEPT AND...THE DIVISION SITE AND...SPATIAL CUES AND THE...SUMMARY: THE CYTOPLAST AND...WHAT NEXT?LITERATURE CITED |
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). 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 actin-based 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).
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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 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 collar-like 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
).
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THE CYTOPLAST CONCEPT AND DIVISION IN HIGHER PLANT CELLS |
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TOPABSTRACTTHE "CYTOPLAST" CONCEPTTHE SPINDLE: DELINEATION AND...THE CYTOPLAST IN DIVIDING...CLEAVAGE AND EVOLUTION OF...THE CYTOPLAST CONCEPT AND...THE DIVISION SITE AND...SPATIAL CUES AND THE...SUMMARY: THE CYTOPLAST AND...WHAT NEXT?LITERATURE CITED |
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: 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 G2 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., p34cdc2: 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
F-actin 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 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.
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THE DIVISION SITE AND PHRAGMOPLAST IN THE CONTEXT OF THE DIVIDING CYTOPLAST |
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TOPABSTRACTTHE "CYTOPLAST" CONCEPTTHE SPINDLE: DELINEATION AND...THE CYTOPLAST IN DIVIDING...CLEAVAGE AND EVOLUTION OF...THE CYTOPLAST CONCEPT AND...THE DIVISION SITE AND...SPATIAL CUES AND THE...SUMMARY: THE CYTOPLAST AND...WHAT NEXT?LITERATURE CITED |
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; 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 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-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).
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; 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).
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