Plant Cytokinesis: Terminology for Structures and Processes

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Opinion Plant Cytokinesis: Terminology for Structures and Processes Andrei Smertenko,1,* Farhah Assaad,2 František Baluška,3 Magdalena Bezanilla,4,23 Henrik Buschmann,5 Georgia Drakakaki,6 Marie-Theres Hauser,7 Marcel Janson,8 Yoshinobu Mineyuki,9 Ian Moore,10 Sabine Müller,11 Takashi Murata,12 Marisa S. Otegui,13 Emmanuel Panteris,14 Carolyn Rasmussen,15 Anne-Catherine Schmit,16 Jozef Šamaj,17 Lacey Samuels,18 L. Andrew Staehelin,19 Daniel Van Damme,20,21 Geoffrey Wasteneys,18 and Viktor Žárský22

Plant cytokinesis is orchestrated by a specialized structure, the phragmoplast. Trends The phragmoplast first occurred in representatives of Charophyte algae and A large number of phragmoplast pro- then became the main division apparatus in land plants. Major cellular activi- teins have been identified. ties, including cytoskeletal dynamics, vesicle trafficking, membrane assembly, and cell wall biosynthesis, cooperate in the phragmoplast under the guidance Electron microscopy/tomography studies have produced nanoscale infor- of a complex signaling network. Furthermore, the phragmoplast combines mation about the architecture of plant-specific features with the conserved cytokinetic processes of animals, phragmoplast and cell plate assembly stages in cryofixed cells. fungi, and protists. As such, the phragmoplast represents a useful system for understanding both plant cell dynamics and the evolution of cytokinesis. We Novel components of the cortical divi- recognize that future research and knowledge transfer into other fields would sion zone and cell plate fusion site have fi been discovered. This information lays bene t from standardized terminology. Here, we propose such a lexicon of a foundation for understanding how terminology for specific structures and processes associated with plant plant cells memorize the division plane cytokinesis. throughout mitosis and how the cell plate is guided to its predetermined attachment site.

Rationale for Updating Plant Cytokinesis Terminology MAP65 and plus end-directed kinesins Current plant cytokinesis terminology was developed using data generated by fluorescence contribute to the maintenance of the antiparallel overlap of phragmoplast fi fi microscopy of live or xed cells, electron microscopy of chemically or cryo xed cells, and microtubules. In addition, the MAP65- genetic strategies. As a consequence of different experimental approaches, and for historic –TRAPPII interaction plays a key role in reasons, the cytokinetic processes and structures are often referred to by different names. cell plate assembly. Because plant cytokinesis has become a useful model system for addressing specific ques- Actin filaments align parallel to micro- tions in plant biochemistry, cell biology, development, etc., we recognize that exchange of tubules in the phragmoplast, while information between scientists working in these diverse fields, as well as further advancement some microfilaments extend from cell of plant cytokinesis research, would benefit from a harmonized terminology. Here, we sum- plate margin to guide its expansion towards the fusion site. marize the steps,[376_TD$IF] and update terminology associated with two major processes in plant cytokinesis: determination of the cell division plane and assembly of the cell plate (Table 1).

We also propose established markers for some processes and structures (Table 2). The 1Institute of Biological Chemistry, terminology proposed here uses cytokinesis in somatic cells as an example and takes Washington State University, Pullman, advantage of available structural information. Division in reproductive organs and specialized WA 99164, USA 2Botany Department, WZW, cell types involves similar processes, which appear to be structurally different [1]. For in-depth Technische Universität München, description of cytokinesis in somatic cells we refer to several recent reviews [2–6]. Terminology Freising, 85354 Germany

3Department of Plant Cell Biology, Table 1. Glossary of Plant[374_TD$IF] Cytokinesis Terminology IZMB, University of Bonn, Term Former terms Description Kirschallee 1, D-53115, Bonn, Germany Actin-depleted Cortical region of cytoplasm with few actin filaments that forms prior to 4Biology Department, University of zone (ADZ) nuclear envelope breakdown and usually coincides with the cortical Massachusetts, Amherst, MA 01003, division zone (CDZ). ADZ can be flanked by domains enriched in actin USA fi laments, known as a twin-peaks structure in some cell types. 5Botany Department, School of Cell division plane Anticipated or true position of the cell plate. Biology and Chemistry, Osnabrück University, Barbarastraße 11, 49076 Cell plate Membrane-polysaccharide compartment that partitions the Osnabrück, Germany cytoplasm after nuclear division. 6Department of Plant Sciences, University of California Davis, Davis, Cell plate assembly General term referring to all processes from cell plate biogenesis to CA 95616, USA maturation. 7Department of Applied Genetics and Cell plate assembly Amorphous scaffold system consisting of proteins and vesicles in Cell Biology, University of Natural matrix (CPAM) the phragmoplast midzone (150 nm thick) that is responsible for Resources and Life Sciences, Vienna, mobilization of protein complexes and vesicles involved in cell plate Austria 8 assembly. Laboratory of Cell Biology, Wageningen University, Cell plate attachment Process of cell plate anchoring and fusion to the parental plasma Droevendaalsesteeg 1, 6708 PB membrane and cell wall. Wageningen, The Netherlands 9 Cell plate biogenesis Generation of a membrane compartment via vesicle tethering and Department of Picobiology, Graduate School of Life Science, University of fusion inside the CPAM. Transport protein particle (TRAPP)II is the Hyogo, Shosha 2167, Himeji 671- predominant tethering complex. 2201, Japan Cell plate expansion Radial increase of the cell plate area during the ring and 10Department of Plant Sciences, discontinuous phragmoplast stages. Characterized by simultaneous University of Oxford, South Parks Rd., presence of different cell plate assembly stages from cytokinetic Oxford, OX1 3RB, UK 11 vesicle fusion to fenestrated sheet. Center for Plant Molecular Biology, University of Tübingen, Auf der Cell plate fusion site Cortical division site Location at which the cell plate fuses with the plasma membrane. Morgenstelle 32, 72076 Tübingen, Cell plate initiation Membrane trafficking and protein/protein interaction processes that Germany 12National Institute for Basic Biology, lead to formation of the cell plate assembly matrix. EXOCYST and Myodaiji, Okazaki 444-8585, Japan TRAPPII are both present. 13Departments of Botany and Cell plate maturation Chemical and structural modifications of the cell plate after Genetics, Laboratories of Cell attachment that result in cross-wall formation. and Molecular Biology, University of Wisconsin–Madison, WI, Central spindle Central region of the anaphase spindle containing antiparallel USA microtubules. 14Department of Botany, School of Cortical[375_TD$IF] division Zone (area) of the plasma membrane and adjacent cell wall that Biology, Aristotle University of zone (CDZ) marks the site where the cell plate will fuse with the cell wall. CDZ is Thessaloniki, Thessaloniki GR-541 24, Macedonia, Greece detectable during preprophase band (PPB) development and 15Center for Plant Cell Biology, persists until completion of cell plate assembly. CDZ possesses a Institute of Integrative Genome dynamic but distinct composition in relation to other plasma Biology, Department of Botany and membrane and cell wall regions. The PPB is a part of the CDZ until Plant Sciences, University of PPB disappears during nuclear envelope breakdown. California, Riverside, CA, USA 16 Cross-wall Cell plate after completing fusion with the parental[39_TD$IF] cell wall. The Institut de Biologie Moléculaire des EXOCYST is the predominant tethering complex during cross-wall Plantes, Centre National de La fi formation. Recherche Scienti que, Université de Strasbourg, F67084, Strasbourg- Cross-wall Chemical and structural modifications of the cross-wall that result in cedex, France transformation formation of the middle lamella. 17Centre of the Region Haná for Biotechnological and Agricultural Cytokinetic vesicles Cell-plate-forming Membrane vesicles that provide material for cell plate assembly. Research, Faculty of Science, Palacký vesicles Principally of Golgi and trans-Golgi network origin and carrying both University, Šlechtitelu 27, 783 71 biosynthetic and endocytic material. Olomouc, Czech Republic 18 Discontinuous Late, asymmetric Phragmoplast after unilateral docking to the plasma membrane. Department of Botany, University of phragmoplast British Columbia, Vancouver, BC, V6T 1Z4, Canada Disk phragmoplast Solid, early, young Phragmoplast at early cytokinetic stages, characterized by even 19Department of Molecular,[356_TD$IF] Cellular distribution of microtubules all over the forming cell plate area. and Developmental Biology, University of Colorado, UCB 347, Boulder, CO Distal phragmoplast Regions of the phragmoplast that face the daughter nuclei. The 80309-0347, USA zone microtubule minus ends are gathered in this zone. 20Department of Plant Biotechnology Division site Region at the cortex defined by CDZ during prophase and cell plate and Bioinformatics, Ghent University, fusion site during cytokinesis. 9052 Ghent, Belgium

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21 Table 1. (continued) Center for Plant Systems Biology, VIB, Technologiepark[359_TD$IF] 927, 9052 Term Former terms Description Ghent, Belgium 22Department of Experimental Plant[360_TD$IF] Fenestrated sheet Cell plate assembly stage characterized by expansion of the tubules Biology, Charles University and within the membrane network and formation of almost continuous Institute of Experimental Botany, membrane sheet. ASCR, Prague, Czech Republic 23 Fusion tubes Membraneous cell plate extensions that connect to the parental Current address: Department of [361_TD$IF] plasma membrane during cell plate attachment. Biological Sciences, Dartmouth College, Hanover, NH 03755-3529, Middle lamella Central layer of the maturing cell plate consisting of mainly pectins, USA. which maintains the structure and integrity of tissues by gluing adjacent cells together. *Correspondence: Leading zone Outer region of the phragmoplast.[340_TD$IF] [email protected] (A. Smertenko). Lagging zone Inner region of the expanding[341_TD$IF] phragmoplast.

Peripheral Growing microtubules at the phragmoplast leading edge that have microtubules not yet reached the midzone.

Preprophase[342_TD$IF] Cortical ring of microtubules, actin ﬁlaments, cytoskeleton- band (PPB) interacting proteins and other components that underlies the CDZ. The PPB is connected to the nucleus by microtubules and cytoplasmic strands and to CDZ by yet unknown membrane-binding proteins. PPBs assemble during G2 phase[34_TD$IF] and disappear during prometaphase.

PPB of microtubules Microtubule component of PPBs; a plant-speciﬁc microtubule array composed of aligned individual microtubules and bundles of mixed polarity.

Phragmoplast Structure composed of cytoskeletal polymers, membranes, and associated cytosolic proteins that functions as the focused secretory module for assembling the cell plate.

Phragmoplast Midzone Phragmoplast middle plane where the cell plate assembly takes place.

Phragmoplast length Distance between the edges of opposite distal zones (Figure S3).

Ring phragmoplast Transitional, torus, Expanding phragmoplast that lacks microtubules in the center, but expanding, mature has not yet established contact with the plasma membrane.

Tubular network Cell plate assembly stage characterized by interconnections between elongated membrane structures. At this stage the cell plate has smoother morphology and lower density of vesicles than the tubulovesicular network.

Tubulovesicular Cell plate assembly stage characterized by appearance of elongated network dumbbell-shaped membrane structures. related to polysaccharide composition of the cell plate [6] and development of plasmodesmata lie outside the scope of this work.

Cell Division Plane Determination Although our knowledge about division plane determination remains scant, we can visualize it using various markers. In a typical somatic vascular plant cell, but also in some cell types of nonvascular plants, the position of the division plane is outlined by the preprophase band (PPB). The PPB consists of a cortical ring of microtubules (Figure 1), actin filaments, organelles [e.g., endoplasmic reticulum (ER) in some species], and numerous proteins that associate with the cytoskeleton or organelles. Generally, the PPB appears as a broad array of microtubules in G2 phase and then narrows during prophase (Figures 2 and S1). Actin in the PPB is comprised of short single microfilaments, which connect adjacent microtubules and facilitate PPB narrowing (Figures 1 and 2; [7,8]). Using drugs that specifically disrupt either actin or tubulin, it appears that actin filaments require microtubules, but microtubules do not require actin to localize to the PPB [9,10].

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Table 2. Markers of Cytokinetic Structures Structure Marker System Refs[34_TD$IF] Comment

Cortical division Actin Allium cepa, root apical meristem, [16,17] Excluded from CDZ zone (CDZ)a Tradescantia virginiana, apical meristems T. virginiana, stamen hair cells

KCBP A. thaliana root apical meristem, [53] tobacco BY-2 cells

RanGAP Arabidopsis thaliana, root apical meristem [48,49][346_TD$IF] After preprophase band (PPB) disassembly A. cepa, root apical meristem Mid- and late prophase Not apparent after PPB disassembly

POK1 A. thaliana, root apical meristem [20] All cell division stages

Myosin VIII Nicotiana tabacum, tissue culture cells [39,43][347_TD$IF] All cell division stages A. thaliana, root apical meristem All cell division stages in branching cells Zea mays, root apical meristem and from anaphase in apical cells Physcomitrella patens protonemal cells

TAN1 A. thaliana, root apical meristem; [21,50][348_TD$IF] All cell division stages Z. mays, leaf epidermis

PHGAP1/2 A. thaliana, root apical meristem [51] Starting from anaphase

TPLATE N. tabacum, tissue culture cells [42,52] Shortly before and After cell plate attachment A. thaliana, root apical meristem

KCA1 N. tabacum, tissue culture cells [18] Excluded from CDZ

Cell plate fusion site AIR9 A. thaliana, root apical meristem [19,53] Also[349_TD$IF] found in PPB

POK1 A. thaliana, root apical meristem [20]

TAN1 A. thaliana, root apical meristem; [21,50][348_TD$IF] Z. mays, leaf epidermis

KCBP A. thaliana, root apical meristem, [53] tobacco BY-2 cells

PHGAP1/2 A. thaliana, root apical meristem [51]

Myosin VIII P. patens protonemal cells [39] All stages in branching cells and from anaphase in apical cells

Phragmoplast midzone MAP65-3 A. thaliana, root apical meristem [32]

Myosin VIII P. patens protonemal cells [39]

Phragmoplast distal zone KCBP A. thaliana, root apical meristem [53]

Katanin A. thaliana, root apical meristem [54]

Cell plate AtTRS120 A. thaliana, root apical meristem [24]

AtTRS130/CLUB A. thaliana, root apical meristem [24]

RAB-A1c, RAB-A1d, A. thaliana, root apical meristem [55–57] RAB-A1e

RAB-A2, RAB-A3 A. thaliana, root apical meristem [23,40,56]

KNOLLE A. thaliana, root apical meristem [58]

KEULE A. thaliana, root apical meristem [25]

Cross-wall EXOCYST A. thaliana, root apical meristem [24,59]

Callose A. thaliana, root apical meristem [37,40,44] aProteins that localize to CDZ after PPB disassembly.

The PPB underlies the plasma membrane region called the cortical division zone (CDZ; Figures 1 and 2). Clathrin-dependent endocytosis in this plasma membrane region is thought to mediate the localized remodeling and maintenance of the distinct molecular composition of the CDZ (Figure 2; [11]). PPB disappears at about the time of nuclear envelope breakdown in prometaphase, whereas CDZ persists throughout the ensuing stages of M phase (Table 2 and[36_TD$IF]

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Figure 1. Stages of Cytokinesis in Somatic Cells. PPB during prophase comprises aligned microtubules of mixed orientation and actin filaments. PPBs mark the CDZ. Phragmoplast initials form during the anaphase-to-telophase transition through establishment of CPAM at the region of antiparallel microtubule overlap. Short actin filaments localize along microtubules and in the CPAM. Actin filaments in the cortical cytoplasm are excluded from the CDZ. Disk phragmoplast contains the midzone occupied by the CPAM where cytokinetic vesicle fusion results in the formation of the tubulovesicular network. Many phragmoplast microtubules interact with the nuclei. Actin filaments connect the midzone to the cell cortex. Ring phragmoplast forms as the consequence of microtubules and CPAM dismantling in the central part of the phragmoplast, where cell plate assembly reaches tubular network stage. New antiparallel microtubule overlaps in the phragmoplast midzone facilitate CPAM formation and cell plate assembly. Actin filaments connect the phragmoplast leading zone with the CDZ and guide phragmoplast expansion. The phragmoplast expands gradually until it reaches the plasma membrane. Discontinuous phragmoplast forms when microtubules are lost at the sites of cell plate attachment to the plasma membrane. Peripheral microtubules come in contact with the cell cortex and then cell plate extensions fusion tubes connect to the cell plate fusion site at the plasma membrane. Cross-wall is the product of cell plate maturation. Primary cell wall synthesis and chemical transformation of cross-wall continues during interphase. Abbreviations: CDZ, cortical division zone; CPAM, cell plate assembly matrix; PPB, preprophase band.

Figure S2). Whether the PPB actively deﬁnes the division plane or just responds to yet unknown cues remains an open question. Recent work shows that in some cell types the PPB of microtubules is dispensable for the determination of the division plane [12], but increases the precision of spindle alignment with the division plane [12,13]. Nuclear location appears to play

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Figure 2. Biogenesis[35_TD$IF] of the PPB of Microtubules. The PPB of microtubules appears as an aligned array in the cortical cytoplasm in late G2 phase (1, 2). During prophase, the PPB narrows down and actin filaments concentrate in the PPB region (3). Protein linkers can attach microtubules to the plasma membrane or actin filaments; clathrin-dependent endocytosis alters the molecular composition of the plasma membrane and the scaffold in the PPB region (zoom of the boxed region). Towards the end of prophase microtubules form a tight band, which lacks actin filaments; actin filaments are present in other parts of the cell cortex (4). Microtubules disappear during prometaphase, leaving behind unique composition of the scaffold and membrane proteins (5). Abbreviations: PPB, preprophase band; CDZ, cortical division zone. an active role in positioning the PPB as displacement of the nucleus by centrifugation causes formation of two PPBs: at the former and new site of the nucleus [14,15].

The CDZ maintains a distinct but dynamic protein composition throughout the remaining stages of cell division. For example, the TPLATE protein associates with the CDZ at the end of cytokinesis, when the cell plate attaches to the plasma membrane and the parental cell wall. TPLATE, a part of an early adaptor complex involved in endocytosis, is proposed to function to confine cell plate proteins, such as the SNARE protein KNOLLE, to the cell plate. Although actin filaments localize to the PPB and, in the same manner as microtubules, form a band that narrows during PPB development, actin filaments become excluded from the CDZ in many cell types in late prophase to form an actin-depleted zone (ADZ) [16,17]. In some types of cells, actin filaments are enriched at either side of the ADZ and called twin peaks. Additionally, other proteins (e.g., kinesin KCA1; [18]) are excluded from the CDZ.

At the end of prophase the nuclear envelope breaks down and this is followed by the formation of an acentriolar spindle. The chromosomes in the metaphase spindle are usually aligned at the division plane. During anaphase, the central spindle forms roughly at the site of the metaphase chromosomes. Thus, the position of both metaphase and anaphase spindles apparently responds to cues that maintain the division plane. The nature of these cues remains unknown, but they are likely produced in the CDZ.

As a cell progresses towards telophase, some division site markers become conﬁned to the future cell plate fusion site (Table 2). Typically, the cell plate fusion site bisects the CDZ. Distinct molecular composition (e.g., TPLATE) suggests that the cell plate fusion site and CDZ represent distinct plasma-membrane domains. In cells treated with herbicide[364_TD$IF] chlorpropham, or in various mutants, including tangled1, phragmoplast orienting kinesin1 (pok1), and pok2, the cell plate attaches at a site that lies outside the CDZ, indicating that cell plate fusion can occur without CDZ components [19–21].

Cell Plate Assembly Cell plate assembly occurs by the cooperation of the post-Golgi endomembrane system and the phragmoplast, which consists of a bipolar array of microtubules and actin ﬁlaments

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(Figures 1 and S3). The phragmoplast originates from antiparallel microtubules of the central spindle (phragmoplast initials in Figure 1) and at early stages looks like a disk in light micrographs (Figure S3A,B).[365_TD$IF] The majority of microtubule plus ends face the central plane of the phragmoplast or phragmoplast midzone, while minus ends are pointed towards the distal zones (Figure S3B). Cell plate assembly starts during the anaphase-to-telophase transition with the formation of an entity called the cell plate assembly matrix (CPAM) at the phragmoplast midzone (Figure 1; [22]). The processes underlying CPAM formation are collectively named cell plate initiation. In electron micrographs of cryofixed cells, CPAM appears as a ribosome-free zone flanking the cell plate (meaning that it might have pores with size <20 nm) within which the cytokinetic vesicles interact and fuse. The structural proteins of the CPAM have not been identified yet, but electron microscopy, live-cell imaging, and immunofluores- cence microscopy show that the phragmoplast midzone contains Golgi- and trans-Golgi network (TGN)-derived vesicles and vesicle-associated molecular machinery, including small Rab GTPases, such as the Rab-A2 subclass, RAB-A3, and RAB-A1d; SNARE proteins, such as the syntaxin KNOLLE; transport protein particle (TRAPP)II; dynamin-related proteins; EXOCYST tethering complexes; and actin. Mutant analysis has pointed to different roles for some of these molecules in cell plate assembly [23–26]. The delivery to, tethering at, and subsequent fusion of cytokinetic vesicles within the[36_TD$IF] CPAM results in cell plate biogenesis and expansion.

Positioning of the CPAM appears to be related to the fact that the CPAM molecules are delivered from both sides of the phragmoplast and, owing presumably to their afﬁnity, assemble where they meet. However, the signal for exactly where the assembly occurs has yet to be identiﬁed. One hypothesis, based on research in Physcomitrella patens, is that CPAM location is determined by overlap of microtubules with opposite polarity (antiparallel microtubules; [27]). Overlapping microtubule plus ends that originate from opposite distal zones become bundled and stabilized by crosslinking protein microtubule-associated protein (MAP)65 and kinesin-4. Initial membrane depositions in the phragmoplast midzone colocalize with MAP65 proteins, which have been shown to interact genetically and/or physically with phospholipids and with the TRAPPII-tethering complex [26–28].

Although MAP65-dependent spatial organization of microtubules in the phragmoplast midzone has been supported by genetics [29–32], electron microscopy [30,33], immunoﬂuorescence [30,34], and live-cell imaging [29,35], using electron tomography no microtubule overlap was observed in the phragmoplast midzone of Arabidopsis root apical meristem cells [36]. Thus, more research is necessary to understand the role of antiparallel microtubule bundling and MAP65 proteins in the phragmoplast organization, CPAM formation, and subsequent stages of cell plate assembly.

Transmission electron microscopy and tomography data reveal four stages of cell plate assembly: (i) accumulation and fusion of vesicles; (ii) tubulovesicular network; (iii) tubular network; and (iv) fenestrated sheet [37]. The ﬁrst two stages take place inside the CPAM. Visualization of microtubules in the phragmoplast with established CPAM using live-cell imaging or immunoﬂuorescence microscopy reveals the phragmoplast midzone as a dark line (Figure S3A). Consistent with the light microscopy images, electron tomography recon- structions demonstrate that only 0.8% of phragmoplast microtubules terminate in the cell plate proximity, while 65% of microtubules terminate inside and 34% outside the CPAM [36], suggesting that the CPAM can capture and stabilize the plus ends of phragmoplast microtubules independently of the antiparallel microtubule overlap regions.

The diameter of the disk phragmoplast approximately equals that of the daughter nuclei. However, cells are generally wider than the nuclei and, in order to complete cell plate synthesis,

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the phragmoplast expands (translocates) centrifugally towards the CDZ and appears as a ring (Figures 1 and S3). Expansion proceeds as microtubules are disassembled in the inner region (lagging zone), and new microtubules polymerize at the outer phragmoplast edge (leading zone). Tubulin released in the lagging zone is recycled for the polymerization of new microtubules in the leading zone. Overlapping antiparallel microtubules crosslinked by MAP65 are continuously formed in the leading zone [38]. These overlap regions structurally stabilize the midzone and initiate CPAM formation where the next round of cell plate assembly takes place. Actin ﬁlaments are largely polymerized at the leading edge, extending out towards the cell cortex and actively attaching at the CDZ. Myosins on the plus ends of microtubules can interact with the actin meshwork thereby guiding phragmoplast expansion to the division site (as exempliﬁed by myosin VIII in P. patens; [39]). Within the central region of the microtubule ring, formed by the breakdown of microtubules in the central region of the disk phragmoplast and assembly of new microtubules at the leading zone, the growing cell plate becomes a callose- rich interconnected tubular network. Remodeling of membrane and cell plate material through clathrin-mediated endocytosis leads to the formation of the planar fenestrated sheet. All stages of cell plate assembly coexist in the expanding phragmoplast. Consequently, the spatiotem- poral distribution of individual membrane proteins that contribute to different cell plate assembly processes in the phragmoplast can be different ([25,40]; compare RAB-A2a and KNOLLE in Figure S2).

As the phragmoplast often forms off cell center, the attachment of the cell plate to the cell plate fusion site first occurs at one site and then progresses to other sites until the fusion is complete (Figures 1 and S3; [41]). Prior to cell plate attachment, peripheral phragmoplast microtubules at the leading zone interact with the cortex. Then finger-shaped membrane structures (fusion tubes) attach the cell plate to the cell plate fusion site [22,37]. This attachment could be important for stabilizing the position of the cell plate; however, it remains unknown whether attachment site selection within CDZ is stochastic or results from a polarized translocation of the phragmoplast towards a specific position at cell plate fusion site. Microtubules depolymer- ize at the attachment site, resulting in a discontinuous phragmoplast (Figures 1 and S3). What signals guide the centrifugal expansion remains unknown, but actin filaments have been shown to play a key role in guiding phragmoplast expansion and final positioning of the cell plate. Depolymerization of microtubules following cell plate fusion events results in fragments of phragmoplast scattered along the perimeter of the cell plate fusion site (Figure S3).

Upon cell plate fusion, the lumen of the cell plate is continuous with the apoplast and its physicochemical properties subsequently mature into primary cell wall. In addition, the attachment allows diffusion of plasma-membrane proteins and lipids from plasma membrane to the cell plate. These changes trigger a novel phase in cell plate assembly, which has been referred to as cell plate maturation. After attachment, the TRAPPII complex disappears from the cell plate and EXOCYST becomes the predominant tethering complex [24]. This marks a change in trafﬁcking to the cell plate. For example, TPLATE and myosin VIII have been shown to be speciﬁcally recruited to the cell plate only after attachment [42,43]. Cell plate maturation results in a cross-wall, which in addition to callose [37,40,44], contains cellulose, hemicellulose, and pectin [45–47]. The cross-wall gradually develops a pectin-rich middle lamella as the cellulose– hemicellulose networks are assembled on the opposite cell plate membranes. However the distinct stages of polysaccharide deposition during cell plate maturation and cross-wall transformation remain to be characterized [6]. Integration of the cross-wall with the parental[39_TD$IF] cell wall involves localized digestion of the cellulose layer of the parental[39_TD$IF] cell wall at the attachment site to enable the middle lamellae of the two walls to form a continuum. The formation of the cross-wall hallmarks completion of the cytokinesis and establishment of the two daughter cells.

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Concluding Remarks Outstanding Questions Exciting research of the past[367_TD$IF] decade has not only significantly boosted understanding of plant How are PPB, ADZ, and cortical divi- cytokinesis, but also demonstrated a high degree of complexity of the self-organization sion zones spatially and temporarily regulated? How are actin filaments processes responsible for both cell plate positioning and formation. The revised terminology depleted from the ADZ? What aims to capture this complexity and highlights emerging concepts in the plant cytokinesis field. changes in membrane proteins and Our[368_TD$IF] work will facilitate addressing the extensive gaps in our knowledge of plant cell cytokinesis lipids accompany CDZ formation? (see Outstanding Questions). Do these changes involve interactions with the cell wall?

Acknowledgments What factors define microtubule and Images of dividing BY-2 cells in Figure S3A were taken by Laining Zhang and Deirdre Fahy; images of TAN1 and tubulin actin filament organization and trigger were provided by Pablo Martinez. We thank Alex Steiner for support with the assembly of Figure[37_TD$IF] S2 panels. This work was their reorganization during phragmo- supported by USDA-NIFA hatch grant WNP00826 and WSU startup fund (to AS). EP is supported by the AUTh Research plast expansion? How and to what fi Committee (grant No. 91913)[370_TD$IF] by funds of Schur Flexibles Group. CR is supported by NSF-MCB#1505848. IM is supported extent do microtubules and actin laments need to cooperate to enable by BBSRC BB/I022996/1. JŠ is supported by grant No. LO1204 (Sustainable development of research in the Centre of the correct guidance of the phragmo- Region Haná) from the National Program of Sustainability I, MEYS. The authors are grateful to Tobias Baskin, Tijs Ketelaar, plast? 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