Cortical Division Zone Research Articles

Plant cell division from the perspective of polarity.

The orientation of cell division is a major determinant of plant morphogenesis. In spite of considerable efforts over the past decades, the precise mechanism of division plane selection remains elusive. The majority of studies on the topic have addressed division orientation from either a predominantly developmental or a cell biological perspective. Thus, mechanistic insights into the links between developmental and cellular factors affecting division orientation are particularly lacking. Here, I review recent progress in the understanding of cell division orientation in the embryo and primary root meristem of Arabidopsis from both developmental and cell biological standpoints. I offer a view of multilevel polarity as a central aspect of cell division: on the one hand, the division plane is a readout of tissue- and organism-wide polarities; on the other hand, the cortical division zone can be seen as a transient polar subcellular plasma membrane domain. Finally, I argue that a polarity-focused conceptual framework and the integration of developmental and cell biological approaches hold great promise to unravel the mechanistic basis of plant cell division orientation in the near future.

Optimal BR signalling is required for adequate cell wall orientation in the Arabidopsis root meristem.

ABSTRACTPlant brassinosteroid hormones (BRs) regulate growth in part through altering the properties of the cell wall, the extracellular matrix of plant cells. Conversely, feedback signalling from the wall connects the state of cell wall homeostasis to the BR receptor complex and modulates BR activity. Here, we report that both pectin-triggered cell wall signalling and impaired BR signalling result in altered cell wall orientation in the Arabidopsis root meristem. Furthermore, both depletion of endogenous BRs and exogenous supply of BRs triggered these defects. Cell wall signalling-induced alterations in the orientation of newly placed walls appear to occur late during cytokinesis, after initial positioning of the cortical division zone. Tissue-specific perturbations of BR signalling revealed that the cellular malfunction is unrelated to previously described whole organ growth defects. Thus, tissue type separates the pleiotropic effects of cell wall/BR signals and highlights their importance during cell wall placement.

IQ67 DOMAIN proteins facilitate preprophase band formation and division-plane orientation.

Spatiotemporal control of cell division is essential for the growth and development of multicellular organisms. In plant cells, proper cell plate insertion during cytokinesis relies on the premitotic establishment of the division plane at the cell cortex. Two plant-specific cytoskeleton arrays, the preprophase band (PPB) and the phragmoplast, play important roles in division-plane orientation and cell plate formation, respectively1. Microtubule organization and dynamics and their communication with membranes at the cortex and cell plate are coordinated by multiple, mostly distinct microtubule-associated proteins2. How division-plane selection and establishment are linked, however, is still unknown. Here, we report members of the Arabidopsis IQ67 DOMAIN (IQD) family3 as microtubule-targeted proteins that localize to the PPB and phragmoplast and additionally reside at the cell plate and a polarized cortical region including the cortical division zone (CDZ). IQDs physically interact with PHRAGMOPLAST ORIENTING KINESIN (POK) proteins4,5 and PLECKSTRIN HOMOLOGY GTPase ACTIVATING (PHGAP) proteins6, which are core components of the CDZ1. The loss of IQD function impairs PPB formation and affects CDZ recruitment of POKs and PHGAPs, resulting in division-plane positioning defects. We propose that IQDs act as cellular scaffolds that facilitate PPB formation and CDZ set-up during symmetric cell division.

Plant Kinesin-12: Localization Heterogeneity and Functional Implications

Kinesin-12 family members are characterized by an N-terminal motor domain and the extensive presence of coiled-coil domains. Animal orthologs display microtubule plus-end directed motility, bundling of parallel and antiparallel microtubules, plus-end stabilization, and they play a crucial role in spindle assembly. In plants, kinesin-12 members mediate a number of developmental processes including male gametophyte, embryo, seedling, and seed development. At the cellular level, they participate in critical events during cell division. Several kinesin-12 members localize to the phragmoplast midzone, interact with isoforms of the conserved microtubule cross-linker MICROTUBULE-ASSOCIATED PROTEIN 65 (MAP65) family, and are required for phragmoplast stability and expansion, as well as for proper cell plate development. Throughout cell division, a subset of kinesin-12 reside, in addition or exclusively, at the cortical division zone and mediate the accurate guidance of the phragmoplast. This review aims to summarize the current knowledge on kinesin-12 in plants and shed some light onto the heterogeneous localization and domain architecture, which potentially conceals functional diversification.

Identification and characterization of the land-plant-specific microtubule nucleation factor MACET4.

Here, we show that the embryophyte (land-plant)-specific protein MACERATOR4 (MACET4) binds microtubules in vitro and in vivo, promotes microtubule polymerization at sub-critical tubulin concentrations, decreases the lag phase in microtubule bulk polymerization assays, and colocalizes with microtubule nucleation sites. Furthermore, we find that MACET4 forms oligomers that induce aster formation in vitro in a manner that is similar to aster formation mediated by centrosomes and TPX2. MACET4 is expressed during cell division and accumulates at the microtubule nucleation regions of the plant-specific cytokinetic microtubule array, the phragmoplast. We found that MACET4 localizes to the preprophase band and the cortical division zone, but not the spindle. MACET4 appears as cytoplasmic foci in vivo and forms octamers in vitro Transient expression in tobacco leaf pavement cells results in labeling of shrinking plus- and minus-ends. MACET4 facilitates microtubule depolymerization by increasing the frequency of catastrophes in vivo and by suppressing rescues in vitro Microtubules formed in the presence of MACET4 in vitro are shorter, most likely due to the depletion of the free tubulin pool. Accordingly, MACET4 knockdown results in longer phragmoplasts. We conclude that the direct activity of MACET4 is in promoting microtubule nucleation.This article has an associated First Person interview with the first author of the paper.

Displacement of the mitotic apparatuses by centrifugation reveals cortical actin organization during cytokinesis in cultured tobacco BY-2 cells

In plant cytokinesis, actin is thought to be crucial in cell plate guidance to the cortical division zone (CDZ), but its organization and function are not fully understood. To elucidate actin organization during cytokinesis, we employed an experimental system, in which the mitotic apparatus is displaced and separated from the CDZ by centrifugation and observed using a global-local live imaging microscope that enabled us to record behavior of actin filaments in the CDZ and the whole cell division process in parallel. In this system, returning movement of the cytokinetic apparatus in cultured-tobacco BY-2 cells occurs, and there is an advantage to observe actin organization clearly during the cytokinetic phase because more space was available between the CDZ and the distantly formed phragmoplast. Actin cables were clearly observed between the CDZ and the phragmoplast in BY-2 cells expressing GFP-fimbrin after centrifugation. Both the CDZ and the edge of the expanding phragmoplast had actin bulges. Using live-cell imaging including the global-local live imaging microscopy, we found actin filaments started to accumulate at the actin-depleted zone when cell plate expansion started even in the cell whose cell plate failed to reach the CDZ. These results suggest that specific accumulation of actin filaments at the CDZ and the appearance of actin cables between the CDZ and the phragmoplast during cell plate formation play important roles in the guidance of cell plate edges to the CDZ.

Putative RopGAPs impact division plane selection and interact with kinesin-12 POK1

Cell shape is defined by the surrounding cell walls in plants. Thus, spatial control over cell division planes and cell expansion polarity are essential to maintain cell morphology. In eukaryotes, cell polarity and expansion are controlled by Rho GTPase signalling, regulating cytoskeletal reorganization and vesicle trafficking(1). However, until now, Rho signalling was not implicated in mitotic events in plants. Here, we report a pair of putative Rho GTPase activating proteins (RhoGAPs) that interact with the mitosis-specific kinesin-12 POK1, a core component of the cortical division zone/site (CDZ/CDS) that is required for division plane maintenance in Arabidopsis(2-4). The designated pleckstrin homology GAPs (PHGAPs) are cytoplasmic and plasma membrane associated in interphase, but during mitosis they additionally localize to the CDZ/CDS in a POK-dependent manner. In contrast to pok1 pok2 mutants, phgap1 phgap2 double mutants show moderate cell wall positioning defects as a consequence of inaccurate positioning of the cortical division zone marker POK1. We conclude that loss of PHGAP function interferes with division plane selection in proliferative cell divisions.

Preprophase band formation and cortical division zone establishment: RanGAP behaves differently from microtubules during their band formation

Correct positioning of the division plane is a prerequisite for plant morphogenesis. The preprophase band (PPB) is a key intracellular structure of division site determination. PPB forms in G2 phase as a broad band of microtubules (MTs) that narrows in prophase and specializes few-micrometer-wide cortical belt region, named the cortical division zone (CDZ), in late prophase. The PPB comprises several molecules, some of which act as MT band organization and others remain in the CDZ marking the correct insertion of the cell plate in telophase. Ran GTPase-activating protein (RanGAP) is accumulated in the CDZ and forms a RanGAP band in prophase. However, little is known about when and how RanGAPs gather in the CDZ, and especially with regard to their relationships to MT band formation. Here, we examined the spatial and temporal distribution of RanGAPs and MTs in the preprophase of onion root tip cells using confocal laser scanning microscopy and showed that the RanGAP band appeared in mid-prophase as the width of MT band was reduced to nearly 7 µm. Treatments with cytoskeletal inhibitors for 15 min caused thinning or broadening of the MT band but had little effects on RanGAP band in mid-prophase and most of late prophase cells. Detailed image analyses of the spatial distribution of RanGAP band and MT band showed that the RanGAP band positioned slightly beneath the MT band in mid-prophase. These results raise a possibility that RanGAP behaves differently from MTs during their band formation.

Arabidopsis KCBP interacts with AIR9 but stays in the cortical division zone throughout mitosis via its MyTH4-FERM domain.

The preprophase band of microtubules performs the crucial function of marking the plane of cell division. Although the preprophase band depolymerises at the onset of mitosis, the division plane is 'memorized' by a cortical division zone to which the phragmoplast is attracted during cytokinesis. Proteins have been discovered that are part of the molecular memory but little is known about how they contribute to phragmoplast guidance. Previously, we found that the microtubule-associated protein AIR9 is found in the cortical division zone at preprophase and returns during cell plate insertion but is absent from the cortex during the intervening mitosis. To identify new components of the preprophase memory, we searched for proteins that interact with AIR9. We detected the kinesin-like calmodulin-binding protein, KCBP, which can be visualized at the predicted cortical site throughout division. A truncation study of KCBP indicates that its MyTH4-FERM domain is required for linking the motor domain to the cortex. These results suggest a mechanism by which minus-end-directed KCBP helps guide the centrifugally expanding phragmoplast to the cortical division site.

3D image analysis of plants using electron tomography and micro-CT.

Precise control of the cell division plane is a prerequisite for plant development. The division site (the position of the division plane insertion) in plant cells is the site along which the cell plate margin joins the parental cell walls. How this division site is determined and established during cell division is an essential question in plant morphogenesis. Herein we demonstrate how computer tomography techniques can aid in understanding nano-machines involved in determination of the division site and in analysing air space development after cytokinesis. The preprophase band (PPB) is a cytokinetic nano-machine used to determine the plant division site. The PPB appears as a broad microtubule (MT) band in the G2 phase and the MT band narrows during the prophase to establish the specialized belt zone in the cell cortex (cortical division zone, [CDZ]). The MT band disappears at prometaphase, but some memories remain in the CDZ throughout the process of cell division, and this is the site of attachment of the newly formed cell plate. We have examined PPB development of high-pressure frozen onion cotyledon epidermis using dual-axis electron tomography. MTs as well as actin microfilaments (MFs) and membrane systems can be preserved well by high-pressure freezing [1]. Since detection of ∼100 vesicles and ∼40 MT ends was possible in a tomogram of the PPB surface (0.25 mm × 0.25 mm) obtained from 250-nm-thick tangential sections of epidermal cells, we were able to quantitatively analyze the frequencies of various types of vesicles and MT ends in the PPB [2]. The results clearly showed that endocytosis is active [2,3] and MTs are very dynamic in the late PPB. Light microscopic studies with fluorescent probes have demonstrated that actins are among the main components of PPB. Electron tomography analysis showed that one actin configuration in the PPB is a relatively short single MFs running parallel to the plasma membrane. The actin MFs connecting two adjacent MTs help to promote MT bundling. Cell plate attachment to the parental wall leads to the fusion of the newly formed middle lamellae in the cell plate to the middle lamella of parental cell wall, and a three-way junction is created. Air space develops from the three-way junction. To determine 3D arrangement of cells and air spaces, we used X-ray micro-CT at the SPring-8 synchrotron radiation facility. Using micro-CT available in BL20XU (8 keV, 0.2 µm/pixel), we were able to elucidate ∼90% of the cortical cell outlines in the hypocotyl-radicle axis of arabidopsis seeds [4] and to analyze cell geometrical properties. As the strength of the system X-ray is too strong for seed survival, we used another beam line BL20B2 (10-15 keV, 2.4-2.7 µm/pixel) to examine air space development during seed imbibition [4,5]. Using this system, we were able to detect air space development at the early imbibition stages of seeds without causing damage during seed germination. AcknowledgmentThe author would like to thank Dr. Ichirou Karahara (Univ. Toyama), Dr. L. Andrew Staehelin (Univ. Colorado), Ms. Naoko Kajimura, Dr. Akio Takaoka (Osaka Univ.), Dr. Kazuyo Misaki, Dr. Shigenobu Yonemura (RIKEN CDB), Dr. Kazuyoshi Murata (NIP), Dr. Kentaro Uesugi, Dr. Akihisa Takeuchi, Dr. Yoshio Suzuki (JASRI), Dr. Miyuki Takeuchi, Dr. Daisuke Tamaoki, Dr. Daisuke Yamauchi, and Ms. Aki Fukuda (Univ. Hyogo) for their collaborations in the work presented here.

The Phragmoplast-Orienting Kinesin-12 Class Proteins Translate the Positional Information of the Preprophase Band to Establish the Cortical Division Zone in Arabidopsis thaliana.

The preprophase band (PPB) is a faithful but transient predictor of the division plane in somatic cell divisions. Throughout mitosis the PPBs positional information is preserved by factors that continuously mark the division plane at the cell cortex, the cortical division zone, by their distinct spatio-temporal localization patterns. However, the mechanism maintaining these identity factors at the plasma membrane after PPB disassembly remains obscure. The pair of kinesin-12 class proteins PHRAGMOPLAST ORIENTING KINESIN1 (POK1) and POK2 are key players in division plane maintenance. Here, we show that POK1 is continuously present at the cell cortex, providing a spatial reference for the site formerly occupied by the PPB. Fluorescence recovery after photobleaching analysis combined with microtubule destabilization revealed dynamic microtubule-dependent recruitment of POK1 to the PPB during prophase, while POK1 retention at the cortical division zone in the absence of cortical microtubules appeared static. POK function is strictly required to maintain the division plane identity factor TANGLED (TAN) after PPB disassembly, although POK1 and TAN recruitment to the PPB occur independently during prophase. Together, our data suggest that POKs represent fundamental early anchoring components of the cortical division zone, translating and preserving the positional information of the PPB by maintaining downstream identity markers.

Potential roles for Kinesins at the cortical division site.

Spatial control of cytokinesis is critical for cell and plant morphology. The plane of cell division is established at G2/M transition and is initially demarcated at the cortex of the cell by the cytoskeletal preprophase band (PPB) and subsequently throughout mitosis by the cortical division zone (CDZ). Few kinesins, belonging to different classes of the superfamily, either display a distinct spatio-temporal localization at the PPB and CDZ, or genetic evidence proposes a specific function there. Protein phosphorylation and degradation are likely directing the cell cycle-dependent localization and activity of some of these kinesins, as indicated by mutation of respective conserved motifs. Furthermore, kinesins are required for continuous recruitment of CDZ identity markers to the CDZ. This review summarizes the limited current knowledge of kinesins potentially involved in the steps required for correctly oriented division planes, considering localization patterns and genetic evidence, and discussing kinesin function in context with interaction partners and cell cycle regulation.

Universal rules for division plane selection in plants

Coordinated cell divisions and cell expansion are the key processes that command growth in all organisms. The orientation of cell divisions and the direction of cell expansion are critical for normal development. Symmetric divisions contribute to proliferation and growth, while asymmetric divisions initiate pattern formation and differentiation. In plants these processes are of particular importance since their cells are encased in cellulosic walls that determine their shape and lock their position within tissues and organs. Several recent studies have analyzed the relationship between cell shape and patterns of symmetric cell division in diverse organisms and employed biophysical and mathematical considerations to develop computer simulations that have allowed accurate prediction of cell division patterns. From these studies, a picture emerges that diverse biological systems follow simple universal rules of geometry to select their division planes and that the microtubule cytoskeleton takes a major part in sensing the geometric information and translates this information into a specific division outcome. In plant cells, the division plane is selected before mitosis, and spatial information of the division plane is preserved throughout division by the presence of reference molecules at a distinct region of the plasma membrane, the cortical division zone. The recruitment of these division zone markers occurs multiple times by several mechanisms, suggesting that the cortical division zone is a highly dynamic region.

Adaptin-like protein TPLATE and clathrin recruitment during plant somatic cytokinesis occurs via two distinct pathways

Plant cytokinesis deploys a transport system that centers cell plate-forming vesicles and fuses them to form a cell plate. Here we show that the adaptin-like protein TPLATE and clathrin light chain 2 (CLC2) are targeted to the expanding cell plate and to the equatorial subregion of the plasma membrane referred to as the cortical division zone (CDZ). Bimolecular fluorescence complementation and immunodetection indicates that TPLATE interacts with clathrin. Pharmacological tools as well as analysis of protein targeting in a mutant background affecting cell plate formation allowed to discriminate two recruitment pathways for TPLATE and CLC2. The cell plate recruitment pathway is dependent on phragmoplast microtubule organization and the formation and transport of secretory vesicles. The CDZ recruitment pathway, on the other hand, is activated at the end of cytokinesis and independent of trans-Golgi-derived vesicle trafficking. TPLATE and CLC2 do not accumulate at a narrow zone central of the CDZ. We have dubbed this subdomain the cortical division site and show that it corresponds precisely with the position where the cell plate merges with the parental wall. These data provide evidence that the plasma membrane is subject to localized endocytosis or membrane remodeling processes that are required for the fusion of the cell plate with a predefined region of the plasma membrane.

Demarcation of the cortical division zone in dividing plant cells

Somatic cytokinesis in higher plants involves, besides the actual construction of a new cell wall, also the determination of a division zone. Several proteins have been shown to play a part in the mechanism that somatic plant cells use to control the positioning of the new cell wall. Plant cells determine the division zone at an early stage of cell division and use a transient microtubular structure, the preprophase band (PPB), during this process. The PPB is formed at the division zone, leaving behind a mark that during cytokinesis is utilized by the phragmoplast to guide the expanding cell plate toward the correct cortical insertion site. This review discusses old and new observations with regard to mechanisms implicated in the orientation of cell division and determination of a cortical division zone.