Narrowing of the preprophase microtubule band is not required for cell division plane determination in cultured plant cells

Summary. In most higher-plant cells, cortical microtubules form a tightly focused preprophase band (PPB) that disappears with the onset of prometaphase, but whose location defines the future location of the cell plate at the end of cytokinesis. It is unclear whether the PPB microtubules themselves designate the precise area where the cell plate will insert, or rather if these microtubules are responding to a hierarchical signal(s). Here we show that narrowing of the microtubules within the PPB zone is not necessary for proper division plane determination. In cultured tobacco BY-2 cells in which PPB microtubules are depolymerized, the phragmoplast can still accurately locate and insert at the proper site. The data do not support a role for PPB microtubule narrowing in focusing the signal that is used later by the phragmoplast to position the cell plate; rather, proper phragmoplast positioning is more likely a consequence of a non-microtubule positional element. Although the PPB microtubules do not directly mark the division site, we show that they are required for accurate spindle positioning, an activity that presets the future growth trajectory of the phragmoplast and is necessary for insuring high-fidelity cell plate positioning. Correspondence and reprints: Department of Biology, Pennsylvania State University, University Park, PA 16802, U.S.A. Present address: Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia, U.S.A.     

Live‐cell imaging reveals sustained centromere binding of CENP‐T via CENP‐A and CENP‐B

At the centromere, a network of proteins, the kinetochore, assembles in order to grant correct chromatin segregation. In this study the dynamics and molecular interactions of the inner kinetochore protein CENP‐T were analyzed employing a variety of fluorescence microscopy techniques in living human cells. Acceptor‐bleaching FRET indicates that CENP‐T directly associates with CENP‐A and CENP‐B. CENP‐T exchange into centromeres is restricted to the S‐phase of the cell cycle as revealed by FRAP, suggesting a coreplicational loading mechanism, as we have recently also demonstrated for CENP‐I. These properties make CENP‐T one of the basic inner kinetochore proteins with most further proteins binding downstream, suggesting a fundamental role of CENP‐T in kinetochore function. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Mechanisms of kinetochore‐microtubule attachment errors in mammalian oocytes

Proper kinetochore‐microtubule attachment is essential for correct chromosome segregation. Therefore, cells normally possess multiple mechanisms for the prevention of errors in kinetochore‐microtubule attachments and for selective stabilization of correct attachments. However, the oocyte, a cell that produces an egg through meiosis, exhibits a high frequency of errors in kinetochore‐microtubule attachments. These attachment errors predispose oocytes to chromosome segregation errors, resulting in aneuploidy in eggs. This review aims to provide possible explanations for the error‐prone nature of oocytes by examining key differences among other cell types in the mechanisms for the establishment of kinetochore‐microtubule attachments.     

Dual localized kinesin‐12 POK2 plays multiple roles during cell division and interacts with MAP65‐3

Kinesins are versatile nano‐machines that utilize variable non‐motor domains to tune specific motor microtubule encounters. During plant cytokinesis, the kinesin‐12 orthologs, PHRAGMOPLAST ORIENTING KINESIN (POK)1 and POK2, are essential for rapid centrifugal expansion of the cytokinetic apparatus, the phragmoplast, toward a pre‐selected cell plate fusion site at the cell cortex. Here, we report on the spatio‐temporal localization pattern of POK2, mediated by distinct protein domains. Functional dissection of POK2 domains revealed the association of POK2 with the site of the future cell division plane and with the phragmoplast during cytokinesis. Accumulation of POK2 at the phragmoplast midzone depends on its functional POK2 motor domain and is fine‐tuned by its carboxy‐terminal region that also directs POK2 to the division site. Furthermore, POK2 likely stabilizes the phragmoplast midzone via interaction with the conserved microtubule‐associated protein MAP65‐3/PLEIADE, a well‐established microtubule cross‐linker. Collectively, our results suggest that dual localized POK2 plays multiple roles during plant cell division.     

B1‐type cyclins control microtubule organization during cell division in Arabidopsis

Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development‐specific requirements of B1‐type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1‐type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1‐type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA‐TUBULIN COMPLEX PROTEIN 3‐INTERACTING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1‐type cyclin complexes and further genetic analyses support a potential role in the regulation of GIP1 by CYCB1s.     

The role of apical cell–cell junctions and associated cytoskeleton in mechanotransduction

Tissues of multicellular organisms are characterised by several types of specialised cell–cell junctions. In vertebrate epithelia and endothelia, tight and adherens junctions (AJ) play critical roles in barrier and adhesion functions, and are connected to the actin and microtubule cytoskeletons. The interaction between junctions and the cytoskeleton is crucial for tissue development and physiology, and is involved in the molecular mechanisms governing cell shape, motility, growth and signalling. The machineries which functionally connect tight and AJ to the cytoskeleton comprise proteins which either bind directly to cytoskeletal filaments, or function as adaptors for regulators of the assembly and function of the cytoskeleton. In the last two decades, specific cytoskeleton‐associated junctional molecules have been implicated in mechanotransduction, revealing the existence of multimolecular complexes that can sense mechanical cues and translate them into adaptation to tensile forces and biochemical signals. Here, we summarise the current knowledge about the machineries that link tight and AJ to actin filaments and microtubules, and the molecular basis for mechanotransduction at epithelial and endothelial AJ.     

Small ubiquitin-related modifier ligase activity of Mms21 is required for maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in Saccharomyces cerevisiae

The SUMO ligase activity of Mms21/Nse2, a conserved member of the Smc5/6 complex, is required for resisting extrinsically induced genotoxic stress. We report that the Mms21 SUMO ligase activity is also required during the unchallenged mitotic cell cycle in Saccharomyces cerevisiae. SUMO ligase-defective cells were slow growing and spontaneously incurred DNA damage. These cells required caffeine-sensitive Mec1 kinase-dependent checkpoint signaling for survival even in the absence of extrinsically induced genotoxic stress. SUMO ligase-defective cells were sensitive to replication stress and displayed synthetic growth defects with DNA damage checkpoint-defective mutants such as mec1, rad9, and rad24. MMS21 SUMO ligase and mediator of replication checkpoint 1 gene (MRC1) were epistatic with respect to hydroxyurea-induced replication stress or methyl methanesulfonate-induced DNA damage sensitivity. Subjecting Mms21 SUMO ligase-deficient cells to transient replication stress resulted in enhancement of cell cycle progression defects such as mitotic delay and accumulation of hyperploid cells. Consistent with the spontaneous activation of the DNA damage checkpoint pathway observed in the Mms21-mediated sumoylation-deficient cells, enhanced frequency of chromosome breakage and loss was detected in these mutant cells. A mutation in the conserved cysteine 221 that is engaged in coordination of the zinc ion in Loop 2 of the Mms21 SPL-RING E3 ligase catalytic domain resulted in strong replication stress sensitivity and also conferred slow growth and Mec1 dependence to unchallenged mitotically dividing cells. Our findings establish Mms21-mediated sumoylation as a determinant of cell cycle progression and maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in budding yeast.     

Formation of division spindles in higher plant meiosis

Depolymerisation of the MT cytoskeleton during late prophase makes it impossible to follow the cytoskeleton cycle in centrosomeless plant meiocytes. This paper describes rearrangements of the MT cytoskeleton during plant meiotic spindle formation in normally dividing pollen mother cells in various higher plant species and forms in which the cytoskeleton does not depolymerise at prophase. In such variants of the wild-type, cytoskeleton rearrangements can be observed at late prophase/early prometaphase. Radial MT bundles coalesce in the meridian plane, reorientate tangentially, curve and give rise to a developed ring-shaped perinuclear cytoskeleton system at the meridian. During nuclear envelope breakdown this ring disintegrates and splits into a set of free MT bundles. Three sub-stages of prometaphase are indicated: early prometaphase (disintegration of perinuclear ring and invasion of MTs into the former nuclear area), middle prometaphase or chaotic stage (formation of bipolar spindle fibres), and late prometaphase (formation of bipolar spindle). Analysis of a range of abnormal phenotypes (disintegrated, multiple, polyarchal, chaotic spindles) reveals two previously unknown processes during late prometaphase: axial orientation and consolidation of the spindle fibres.     

Effects of auxin on the spatial distribution of cell division and xylogenesis in lettuce pith explants

Summary Cylinders of pith parenchyma were tissue-cultured with their opposite ends on media which differed only in content of the morphogens auxin (IAA), sucrose, or zeatin. A range of concentrations of each of these morphogens applied at one end (none at the other end) resulted in distribution patterns of cell division and xylogenesis that were attributable to interaction between inductive levels and morphogen mobility. Auxin was crucial for tracheary patterns: large tracheary elements formed by direct differentiation of pith cells near the auxin source, smaller but still roughly isodiametric tracheary elements formed after cell division, and tracheary strands developed where, presumably, auxin transport had become polarized and then canalized. Xylogenesis was confined to regions within millimeters of the auxin source, and [¹⁴C]IAA studies showed a steep logarithmic concentration gradient along the cylinder. Patterns of tracheary strands and rings revealed that the pith explants retained some polarity from the stem from which they had been excised. However, the direction of flow of applied auxin was more effective than original polarity in controlling the orientation of tracheary strands and their constituent tracheary elements. It seems that, in tissues with little or no polarity, diffusive flow of auxin gradually induces polar flow in the same direction, together with an associated bioelectric current, and that this orients the cortical microtubules that in turn determine the orientations of cell elongation and of the secondary wall banding in tracheary elements.Abbreviations IAA indoleacetic acid - NAA naphthaleneacetic acid - TIBA triiodobenzoic acid Dedicated to the memory of Professor John G. Torrey     

Cell division in caffeine-induced binucleate protonemal cells ofAdiantum. II. Formation of the preprophase band and cell division in centrifuged and non-centrifuged cells

Organization of microtubules (MTs) in relation to the behavior of nuclei was examined in dividing binucleate cells ofAdiantum capillus-veneris L. To induce binucleate cells, caffeine, an inhibitor of formation of the cell plate, was applied at 4 mM to synchronously dividing protonemal cells during cytokinesis (Murata and Wada 1993). Formation of the preprophase band (PPB) during the next cell cycle was examined in non-centrifuged and centrifuged cells. The two nuclei were separated or associated with one another in both non-centrifuged and centrifuged cells, although the location of the nuclei in the cylindrical protonemal cells was different (Murata and Wada 1993). Irrespective of centrifugation, a single PPB was formed around the nuclei in cells with associated nuclei. Two PPBs were formed in cells with separated nuclei in centrifuged cells. Patterns of mitosis and cytokinesis varied, depending on the location of the PPB and the distribution of the nuclei. The role of the nucleus in formation of the PPB is discussed.     

Kinesin-7 CENP-E regulates cell division,gastrulation and organogenesis in development

Kinesin-7 CENP-E motor protein is essential for chromosome alignment and kinetochore-microtubule attachment in cell division. Human CENP-E has recently identified to be linked with the microcephalic primordial dwarfism syndromes associated with a smaller head, brain malformations and a prominent nose. However, the roles of CENP-E in embryonic development remain largely unknown. In this study, we find that zebrafish CENP-E inhibition results in defects in early zygote cleavage, including asymmetric cell division, cell cycle arrest and the developmental abnormalities. We also demonstrate that CENP-E ablation in cultured cells leads to chromosome misalignment, spindle abnormalities and interruptions of the cell cycle. These observations suggest that CENP-E plays a key role in early cell division and cell cycle progression. Furthermore, we also find that CENP-E inhibition results in the defects in the epiboly, the developmental arrest, the smaller head and the abnormal embryo during zebrafish embryogenesis. Our data demonstrate new functions of CENP-E in development and provide insights into its essential roles in organogenesis.     

Probing microtubule organizing centres with MPM-2 in dividing cells of higher plants using immunofluorescence and immunogold techniques

Summary The localization in higher plant cells of phosphorylated proteins recognized by the monoclonal antibody MPM-2 was investigated, with particular attention to putative microtubule organizing centres (MTOCs). Immunofluorescence and immunogold electron microscopy showed that MPM-2 did not localize with most putative MTOCs in cells and protoplasts of the gymnospermPicea glauca and in cells of the angiospermVicia faba. The distribution of phosphoproteins detected by MPM-2 was similar during mitosis in both species. At late interphase and early prophase MPM-2 preferentially labelled nucleoli and the region around the condensing chromosomes but not the cytoplasm. General labelling of the cytoplasm followed dissolution of the nuclear envelope and by prometaphase centromeres stained strongly. At metaphase and very early anaphase kinetochores stained strongly by immunofluorescence but only weakly using immunogold; spindle microtubules (MTs) showed little staining. Kinetochore staining disappeared during anaphase and by telophase centromeres and loose regions of chromatin in reforming nuclei were labelled. Treatment with the anti-microtubular drug amiprophosmethyl (APM) showed that the phosphorylation/dephosphorylation cycle detected by MPM-2 proceeded independently of the mitotic spindle. Staining of centromeres/kinetochores with MPM-2 suggests that phosphorylation and dephosphorylation of this region of mitotic chromosomes may be involved in chromosome organization, chromatid separation and MT nucleation and/or attachment.Abbreviations APM amiprophos-methyl - DAPI 4,6-diamidino-2-phenylindole - EGTA ethylene glycol-bis(-aminoethyl ether) - FITC fluorescein isothiocyanate - MT microtubule - MTOC microtubule organizing centre - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline - PBSB phosphate buffered saline with bovine serum albumin - PIPES piperazine-N,N-bis (2-ethanesulfonic acid) - PPB preprophase band - SPB spindle pole body - TRITC tetramethylrhodamine isothiocyanate     

不同温度下酿酒酵母细胞分裂周期蛋白Cdc5在有丝分裂中的分子动力学研究

[目的] 探究不同温度下酿酒酵母细胞分裂周期蛋白Cdc5蛋白在有丝分裂中的分子动力学变化。[方法] 本研究以酿酒酵母（Saccharomyces cerevisiae）为材料,采用活细胞成像的方法,探究Cdc5蛋白在不同温度下在酿酒酵母有丝分裂过程中的精细分子动力学变化;通过测量OD₅₉₅绘制生长曲线图,看其宏观的分裂情况是否与微观下Cdc5蛋白的分子动力学变化一致;利用流式细胞术检测细胞的细胞周期变化的情况。[结果] 在胞质分裂时,Cdc5蛋白从母细胞进入子细胞,并在芽颈处发生聚集。25℃条件下细胞中Cdc5蛋白在芽颈处的聚集时间长,37℃条件下Cdc5蛋白在芽颈处聚集时间短,两者间存在显著差异;但两个温度下,细胞中Cdc5蛋白的表达量没有显著性差异。同时,温度也会影响Cdc5蛋白在降解过程中的动力学行为,包括Cdc5蛋白在母细胞与子细胞中荧光强度峰值出现的次数和时间。生长曲线结果显示,酿酒酵母单一细胞分裂周期的变化影响了其宏观的细胞生长,且酵母分裂速度越快,子细胞长宽比越小;细胞周期结果表明,37℃下Cdc5蛋白的动力学变化与酿酒酵母细胞周期变化一致,酿酒酵母细胞周期从G₀/G₁期进入S期,亦加速了酿酒酵母的分裂。[结论] 本研究首次探究了不同温度下酿酒酵母有丝分裂中Cdc5蛋白的精细分子动力学及对应的酵母的宏观生长情况,结果表明温度会对Cdc5蛋白的动力学产生影响,且其精细分子动力学与酿酒酵母的分裂速度成正相关,该结果为进一步研究其在细胞有丝分裂中的功能提供了前期研究基础。     

Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.     

Pole reversal and the development of cell asymmetry during division in cryptomonad flagellates

Summary A unique form of cell division is reported for the cellsKomma caudata andCryptomonas ovata (Cryptophyceae). During cytokinesis, the posterior tail-like region of each daughter cell develops from the anterior region of the parental cell. This process, termed pole reversal, involves a major realignment in overall cell polarity as well as alterations to cytoplasmic and surface components. Pole reversal may be a consequence of flagellar apparatus transformation and reorientation during division, and pole reversal may facilitate the development of the asymmetric cell shape in daughter cells.     

Cytoskeletal dynamics ofApedinella radians (Pedinellophyceae) II. Cell division and maintenance of cell polarity and symmetry

Summary The dynamics of the cytoskeletal proteins centrin, actin, and tubulin were followed during cell division in the unicellular phytoflagellateApedinella radians (Pedinellophyceae). Three centrin, or centrin-like, components appear to coordinate independent developmental events during cell division. The first component, basal body centrin, maintains a physical link between basal bodies and the anterior nuclear membrane. Basal body centrin divides in two at metaphase, and each portion segregates with two basal bodies at anaphase. As the positioning of basal bodies defines the anterior region of the cell, basal body centrin appears to play a role in maintaining cell polarity throughout the cell cycle. The second centrin component consists of an array of filamentous bundles arranged as a six-pointed star. During cell division, the star undergoes a conformational change resulting in two distinct centrin triangles, one distributed to each daughter cell, suggesting that centrin filamentous bundles are involved in maintaining cell (radial) symmetry. The third centrin component is transient and associates with the spindle poles, emerging prior to mitosis and remaining until late anaphase/early telophase. Spindle pole centrin establishes temporary horizontal bipolarity, thereby establishing the spindle axis. Unlike centrin filamentous bundles, actin filamentous bundles depolymerize prior to mitosis, indicating they do not influence cell symmetry during cell division. Mitosis is described for the first time in a pedinellid and features a closed spindle, the absence of rhizoplasts and a persistent spindle.     

Ultrastructure of cell division in the marine red algaLomentaria baileyana

Summary Mitosis in the marine red algaLomentaria baileyana (Rhodymeniales, Rhodophyta) was studied with the electron microscope. Nucleus associated organelles known as polar rings (PRs) migrate to establish the division poles at prophase. At prometaphase, shallow invaginations in the nuclear envelope (NE) form on two sides of each PR and soon rupture. The gaps that are consequently formed contain several small fragments of NE. A larger region of NE remains intact between the two gaps. By metaphase several cisternae of perinuclear endoplasmic reticulum (PER) have enclosed most of the nucleus but remain absent from the polar regions. The nucleolus disperses partially and a typical metaphase plate of chromosomes is formed. Each PR has disjoined into separate proximal and distal portions. MTs converge widely on all regions of the polar area, but do not extend into the cytoplasm. Some MTs end near or at the chromosomes while others extend slightly farther past the chromosomes or diagonally to the NE. As chromosomes move to opposite poles at anaphase, they are accompanied by nucleolar material. An interzonal midpiece (IZM) is created as the pole to pole distance increases and the NE remains intact except for the polar gaps. Following detachment from the IZM, the daughter nuclei are separated by a large central vacuole as a cleavage furrow develops and eventually constricts to form two cells following pit connection formation. It is suggested that mitosis inLomentaria represents an evolutionary intermediate between that seen in the higher and lower groups of red algae. This conclusion is in agreement with conventional morphological and light microscopic criteria used to placeLomentaria in theRhodymeniales, which is considered to be the next to most advanced order in theRhodophyta.