Microtubule-associated AIR9 recognizes the cortical division site at preprophase and cell-plate insertion

In plants, the preprophase band (PPB) of microtubules marks the cortical site where the cross-wall will fuse with the parental wall during cytokinesis . This band disappears before metaphase, and it is not known how the division plane is "memorized". One idea is that the PPB leaves behind molecules involved in the maturation of the cell plate . Here, we report on the proteomic isolation of a novel 187 kDa microtubule-associated protein, AIR9, conserved in land plants and trypanosomatid parasites. AIR9 decorates cortical microtubules and the PPB but is downregulated during mitosis. AIR9 reappears at the former PPB site precisely when the cortex is contacted by the outwardly growing cytokinetic apparatus. AIR9 then moves inward on the new cross-wall and thus forms a torus. Truncation studies show that formation of the torus requires a repeated domain separate from AIR9's microtubule binding site. Cell plates induced to insert outside the predicted division site do not elicit an AIR9 torus, suggesting that AIR9 recognizes a component of the former PPB. Such misplaced walls remain immature, based on their prolonged staining for the cell-plate polymer callose. We propose that AIR9 may be part of the mechanism ensuring the maturation of those cell plates successfully contacting the "programmed" cortical division site.     

The effect of taxol onTriticum preprophase root cells: preprophase microtubule band organization seems to depend on new microtubule assembly

Summary To examine whether preprophase microtubule band (PPB) organization occurs by rearrangement of pre-existing, or by assembly of new microtubules (Mts), we treated root cells ofTriticum turgidum with taxol, which stabilizes pre-existing Mts by slowing their depolymerization. With taxol early preprophase cells failed to form a normal PPB and PPB narrowing was prevented in cells that had already formed a wide one. The PPB became persistent in prometaphase cells and the formation of multipolar prophase-prometaphase spindles was induced. These data favour the suggestion that PPB formation and narrowing, as well as prophase spindle development, are dynamic processes depending on continuous Mt assembly at the PPB site and in the perinuclear cytoplasm.Abbreviations Mt microtubule - MTOC microtubule organizing centre - PPB preprophase microtubule band - DMSO dimethyl sulfoxide     

Arabidopsis TANGLED identifies the division plane throughout mitosis and cytokinesis

BACKGROUND: In premitotic plant cells, the future division plane is predicted by a cortical ring of microtubules and F-actin called the preprophase band (PPB). The PPB persists throughout prophase, but is disassembled upon nuclear-envelope breakdown as the mitotic spindle forms. Following nuclear division, a cytokinetic phragmoplast forms between the daughter nuclei and expands laterally to attach the new cell wall at the former PPB site. A variety of observations suggest that expanding phragmoplasts are actively guided to the former PPB site, but little is known about how plant cells "remember" this site after PPB disassembly. RESULTS: In premitotic plant cells, Arabidopsis TANGLED fused to YFP (AtTAN::YFP) colocalizes at the future division plane with PPBs. Strikingly, cortical AtTAN::YFP rings persist after PPB disassembly, marking the division plane throughout mitosis and cytokinesis. The AtTAN::YFP ring is relatively broad during preprophase/prophase and mitosis; narrows to become a sharper, more punctate ring during cytokinesis; and then rapidly disassembles upon completion of cytokinesis. The initial recruitment of AtTAN::YFP to the division plane requires microtubules and the kinesins POK1 and POK2, but subsequent maintenance of AtTAN::YFP rings appears to be microtubule independent. Consistent with the localization data, analysis of Arabidopsis tan mutants shows that AtTAN plays a role in guidance of expanding phragmoplasts to the former PPB site. CONCLUSIONS: AtTAN is implicated as a component of a cortical guidance cue that remains behind when the PPB is disassembled and directs the expanding phragmoplast to the former PPB site during cytokinesis.     

Actomyosin is Involved in the Organization of the Microtubule Preprophase Band in Arabidopsis Suspension Cultured Cells

The microtubule preprophase bands （PPBs） participate in the sequence of events to position cell plates in most plants. However, the mechanism of PPB formation remains to be clarified. In the present study, the organization of PPBs in Arabidopsis suspension cultured cells was investigated by confocal laser scanning microscopy combined with pharmacological treatments of reagents specific for the cytoskeleton elements. Double staining of F-actin and microtubules （MTs） showed that actin filaments were arranged randomly and no colocalization with cortical MTs was observed in the interphase cells. However, cortical actin filaments showed colocalization with MTs during the formation of PPBs. A broad actin band formed with the broad MT band in the initiation of PPB and narrowed down together with the MT band to form the PPB. Nevertheless, broad MT bands were formed but failed to narrow down in cells treated with the F-actin disruptor latrunculin A. In contrast, in the presence of the F-actin stabilizer phalloidin, PPB formation did not exhibit any abnormality. Therefore, the integrity, but not the dynamics, of the actin cytoskeleton is necessary for the formation of normal PPBs. Treatment with 2, 3-butanedine monoxime, a myosin inhibitor, also resulted in the formation of broad MT bands, indicating that actomyosin may be involved in the rearrangement of MTs to form the PPBs. Double staining of MTs and myosin revealed that myosin concentrated on the PPB region during PPB formation. It is suggested that the actin cytoskeleton at the PPB site may serve as a rack to transport cortical MTs by using myosin when the broad MT band narrows down to form the PPB.     

Microtubules Orient the Mitotic Spindle in Yeast through Dynein-dependent Interactions with the Cell Cortex

Proper orientation of the mitotic spindle is critical for successful cell division in budding yeast. To investigate the mechanism of spindle orientation, we used a green fluorescent protein (GFP)–tubulin fusion protein to observe microtubules in living yeast cells. GFP–tubulin is incorporated into microtubules, allowing visualization of both cytoplasmic and spindle microtubules, and does not interfere with normal microtubule function. Microtubules in yeast cells exhibit dynamic instability, although they grow and shrink more slowly than microtubules in animal cells. The dynamic properties of yeast microtubules are modulated during the cell cycle. The behavior of cytoplasmic microtubules revealed distinct interactions with the cell cortex that result in associated spindle movement and orientation. Dynein-mutant cells had defects in these cortical interactions, resulting in misoriented spindles. In addition, microtubule dynamics were altered in the absence of dynein. These results indicate that microtubules and dynein interact to produce dynamic cortical interactions, and that these interactions result in the force driving spindle orientation.     

Ameiotic, a gene that controls meiotic chromosome and cytoskeletal behavior in maize.

Microtubule organization during the novel cell division of ameiotic microsporocytes was examined using indirect immunofluorescence microscopy. A recessive mutation of the maize gene Ameiotic causes the replacement of meiosis I with a synchronized mitotic division (Palmer, R. G. (1971). Chromosoma 35, 233-246). All identifiable cytological features of this division, including chromosome behavior and microtubule organization, were typical of somatic cell division. Significantly, a cortical microtubule band was observed during prophase in ameiotic cells. In most somatic plant cells, a preprophase band of microtubules (PPB) predicts the cortical site where the future cell plate will join the sidewall. Similar structures, however, are absent in all meiotic and postmeiotic reproductive cells examined to date. These disruptions are consistent with a model where the wild-type Ameiotic gene encodes a product which acts during or before G2 and is necessary for initiating several independent meiotic processes, including both meiotic chromosome behavior and microtubule organization. The ameiotic mutation provides additional evidence that aspects of cytoskeletal organization unique to meiosis are genetically controlled. Finally, the presence of a PPB during the ameiotic division supports a model whereby multiple mechanisms are used to determine and maintain division plane polarity during normal meiosis.     

Live-cell analysis of mitotic spindle formation in taxol-treated cells

Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-alpha tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 "dynamitin" does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible-but before NEB is complete-results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation.     

Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration

Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. Here we show that the Drosophila katanin Dm-Kat60 functions to generate a dynamic cortical-microtubule interface in interphase cells. Dm-Kat60 concentrates at the cell cortex of S2 Drosophila cells during interphase, where it suppresses the polymerization of microtubule plus-ends, thereby preventing the formation of aberrantly dense cortical arrays. Dm-Kat60 also localizes at the leading edge of migratory D17 Drosophila cells and negatively regulates multiple parameters of their motility. Finally, in vitro, Dm-Kat60 severs and depolymerizes microtubules from their ends. On the basis of these data, we propose that Dm-Kat60 removes tubulin from microtubule lattice or microtubule ends that contact specific cortical sites to prevent stable and/or lateral attachments. The asymmetric distribution of such an activity could help generate regional variations in microtubule behaviours involved in cell migration.     

Microtubule reorganization accompanying preprophase band formation in guard mother cells ofAvena sativa L.

Summary In order to study developmental changes in microtubule organization attending the formation of a longitudinally oriented preprophase band, the guard mother cells ofAvena were examined using a new procedure for anti-tubulin immunocytochemistry on large epidermal segments. We found that the interphase band (IMB) of transverse cortical microtubules present in these cells following asymmetric division is replaced after subsidiary cell formation by mesh-like to radial microtubules that extend throughout the cytoplasm. Many of the Mts are also grouped in bundles. Gradually, this intermediate array is succeeded by longitudinal elements of the PPB. Thus, preprophase band formation is accompanied by a 90° shift in Mt orientation, with a radial arrangement serving as an intermediate stage. The micrographs are most consistent with the rearrangement of intact Mts, although changes in Mt assembly are possible as well. The role of the IMB in guard mother cells is also discussed.Abbreviations GMC guard mother cell - IMB interphase microtubule band - Mt microtubule - PPB preprophase band     

Establishment of polarity during organization of the acentrosomal plant cortical microtubule array

The plant cortical microtubule array is a unique acentrosomal array that is essential for plant morphogenesis. To understand how this array is organized, we exploited the microtubule (+)-end tracking activity of two Arabidopsis EB1 proteins in combination with FRAP (fluorescence recovery after photobleaching) experiments of GFP-tubulin to examine the relationship between cortical microtubule array organization and polarity. Significantly, our observations show that the majority of cortical microtubules in ordered arrays, within a particular cell, face the same direction in both Arabidopsis plants and cultured tobacco cells. We determined that this polar microtubule coalignment is at least partially due to a selective stabilization of microtubules, and not due to a change in microtubule polymerization rates. Finally, we show that polar microtubule coalignment occurs in conjunction with parallel grouping of cortical microtubules and that cortical array polarity is progressively enhanced during array organization. These observations reveal a novel aspect of plant cortical microtubule array organization and suggest that selective stabilization of dynamic cortical microtubules plays a predominant role in the self-organization of cortical arrays.     

Mechanisms of Self-Organization of Cortical Microtubules in Plants Revealed by Computational Simulations

Microtubules confined to the two-dimensional cortex of elongating plant cells must form a parallel yet dispersed array transverse to the elongation axis for proper cell wall expansion. Some of these microtubules exhibit free minus-ends, leading to migration at the cortex by hybrid treadmilling. Collisions between microtubules can result in plus-end entrainment (“zippering”) or rapid depolymerization. Here, we present a computational model of cortical microtubule organization. We find that plus-end entrainment leads to self-organization of microtubules into parallel arrays, whereas catastrophe-inducing collisions do not. Catastrophe-inducing boundaries (e.g., upper and lower cross-walls) can tune the orientation of an ordered array to a direction transverse to elongation. We also find that changes in dynamic instability parameters, such as in mor1-1 mutants, can impede self-organization, in agreement with experimental data. Increased entrainment, as seen in clasp-1 mutants, conserves self-organization, but delays its onset and fails to demonstrate increased ordering. We find that branched nucleation at acute angles off existing microtubules results in distinctive sparse arrays and infer either that microtubule-independent or coparallel nucleation must dominate. Our simulations lead to several testable predictions, including the effects of reduced microtubule severing in katanin mutants.     

Dynamic Behavior of Microtubules during Dynein-dependent Nuclear Migrations of Meiotic Prophase in Fission Yeast

During meiotic prophase in fission yeast, the nucleus migrates back and forth between the two ends of the cell, led by the spindle pole body (SPB). This nuclear oscillation is dependent on astral microtubules radiating from the SPB and a microtubule motor, cytoplasmic dynein. Here we have examined the dynamic behavior of astral microtubules labeled with the green fluorescent protein during meiotic prophase with the use of optical sectioning microscopy. During nuclear migrations, the SPB mostly follows the microtubules that extend toward the cell cortex. SPB migrations start when these microtubules interact with the cortex and stop when they disappear, suggesting that these microtubules drive nuclear migrations. The microtubules that are followed by the SPB often slide along the cortex and are shortened by disassembly at their ends proximal to the cortex. In dynein-mutant cells, where nuclear oscillations are absent, the SPB never migrates by following microtubules, and microtubule assembly/disassembly dynamics is significantly altered. Based on these observations, together with the frequent accumulation of dynein at a cortical site where the directing microtubules interact, we propose a model in which dynein drives nuclear oscillation by mediating cortical microtubule interactions and regulating the dynamics of microtubule disassembly at the cortex.     

Cortical microtubule labeling: Insight of AFH14 in non-dividing cells

We recently reported that AFH14 participated in microtubule and actin filament interaction in cell division, and the AFH14 (FH1FH2) was important to the directly binding activity of microtubules and microfilaments. To preliminarily understand the function and localization of AFH14 in non-dividing cells, we overexpressed FH1FH2-RFP in onion epidermal cells, and found a fluorescence labeled filamentous network. The result of double labeling with different cytoskeleton reporter proteins indicated that FH1FH2-RFP co-localized with cortical microtubules. Treatment of cells expressing FH1FH2-RFP with cytoskeleton disrupting drugs confirms that FH1FH2-RFP binds to microtubules. Moreover, the binding of FH1FH2-RFP to microtubules were revealed to be dynamic by fluorescence recovery after photobleaching (FRAP) experiment. Time-lapse confocal microscopy showed that FH1FH2-RFP could display a dynamics similar to the microtubule dynamic instability. These data suggest that FH1FH2 domain may lead AFH14 function on cortical microtubules in non-dividing cells, and FH1FH2-RFP may be utilized as a microtubule reporter protein in living onion epidermal cells.Key words: cortical microtubule, AFH14, non-dividing cell, microtubule dynamics, FRAP     

Gamma-tubulin distribution during cortical microtubule reorganization at the M/G1 interface in tobacco BY-2 cells

Cortical microtubules are considered to regulate the direction of cellulose microfibril deposition. Despite their significant role in determining cell morphology, cortical microtubules completely disappear from the cell cortex during M phase and become reorganized at G1 phase. The mechanism by which these microtubules become properly formed again is, however, still unclear. We have proposed that the origin of cortical microtubules is on the daughter nuclear surface, but further cortical microtubule reorganization occurs at the cell cortex. Hence it is probable that the locations of microtubule organizing centers (MTOCs) are actively changing. However, the actual MTOC sites of cortical microtubules were not clearly determined. In this paper, we have examined the distribution of gamma-tubulin, one of the key molecules of MTOCs in various organisms, during cortical microtubule reorganization using both immunofluorescence and a GFP reporter system. Using a monoclonal antibody (clone G9) that recognizes highly conserved residues in y-tubulin, y-tubulin was found to be constitutively expressed and to be clearly localized to microtubule structures, such as the preprophase bands, spindles, and phragmoplasts, specific to each cell cycle stage. This distribution pattern was confirmed by the GFP reporter system. During cortical microtubule reorganization at the M to G1 transition phase, gamma-tubulin first accumulated at the daughter nuclear surfaces, and then seemed to spread onto the cell cortex along with microtubules elongating from the daughter nuclei. Based on the results, it was confirmed that daughter nuclear surfaces acted as origins of cortical microtubules, and that further reorganization occurred on the cell cortex.     

Evidence for a role of capillary pericytes in vascular growth of the developing ovine corpus luteum

The role of microfilaments, microtubules, and mitogen-activated protein (MAP) kinase in regulation of several important dynamic events of porcine oocyte maturation and fertilization is described. Fluorescently labeled microfilaments, microtubules, and cortical granules were visualized using either epifluorescence microscopy or laser scanning confocal microscopy. Mitogen-activated protein kinase phosphorylation was revealed by Western immunoblotting. We showed that 1) microfilament disruption did not affect meiosis resumption and metaphase I meiotic apparatus formation but inhibited further cell cycle progression (chromosome separation) even though MAP kinase was phosphorylated; 2) cortical granule (CG) migration was driven by microfilaments (but not microtubules), and once the chromosomes and CGs were localized beneath the oolemma their anchorage to the cortex was independent of either microfilaments or microtubules; 3) neither microfilaments nor microtubules were involved in CG exocytosis during oocyte activation; 4) sperm incorporation was mediated by microfilaments, while pronuclear (PN) syngamy was controlled by microtubules rather than microfilaments; 5) spindle microtubule organization was temporally correlated with MAP kinase phosphorylation, while the extensive microtubule organization in the sperm aster that is required for PN apposition and syngamy occurred in the absence of MAP kinase activation; and 6) MAP kinase phosphorylation did not change either when microtubules were disrupted by nocodazole or when cytoplasmic microtubule asters were induced by taxol. The present study suggests that the role of the cytoskeleton during porcine oocyte maturation is similar to that of rodents, while the mechanisms of fertilization in pig resemble those of lower vertebrates.     

The microtubule cycle during successive mitotic waves in the syncytial female gametophyte of ginkgo

Plant morphogenesis is driven by a surprising number of microtubule arrays. The four arrays of vegetative tissues are hoop-like cortical, preprophase band (PPB), spindle, and phragmoplast. When syncytia occur during the reproductive phase of the plant life cycle, neither hoop-like corticals nor PPBs are present, and functional phragmoplasts fail to form following the proliferative mitoses that give rise to the multinucleate cytoplasm. Instead, the interphase microtubules are radial microtubule systems (RMSs) that emanate from the nuclei. These RMSs organize the cytoplasm into nascent cells and ultimately trigger phragmoplast formation at their boundaries. During investigations of the syncytial stage that initiates development of the female gametophyte in gymnosperms, we studied the large (3–4 mm) female gametophyte of Ginkgo biloba. Here we describe the microtubule cycle correlated with successive mitotic waves and discuss the importance of this system in studying the acentrosomal nucleation and organization of cycling microtubule arrays. Electronic Publication     

Preprophasic microtubule systems and development of the mitotic spindle in hornworts (Bryophyta)

Summary Studies of monoplastidic mitosis in hornworts (Bryophyta) using transmission electron microscopy and indirect immunofluorescence staining of microtubules have revealed that two mutually perpendicular microtubule systems predict division polarity in preprophase. Events of cytoplasmic reorganization in preparation for division occur in the following order: migration of the single plastid to a position perpendicular to the division site, constriction of the plastid where its midpoint intersects the division site, development of an axial system of microtubules parallel to the elongating plastid isthmus, and appearance of an atypical preprophase band of microtubules (PPB). The PPB is asymmetrical with a tight band of microtubules on the side over the plastid isthmus and a broad band of widely spaced microtubules over the nucleus. The axial system contributes directly to development of the spindle. In prometaphase, the axial system separates at the equator and additional microtubule bundles project from polar regions, creating two opposing halfspindles. The PPB is still present during asymmetrical organization of the spindle and microtubules extending from the broad portion of the PPB to poles appear to be incorporated into the developing spindle. Dynamic changes in the microtubular cytoskeleton demonstrate (1) intimate relationship of plastid and nuclear division, (2) contribution of preprophase/prophase microtubule systems to spindle development in monoplastidic cells, and (3) dynamic reorientation of microtubules from one system to another.     

The contributions of microtubule stability and dynamic instability to adenovirus nuclear localization efficiency

Adenoviruses (Ads) utilize host cell microtubules to traverse the intracellular space and reach the nucleus in a highly efficient manner. Previous studies have shown that Ad infection promotes the formation of stable, posttranslationally modified microtubules by a RhoA-dependent mechanism. Ad infection also shifts key parameters of microtubule dynamic instability by a Rac1-dependent mechanism, resulting in microtubules with lower catastrophe frequencies, persistent growth phases, and a bias toward net growth compared to microtubules in uninfected cells. Until now it was unclear whether changes in RhoGTPase activity or microtubule dynamics had a direct impact on the efficiency of Ad microtubule-dependent nuclear localization. Here we have performed synchronous Ad infections and utilized confocal microscopy to analyze the individual contributions of RhoA activation, Rac1 activation, microtubule stability, dynamic behavior, and posttranslational modifications on Ad nuclear localization efficiency (NLE). We found that drug-induced suppression of microtubule dynamics impaired Ad NLE by disrupting the radial organization of the microtubule array. When the microtubule array was maintained, the suppression or enhancement of microtubule turnover did not significantly affect Ad NLE. Furthermore, RhoA activation or the formation of acetylated microtubules did not enhance Ad NLE. In contrast, active Rac1 was required for efficient Ad nuclear localization. Because Rac1 mediates persistent growth of microtubules to the lamellar regions of cells, we propose that Ad-induced activation of Rac1 enhances the ability of microtubules to "search and capture" incoming virus particles.     

Complementary roles for dynein and kinesins in the Xenopus egg cortical rotation

Aligned vegetal subcortical microtubules in fertilized Xenopus eggs mediate the "cortical rotation", a translocation of the vegetal cortex and of dorsalizing factors toward the egg equator. Kinesin-related protein (KRP) function is essential for the cortical rotation, and dynein has been implicated indirectly; however, the role of neither microtubule motor protein family is understood. We examined the consequence of inhibiting dynein--dynactin-based transport by microinjection of excess dynamitin beneath the vegetal egg surface. Dynamitin introduced before the cortical rotation prevented formation of the subcortical array, blocking microtubule incorporation from deeper regions. In contrast, dynamitin injected after the microtubule array was fully established did not block cortical translocation, unlike inhibitory-KRP antibodies. During an early phase of cortical rotation, when microtubules showed a distinctive wavy organization, dynamitin disrupted microtubule alignment and perturbed cortical movement. These findings indicate that dynein is required for formation and early maintenance of the vegetal microtubule array, while KRPs are largely responsible for displacing the cortex once the microtubule tracks are established. Consistent with this model for the cortical rotation, photobleach analysis revealed both microtubules that translocated with the vegetal cytoplasm relative to the cortex, and ones that moved with the cortex relative to the cytoplasm.     

Aurora B Kinase Activity is Required to Prevent Polar Cortical Ingression during Cytokinesis

Cytokinesis requires proper regulation of microtubule dynamics. It has been suggested that dynamic astral microtubules prevent cortical ingression. However, it remains unknown how astral microtubules maintain their dynamic state. Here we show that aurora B kinase, a component of the chromosome passenger complex, is required to sustain the dynamic state of astral microtubules during cytokinesis. Treatment of HeLa cells with Hesperadin, an inhibitor of aurora B kinase, caused abnormal cortical protrusion, leading to cortical ingression in the protruding region and cytokinesis failure. Actin filaments, myosin II, and RhoA failed to localize at the equator but instead distributed along the lateral and/or polar cortex in cells treated with Hesperadin. Time-lapse analyses of microtubule dynamics showed that, in cells treated with Hesperadin, abnormally bundled astral microtubules targeted the protruding region. Mitotic kinesin-like protein 1 (MKLP1), a component of the spindle midzone required for bundling of microtubules, was not detected along bundled astral microtubules in cells treated with Hesperadin, suggesting that factors other than MKLP1 may be involved in this process. Our results suggest that aurora B kinase activity is required for proper regulation of microtubule dynamics to ensure that cytokinesis occurs precisely at the cell equator.