Band Of Microtubules Research Articles

A cytoskeletal 50 kDa protein in higher plants that forms intermediate-sized filaments and stabilizes microtubules

Filamentous structures of 7–10 nm in diameter were regenerated in vitro from a soluble 50 kDa protein (p50) that had been isolated from mung bean seedlings and from cultured tobacco cells. The polymerization of p50 in vitro was dependent on the presence of guanosine nucleotides, in particular, guanosine monophosphate (GMP). Unlike tubulin, p50 is a stable basic protein with the ability to polymerize even after heat treatment for 1 min at 70 °C. Furthermore, the freeze-dried powder of p50 retained the ability to regenerate filamentous structures after it had been dissolved in polymerization buffer to which GMP was then added. Two monoclonal antibodies against p50 were obtained. These antibodies stained the filamentous structures that extended from the surface of the nucleus to the cell periphery in interphase tobacco cells. They stained spindles and phragmoplasts as did tubulin-specific antibodies. They also stained fibrillar structures that were present around the spindle poles and the telophase daughter nuclei in which no microtubules were present. These results suggest that a part of the cell's complement of p50 may be associated with microtubules in dividing cells while the rest may itself form unique fibrillar structures. The antibodies against p50 did not stain cortical microtubules or the pre-prophase band of microtubules. The antibody against p50 also stained intermediate filament-like structures in cultured animal cells. The formation of microtubules in vitro was markedly stimulated and the assembled microtubules were greatly stabilized by p50. Further investigation of p50 is indispensable for the understanding of properties and function of intermediate-sized filaments in higher plant cells.

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

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.

Identification of a novel ca2+-regulated protein that is associated with the marginal band and centrosomes of chicken erythrocytes

We have identified a novel Ca(2+)-regulated protein, p23, that is expressed specifically in avian erythrocyte and thrombocyte lineages. Sequence analysis of this 23 kDa protein reveals that it bears no homology to any known sequence. In mature definitive erythrocytes p23 exists in equilibrium between a soluble and a cytoskeletal bound pool. The cytoskeletal fraction is associated with the marginal band of microtubules, centrosomes and nuclear membrane under conditions of low free [Ca2+]. An increase in free [Ca2+] to 10(-6) M is sufficient to induce dissociation of > 95% of bound p23 from its target cytoskeletal binding sites, yet this [Ca2+] has little effect on calmodulin-mediated MB depolymerization. Analysis of p23 expression and localization during erythropoiesis together with results from heterologous p23 expression in tissue cultured cells demonstrated that this protein does not behave as a bone fide microtubule-associated protein. In addition, the developmental analysis revealed that although p23 is expressed early in definitive erythropoeisis, its association with the MB, centrosome and nuclear membrane occurs only in the final stages of differentiation. This cytoskeletal association correlates with marked p23 stabilization and accumulation at a time p23 expression is being markedly downregulated. We hypothesize that the mechanism of p23 association to the MB and centrosomes may be induced in part by a decrease in intracellular [Ca2+] during the terminal stages of definitive erythropoiesis.

Arrangement of microtubules during spore formation in Equisetum arvense (Equisetaceae)

The arrangement of cortical microtubules (MTs) during spore formation in Equisetum arvense was examined by immunofluorescence microscopy. The arrangement of MTs was observed to change during sporoderm formation. During exospore formation, the cortical MTs of the tapetum appeared along the tapetal plasma membrane that enclosed each developing spore cell. After exospore formation, the arrangement of the cortical MTs changed into one of separate bands of MTs arranged spirally (spiral bands of MTs). The spiral bands of MTs were superimposed on the developing elaters. This new pattern corresponded to the pattern of cellulose microfibrils deposited in the inner layer of the elater, suggesting that these spiral bands are involved in the deposition of the cellulose microfibrils in the elater. We conclude that the spiral bands of MTs are functionally equivalent to cortical MTs in secondary wall formation.

Cytokinesis in Spirogyra: Integration of Cleavage and Cell-Plate Formation

In Spirogyra, cytokinesis is initiated by a cleavage furrow. When the furrow contacts the elongating interzonal fibers of the telophase spindle, a small phragmoplast appears at the zone of contact, inside of which a cell-plate appears to complete cytokinesis. The use of two sequential mechanisms for achieving cytokinesis may be an intermediate, and possibly a transitional, state in the evolution of the phragmoplast found in higher plants. We tested the relative contribution of the two types of cytokinetic mechanisms by observing the effects of certain drugs on living cells, recorded in time-lapse. The antimicrotubule drug oryzalin causes the spindle to break down rapidly. When applied during early to midcytokinesis, the interzonal spindle collapses onto the cleavage furrow, which continues to grow inward. However, cytokinesis is not complete; examination of such cells 8-12 h later, after the nuclei separate, reveals a small aperture in the center of the cross-wall. This result shows that the phragmoplast/cell-plate is essential for complete cytokinesis, which apparently cannot be accomplished by cleavage alone, and that inward growth of the cleavage furrow is not dependent on microtubules. The antiactin drug cytochalasin D stops cleavage quite rapidly, and the eventual consequences of treatment depend on the stage reached in cleavage. If the cytochalasin is applied early in cytokinesis, cell division is unable to proceed further. Between telophase nuclei the cell does generate a large mass of cytoplasm that contains typical proliferating phragmoplast fibers; cell-plate formation is initiated in this mass but is never able to proceed further, and no coalescence of material into a cross-wall has been recorded. If cytochalasin is applied later, after the cleavage furrow has contacted the interzonal spindle fibers, cleavage stops but now cell-plate formation proceeds and cytokinesis is completed, albeit slowly. This result demonstrates that an interaction between the cleavage furrow and the forming phragmoplast is necessary for the latter to operate normally. These results support our suggestion that Spirogyra illustrates an intermediate stage in the evolution of the phragmoplast, since both cleavage and cell-plate formation are required for cytokinesis. (This suggestion does not require Spirogyra to be on the line of evolution leading to higher plants; rather, Spirogyra appears to be undergoing an evolutionary process similar to what might have occurred in their progenitors.) Furthermore, interaction of the cleavage furrow with the forming cell-plate/phragmoplast is required for cross-wall completion. These results may have significance in considering mechanisms by which higher plants locate their cellplates properly, an ability associated with the evolution and possible function(s) of the preprophase band of microtubules.

Endosperm Development in Barley: Microtubule Involvement in the Morphogenetic Pathway.

An immunofluorescence study of sectioned barley endosperm imaged by confocal laser scanning microscopy provided three-dimensional data on the relationship of microtubules to the cytoplasm, nuclei, and cell walls during development from 4 to 21 days after pollination (DAP). Microtubules play an important role throughout endosperm ontogeny. The syncytium is organized into units of nuclear-cytoplasmic domains by nuclear-based radial microtubule systems that appear to control the pattern of the first anticlinal walls at 5 to 6 DAP. After 7 DAP, phragmoplasts of two origins (interzonal and cytoplasmic) guide wall formation. Large compartments formed by the "free growing" walls in association with cytoplasmic phragmoplasts formed adventitiously at interfaces of opposing microtubule systems are subsequently subdivided by interzonal phragmoplast/cell plates to give rise to the starchy endosperm. During development of the aleurone layer from 8 to 21 DAP, the microtubule cycle is typical of plant histogenesis; cortical microtubules are hooplike, and preprophase bands of microtubules predict the division plane.

Embryo sac development in Arabidopsis thaliana II. The cytoskeleton during megagametogenesis

The microtubular and actin cytoskeletons have been investigated during megagametogenesis in Arabidopsis thaliana using immunofluorescence labelling of isolated coenocytic and mature embryo sacs. We found both actin and microtubules (MTs) to occur in abundance throughout megagametogenesis and in all constituent cells of the mature embryo sac. During many stages, the patterns of distribution of these cytoskeletal elements are congruent and may prove to be co-aligned. Many changes in the arrays of MTs and microfilaments take place and indicate varying roles of the cytoskeleton in the different stages and cell types of megagametogenesis. Two major populations of MTs recur throughout embryo sac formation: (1) Elaborate nuclear-based networks are found during the two-nucleate and four-nucleate developmental stages as well as in the egg cell. These arrays may function in positioning the nuclei. (2) Cytoplasmic MTs in longitudinal orientation in the two-nucleate embryo sac, synergids and part of the egg cell, or in a reticulate pattern in the four-nucleate embryo sac, egg and central cell probably participate in organization of the cytoplasm. Synergid MTs converge at the filiform apparatus. Preprophase bands of MTs are absent throughout megagametogenesis but phragmoplast arrays occur during cellularization of the embryo sac. Well developed arrays of cortical MTs are restricted to the antipodal cells. A large concentration of MTs in the part of the egg cell adjacent to the synergids is well placed for being involved with sperm cell movement within the degenerative synergid. On the basis of the morphology of the cytoskeleton, we concur with views that the shape of megagametophyte is largely determined by the surrounding tissues, including the integumentary tapetum.

Assembly of Marginal Band of Microtubules in the Immunocyte of Blattella germanica

Assembly of Marginal Band of Microtubules in the Immunocyte of Blattella germanica

Morphogenetic Plastid Migration and Microtubule Arrays in Mitosis and Cytokinesis in the Green Alga Coleochaete orbicularis

This study provides data on cell division in Coleochaete orbicularis, an important taxon in evolutionary theories deriving land plants from green algae. Vegetative growth in discoid species of Coleochaete results from marginal cell division in two planes—radial and circumferential. Like many algae and certain of the simple land plants, Coleochaete is monoplastidic. Prior to mitosis, the single plastid migrates to a position where it will divide and be distributed into the daughter cells. Unlike monoplastidic cell division in hornworts, mosses, and lycopsids; microtubule nucleation is not intimately associated with the plastids. Instead, microtubule organization is associated with centriolar centrosomes throughout the cell cycle, as is common in algae. The cytokinetic apparatus lacks preprophase bands of microtubules, but includes typical phragmoplasts consisting of brushlike arrays of microtubules on either side of a dark zone. However, the origin and role of phragmoplasts is unusual. Phragmoplasts appear to develop among microtubules that emanate from the polar centrosomes rather than from nuclear envelopes and/or plastids. The function of phragmoplasts in Coleochaete is unclear, as the process of cytokinesis is not strictly centrifugal. Some infurrowing occurs in radial division, and cytokinesis appears to be entirely centripetal by infurrowing in circumferential division. The cortical arrays of microtubules differ from those typical of land plants in that they develop as a network in association with centrosomes after mitosis.

Localization of the Functional p34cdc2 Homolog of Maize in Root Tip and Stomatal Complex Cells: Association with Predicted Division Sites.

We have used an antibody against the functional homolog of the cdc2 kinase of maize to localize the p34cdc2 protein within dividing cells of the root apex and the stomatal complex of leaf epidermis. The microtubule cytoskeletal structure of plant cells was visualized concomitantly with a monoclonal antibody specific for [alpha]-tubulin. We found that the cdc2 protein is localized mainly to the nucleus in plant cells at interphase and early prophase. This finding contrasts markedly with the predominantly cytoplasmic staining obtained using antibody to the PSTAIRE motif, which is common to cdc2 and numerous cdc2-like proteins. In a subpopulation of root cells at early prophase, the p34cdc2 protein is also distributed in a band bisecting the nucleus. Double labeling with the maize p34cdc2Zm antibody and tubulin antibody revealed that this band colocalizes with the preprophase band (PPB) of microtubules, which predicts the future division site. Root cells in which microtubules had been disrupted with oryzalin did not contain this band of p34cdc2 protein, suggesting that formation of the microtubule PPB is necessary for localization of the p34cdc2 kinase to the plane of the PPB. The p34cdc2 protein is also localized to the nucleus and PPB in cells that give rise to the stomatal complex, including those cells preparing for the highly asymmetrical divisions that produce subsidiary cells. Association of the p34cdc2 protein with the PPB suggests that the cdc2 kinase has a role in establishing the division site of plant cells and, therefore, a role in plant morphogenesis.

The formation of aplastidic abscission (tmema) cells and protonemal disruption in the mossBryum tenuisetum Limpr. is associated with transverse arrays of microtubules and microfilaments

When grown on nutrient agar, protonemata ofBryum tenuisetum produce aerial filaments containing several abscission or tmema cells (TC). Basipetal migration of the nucleus and some of the chloroplasts signals the onset of TC formation. This is followed by the creation of a plastid-free zone at the base of the mother cell. The ensuing cytokinesis produces a very short aplastidic TC. This expands without the deposition of new wall material. Eventually the wall ruptures around the equator thus disrupting the protonemal filament. The site of wall breakdown is marked by a narrow band of cortical cytoplasm containing colocalized circumferential rings of actin filaments and microtubules. A transverse band of microtubules appears at the extreme basal end of the tmema mother cell. This band, which is not colocalized with actin filaments, migrates distally over the surface of the nucleus. Intimate spatial and developmental correlations suggest that this transverse array of the microtubules has a key role in excluding plastids from the TC. It is therefore considered not to be homologous with a preprophase band.

Studies on the Sporogenous Lineage in the Moss Timmiella barbuloides

AbstractThis correlated immunofluorescence and electron microscope study reveals that the microtubule arrays during meiosis in Timmiella barbuloides mirror those in other mosses but the organization of the metaphase I spindle is quite different. In other mosses the sagittiform metaphase I spindle initially contains four bands of microtubules derived from the tetrahedral system present at prophase. These bands converge towards the division axis and each half spindle contains two focal points of microtubules straddling a cleavage furrow. In Timmiella the sagittiform spindle also contains four microtubular foci derived from the preprophasic tetrahedron. However, one of these contributes to one half spindle, the other half deriving from the three remaining foci orientated at approximately 120° to each other. In contrast to other mosses the sporocytes in Timmiella are hardly lobed, the cleavage‐furrows ill‐defined, the prophasic plastid positioning in the lobes is also more variable and the organelle band in meiocytes comprises mitochondria alone.

Interphase and preprophase microtubule organization in some polarized cell types of the liverwort Marchantia paleacea Bert.

SUMMARYThe organization of interphase and preprophase‐prophase microtubules was studied in photosynthetic filament mother cells (PFMCs), photosynthetic filament cells (PFCs), mucilage papilla mother cells (MPMCs) and mucilage papillae (MP) of Marchantia paleacea Bert. These cell types exhibit polarized growth resulting, (a) in the formation of cell outgrowths followed by asymmetrical division (PFMCs and MPMCs), or (b) in the development of a tubular cell shape (PFCs and MP). The above cell types display an interphase cortical microtubule ring perpendicular to the axis of growth. In PFMCs and MPMCs it resides at the base of the cell outgrowth, while in PFCs and MP it has a median location. This microtubule ring is involved in the deposition of transverse, circumferentially aligned, cellulose microfibrils, defines the site where cell protrusion formation occurs and affects cell morphogenesis. In differentiated MP microtubules are rearranged in longitudinal or oblique cortical arrays. PFCs and MP also contain a prominent system of endoplasmic microtubules which may be involved in cytoplasmic polarization. In PFMCs, PFCs and MPMCs the interphase microtubule ring seems to function as a preprophase microtubule band (PPB). In the asymmetrically dividing PFMCs and MPMCs the PPB is complete but in symmetrically dividing PFCs it may be interrupted. The cell plate meets the parent wall at sites adjacent to the PPB cortical zone. These observations reveal further peculiarities in cortical microtubule organization and particularly in the PPBs of M. paleacea. Possible factors underlying the formation of complete or interrupted PPBs are discussed.

Arrangement of cortical microtubules during formation of bordered pit in the tracheids ofTaxus

The arrangement of cortical microtubules during the development of the secondary wall and bordered pits in the tracheids ofTaxus was examined by immunofluorescence and electron microscopy. The cambial region of radial longitudinal sections of developing young shoots (2–3 years old) contains cells at various stages of differentiation from cambial cells to tracheids. At the early stage of formation of bordered pits, circular bands of microtubules were seen to be associated with the inner edge of the border of the developing pit. In other regions than the pit secondary wall of uniform thickness was laid down, and obliquely oriented cortical microtubules ran parallel to one another. These cortical microtubules also covered the surface of the border of the developing pit on the side facing the center of the cell. As the border of the pit developed, a circular band of MTs remained associated with the inner edge of border, suggesting that the MTs were involved in the formation of the rim of the bordered pit, extending the initial border thickening, which consisted of concentrically oriented cellulose microfibrils. After completion of the formation of the bordered pit, helical thickenings became apparent. The obliquely oriented microtubules were organized in bands parallel to one another, being superimposed on the helical thickenings. The involvement of MTs in the formation of bordered pits and helical thickening is discussed.

Microtubules and cell shaping in the mesophyll ofNigella damascena L.

Cell shaping in the mesophyll ofNigella damascena was investigated with the aim of determining the origin of the arm-like protrusions, which are characteristic of, e.g., arm-palisade cells. It was found that hoops of cell wall were deposited during the early stages of cell expansion. The hoops were interconnected, thus embracing the cells with a wide-meshed net of local wall reinforcement. The pattern of wall deposition in the extra-cellular matrix correlated with a pattern of bands of microtubules in the cortical cytoplasm of the cells. During lateral expansion bulges were forced through the comparatively thin walls of spaces between the meshes, giving rise to the arm-like protrusions. After establishing the cell shape the bands of microtubules disintegrated and cell wall was uniformly deposited. The results are discussed in the context of the mode of cell shaping observed in the mesophyll of other systems and of a previous, classical hypothesis on the origin of arms in mesophyll cells.

Microtubules in dividing root cells of the conifer pinus radiata and the cycad zamia furfuracea

AbstractMicrotubules in dividing root cells of the gymnosperms Pinus radiata (conifer) and Zamia furfuracea (cycad) were examined using immunofluorescence techniques. Root tip squashes were prepared to visualize the 3‐dimensional organization of microtubules in intact cells while sections of methacrylate embedded roots revealed microtubules in situ. Both species were characterized by well developed preprophase bands (PPB) of microtubules and highly focused spindle poles at prophase and anaphase. The metaphase spindle and telophase phragmoplast appeared typical of flowering plants.

The evolution, structure and functioning of stomata

Summary The anatomy and ultrastructure of guard cells from a range of species varying from the primitive types, such as mosses, to the advanced grasses and orchids are described. An attempt is made to trace the lines along which stomata developed and to define what might be considered advanced stomata. Additionally, the differentiation of guard cells from guard mother cells is discussed. Of particular note is the preprophase band of microtubules which marks the zone where the future cell wall will form and the movement of the spindle and developing cell plate through 45 degrees. The structure and function of guard cells are intimately linked. Stomata are turgor regulated valves; the osmotica for absorbing water during opening are K+, Cl- and malate anions which accumulate in the guard cell vacuoles. Upon stomatal closure, K+ and Cl- exit from the guard cells while at least some of the carbon from malate is channelled into starch and there is a resultant loss of guard cell turgor. The Calvin cycle may be a...

Microtubule and F-actin dynamics at the division site in living Tradescantia stamen hair cells

ABSTRACT We have visualised F-actin and microtubules in living Tradescantia virginiana stamen hair cells by confocal laser scanning microscopy after microinjecting rhodamine-phalloidin or carboxyfluorescein-labelled brain tubulin. We monitored these components of the cytoskeleton as the cells prepared for division at preprophase and progressed through mitosis to cytokinesis. Reorganisation of the interphase cortical cytoskeleton results in preprophase bands of both F-actin and microtubules that coexist in the cell cortex, centred on the site at which the future cell plate will fuse with the parent cell wall. The preprophase band of microtubules is formed from microtubules that polymerise and incorporate tubulin during prophase. The preprophase band of actin may form either by reorganisation of pre-existing filaments or by de novo polymerisation. Both cytoskeletal components disappear from the future division site approximately five minutes prior to the breakdown of the nuclear envelope. Cortical microtubules are undetectable throughout mitosis and cytokinesis, whereas cortical F-actin remains abundant, although it is notably excluded from the division site. The phragmoplast, containing both F-actin and microtubules, expands towards the cortical actin exclusion-zone through a region that has no detectable microtubules or F-actin. The phragmoplast comes to rest in the predefined region of the cortex that is devoid of F-actin. It is proposed that cortical F-actin may act as a “negative” template which could position the phragmoplast and cell plate correctly. This is the first in vivo documentation of F-actin dynamics at the division site in living plant cells.

Pollen development in orchids 4. Cytoskeleton and ultrastructure of the unequal pollen mitosis inPhalaenopsis

The unequal first mitosis in pollen ofPhalaenopsis results in a small generative cell cut off at the distal surface of the microspore and a large vegetative cell. No preprophase band of microtubules is present, but polarization of the microspore prior to this critical division is well marked. A generative pole microtubule system (GPMS) marks the path of nuclear migration to the distal surface, and the organelles become unequally distributed. Mitochondria, plastids and dictyosomes are concentrated around the vegetative pole in the center of the microspore and are almost totally excluded from the generative pole. The prophase spindle is multipolar with a dominant convergence center at the GPMS site. The metaphase spindle is disc-shaped with numerous “minipoles” terminating in broad polar regions. In anaphase, the spindle becomes cone-shaped as the spindle elongates and the vegetative pole narrows. These changes in spindle architecture are reflected in the initial shaping of the telophase chromosome groups. F-actin is coaligned with microtubules in the spindle and is also seen as a network in the cytoplasm. An outstanding feature of orchid pollen mitosis is the abundance of endoplasmic reticulum (ER) associated with the spindle. ER extends along the kinetochore fibers, and the numerous foci of spindle fibers at the broad poles terminate in a complex of ER.

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.