Organization of cortical microtubules and microfibril deposition in response to blue-light-induced apical swelling in a tip-growing Adiantum protonema cell

The arrangements of cortical microtubules (MTs) in a tip-growing protonemal cell of Adiantum capillus-veneris L. and of cellulose microfibrils (MFs) in its wall were examined during blue-light (BL)-induced apical swelling. In most protonemal cells which had been growing in the longitudinal direction under red light, apical swelling was induced within 2 h of the onset of BL irradiation, and swelling continued for at least 8 h. During the longitudinal growth under red light, the arrangement of MFs around the base of the apical hemisphere (the subapical region) was perpendicular to the cell axis, while a random arrangement of MFs was found at the very tip, and a roughly axial arrangement was observed in the cylindrical region of most cells. This orientation of MFs corresponds to that of the cortical MTs reported previously (Murata et al. 1987, Protoplasma 141, 135–138). In cells irradiated with BL, a random rather than transverse arrangement of both MTs and MFs was found in the subapical region. Time-course studies showed that this reorientation occurred within 1 h after the onset of the BL irradiation, i.e. it preceded the change in growth pattern. These results indicate that the orientation of cortical MTs and of cellulose MFs is involved in the regulation of cell diameter in a tip-growing Adiantum protonemal cell.Abbreviations BL blue light - MF(s) microfibril(s) - MT(s) microtubule(s)     

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.     

Arrangement of cortical microtubules in the shoot apex of Vinca major L.

The arrangements of cortical microtubules (MTs) and of cellulose microfibrils in the median longitudinal cryosections of the vegetative shoot apex of Vinca major L., were examined by immunofluorescence microscopy and polarizing microscopy, respectively. The arrangement of MTs was different in the various regions of the apex: the MTs tended to be arranged anticlinally in tunica cells, randomly in corpus cells, and transversely in cells of the rib meristem. However, in the inner layers of the tunica in the flank region of the apex, cells with periclinal, oblique or random arrangements of MTs were also observed. In leaf primordia, MTs were arranged anticlinally in cells of the superficial layers and almost randomly in the inner cells. Polarizing microscopy of cell walls showed that the arrangement of cellulose microfibrils was anticlinal in tunica cells, random in corpus cells, and transverse in cells of the rib meristem; thus, the patterns of arrangement of microfibrils were the same as those of MTs in the respective regions. These results indicate that the different patterns of arrangement of MTs and microfibrils result in specific patterns of expansion in the three regions. These differences may be necessary to maintain the organization of the tissues in the shoot apex.Abbreviations MT(s) microtubule(s) - lp length of the youngest leaf primordium     

Temperature-dependent changes of cell shape during heterophyllous leaf formation in <Emphasis Type="Italic">Ludwigia </Emphasis><Emphasis Type="Italic">arcuata</Emphasis> (Onagraceae)

Although elongation of epidermal cells in submerged leaves is thought to be a common feature of heterophyllous aquatic plants, such elongation has not been observed in Ludwigia arcuata Walt. (Onagraceae). In this study we found that reduced culture temperature induced the elongation of epidermal cells of submerged leaves in L. arcuata. Since such submerged leaves also showed a reduction in the number of epidermal cells aligned across the leaf transverse axis, these data indicate that heterophyllous leaf formation in L. arcuata is partially temperature sensitive, i.e., the elongation of epidermal cells was temperature sensitive while the reduction in the number of epidermal cells did not show such temperature sensitivity. To clarify the mechanisms that cause such temperature sensitivity, we examined the effects of ethylene, which induced the formation of submerged-type leaves on aerial shoots at the relatively high culture-temperature of 28 degrees C. At 23 degrees C, ethylene induced both cell elongation and reduction in the number of epidermal cells across the leaf transverse axis, while cell elongation was not observed at 28 degrees C. Moreover, both submergence and ethylene treatment induced a change in the arrangement of cortical microtubules (MTs) in epidermal cells of developing leaves at 23 degrees C. Such changes in the arrangement of MTs was not induced at 28 degrees C. Factors involved in the temperature-sensitive response to ethylene would be critical for temperature-sensitive heterophyllous leaf formation in L. arcuata.     

Alteration of oriented deposition of cellulose microfibrils by mutation of a katanin-like microtubule-severing protein

It has long been hypothesized that cortical microtubules (MTs) control the orientation of cellulose microfibril deposition, but no mutants with alterations of MT orientation have been shown to affect this process. We have shown previously that in Arabidopsis, the fra2 mutation causes aberrant cortical MT orientation and reduced cell elongation, and the gene responsible for the fra2 mutation encodes a katanin-like protein. In this study, using field emission scanning electron microscopy, we found that the fra2 mutation altered the normal orientation of cellulose microfibrils in walls of expanding cells. Although cellulose microfibrils in walls of wild-type cells were oriented transversely along the elongation axis, cellulose microfibrils in walls of fra2 cells often formed bands and ran in different directions. The fra2 mutation also caused aberrant deposition of cellulose microfibrils in secondary walls of fiber cells. The aberrant orientation of cellulose microfibrils was shown to be correlated with disorganized cortical MTs in several cell types examined. In addition, the thickness of both primary and secondary cell walls was reduced significantly in the fra2 mutant. These results indicate that the katanin-like protein is essential for oriented cellulose microfibril deposition and normal cell wall biosynthesis. We further demonstrated that the Arabidopsis katanin-like protein possessed MT-severing activity in vitro; thus, it is an ortholog of animal katanin. We propose that the aberrant MT orientation caused by the mutation of katanin results in the distorted deposition of cellulose microfibrils, which in turn leads to a defect in cell elongation. These findings strongly support the hypothesis that cortical MTs regulate the oriented deposition of cellulose microfibrils that determines the direction of cell elongation.     

Reorganization of cortical microtubules and cellulose deposition during leaf formation in Graptopetalum paraguayense

In the regeneration of a shoot from a leaf of the succulent, Graptopetalum paraguayense E. Walther the first new organs are leaf primordia. The original arrangement of cellulose microfibrils and of microtubules (MTs) in the epidermis of the leaf-forming site is one of parallel, straight lines. In the new primordium both structures still have a congruent arrangement but it is roughly in the form of concentric circles that surround the new cylindrical organ. The regions which undergo the greatest shift in orientation (90°) were studied in detail. Departures from the original cellulose alignment are detected in changes in the polarized-light image. Departures from the original cortical MT arrangement are detected using electron microscopy. The over-all reorganization of the MT pattern is followed by the tally of MT profiles, the various regions being studied in two perpendicular planes of section. This corrects for the difference in efficiency in counting transverse versus longitudinal profiles of MTs. Reorientation takes place sporadically, cell by cell, for both the cellulose microfibrils and the MTs, indicating a coordinated reorientation of the two structures. That MTs and cellulose microfibrils reorient jointly in individual cells was shown by reconstruction of the arrays of cortical MTs in paradermal sections of individual cells whose recent change in the orientation of cellulose deposition had been detected with polarized light. Closeness of the two alignments was also indicated by images where the MT and microfibril alignments co-varied within a single cell. The change-over in alignment of the MTs appears to involve stages where arrays of contrasting orientation co-exist to give a criss-cross image. During this critical reorganization, the frequency of the MTs is high. It falls during subsequent enlargement of the organ. It was found that the rearrangement of the cortical MTs to approximate a series of concentric circles on the residual meristem occurred before the emergence of leaf primordia. Through their apparent influence on microfibril alignments, the changes in MT disposition, described here, have the potential to generate major biophysical changes that accompany organogenesis.Abbreviation MT(s) microtubule(s)     

Animal cell shape changes and gene expression.

Cell shape and cell contacts are determined by transmembrane receptor-mediated associations of the cytoskeleton with specific extracellular matrix proteins and with ligands on the surface of adjacent cells. The cytoplasmic domains of these microfilament-membrane associations at the adherens junction sites, also localize a variety of regulatory molecules involved in signal transduction and gene regulation. The stimulation of cells with soluble polypeptide factors leads to rapid changes in cell shape and microfilament component organization. In addition, this stimulation also activates the phosphoinositide signaling pathway. Recently, a linkage between actin-binding proteins and the phosphoinositide signaling pathway, was discovered. It is suggested that by the association with the second messenger system, and/or by controlling the localization of regulatory molecules, the cytoskeleton may regulate gene expression.     

Nanoscale and geometric influences on the microtubule cytoskeleton in plants: thinking inside and outside the box

The dynamic microtubule (MT) cytoskeleton found in the cell cortex of plants drives cell expansion via cell wall modifications. In the last decade, live cell imaging studies employing green fluorescent protein have helped unravel the mechanisms behind how cells arrange cortical MTs into complex arrays and shape cell expansion. In this review, we explore the reverse scenario: how cell geometry and organelles influence and constrain the organization and behavior of cortical MTs. This newly emerging principle explains how cells perceive local nanoscale structural input from MT-organizing centers, such as the nucleus, endomembranes, and cell edges, and translate this into global cell-wide order via MT self-organization. Studies primarily using the model plant Arabidopsis thaliana and tobacco BY-2 suspension cultures have broadened our understanding of how cells form not only elegant parallel arrays but also more complex MT configurations, including the prominent MT bundles found in preprophase bands, leaf epidermal cells, and developing xylem.     

Nanoscale and geometric influences on the microtubule cytoskeleton in plants: thinking inside and outside the box

The dynamic microtubule (MT) cytoskeleton found in the cell cortex of plants drives cell expansion via cell wall modifications. In the last decade, live cell imaging studies employing green fluorescent protein have helped unravel the mechanisms behind how cells arrange cortical MTs into complex arrays and shape cell expansion. In this review, we explore the reverse scenario: how cell geometry and organelles influence and constrain the organization and behavior of cortical MTs. This newly emerging principle explains how cells perceive local nanoscale structural input from MT-organizing centers, such as the nucleus, endomembranes, and cell edges, and translate this into global cell-wide order via MT self-organization. Studies primarily using the model plant Arabidopsis thaliana and tobacco BY-2 suspension cultures have broadened our understanding of how cells form not only elegant parallel arrays but also more complex MT configurations, including the prominent MT bundles found in preprophase bands, leaf epidermal cells, and developing xylem.     

Microtubules regulate the organization of actin filaments at the cortical region in root hair cells ofHydrocharis

Summary We studied the mechanism controlling the organization of actin filaments (AFs) inHydrocharis root hair cells, in which reverse fountain streaming occurs. The distribution of AFs and microtubules (MTs) in root hair cells were analyzed by fluorescence microscopy and electron microscopy. AFs and MTs were found running in the longitudinal direction of the cell at the cortical region. AFs were observed in the transvacuolar strand, but not MTs. Ultrastructural studies revealed that AFs and MTs were colocalized and that MTs were closer to the plasma membrane than AFs. To examine if MTs regulate the organization of AFs, we carried out a double inhibitor experiment using cytochalasin B (CB) and propyzamide, which are inhibitors of AFs and MTs, respectively. CB reversibly inhibited cytoplasmic streaming while propyzamide alone had no effect on it. However, after treatment with both CB and propyzamide, removal of CB alone did not lead to recovery of cytoplasmic streaming. In these cells, AFs showed a meshwork structure. When propyzamide was also removed, cytoplasmic streaming and the original organization of AFs were recovered. These results strongly suggest that MTs are responsible for the organization of AFs inHydrocharis root hair cells.     

Fate of nascent microtubules organized at the M/G1 interface, as visualized by synchronized tobacco BY-2 cells stably expressing GFP-tubulin: time-sequence observations of the reorganization of cortical microtubules in living plant cells

Transgenic BY-2 cells stably expressing a GFP (green fluorescent protein)-tubulin fusion protein (BY-GT16) were subcultured in a modified Linsmaier and Skoog medium. The BY-GT16 cells could be synchronized by aphidicolin and the dynamics of their microtubules (MTs) were monitored by the confocal laser scanning microscopy (CLSM). We have succeeded in investigating the mode of reorganization of cortical MTs at the M/G1 interface. The cortical MTs were initially organized in the perinuclear regions and then they elongated to reach the cell cortex, forming the bright spots there. Subsequently, the first cortical MTs rapidly elongated from the spots and they were oriented parallel to the long axis towards the distal end of the cells. Around the time when the tips of the parallel MTs reached the distal end, the formation of transverse cortical MTs followed in the cortex near the division site, as we had previously suggested [Hasezawa and Nagata (1991) Bot. Acta 104: 206, Nagata et al. (1994) Planta 193: 567]. It was confirmed in independent observations that the appearance of the parallel MTs was followed by the appearance of the transverse MTs in each cell. We found that the transverse MTs spread through the whole cell cortex within about 20-30 min, while the parallel MTs disappeared. The significance of these observations on the mode of cortical MT organization is discussed.     

The chloroplast clpP gene, encoding a proteolytic subunit of ATP-dependent protease, is indispensable for chloroplast development in tobacco

ClpP is a proteolytic subunit of the ATP-dependent Clp protease, which is found in chloroplasts in higher plants. Proteolytic subunits are encoded both by the chloroplast gene, clpP, and a nuclear multi gene family. We insertionally disrupted clpP by chloroplast transformation in tobacco. However, complete segregation was impossible, indicating that the chloroplast-encoded clpP gene has an indispensable function for cell survival. In the heteroplasmic clpP disruptant, the leaf surface was rough by clumping, and the lateral leaf expansion was irregularly arrested, which led to an asymmetric, slender leaf shape. Chloroplasts consisted of two populations: chloroplasts that were similar to the wild type, and small chloroplasts that emitted high chl fluorescence. Ultrastructural analysis of chloroplast development suggested that clpP disruption also induced swelling of the thylakoid lumen in the meristem plastids and inhibition of etioplast development in the dark. In mature leaves, thylakoid membranes of the smaller chloroplast population consisted exclusively of large stacks of tightly appressed membranes. These results indicate that chloroplast-encoded ClpP is involved in multiple processes of chloroplast development, including a housekeeping function that is indispensable for cell survival.     

The cell morphogenesis gene ANGUSTIFOLIA encodes a CtBP/BARS-like protein and is involved in the control of the microtubule cytoskeleton

The ANGUSTIFOLIA (AN) gene is required for leaf hair (trichome) branching and is also involved in polarized expansion underlying organ shape. Here we show that the AN gene encodes a C-terminal binding proteins/brefeldin A ADP-ribosylated substrates (CtBP/BARS) related protein. AN is expressed at low levels in all organs and the AN protein is localized in the cytoplasm. In an mutant trichomes, the organization of the actin cytoskeleton is normal but the distribution of microtubules is aberrant. A role of AN in the control of the microtubule cytoskeleton is further supported by the finding that AN genetically and physically interacts with ZWICHEL, a kinesin motor molecule involved in trichome branching. Our data suggest that CtBP/BARS-like protein function in plants is directly associated with the microtubule cytoskeleton.     

Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons.

Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.     

Cytoskeleton and morphogenesis in brown algae

BACKGROUND: Morphogenesis on a cellular level includes processes in which cytoskeleton and cell wall expansion are strongly involved. In brown algal zygotes, microtubules (MTs) and actin filaments (AFs) participate in polarity axis fixation, cell division and tip growth. Brown algal vegetative cells lack a cortical MT cytoskeleton, and are characterized by centriole-bearing centrosomes, which function as microtubule organizing centres. SCOPE: Extensive electron microscope and immunofluorescence studies of MT organization in different types of brown algal cells have shown that MTs constitute a major cytoskeletal component, indispensable for cell morphogenesis. Apart from participating in mitosis and cytokinesis, they are also involved in the expression and maintenance of polarity of particular cell types. Disruption of MTs after Nocodazole treatment inhibits cell growth, causing bulging and/or bending of apical cells, thickening of the tip cell wall, and affecting the nuclear positioning. Staining of F-actin using Rhodamine-Phalloidin, revealed a rich network consisting of perinuclear, endoplasmic and cortical AFs. AFs participate in mitosis by the organization of an F-actin spindle and in cytokinesis by an F-actin disc. They are also involved in the maintenance of polarity of apical cells, as well as in lateral branch initiation. The cortical system of AFs was found related to the orientation of cellulose microfibrils (MFs), and therefore to cell wall morphogenesis. This is expressed by the coincidence in the orientation between cortical AFs and the depositing MFs. Treatment with cytochalasin B inhibits mitosis and cytokinesis, as well as tip growth of apical cells, and causes abnormal deposition of MFs. CONCLUSIONS: Both the cytoskeletal elements studied so far, i.e. MTs and AFs are implicated in brown algal cell morphogenesis, expressed in their relationship with cell wall morphogenesis, polarization, spindle organization and cytokinetic mechanism. The novelty is the role of AFs and their possible co-operation with MTs.     

A mycorrhizal fungus changes microtubule orientation in tobacco root cells

Summary Cortical cells of mycorrhizal roots undergo drastic morphological changes, such as vacuole fragmentation, nucleus migration, and deposition of cell wall components at the plant-fungus interface. We hypothesized that the cytoskeleton is involved in these mechanisms leading to cell reorganization. We subjected longitudinal, meristem to basal zone, sections of uninfectedNicotiana tabacum roots to immunofluorescence methods to identify the microtubular (MT) structures associated with root cells. Similar sections were obtained from tobacco roots grown in the presence ofGigaspora margarita, an arbuscular mycorrhizal fungus which penetrates the root via the epidermal cells, but mostly develops in the inner cortical cells. While the usual MT structures were found in uninfected roots (e.g., MTs involved in mitosis in the meristem and cortical hoops in differentiated parenchyma cells), an increase in complexity of MT structures was observed in infected tissues. At least three new systems were identified: (i) MTs running along large intracellular hyphae, (ii) MTs linking hyphae, (iii) MTs binding the hyphae to the host nucleus. The experiments show that mycorrhizal infection causes reorganization of root MTs, suggesting their involvement in the drastic morphological changes shown by the cortical cells.     

Immunofluorescence Microscopy of Microtubule Arrangement in Root Cells of Pisum sativum L. var Alaska

The arrangement of wall microtubules (MTs) in Pisum sativumroots was viewed immunofluorescently using cryosectioning. Mostcells in the tip region of pea roots (0–2 mm from tip)had wall MTs arranged transversely to the root axis. In theregion elongating at a higher rate (2–4 mm), wall MTsof epidermal, cortical and stelar cells were all transverselyarranged. In the region of about 5 mm from the tip, in whichcell elongation had already ceased, wall MTs in cortical cellschanged from a transverse to an oblique arrangement in relationto the root axis. Some cells had a crossed arrangement of wallMTs, which was interpreted as representing two sets of unidirectional,oblique wall MTs in opposite cell cortices of a single cell.This change was completed within a region of 1-mm width. Sinceroots elongated at a rate of 0.6 mm h^–1, it means thatthe arrangement of wall MTs changed within 2 h. An oblique arrangementof wall MTs was also observed in stelar cells. As the cellsaged, the oblique arrangement tended to change to a steeperor even a longitudinal one. (Received January 24, 1986; Accepted May 15, 1986)     

Generation of a stable, posttranslationally modified microtubule array is an early event in myogenic differentiation

Microtubules (MTs) have been implicated to function in the change of cell shape and intracellular organization that occurs during myogenesis. However, the mechanism by which MTs are involved in these morphogenetic events is unclear. As a first step in elucidating the role of MTs in myogenesis, we have examined the accumulation and subcellular distribution of posttranslationally modified forms of tubulin in differentiating rat L6 muscle cells, using antibodies specific for tyrosinated (Tyr), detyrosinated (Glu), and acetylated (Ac) tubulin. Both Glu and Ac tubulin are components of stable MTs, whereas Tyr tubulin is the predominant constituent of dynamic MTs. In proliferating L6 myoblasts, as in other types of proliferating cells, the level of Glu tubulin was very low when compared with the level of Tyr tubulin. However, when we shifted proliferating L6 cells to differentiation media, we observed a rapid accumulation of Glu tubulin in cellular MTs. By immunofluorescence, the increase in Glu tubulin was first detected in MTs of prefusion myoblasts and was specifically localized to MTs that were associated with elongating portions of the cell. MTs in the multinucleated myotubes observed at later stages of differentiation maintained the elevated level of Glu tubulin that was observed in the prefusion myoblasts. When cells at early stages of differentiation (less than 1 d after switching the culture medium) were immunostained for Glu tubulin and the muscle-specific marker, muscle myosin, we found that the increase in Glu tubulin preceded the accumulation of muscle myosin. Thus, the elaboration of Glu MTs is one of the very early events in myogenesis. Ac tubulin also increased during L6 myogenesis; however, the increase in acetylation occurred later in myogenesis, after fusion had already occurred. Because detyrosination was temporally correlated with early events of myogenesis, we examined the mechanism responsible for the accumulation of Glu tubulin in the MTs of prefusion myoblasts. We found that an increase in the stability of L6 cell MTs occurred at the onset of differentiation, suggesting that the early increase in detyrosination that we observed is a manifestation of a decrease in MT dynamics in elongating myoblasts. We conclude that the establishment of an oriented array of microtubules heightened in its stability and its level of posttranslationally modified subunits may be involved in the subcellular remodeling that occurs during myogenesis.     

Characterization of a member of the AN subfamily, IAN, from Ipomoea nil

ANGUSTIFOLIA (AN) is the first C-terminal binding protein (CtBP) gene from plants and controls leaf width and pattern of trichome branching in Arabidopsis thaliana (L.) Heynh. We characterized an ortholog of AN from Ipomoea nil (L.) Roth (Japanese morning glory) and designated it Ipomoea nil's AN (IAN). IAN is a single-copy gene in the genome and is expressed ubiquitously in various organs of I. nil. IAN contains not only a D2-HDH motif, which is highly conserved within the CtBP family, but also LXCXE, NLS and PEST motifs, which are specific to the AN subfamily. The expression of IAN cDNA driven by the cauliflower mosaic virus 35S promoter restored a defect in leaf expansion in the leaf width direction in the angustifolia-1 (an-1) mutant of Arabidopsis, suggesting that IAN retains a common function with AN. In contrast, the complementation by IAN of a defect in the trichome branching pattern on the leaf surface of the an-1 mutant was less effective than that observed for leaf shape. These results suggest that the mechanisms by which AN regulates leaf width and trichome branching are separable.