Organization of cortical microtubules at the plasma membrane in Arabidopsis

 To understand the role of microtubules in the regulation of cell elongation, we characterized microtubule patterns in fass, a cell shape mutant of Arabidopsis thaliana (L.) Heynh. Examining microtubule patterns via immunocytochemistry, we found that fass cells were able to organize their microtubules into mitotic spindles and phragmoplasts. During interphase or preprophase, fass cells had cortical microtubules, verified by transmission electron microscopy, but these microtubules were not organized into the cortical array or preprophase band. Using chromatin condensation and tubulin localization on the nuclear envelope as preprophase stage markers, we found that although fass cells lacked the preprophase band and cortical array, their cell division cycle appeared normal. To pinpoint the defect in fass cells, we delineated the sequential events leading to cortical array formation in Arabidopsis cells and found that fass cells initiated and recolonized cortical microtubules in the same manner as wild-type cells, but failed to order them into the cortical array. Taken together, these results suggest fass cells are impaired in a component of the microtubule organizing center(s) required for the proper ordering of cortical microtubules at the plasma membrane. Received: 23 August 1996 / Accepted: 25 September 1996     

How microtubules get fluorescent speckles.

The dynamics of microtubules in living cells can be seen by fluorescence microscopy when fluorescently labeled tubulin is microinjected into cells, mixing with the cellular tubulin pool and incorporating into microtubules. The subsequent fluorescence distribution along microtubules can appear "speckled" in high-resolution images obtained with a cooled CCD camera (Waterman-Storer and Salmon, 1997. J. Cell Biol. 139:417-434). In this paper we investigate the origins of these fluorescent speckles. In vivo microtubules exhibited a random pattern of speckles for different microtubules and different regions of an individual microtubule. The speckle pattern changed only after microtubule shortening and regrowth. Microtubules assembled from mixtures of labeled and unlabeled pure tubulin in vitro also exhibited fluorescent speckles, demonstrating that cellular factors or organelles do not contribute to the speckle pattern. Speckle contrast (measured as the standard deviation of fluorescence intensity along the microtubule divided by the mean fluorescence intensity) decreased as the fraction of labeled tubulin increased, and it was not altered by the binding of purified brain microtubule-associated proteins. Computer simulation of microtubule assembly with labeled and unlabeled tubulin showed that the speckle patterns can be explained solely by the stochastic nature of tubulin dimer association with a growing end. Speckle patterns can provide fiduciary marks in the microtubule lattice for motility studies or can be used to determine the fraction of labeled tubulin microinjected into living cells.     

The microtubular system and posttranslationally modified tubulin during spermatogenesis in a parasitic nematode with amoeboid and aflagellate spermatozoa

Using transmission electron microscopy and immunologic approaches with various antibodies against general tubulin and posttranslationally modified tubulin, we investigated microtubule organization during spermatogenesis in Heligmosomoides polygyrus, a species in which a conspicuous but transient microtubular system exists in several forms: a cytoplasmic network in the spermatocyte, the meiotic spindle, a perinuclear network and a longitudinal bundle of microtubules in the spermatid. This pattern differs from most nematodes including Caenorhabditis elegans, in which spermatids have not microtubules. In the spermatozoon of H. polygyrus, immunocytochemistry does not detect tubulin, but electron microscopy reveals two centrioles with a unique structure of 10 singlets. In male germ cells, microtubules are probably involved in cell shaping and positioning of organelles but not in cell motility. In all transient tubulin structures described in spermatocytes and spermatids of H. polygyrus, detyrosination, tyrosination, and polyglutamylation were detected, but acetylation and polyglycylation were not. The presence/absence of these posttranslational modifications is apparently not stage dependent. This is the first study of posttranslationally modified tubulin in nematode spermatogenesis. Mol. Reprod. Dev. 49:150–167, 1998. © 1998 Wiley-Liss, Inc.     

Cortical Microtubule Organization and Internodal Cell Maturation in Chara corallina

The arrangement of cortical microtubules, as documented by immunofluorescence and immunogold-silver enhancement, changes during the growth and maturation of giant internodal cells of Chara corallina, a process taking approximately 45 days. Transverse microtubules are found throughout growth along with a subset of distinctly non-transverse microtubules. During the second half of the growing period, when relative growth rates are diminishing, these non-transverse microtubules become more abundant but a few days prior to growth cessation, they are mostly absent. At about the time of growth cessation the microtubules, while retaining their locally parallel alignment, begin to show increasing deviation from the transverse axis. Eventually, a mosaic of locally parallel yet variably oriented fields of microtubules forms. Many days after growth stops, microtubules become shorter and less numerous and lose parallel alignment, leading to the formation of a random MT pattern.     

Microtubule configurations and post-translational alpha-tubulin modifications during mammalian spermatogenesis

The mechanisms underlying cell cycle progression and differentiation are tightly entwined with changes associated in the structure and composition of the cytoskeleton. Mammalian spermatogenesis is a highly intricate process that involves differentiation and polarization of the round spermatid. We found that pachytene spermatocytes and round spermatids have most of the microtubules randomly distributed in a cortical network without any apparent centrosome. The Golgi apparatus faces the acrosomal vesicle and some microtubules contact its surface. In round spermatids, at step 7, there is an increase in short microtubules around and over the nucleus. These microtubules are located between the rims of the acrosome and may be the very first sign in the formation of the manchette. This new microtubular configuration is correlated with the beginning of the migration of the Golgi apparatus from the acrosomal region towards the opposite pole of the cell. Next, the cortical microtubules form a bundle running around the nucleus perpendicular to the main axis of the cell. At later stages, the nuclear microtubules increase in size and a fully formed manchette appears at stage 9. On the other hand, acetylated tubulin is present in a few microtubules in pachytene spermatocytes and in the axial filament (precursor of the sperm tail) in round spermatids. Our results suggest that at step 7, the spermatid undergoes a major microtubular reordering that induces or allows organelle movement and prepares the cell for the formation of the manchette and further nuclear shaping. This new microtubular configuration is associated with an increase in short microtubules over the nucleus that may correspond to the initial step of the manchette formation. The new structure of the cytoskeleton may be associated with major migratory events occurring at this step of differentiation.     

Reorganization of interphase microtubules in root cells of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. during acclimation to osmotic and salt stress

We examined the organization of microtubule system of interphase cells in roots of Medicago sativa L. during acclimation to salt and osmotic stress at different concentrations of NaCl, Na₂SO₄, and mannitol. We identified morphological changes of tubulin cytoskeleton in different root tissues during the acclimation to salt and osmotic stress: (1) decreased density of the cortical microtubule network, (2) random orientation of cortical microtubule bundles, (4) thickening of the bundles, (3) nonuniform density of the bundles, (4) fragmentation of the bundles, and (5) formation of microtubule converging centers. Network thinning and thickening of the bundles were observed both under osmotic and salt stress. Random orientation of cortical microtubules was visualized under osmotic stress but not during salt stress. Fragmentation of microtubule bundles took place under salt stress with a high concentration of mannitol. Formation of microtubule converging centers was common under prolonged action of sodium sulfate, less evident under sodium chloride, and not found after mannitol treatment. Our data show that, in alfalfa root cells, cortical microtubules rearrange not only in response to different ions, but also to osmotic pressure. Thus, the signaling pathways and molecular mechanisms inducing reorganization of the microtubule system may be triggered by sodium cations, as well as by sulfate and chloride anions at concentrations that do not cause irreversible cell damage.     

Microtubule reorganization as a response to implementation of NO signals in plant cells

The effects of an exogeneous NO donor, sodium nitroprusside, on the orientation and organization of cortical microtubules in Arabidopsis thaliana root cells expressing GFP-MAP4 were studied in vivo. It was found that sodium nitroprusside treatment (10–500 μM, 24 h) caused the acceleration of primary root growth and enhanced initiation of root hairs in the differentiation zone. The influence of sodium nitroprusside revealed in changes in the orientation and organization of cortical microtubules in different types of cells of A. thaliana root. The most sensitive to sodium nitroprusside exposure were microtubules in epidermal cells of the elongation zone, where native transverse orientation of cortical microtubules turned into random, oblique, or longitudinal relative to the primary root axis. We suggest that NO, as one of the intracellular secondary messengers, triggers cell differentiation by reorientation of cortical microtubules, possibly via tubulin nitrotyrosination.     

Rapid and reversible tubulin tyrosination in human neutrophils stimulated by the chemotactic peptide,fMet-Leu-Phe

Neutrophil activation by specific stimuli, such as the oligopeptide chemotactic factor fMet-Leu-(fMLF), is associated with an increased enzymatic addition of tyrosine to tubulin α -subunits, as measured by ¹⁴C tyrosine uptake. In studies using immunoblots we have found that this increased tyrosine uptake into tubulin in activated neutrophils reflects an increase in the proportion of cellular tubulin that is tyrosinated rather than simply an increase in the turnover of tyrosinated subunits. However, the increased accumulation of tyrosinated tubulin was also found to follow an initial depletion of tyrosinated tubulin and concomitant increase in detyrosinated tubulin between 0 and 60 sec following stimulation of neutrophils with fMLF. Immunogold electron microscopy studies of intact micro tubules recovered from activated neutrophils demonstrated that these rapid changes in the relative content of tubulin isoforms in the cells were not associated with the formation or disappearance of microtubule microdomains composed of only one form of tubulin. Previously, we have shown that under conditions of fMLF-stimulated exocytosis there is an increased binding of neutrophil granules to endogenous microtubules. Since neutrophil activation by fMLF is associated with increased tyrosination of α -tubulin subunits, we speculated that rapid changes in the levels of tyrosinated tubulin in the microtubules of activated neutrophils might have a role in the regulation of granule-microtubule interactions. When the binding of purified neutrophil granules to reconstituted rat brain microtubules containing approximately 50% tyrosinated tubulin was measured by electron microscopy and compared with granule binding to microtubules that contained no detectable tyrosinated tubulin, granule-microtubule associations were found to be significantly favored by detyrosinated vs. tyrosinated tubulin. These findings indicate that interactions between cytoplasmic granules and microtubules in activated neutrophils may be modulated by rapid changes in the relative content of detyrosinated and tyrosinated tubulin in the microtubule network of the cells. © 1993 Wiley-Liss, Inc.     

Microtubule orientation and dynamics in elongating characean internodal cells following cytosolic acidification, induction of pH bands, or premature growth arrest

Summary Cortical microtubules (MTs) at indifferent zones in immatureNitella internodes were investigated by injection of fluorescently tagged sheep brain tubulin into living cells and by immunofluorescence on fixed material. Nearly identical MT patterns and numbers were detected with the two techniques, indicating that sheep brain tubulin incorporated into all cortical MTs. MTs were aligned transversely to the long axis of the cell and approximately one MT was present every micrometer of longitudinal cell distance. Treatment of internodes with propionic acid to acidify cytosolic pH caused depolymerization of MTs and an increase in the unpolymerized tubulin pool. Transfer of young, vigorously elongating cells to media inducing premature growth cessation resulted in a slight decrease in microtubule numbers but did not significantly alter microtubule orientation patterns or microtubule lifespans. MTs remained transverse for days following growth cessation before finally assuming a more random alignment characteristic of mature, non-growing internodes. No differences in MT numbers, orientation, or dynamics were detected between acid and alkaline bands in internodes incubated in a band-inducing medium. Thus, properties of cortical MT arrays were not closely coupled to growth status or to regional differences in cellular physiology associated with pH banding.Abbrevations BIM band-inducing medium - CCM Chara culture medium - CF carboxyfluorescein - FRAP fluorescence redistribution after photobleaching - MT microtubule     

Injury toNitella internodal cells alters microtubule organization but microtubules are not involved in the wound response

Summary The arrangement and relative stability of cortical microtubules during and after wound induction in internodal cells ofNitella flexilis andNitella pseudoflabellata were examined by immunofluorescence and by microinjection of fluorescently tagged tubulin. The formation of cellulosic wall appositions (wound walls), induced by treatment with 5×10^–2MCaCl₂, was identicalin young, growing cells and older non-growing internodes, suggesting that the initial microtubule pattern, which differs in growing and non-growing cells, does not influence wound wall formation. Depolymerization of microtubules with oryzalin did not alter wound wall morphology and microtubules were not detected during wound wall formation. After cessation of wound wall growth, microtubules were once again found in the wound site but these were always randomly oriented, even in young cells where the surrounding microtubules were organized into transverse arrays. Microtubules were similarly randomized in chloroplast-free windows induced by laser irradiation. Analysis of microtubule organization in living cells revealed that the microtubules in wound sites are less stable than the microtubules of adjacent transversely oriented arrays. The results indicate that although wounding can alter the relative stability and spatial organization of cortical microtubules, microtubules are neither involved in vesicle transport nor the construction of cellulosic wound walls.Abbreviations AFW artificial fresh water - BSA bovine serum albumin - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline     

Organization of the cytoskeleton in pollen tubes ofPyrus communis: a study employing conventional and freeze-substitution electron microscopy,immunofluorescence, and rhodamine-phalloidin

Summary The structure and organization of the cytoskeleton in the vegetative cell of germinated pollen grains and pollen tubes ofPyrus communis was examined at the ultrastructural level via chemical fixation and freeze substitution, and at the light microscopic level with the aid of immunofluorescence of tubulin and rhodamine-phalloidin.Results indicate that cortical microtubules and microfilaments, together with the plasma membrane, form a structurally integrated cytoskeletal complex. Axially aligned microtubules are present in cortical and cytoplasmic regions of the pollen grain portion of the cell and the distal region of the pollen tube portion. Cytoplasmic bundles of microfilaments are found in association with elements of endoplasmic reticulum and vacuoles. Axially aligned microfilaments are also found in this region, associated with and independent of the microtubules. Microtubules are lacking in the subapical region where short, axially aligned microfilaments are found in the cell cortex. In the apical region, which also lacks microtubules, a 3-dimensional network of short microfilaments occurs. Microfilaments, but not microtubules, appear to be associated with the vegetative nucleus.     

Changes in the density of microtubular networks in mesophyll cells and mesophyll derived protoplasts of Nicotiana and Triticum during leaf development

Changes in the density of microtubular mesh-works were analysed in mesophyll cells and mesophyll derived protoplasts of Nicotiana tabacum L. and Triticum aestivum L. during leaf development. The main purpose of this study was to test whether the low density, if not lack, of microtubular networks recently described in protoplasts that had been isolated from fully differentiated mesophyll cells happened during protoplast isolation or whether the loss of microtubules actually occurred during differentiation of the leaf tissue. Immunofluorescence microscopy showed that the density of the microtubular cytoskeleton in the leaf tissue decreased steadily after cessation of cell growth in both species. Nevertheless, in Triticum microtubule disappearance was swifter and occurred along a gradient from the base to tip of the leaf, a phenomenon reflecting the differences in the ontogeny between the dicotyledonous Nicotiana and the mono-cotyledonous Triticum leaves. Protein extraction from leaf tissues and Western blot analysis indicated that in both species the disappearance of microtubules was the result of a degradation of tubulin and not only due to a depolymerisation into tubulin subunits. When the cell walls were removed from live cells and the protoplasts released, the original patterns of the microtubules became obscured and, particularly in differentiated cells, the integrity and density of the microtubule strands deteriorated. The potential application of the density of the microtubular cytoskeleton as a marker in studies on differentiation and dedifferentiation in mesophyll cells and protoplasts is discussed.We wish to thank Silke Heichel for excellent technical assistance. We also express our thanks to the group of A.M. Lambert at CNRS, Strasbourg, France, for advice during establishment of our Western blot system. The work was supported by a grant of the German Ministry of Science and Technology (BMFT).     

Microtubule organization and cell division in embryogenie protoplast cultures of white spruce (Picea glauca)

Summary Immunofluorescence methods were developed for examining the distribution of microtubules in freshly isolated and cultured protoplasts and regenerated somatic embryos of white spruce (Picea glauca). Freshly isolated protoplasts consisted of both uniand multinucleate types. Uninucleate protoplasts established parallel cortical microtubules during cell wall formation and cell shaping, divided within 24 h and developed into somatic embryos in culture. Dividing cells were characterized by preprophase bands (PPBs) of microtubules, atypical spindle microtubules focused at the poles and a typical phragmoplast at telophase. Multinucleate protoplasts also established parallel arrays of cortical microtubules during cell wall formation. In addition their nuclei divided synchronously within 4 days, then cell walls formed between the daughter nuclei. Individual multinucleate protoplast-derived colonies subsequently gave rise to elongate suspensor cells thereby forming embryo-like structures by 7 days.     

The Homology of Cortical Microtubules in Platyhelminth Spermatozoa: a Comparative Immunocytochemical Study of Acetylated Tubulin

Spermatozoa of certain acoels, a group of primitive Platyhelminthes, and spermatozoa of the most derived Platyhelminthes, the Cercomeridea (parasitic Platyhelminthes), show a general morphological resemblance in that they are long filiform cells with two incorporated axonemes and longitudinal cortical microtubules. A possible way to test the homology of these cortical microtubules in the different groups is to analyze the presence/absence of post-translational modifications of tubulin. An indirect immunofluorescence study showed that the doublet microtubules of the sperm axonemes are labelled by an anti acetylated-alpha-tubulin antibody in all groups, irrespective of the axoneme pattern (9 + 0, 9 + 2 and non-trepaxonematan 9 + “1” in various acoels, and trepaxonematan 9 + “1” of the temnocephalid Troglocaridicola sp., the digenean Echinostoma caproni and the monopisthocotylean monogenean Pseudodactylogyrus sp.). Significant differences are found in the sperm cortical microtubules: they are acetylated in the acoel Actinoposthia beklemischevi, but not in the digenean E. caproni and the temnocephalid Troglocaridicola sp. These results suggest that the sperm cortical microtubules of the acoels are not homologous with the morphologically similar elements found in the higher Platyhelminthes.     

Structure and function of the microtubular cytoskeleton during megasporogenesis and embryo sac development in Gasteria verrucosa (Mill.) H. Duval

Summary Using immunocytochemical techniques, tubulin distribution in various stages of meiosis and embryo sac development was studied. In the archespore cell some microtubules appeared to be randomly oriented. During zygotene and pachytene, when the cell volume increases, a large number of microtubules in dispersed configurations and bundles were observed. During this stage the nucellar cells divide, and their parallel cortical microtubules play an important role in preparing the direction of cell enlargement. The protoderm cells show anticlinal-directed cortical microtubules. It can be concluded that the enlargement of the meiocyte during these early meiotic stages is influenced both by its own cytoskeleton and by growth of the nucellus. Thereafter, the microtubules function directly in meiosis and disappear for the greater part until the two-nucleate coenocyte is formed. In a four-nucleate coenocyte microtubules reappear around the nucleus; in a young synergid, randomly oriented microtubules are involved in cell shaping during the formation of the filiform apparatus; in the synergids of the mature embryo sac, many parallel arrays of microtubules are present. Microtubules are less abundant in other cells. It is concluded that the cytomorphogenesis of the developing coenocyte and embryo sac are due to cell growth of the nucellar cells together with vacuolation of the coenocyte.     

Dynein supports motility of endoplasmic reticulum in the fungus Ustilago maydis

The endoplasmic reticulum (ER) of most vertebrate cells is spread out by kinesin-dependent transport along microtubules, whereas studies in Saccharomyces cerevisiae indicated that motility of fungal ER is an actin-based process. However, microtubules are of minor importance for organelle transport in yeast, but they are crucial for intracellular transport within numerous other fungi. Herein, we set out to elucidate the role of the tubulin cytoskeleton in ER organization and dynamics in the fungal pathogen Ustilago maydis. An ER-resident green fluorescent protein (GFP)-fusion protein localized to a peripheral network and the nuclear envelope. Tubules and patches within the network exhibited rapid dynein-driven motion along microtubules, whereas conventional kinesin did not participate in ER motility. Cortical ER organization was independent of microtubules or F-actin, but reformation of the network after experimental disruption was mediated by microtubules and dynein. In addition, a polar gradient of motile ER-GFP stained dots was detected that accumulated around the apical Golgi apparatus. Both the gradient and the Golgi apparatus were sensitive to brefeldin A or benomyl treatment, suggesting that the gradient represents microtubule-dependent vesicle trafficking between ER and Golgi. Our results demonstrate a role of cytoplasmic dynein and microtubules in motility, but not peripheral localization of the ER in U. maydis.     

αTubulin 67C and Ncd Are Essential for Establishing a Cortical Microtubular Network and Formation of the Bicoid mRNA Gradient in Drosophila

The Bicoid (Bcd) protein gradient in Drosophila serves as a paradigm for gradient formation in textbooks. To explain the generation of the gradient, the ARTS model, which is based on the observation of a bcd mRNA gradient, proposes that the bcd mRNA, localized at the anterior pole at fertilization, migrates along microtubules (MTs) at the cortex to the posterior to form a bcd mRNA gradient which is translated to form a protein gradient. To fulfil the criteria of the ARTS model, an early cortical MT network is thus a prerequisite. We report hitherto undiscovered MT activities in the early embryo important for bcd mRNA transport: (i) an early and omnidirectional MT network exclusively at the anterior cortex of early nuclear cycle embryos showing activity during metaphase and anaphase only, (ii) long MTs up to 50 µm extending into the yolk at blastoderm stage to enable basal-apical transport. The cortical MT network is not anchored to the actin cytoskeleton. The posterior transport of the mRNA via the cortical MT network critically depends on maternally-expressed αTubulin67C and the minus-end motor Ncd. In either mutant, cortical transport of the bcd mRNA does not take place and the mRNA migrates along another yet undisclosed interior MT network, instead. Our data strongly corroborate the ARTS model and explain the occurrence of the bcd mRNA gradient.     

Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes

Microtubule assembly in vivo was studied by hapten-mediated immunocytochemistry. Tubulin was derivatized with dichlorotriazinylaminofluorescein (DTAF) and microinjected into living, interphase mammalian cells. Sites of incorporation were determined at the level of individual microtubules by double-label immunofluorescence. The haptenized tubulin was localized by an anti-fluorescein antibody and a second antibody conjugated with fluorescein. Total microtubules were identified by anti-tubulin and a secondary antibody conjugated with rhodamine. Contrary to recent studies (Salmon, E. D., et al., 1984, J. Cell Biol., 99:2165-2174; Saxton, W. M., et al., 1984, J. Cell Biol., 99:2175-2186) which suggest that tubulin incorporates all along the length of microtubules in vivo, we found that microtubule assembly in interphase cells was in vivo, as in vitro, an end-mediated process. Microtubules that radiated out toward the cell periphery incorporated the DTAF-tubulin solely at their distal, that is, their plus ends. We also found that a proportion of the microtubules connected to the centrosomes incorporated the DTAF-tubulin along their entire length, which suggests that the centrosome can nucleate the formation of new microtubules.