Microtubules,protoplasts and plant cell shape

Indirect immunofluorescence has been used to study the function of cytoplasmic microtubules in controlling the shape of elongated carrot cells in culture. Using a purified wall-degrading preparation, the elongated cells are converted to spherical protoplasts and the transverse hoops of bundled microtubules are disorganised but not depolymerised in the process. Since microtubules remain attached to fragments of protoplast membrane adhering to coverslips and are still seen to be organised laterally in bundles, it would appear that re-orientation of the transverse bundles is due to loss of cell wall and not to the cleavage of microtubule bridges. After 24 h treatment in 10^-3 M colchicine, microtubules are depolymerised in elongated cells but, at this time, the cells retain their elongated shape. This suggests that wall which was organised in the presence of transverse microtubule bundles can retain asymmetric shape for short periods in the absence of those tubules. However, after longer periods of time the cells become spherical in colchicine. Neither wall nor tubules therefore exert individual control on continued cellular elongation and so we emphasize the fundamental nature of wall/microtubule interactions in shape control. It is concluded that the observations are best explained by a model in which hooped bundles of microtubules—which are directly or indirectly associated with molecules involved with cellulose biosynthesis at the cell surface—act as an essential template or scaffolding for the orientated deposition of cellulose.     

Microtubule networks for plant cell division

During cytokinesis the cytoplasm of a cell is divided to form two daughter cells. In animal cells, the existing plasma membrane is first constricted and then abscised to generate two individual plasma membranes. Plant cells on the other hand divide by forming an interior dividing wall, the so-called cell plate, which is constructed by localized deposition of membrane and cell wall material. Construction starts in the centre of the cell at the locus of the mitotic spindle and continues radially towards the existing plasma membrane. Finally the membrane of the cell plate and plasma membrane fuse to form two individual plasma membranes. Two microtubule-based cytoskeletal networks, the phragmoplast and the pre-prophase band (PPB), jointly control cytokinesis in plants. The bipolar microtubule array of the phragmoplast regulates cell plate deposition towards a cortical position that is templated by the ring-shaped microtubule array of the PPB. In contrast to most animal cells, plants do not use centrosomes as foci of microtubule growth initiation. Instead, plant microtubule networks are striking examples of self-organizing systems that emerge from physically constrained interactions of dispersed microtubules. Here we will discuss how microtubule-based activities including growth, shrinkage, severing, sliding, nucleation and bundling interrelate to jointly generate the required ordered structures. Evidence mounts that adapter proteins sense the local geometry of microtubules to locally modulate the activity of proteins involved in microtubule growth regulation and severing. Many of the proteins and mechanisms involved have roles in other microtubule assemblies as well, bestowing broader relevance to insights gained from plants.     

Microtubule reorganization,cell wall synthesis and establishment of the axis of elongation in regenerating protoplasts of the algaMougeotia

Summary Microtubule reorganization and cell wall deposition have been monitored during the first 30 hours of regeneration of protoplasts of the filamentous green algaMougeotia, using immunofluorescence microscopy to detect microtubules, and the cell-wall stain Tinopal LPW to detect the orientation of cell wall microfibrils. In the cylindrical cells of the alga, cortical microtubules lie in an ordered array, transverse to the long axis of the cells. In newly formed protoplasts, cortical microtubules exhibit some localized order, but within 1 hour microtubules become disordered. However, within 3 to 4 hours, microtubules are reorganized into a highly ordered, symmetrical array centered on two cortical foci. Cell wall synthesis is first detected during early microtubule reorganization. Oriented cell wall microfibrils, co-aligned with the microtubule array, appear subsequent to microtubule reorganization but before cell elongation begins. Most cells elongate in the period between 20 to 30 hours. Elongation is preceded by the aggregation of microtubules into a band intersecting both foci, and transverse to the incipient axis of elongation. The foci subsequently disappear, the microtubule band widens, and microfibrils are deposited in a band which is co-aligned with the band of microtubules. It is proposed that this band of microfibrils restricts lateral expansion of the cells and promotes elongation. Throughout the entire regeneration process inMougeotia, changes in microtubule organization precede and are paralleled by changes in cell wall organization. Protoplast regeneration inMougeotia is therefore a highly ordered process in which the orientation of the rapidly reorganized array of cortical microtubules establishes the future axis of elongation.     

Secondary cell wall patterning during xylem differentiation

Xylem cell differentiation involves temporal and spatial regulation of secondary cell wall deposition. The cortical microtubules are known to regulate the spatial pattern of the secondary cell wall by orientating cellulose deposition. However, it is largely unknown how the microtubule arrangement is regulated during secondary wall formation. Recent findings of novel plant microtubule-associated proteins in developing xylem vessels shed new light on the regulation mechanism of the microtubule arrangement leading to secondary wall patterning. In addition, in vitro culture systems allow the dynamics of microtubules and microtubule-associated proteins during secondary cell wall formation to be followed. Therefore, this review focuses on novel aspects of microtubule dynamics leading to secondary cell wall patterning with a focus on microtubule-associated proteins.     

Microtubule cortical array organization and plant cell morphogenesis

Plant cell cortical microtubule arrays attain a high degree of order without the benefit of an organizing center such as a centrosome. New assays for molecular behaviors in living cells and gene discovery are yielding insight into the mechanisms by which acentrosomal microtubule arrays are created and organized, and how microtubule organization functions to modify cell form by regulating cellulose deposition. Surprising and potentially important behaviors of cortical microtubules include nucleation from the walls of established microtubules, and treadmilling-driven motility leading to polymer interaction, reorientation, and microtubule bundling. These behaviors suggest activities that can act to increase or decrease the local level of order in the array. The SPIRAL1 (SPR1) and SPR2 microtubule-localized proteins and the radial swollen 6 (rsw-6) locus are examples of new molecules and genes that affect both microtubule array organization and cell growth pattern. Functional tagging of cellulose synthase has now allowed the dynamic relationship between cortical microtubules and the cell-wall-synthesizing machinery to be visualized, providing direct evidence that cortical microtubules can organize cellulose synthase complexes and guide their movement through the plasma membrane as they create the cell wall.     

Mechanisms for shaping,orienting, positioning and patterning plant secondary cell walls

Xylem vessels are cells that develop a specifically ornamented secondary cell wall to ensure their vascular function, conferring both structural strength and impermeability. Further plasticity is given to these vascular cells by a range of different patterns described by their secondary cell walls that—as for the growth of all plant organs—are developmentally regulated. Microtubules and their associated proteins, named MAPs, are essential to define the shape, the orientation, the position and the overall pattern of these secondary cell walls. Key actors in this process are the land-plant specific MAP70 proteins which not only allow the secondary cell wall to be positioned at the cell cortex but also determine the overall pattern described by xylem vessel secondary cell walls.Key words: xylem/wood vessels, tracheary elements, secondary cell wall, cell wall patterning, microtubules, microtubule-associated proteins, MAP70Xylem formation has been one of the key steps of plant evolution. These physically strong tube cells allowed plants to colonize land by reinforcing their upright position against gravity and resisting desiccation by permitting water conduction throughout the plant body. This double role is fulfilled by specific conducting wood cells—the tracheary elements (TEs). These cells represent the cellular units of the adjustable plant vasculature, which relies on the three structural characteristics of TEs: (1) these cells develop a secondary cell wall to resist pressure exerted by the sap they will conducted, (2) these cells undergo programmed cell death (PCD) to hollow out their entire cytoplasmic content to form a conduit for the sap and (3) these cells will undergo a terminal perforation at their basal end (with respect to the corresponding meristem) to form a complete functional vascular cylinder which will connect with the underlying vascular vessels once terminally differentiated.^1,2 TEs are further characterized by a diversity of organizational pattern described by their secondary cell wall, which can be annular or spiral (referred to as protoxylem-type ornamentations) reticulate or pitted (referred to as metaxylem-type ornamentations).^3,4 These differently ornamented TEs are developmentally regulated and for protoxylemtype TEs appear during the development of early primary tissues (annular TEs are mostly observed in developing embryos) while metaxylem-type TEs appear in the later development of primary and secondary tissues (they represent the TEs present in wood). Annular and spiral TEs are first formed in organs undergoing primary growth and are considered to be “extendable” (their pattern in rings and spirals does not oppose further extension of the TE cell) during the growth of this organ. Once the growing organ has attained a certain size these TEs will be crushed by the surrounding tissue whilst the more heavily reinforced reticulate and pitted TEs will form to insure the vascular flow and strengthen the entire organ. In short, the modularity and plasticity of this plant vascular system is directly dependant on the differentiation and the type of cell wall ornamentation of its constituent TEs. The establishment of such regular patterning of secondary cell walls has been attributed to the underlying cortical microtubule array that predefines the cell wall depositions (reviewed in ref. ²). Pharmacological modulation of microtubule properties in both whole plants and in vitro TE differentiating systems leads to severe defects in the patterning, orientation, smoothness and deposition of TE secondary cell walls (reviewed in ref. ²).     

Interaction of auxin, light, and mechanical stress in orienting microtubules in relation to tropic curvature in the epidermis of maize coleoptiles

Summary Changes in the orientation of cortical microtubules (longitudinal vs. transverse with respect to the long cell axis) at the outer epidermal wall of maize coleoptile segments were induced by auxin, red or blue light, and mechanical stresses (cell extension or compression produced by bending). Immunofluorescent techniques were used for the quantitative determination of frequency distributions of microtubule orientation. Detailed kinetic studies showed that microtubule reorientations are temporally correlated with the simultaneously measured changes in growth rate elicited by auxin, red light, or blue light. Growth inhibition induced by depletion of endogenous auxin produces a longitudinal microtubule pattern that can be changed into a transverse pattern in a dose-dependent manner by applying exogenous auxin. A mid-point pattern with equal frequencies of longitudinal and transverse microtubules was adjusted at 2 mol/1 auxin. Bending stress applied under these conditions adjusts permanent, maximally longitudinal and transverse microtubule orientations at the compressed and extended segment sides, respectively, quantitatively mimicking the responses to differential flank growth during phototropic and gravitropic curvature. During tropic curvature the changes in microtubule pattern reflect the distribution of growth rather than the distribution of auxin. The microtubule pattern responds to auxin-dependent growth changes and mechanical stress in a synergistic manner, confirming the functional equivalence of these factors in affecting microtubule orientation. Similar results were obtained when segment growth was altered by blue or red light instead of auxin in the presence or absence of mechanical stress. It is concluded from these results that growth changes, elicited by auxin, light, etc., and mechanical stress affect microtubule orientation through a common signal perception and transduction chain.Abbreviations IAA indole-3-acetic acid (auxin) - MT cortical microtubule     

Microtubule organization in the differentiating transfer cells of the placenta inLilium spp.

Summary Placental cells in the ovarian transmitting tissue ofLilium spp. are organized as transfer cells with inbuddings facing the ovarian locule. A detailed analysis of microtubule (MT) organization during development of these polarized cells is reported here. Formation of wall projections occurs at the apical part of the cell starting on the day of anthesis, and a fully mature secretion zone is found four days after anthesis. MTs are organized into distinct cortical and central arrays. The cortical array undergoes a unique transition at anthesis. MTs in the basal half of the cell remain in longitudinal bundles while in the apical half of the cell their longitudinal orientation is replaced by a transverse alignment. One day after anthesis, these transverse bundles become a meshwork of short, randomly organized MTs, while MTs in the basal half of the cell retain their longitudinal alignment. The realignment of MTs in the apical half of the cell coincides with the deposition of the secondary cell wall. The central array is composed of short, randomly arranged strands of MTs in the cytoplasm between the nucleus and the apical and basal periclinal walls of the cell. This array first appears as solitary strands in the apical part of the cell one day before anthesis. The central array extends during development and is eventually seen in the basal half of the cell. We propose that MTs in the cortical region near the apical wall act as templates for the deposition of cellulose microfibrils in the secondary cell wall. MTs in the central array in these transfer cells may be involved in the trafficking of vesicles and/or positioning of organelles near the secretion zone.Abbreviations MT microtubule - daa day after anthesis - dba day before anthesis     

Microheterogeneity of microtubule-associated proteins, MAP-1 and MAP-2, and differential phosphorylation of individual subcomponents

High molecular weight microtubule-associated proteins 1 and 2 (MAP-1 and MAP-2), prepared by copolymerization with tubulin, were electrophorectically separated into three and two major subcomponents, respectively, using 5% sodium dodecyl sulfate-polyacrylamide gels. By two-dimensional gel electrophoresis, all five MAP components were shown to possess a pI of around 5. Four of these proteins, MAP-1A, MAP-1C, MAP-2A, and MAP-2B, present in comparable amounts, were iodinated after electrophoretic separation and analyzed by two-dimensional peptide mapping. With both trypsin and V8 protease, almost identical patterns were obtained from MAP-2A and MAP-2B. MAP-1A and MAP-1C, too, gave similar digestion patterns, although some differences were noted. Incubation with [gamma-32P]ATP demonstrated that endogeneous protein kinase activities phosphorylated individual subcomponents at different rates. MAP-2A, the highest labeled component, was phosphorylated 2.5-fold compared to MAP-2B both in the presence and the absence of cAMP. Labeling of MAP-1 subcomponents was 4 times less than that of MAP-2A in the absence and 16 times less in the presence of cAMP. 32P-labeled MAP-2A and MAP-2B bands were indistinguishable by one-dimensional peptide mapping, as were the three MAP-1 bands. For both MAP-1 and MAP-2 subcomponents, cAMP induced phosphorylation at new molecular sites. Incubation of radiolabeled microtubule proteins with 1 mM ATP effected, upon electrophoresis, a clear shift of MAP-2A and MAP-2B bands to positions of higher apparent molecular weights, while only slightly affecting MAP-1 bands.     

Expression of biotin-binding proteins,avidin and streptavidin,in plant tissues using plant vacuolar targeting sequences

Tobacco plants have been developed which constitutively express high levels of the biotin-binding proteins, avidin and streptavidin. These plants were phenotypically normal and produced fertile pollen and seeds. The transgene was expressed and its product located in the vacuoles of most cell types in the plants. Targeting was achieved by use of N-terminal vacuolar targeting sequences derived from potato proteinase inhibitors which are known to target constitutively to vacuoles in potato tubers and, under wound-induction, in tomato leaves. Avidin was located in protein body-like structures within the vacuole and transgene protein levels remained relatively constant throughout the lifetime of the leaf. We describe two chimeric constructs with similar levels of expression. One comprised a potato proteinase inhibitor I signal peptide cDNA sequence attached to an avidin cDNA and the second a potato proteinase inhibitor II signal peptide genomic sequence (including an intron) attached to a core streptavidin synthetic sequence. We were unable to regenerate plants when transformation used constructs lacking the targeting sequences. The highest levels observed (up to 1.5% of total leaf protein) confirm the vacuole as the organelle of choice for stable storage of plant-toxic transgene products. The efficient targeting of these proteins did not result in any measured changes in plant biotinmetabolism.     

Microtubule Associated Proteins in Plants and the Processes They Manage

Microtubule associated proteins （MAPs） are proteins that physically bind to microtubules in eukaryotes. MAPs play important roles in regulating the polymerization and organization of microtubules and in using the ensuing microtubule arrays to carry out a variety of cellular functions. In plants, MAPs manage the construction, repositioning, and dismantling of four distinct microtubule arrays throughout the cell cycle. Three of these arrays, the cortical array, the preprophase band, and the phragmoplast, are prominent to plants and are responsible for facilitating cell wall deposition and modification, transducing signals, demarcating the plane of cell division, and forming the new cell plate during cytokinesis. This review highlights important aspects of how MAPs in plants establish and maintain microtubule arrays as well as regulate cell growth, cell division, and cellular responses to the environment.     

Differential distribution of microtubule-associated proteins MAP-1 and MAP-2 in neurons of rat brain and association of MAP-1 with microtubules of neuroblastoma cells (clone N2A).

To study the individual location of the microtubule proteins MAP-1 and MAP-2 in neuronal tissues and cells, antisera to electrophoretically purified MAP-1 and MAP-2 components were raised in rabbits. When frozen sections through rat brain were examined by immunofluorescence microscopy the antibodies to MAP-1 strongly stained a variety of nerve cells including dendrites and myelinated axons in the cerebrum and cerebellum. Antibodies to MAP-2 showed similar staining patterns, except that myelinated axons were unstained. These results were confirmed by immunoelectron microscopy of frozen sections through cerebellum using the peroxidase technique. Thereby, the association of MAP-1 with microtubules was also clearly demonstrated. When cultured mouse neuroblastoma N2A cells were examined by immunofluorescence microscopy the antiserum to MAP-1 brightly stained filamentous structures resembling microtubules, whereas relatively weak and diffuse staining of the cytoplasm was observed with the antiserum to MAP-2. In agreement with the immunolocalization, MAP-1, but not MAP-2, was found as a prominent component of microtubules proteins polymerized in vitro by taxol from soluble N2A cell extracts. Together these results indicate that neuronal microtubules are preferentially associated with distinct high mol. wt. polypeptides. Therefore, they support the concept that different complements of associated proteins determine distinct functions of microtubules.     

Proteomic Analysis of Microtubule Interacting Proteins over the Course of Xylem Tracheary Element Formation in Arabidopsis

Plant vascular cells, or tracheary elements (TEs), rely on circumferential secondary cell wall thickenings to maintain sap flow. The patterns in which TE thickenings are organized vary according to the underlying microtubule bundles that guide wall deposition. To identify microtubule interacting proteins present at defined stages of TE differentiation, we exploited the synchronous differentiation of TEs in Arabidopsis thaliana suspension cultures. Quantitative proteomic analysis of microtubule pull-downs, using ratiometric ¹⁴N/¹⁵N labeling, revealed 605 proteins exhibiting differential accumulation during TE differentiation. Microtubule interacting proteins associated with membrane trafficking, protein synthesis, DNA/RNA binding, and signal transduction peaked during secondary cell wall formation, while proteins associated with stress peaked when approaching TE cell death. In particular, CELLULOSE SYNTHASE-INTERACTING PROTEIN1, already associated with primary wall synthesis, was enriched during secondary cell wall formation. RNAi knockdown of genes encoding several of the identified proteins showed that secondary wall formation depends on the coordinated presence of microtubule interacting proteins with nonoverlapping functions: cell wall thickness, cell wall homogeneity, and the pattern and cortical location of the wall are dependent on different proteins. Altogether, proteins linking microtubules to a range of metabolic compartments vary specifically during TE differentiation and regulate different aspects of wall patterning.     

MAPs in plant cells: delineating microtubule growth dynamics and organization

Microtubule-associated proteins (MAPs) serve a wide variety of functions, from constructing and maintaining the microtubule cytoskeleton to using this cytoskeleton to transport cargo and to tether molecules that are involved in numerous cellular processes. Throughout the cell cycle, distinct microtubule arrays carry out specific roles in cytokinesis, karyokinesis, and cell expansion. Recent findings have shed new light on the importance of MAPs in controlling microtubule growth dynamics as well as in cross-linking microtubules to facilitate the formation and function of these cytoskeletal arrays.     

Progress in understanding the role of microtubules in plant cells

Microtubules have long been known to play a key role in plant cell morphogenesis, but just how they fulfill this function is unclear. Transverse microtubules have been thought to constrain the movement of cellulose synthase complexes in order to generate transverse microfibrils that are essential for elongation growth. Surprisingly, some recent studies demonstrate that organized cortical microtubules are not essential for maintaining or re-establishing transversely oriented cellulose microfibrils in expanding cells. At the same time, however, there is strong evidence that microtubules are intimately associated with cellulose synthesis activity, especially during secondary wall deposition. These apparently conflicting results provide important clues as to what microtubules do at the interface between the cell and its wall. I hypothesize that cellulose microfibril length is an important parameter of wall mechanics and suggest ways in which microtubule organization may influence microfibril length. This concept is in line with current evidence that links cellulose synthesis levels and microfibril orientation. Furthermore, in light of new evidence showing that a wide variety of proteins bind to microtubules, I raise the broader question of whether a major function of plant microtubules is in modulating signaling pathways as plants respond to sensory inputs from the environment.     

Microtubule organization and morphogenesis of stomata in caffeine-affected seedlings ofZea mays

Summary Treatment ofZea mays seedlings with a 5 mM caffeine solution inhibits cytokinesis in guard cell mother cells (GMCs), producing unicellular, binucleate aberrant stomata (a-stomata). Ventral wall (VW) strips of limited length, which usually meet the wall portions of GMCs adjoining the cortical zone of the preprophase microtubule band (PMB), are laid down in many a-stomata.In a-stomata with or without VW-strips, the periclinal walls are lined by numerous microtubules (Mts) converging on their mid-region, where local wall thickenings are deposited. When the VW-strips reach the mid-region of the periclinal walls, thickenings lined by numerous Mts rise at their free margins. In certain a-stomata an anticlinal wall column, surrounded by a dense Mt bundle, grows centripetally from either or both of the periclinal wall thickenings. In wall thickenings, the cellulose microfibrils are co-aligned with the adjacent Mts. Pore formation is initiated in all a-stomata. Deposition of an electron dense intra-wall material followed by lysis precedes pore opening. This process is closely related to the a-stornata morphogenesis. These observations show that the primary morphogenetic phenomenon in a-stomata is the establishment of an intense and stable polarity in the cytoplasm abutting on the mid-region of the periclinal walls and/or the adjacent plasmalemma area. Prime morphogenetic factor(s), including microtubule organizing centres (MTOCs), seem to function in these sites. Morphogenesis in a-stomata is a Mt-dependent process that is carried out as in normal stomata but in the absence of a VW.Abbreviations a-stomata unicellular binucleate aberrant stomata - CIPC chlorisopropyl-N-phenyl carbamate - GC guard cell - GMC guard cell mother cell - Mt microtubule - MTOC microtubule organizing centre - PMB preprophase microtubule band - VW ventral wall     

Molecular encounters at microtubule ends in the plant cell cortex

The cortical arrays that accompany plant cell division and elongation are organized by a subtle interplay between intrinsic properties of microtubules, their self-organization capacity and a variety of cellular proteins that interact with them, modify their behaviour and drive organization of diverse, higher order arrays during the cell cycle, cell growth and differentiation. As a polar polymer, the microtubule has a minus and a plus end, which differ in structure and dynamic characteristics, and to which different sets of partners and activities associate. Recent advances in characterization of minus and plus end directed proteins provide insights into both plant microtubule properties and the way highly organized cortical arrays emerge from the orchestrated activity of individual microtubules.     

Shifts in plant cell axiality: histogenetic influences on cellulose orientation in the succulent, Graptopetalum

The elongation of typical plant meristems can be explained, biophysically, by the cellulosic hoop reinforcement in the longitudinal walls of most of the cells. A cortical microtubule array is believed to govern the reinforcement alignment. The orientation of the reinforcement is studied here with polarized light. Transverse orientation appears to be invariant, regardless of division direction, in interior cells of at least some established meristems. Exceptional longitudinal reinforcement (i.e., in the direction of growth) is occasionally seen, however, in the outer epidermal wall of established Graptopetalum roots, leaves, and stems. It is found in pairs of elongate cells that arise from a longitudinal division. This behavior is prominent when the epidermis is shifting reinforcement to initiate new organs. Usually the division direction and the new cell long axis coincide; reinforcement parallels both. When these two factors are in marked opposition, as in certain longitudinal divisions of very broad cells, the reinforcement can follow the new cell's long axis even though this orients alignment perpendicular to the new wall. The normal prevailing effect of the division direction may relate to the formation of the cortical microtubule array parallel to the orientation of the preprophase band. The normally synergistic effect of the new cell's long axis may stem from the fact that when the band-like array occupies the smaller pair of anticlinal faces of the cuboidal cell (and hence parallels the cell's long dimension in surface view), it will have the densest and presumably most stable structure. Reinforcement orientation is seen as a function of at least two factors which bear on the stability of the newly forming array.     

Identification of microtubule-associated proteins in the centrosome, spindle, and kinetochore of the early Drosophila embryo

We have developed affinity chromatography methods for the isolation of microtubule-associated proteins (MAPs) from soluble cytoplasmic extracts and have used them to analyze the cytoskeleton of the early Drosophila embryo. More than 50 Drosophila embryo proteins bind to microtubule affinity columns. To begin to characterize these proteins, we have generated individual mouse polyclonal antibodies that specifically recognize 24 of them. As judged by immunofluorescence, some of the antigens localize to the mitotic spindle in the early Drosophila embryo, while others are present in centrosomes, kinetochores, subsets of microtubules, or a combination of these structures. Since 20 of the 24 antibodies stain microtubule structures, it is likely that most of the proteins that bind to our columns are associated with microtubules in vivo. Very few MAPS seem to be identically localized in the cell, indicating that the microtubule cytoskeleton is remarkably complex.     

Cell proliferation,cell shape,and microtubule and cellulose microfibril organization of tobacco BY-2 cells are not altered by exposure to near weightlessness in space

The microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant’s final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g. Likewise, at near weightlessness cortical microtubules in protoplasts have difficulty organizing into parallel arrays, which are required for proper plant cell elongation. However, intact plants do grow in space and therefore should have a normally functioning microtubule cytoskeleton. Since the main difference between protoplasts and plant cells in a tissue is the presence of a cell wall, we studied single, but walled, tobacco BY-2 suspension-cultured cells during an 8-day space-flight experiment on board of the Soyuz capsule and the International Space Station during the 12S mission (March–April 2006). We show that the cortical microtubule density, ordering and orientation in isolated walled plant cells are unaffected by near weightlessness, as are the orientation of the cellulose microfibrils, cell proliferation, and cell shape. Likely, tissue organization is not essential for the organization of these structures in space. When combined with the fact that many recovering protoplasts have an aberrant cortical microtubule cytoskeleton, the results suggest a role for the cell wall, or its production machinery, in structuring the microtubule cytoskeleton.