Dynamics of microtubule reassembly and reorganization in the coenocytic green alga Ernodesmis verticillata (Kützing) Børgesen

The dynamics of microtubule (MT) disassembly and reassembly were studied in the green alga Ernodesmis verticillata, using indirect immunofluorescent localization of tubulin. This alga possesses two distinct MT arrays: highly-ordered, longitudinally-oriented cortical MTs, and shorter perinuclear MTs radiating from nuclear surfaces. Perinuclear MTs are very labile, completely disassembling in the cold (cells on ice) within 5–10 min or in 25 μM amiprophos-methyl (APM) within 15–30 min. Although cortical MTs are generally absent after 3 h in APM, it takes 45–60 min before any cold-induced depolymerization is apparent, and some cortical MTs persist after 6 h of cold treatment. The extent of immunofluorescence of cytoplasmic (depolymerized?) tubulin is inversely proportional to the abundance of cortical MTs. Recovery of MT arrays upon warming or upon removal of APM occurs within 30–60 min for the perinuclear MTs, but the cortical arrays take much longer to regain their normal patterns. The cortical MTs initially reappear in a random distribution with respect to the cell axis, but within 3–4 d of warming (or 24–36 h of removing APM) they are nearly parallel to each other and to the cell's longitudinal axis. Thus, although the timing differs, the actual patterns of depolymerization and recovery are similar, irrespective of whether physical or chemical agents are used. Longer-term treatments in 1 μM APM indicate that despite the rapid disappearance of perinuclear MTs, a loss of the uniform nuclear spacing occurs gradually over 1–6 d. Similar disorganization of nuclei is obtained with long-term treatment with 1 μM taxol, where a gradual loss of perinuclear MTs is accompanied by an increased abundance of mitotic spindles. This implies that perinuclear MTs can disassemble in vivo in the presence of taxol, and that they are not the sole components involved in maintaining nuclear spacing in these coenocytes. The results indicate that both nuclear and cortical sites of MT nucleation may exist in this organism, and that MT reassembly and re-organization are temporally distinct events in cells that have highly-ordered arrays of long MTs.     

Induction of random microtubule polymerization in cold and drug-treated PtK1 cells following hyperosmotic shock treatment

The effects of hypertonic sucrose on spindle and interphase microtubule (MT) arrays of PtK1 cells were investigated by incubating cells in complete culture medium at 4 degrees or 37 degrees C, with or without hypertonic sucrose, nocodazole or vinblastine (VLB). Results from anti-tubulin immunofluorescence showed that sucrose-induced alterations of spindle morphology seen at 37 degrees C did not occur at cold temperatures, but cold-induced MT loss was diminished. Application of warm hypertonic sucrose following depolymerization of MTs by nocodazole or cold resulted in the formation of a "feltwork" of randomly oriented, short MTs throughout the cytoplasm. These results, and those obtained substituting VLB for nocodazole, suggest that the effects of sucrose depend on the cytoplasmic concentration of soluble tubulin and support the hypothesis that osmotic factors are involved in effects of hypertonic sucrose on MT organization.     

Metabolic inhibitors and mitosis: II. Effects of dinitrophenol/deoxyglucose and nocodazole on the microtubule cytoskeleton

Summary To examine the effects exerted on the microtubule (MT) cytoskeleton by dinitrophenol/deoxyglucose (DNP/DOG) and nocodazole, live PtK₁ cells were treated with the drugs and then fixed and examined by immunofluorescence staining and electronmicroscopy. DNP/DOG had little effect on interphase MTs. In mitotic cells, kinetochore and some astral fibers were clearly shortened in metaphase figures by DNP/DOG. Nocodazole rapidly broke down spindle MTs (except those in the midbody), while interphase cells showed considerable variation in the susceptibility of their MTs. Nocodazole had little effect on MTs in energy-depleted (DNP/DOG-treated) cells. When cytoplasmic MTs had all been broken down by prolonged nocodazole treatment and the cells then released from the nocodazole block into DNP/DOG, some MT reassembly occurred in the ATP-depleted state. MTs in permeabilized, extracted cells were also examined with antitubulin staining; the well-preserved interphase and mitotic arrays of MTs showed no susceptibility to nocodazole. In contrast, MTs suffered considerable breakdown by ATP, GTP and ATPS; AMPPNP had little effect. This susceptibility of extracted MT cytoskeleton to nucleotide phosphates was highly variable; some interphase cells lost all MTs, most were severely affected, but some retained extensive MT networks; mitotic spindles were diminished but structurally coherent and more stable than most interphase MT arrays.We suggest that: 1. in the living cell, ATP or nucleotide triphosphates (NTPs) are necessary for normal and nocodazole-induced MT disassembly; 2. the NTP requirement may be for phosphorylation; 3. shortening of kinetochore fibers may be modulated by compression and require ATP; 4. many of these results cannot be accomodated by the dynamic equilibrium theory of MT assembly/disassembly; 5. the use and role of ATP on isolated spindles may have to be reevaluated due to the effects ATP has on the spindle cytoskeleton of permeabilized cells.     

Complete disintegration of the microtubular cytoskeleton precedes its auxin-mediated reconstruction in postmitotic maize root cells

The inhibitory action of 0.1 microM auxin (IAA) on maize root growth was closely associated with a rapid and complete disintegration of the microtubular (MT) cytoskeleton, as visualized by indirect immunofluorescence of tubulin, throughout the growth region. After 30 min of this treatment, only fluorescent spots were present in root cells, accumulating either around nuclei or along cell walls. Six h later, in addition to some background fluorescence, dense but partially oriented oblique or longitudinal arrays of cortical MTs (CMTs) were found in most growing cells of the root apex. After 24 h of treatment, maize roots had adapted to the auxin, as inferred from the slowly recovering elongation rate and from the reassembly of a dense and well-ordered MT cytoskeleton which showed only slight deviations from that of the control root cells. Taxol pretreatment (100 microM, 24 h) prevented not only the rapid auxin-mediated disintegration of the MT cytoskeleton but also a reorientation of the CMT arrays, from transversal to longitudinal. The only tissue to show MTs in their cells throughout the auxin treatment was the epidermis. Significant resistance of transverse CMT arrays in these cells towards auxin was confirmed using a higher auxin concentration (100 microM, 24 h). The latter auxin dose also revealed inter-tissue-specific responses to auxin: outer cortical cell files reoriented their CMTs from the transversal to longitudinal orientation, whereas inner cortical cell files lost their MTs. This high auxin-mediated response, associated with the swelling of root apices, was abolished with the pretreatment of maize root with taxol.     

Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis

In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells.     

Cytoplasmic dynein is involved in the retention of microtubules at the centrosome in interphase cells

Cytoplasmic dynein is known to be involved in the establishment of radial microtubule (MT) arrays. During mitosis, dynein activity is required for tethering of the MTs at the spindle poles. In interphase cells, dynein inhibitors induce loss of radial MT organization; however, the exact role of dynein in the maintenance of MT arrays is unclear. Here, we examined the effect of dynein inhibitors on MT distribution and the centrosome protein composition in cultured fibroblasts. We found that while these inhibitors induced rapid ( t _1/2 ∼ 20 min) loss of radial MT organization, the levels of key centrosomal proteins or the rates of MT nucleation did not change significantly in dynein-inhibited cells, suggesting that the loss of dynein activity does not affect the structural integrity of the centrosome or its capacity to nucleate MTs. Live observations of the centrosomal activity showed that dynein inhibition enhanced the detachment of MTs from the centrosome. We conclude that the primary role of dynein in the maintenance of a radial MT array in interphase cells consists of retention of MTs at the centrosome and hypothesize that dynein has a role in the MT retention, separate from the delivery to the centrosome of MT-anchoring proteins.     

Experimental Studies on the Stability of the Cortical Microtubule Cytoskeleton in Relation to Polarity and Cell Elongation in the Coenocytic Green Alga, Chaetomorpha moniligera

The stability and ordered assembly of cytoskeletal microtubules(MTs) and the relationship between cell growth and MT cytoskeletonin the coenocytic green alga, Chaetomorpha moniligera Kjellmanwere examined. The cytoplasm of cylindrical growing cells ofChaetomorpha is covered with dense arrays of longitudinallyarranged cortical MTs which constitute the MT cytoskeleton.Seventy-five percent of MTs of the cytoskeleton disappearedwithin 4 h, with 25% remaining after 20 h following cold treatment.On terminating MT assembly with amiprophos-methyl (APM), thenumber of MTs decreased by 75% within 4 h. The remaining MTsdisappeared gradually within 24 h. The MT cytoskeleton of Chaetomorphawould thus appear to be composed of at least two kinds of MTsdiffering in stability. The MT cytoskeleton returned to normalafter treatment with APM for less than 48 h. However, this didnot occur after treatment with APM for more than 48 h, and theMT arrays became random. Cell elongation ceased completely within24 h after treatment with APM for less than 48 h but was restoredwithin 24 h after removing APM. The restoration of cell elongationwas no longer evident after removaI of APM for more than 48h. The results indicate that assembly of MTs into ordered arraysdepends on cell polarity and that in turn cell elongation isdependent on the polar-dependent arrays of MTs.Copyright 1994,1999 Academic Press Cell polarity, Chaetomorpha moniligera, coenocytic green alga, cold treatment, immunofluorescence, microtubule     

Centrosomal and non-centrosomal microtubules.

While microtubule (MT) arrays in cells are often focused at the centrosome, a variety of cell types contain a substantial number of non-centrosomal MTs. Epithelial cells, neurons, and muscle cells all contain arrays of non-centrosomal MTs that are critical for these cells' specialized functions. There are several routes by which non-centrosomal MTs can arise, including release from the centrosome, cytoplasmic assembly, breakage or severing, and stabilization from non-centrosomal sites. Once formed, MTs that are not tethered to the centrosome must be organized, which can be accomplished by means of self-organization or by capture and nucleation of MTs where they are needed. The presence of free MTs requires stabilization of minus ends, either by MT-associated proteins or by an end-capping complex. Although some of the basic elements of free MT formation and organization are beginning to be understood, a great deal of work is still necessary before we have a complete picture of how non-centrosomal MT arrays are assembled in specific cell types.     

Microtubule organization during development of stomatal complexes inLolium rigidum

Summary Microtubule (MT) arrays in stomatal complexes ofLolium have been studied using cryosectioning and immunofluorescence microscopy. This in situ analysis reveals that the arrangement of MTs in pairs of guard cells (GCs) or subsidiary cells (SCs) within a complex is very similar, indicating that MT deployment is closely coordinated during development. In premitotic guard mother cells (GMCs), MTs of the transverse interphase MT band (IMB) are reorganized into a longitudinal array via a transitory array in which the MTs appear to radiate from the cell edges towards the centre of the walls. Following the longitudinal division of GMCs, cortical MTs are reinstated in the GCs at the edge of the periclinal and ventral walls. The MTs become organized into arrays which radiate across the periclinal walls, initially from along the length of the ventral wall and later only from the pore site. As the GCs elongate, the organization of MTs and the patterns of wall expansion differ on the internal and external periclinal walls. A final reorientation of MTs from transverse to longitudinal is associated with the elongation and constriction of GCs to produce mature complexes. During cytokinesis in the subsidiary mother cells (SMCs), MTs appear around the reforming nucleus in the daughter epidermal cells but appear in the cortex of the SC once division is complete. Our results are thus consistent with the idea that interphase MTs are nucleated in the cell cortex in all cells of the stomatal complex but not in adjacent epidermal cells.Abbreviations GMC guard mother cell - GC guard cell - IMB interphase microtubule band - MT microtubule - PPB preprophase band - SMC subsidiary mother cell - SC subsidiary cell     

Amiprophos-methyl (APM): A rapid,reversible, anti-microtuble agent for plant cell cultures

Summary In plant cell suspension cultures sensitive to the herbicide amiprophos-methyl (APM), 1 to 3 M APM completely depolymerized both cortical and mitotic microtubule (MT) arrays in 1 hour. In comparison, a 2 hour application of 3 mM colchicine had no effect on MT arrays. Recovery from APM treatment occurred as early as 5 minutes after removal of APM. Short, cortical MTs were visible in 3 hours and complete MT arrays were found within 22 hours after drug removal.Sensitivity to APM-induced MT depolymerization varied according to species but was increased or decreased by varying the mitotic rate in cultures. The results indicated APM sensitivity was related to lowered stability of MT arrays in rapidly cycling cells. APM treatment may help distinguish stabilized cortical MTs in elongating cells and nonstabilized cortical MTs in rapidly dividing cells.Abbreviations MT microtubule - APM amiprophos-methyl - DMSO dimethyl sulfoxide - PBS phosphate buffered saline     

Microtubule cytoskeleton in intact and wounded coenocytic green algae

Microtubule (MT) arrangements were investigated, with immunofluorescence and electron microscopy, in two related species of coenocytic green algae. Intact cells of both Ernodesmis verticillata (Kützing) Boergesen and Boergesenia forbesii (Harvey) Feldmann have two morphologically distinct populations of MTs: a highly regular cortical array consisting of a single layer of parallel, longitudinal MTs; and perinuclear MTs radiating from the surface of the envelope of each interphase nucleus. In both algae, mitotic figures lack perinuclear MTs around them. Pre-incubation with taxol does not alter the appearance of these arrays. The cortical and nuclear MTs appear to coexist throughout the nuclear cycle, unlike the condition in most plant cells. At the cut/contracting ends of wounded Ernodesmis cells, cortical MTs exhibit bundling and marked convolution, with some curvature and slight bundling of MTs throughout the cell cortices. In Boergesenia, wound-induced reticulation and separation of the protoplasm into numerous spheres also involves a fasciation of MTs within the attenuating regions of the cytoplasm. Although some cortical MTs are fairly resistant to cold and amiprophos-methyl-induced depolymerization, the perinuclear ones are very labile, depolymerizing in 5–10 min in the cold. The MT cytoskeleton is not believed to be directly involved in wound-induced motility in these plants because amiprophos-methyl and cold depolymerize most cortical MTs without inhibiting motility. Also, the identical MT distributions in intact cells of these two algae belie the very different patterns of cytoplasmic motility. Although certain roles of the MT arrays may be ruled out, their exact functions in these plants are not known.Abbreviations APM amiprophos-methyl - DIC differential interference contrast - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MT(s) microtubule(s) - PBS phosphate-buffered saline     

Centrosome fragments and microtubules are transported asymmetrically away from division plane in anaphase

Spinning disc confocal microscopy of LLCPK1 cells expressing GFP-tubulin was used to demonstrate that microtubules (MTs) rapidly elongate to the cell cortex after anaphase onset. Concurrently, individual MTs are released from the centrosome and the centrosome fragments into clusters of MTs. Using cells expressing photoactivatable GFP-tubulin to mark centrosomal MT minus ends, a sevenfold increase in MT release in anaphase is documented as compared with metaphase. Transport of both individually released MTs and clusters of MTs is directionally biased: motion is directed away from the equatorial region. Clusters of MTs retain centrosomal components at their focus and the capacity to nucleate MTs. Injection of mRNA encoding nondegradable cyclin B blocked centrosome fragmentation and the stimulation of MT release in anaphase despite allowing anaphase-like chromosome segregation. Biased MT release may provide a mechanism for MT-dependent positioning of components necessary for specifying the site of contractile ring formation.     

Microtubule organization in root cortical cells ofHyacinthus orientalis

Summary Overall cellular arrangement of cortical microtubules (MTs) is studied by reconstruction of MT images on serial thin sections. The mature root cortex ofHyacinthus orientalis L. cv. Delft blue is composed of elongate, highly vacuolate nondividing parenchyma cells. In longitudinal sections in these cells, MTs generally form parallel arrays at oblique angles to longitudinal cell axes. These MTs extend towards the transverse face of the cell where they appear in localized parallel arrays as well as in crisscross patterns. Repeated observations of oblique parallel arrays of MTs along the length of the cell and the continuity of MT bundles in serial sections suggest that MTs form a single helix in the cell. MTs in neighboring cells appear in sections either as parallel or as herringbone patterns, suggesting that the MT helices in these cells may spiral in the same or the opposite directions.Abbreviations MT Microtubule - MF microfibil - EM electron microscopy     

Straighten up and fly right: microtubule dynamics and organization of non-centrosomal arrays in higher plants

Live cell imaging and genetic studies are demonstrating that cortical microtubule arrays in plant cells are dynamic structures in which microtubule (MT) bundles play a key role in creating array organization and function. Steps important for creating and organizing these arrays include recruitment of nucleation complexes to the cell cortex and to the lattices of previously established MTs, association of newly created MTs to the cell cortex, release of MTs from sites of nucleation, transport of released MTs by polymer treadmilling, and subsequent interactions between treadmilling MTs. The results of MT interactions include induced catastrophe, severing, and the capture and reorientation of growing polymer ends by bundling interactions. Together, these properties predict a capacity for self-ordering that is likely to play an important role in establishing the parallel organization of the arrays.     

Establishment of a stable, acetylated microtubule bundle during neuronal commitment

Two posttranslational modifications of alpha-tubulin, acetylation and detyrosination, are associated with stable microtubule (MT) populations, including those of neuronal processes. We have used a pluripotent embryonal carcinoma cell line, P19, to investigate changes in MT isotype and stability found in MT arrays during neurogenesis. This cell line has an advantage in that both commitment- and differentiation-related events can be observed. Uncommitted P19 cells have minimal arrays of acetylated and detyrosinated MTs. Following neuronal induction with retinoic acid (RA), indirect immunofluorescence microscopy shows that the first MT modifications occur during commitment and before any morphological change is observed. RA-induced cells initially polymerize a temporarily enlarged population of MTs. Included in this population is a new array of acetylated MTs arranged in a bundle of parallel MTs. This bundle is colchicine-stable, although no MT-associated proteins (MAPs) are detectable using a battery of anti-MAP antibodies. Observation of MT arrays with patterns that are intermediate between the early bundles and short neurites suggests that the acetylated MT bundle subsequently extends to form a neurite. MAP 2 is first detected at about the time of neurite extension. However, at this early stage of differentiation, MAP 2 is not yet limited to dendritic processes. This report provides the first evidence that the stable MTs of mature neurons may be initiated during neuronal commitment.     

Assembly of cortical microtubules during cold treatment of the coenocytic green alga,Chaetomorpha moniligera

The effects of cold treatment on the cortical microtubules (MTs) of Chaetomorpha moniligera Kjellman were investigated by immunofluorescence microscopy. Cortical MTs in Chaetomorpha thallus are arranged longitudinally. In this study, 70–75% of MTs disassembled within 4 h on ice while the others remained stable under these conditions. Reticulate background immunofluorescence, assumed to indicate the presence of a tubulin monomer, was distributed about the stable MTs. Immunofluorescence was prominent in only 50% of the cells. Tubulin polymerization was noted where the background and MT immunofluorescence was strong. New MTs grew transversely as single strings or clusters from the sides of MTs after cold treatment for 4 h and elongated with time to take on a reticulate form at 24 h. The significance of this tubulin polymerization under cold treatment is discussed.Abbreviations MT microtubule - MTOC microtubule-organizing center     

A Highlights from MBoC Selection: MAP65/Ase1 promote microtubule flexibility

Microtubules (MTs) are dynamic cytoskeletal elements involved in numerous cellular processes. Although they are highly rigid polymers with a persistence length of 1–8 mm, they may exhibit a curved shape at a scale of few micrometers within cells, depending on their biological functions. However, how MT flexural rigidity in cells is regulated remains poorly understood. Here we ask whether MT-associated proteins (MAPs) could locally control the mechanical properties of MTs. We show that two major cross-linkers of the conserved MAP65/PRC1/Ase1 family drastically decrease MT rigidity. Their MT-binding domain mediates this effect. Remarkably, the softening effect of MAP65 observed on single MTs is maintained when MTs are cross-linked. By reconstituting physical collisions between growing MTs/MT bundles, we further show that the decrease in MT stiffness induced by MAP65 proteins is responsible for the sharp bending deformations observed in cells when they coalign at a steep angle to create bundles. Taken together, these data provide new insights into how MAP65, by modifying MT mechanical properties, may regulate the formation of complex MT arrays.     

Severing at sites of microtubule crossover contributes to microtubule alignment in cortical arrays

The cortical microtubule (MT) array and its organization is important in defining the growth axes of plant cells. In roots, the MT array exhibits a net-like configuration in the division zone, and a densely-packed transverse alignment in the elongation zone. This transition is essential for anisotropic cell expansion and consequently has been the subject of intense study. Cotyledons exhibit a net-like array in pavement cells and a predominantly aligned array in the petioles, and provide an excellent system for determining the basis of plant MT organization. We show that in both kinds of MT array, growing MTs frequently encounter existing MTs. Although some steep-angled encounters result in catastrophes, the most frequent outcome of these encounters is successful negotiation of the existing MT by the growing MT to form an MT crossover. Surprisingly, the outcome of such encounters is similar in both aligned and net-like arrays. In contrast, aligned arrays exhibit a much higher frequency of MT severing events compared with net-like arrays. Severing events occur almost exclusively at sites where MTs cross over one another. This process of severing at sites of MT crossover results in the removal of unaligned MTs, and is likely to form the basis for the difference between a net-like and an aligned MT array.     

A planar microtubule-organizing zone in guard cells of Allium: experimental depolymerization and reassembly of microtubules

The generation of the unique radial array of microtubules (MTs) in stomatal guard cells raises questions about the location and activities of relevant MT-organizing centers. By using tubulin immunofluorescence microscopy, we studied the pattern of depolymerization and reassembly of MTs in guard cells of Allium cepa L. Chilling at 0°C reduces the MTs to small remnants that surround the nuclear surface of cells in the early postcytokinetic stage, or form a dense layer along the central portion of the ventral wall in older guard cells. A rapid reassembly on rewarming restores either MTs extending from the nuclear surface randomly throughout the cytoplasm in very young cells, or an array of MTs radiating from the dense layer at the ventral wall later in development. A similar pattern of depolymerization and reassembly is achieved by incubation with 100 M colchicine followed by a brief irradiation with ultraviolet (UV) light. Incubation with 200 M colchicine leads to a complete depolymerization that leaves only a uniform, diffuse cytoplasmic fluorescence. Nonetheless, UV irradiation of developing guard cells induces the regeneration of a dense layer of MTs at the ventral wall. The layer is again positioned centrally along the wall, even if the nucleus has been displaced by centrifugation in the presence of cytochalasin D. Neither the regenerated layer nor the perinuclear MTs seen earlier are related to the staining pattern of serum 5051, which reportedly binds to centrosomal material in animal and plant cells. The results support the view that, soon after cytokinesis, a planar MT-organizing zone is established in the cortex along the central portion of the ventral wall, which then generates the radial MT array.Abbreviations GC guard cell - MT microtubule - MTOC microtubule-organizing center - UV ultraviolet To whom correspondence should be addressed.     

Reinstatement of Microtubule Arrays from Cortical Nucleating Sites in Stomatal Complexes of Lolium rigidum Following Depolymerization of Microtubules by Oryzalin and High Pressure

Immunofluorescence visualization of microtubule (MT) arraysin stomatal complexes of Lolium rigidum shows that disassemblyof the arrays can be successfully achieved using oryzalin orhigh pressure treatments. Under conditions allowing for MT recovery,MTs reappear within an hour after oryzalin or within 5 min afterhigh pressure treatment. During recovery guard mother cells(GMCs) nucleate MTs at sites distributed randomly in the cellcortex. Even after 22 h of recovery the MTs are not arrangedinto any configuration found in untreated tissue. This inabilityto reorganize their MTs after treatment makes GMCs more sensitiveto the loss of MTs than are other cells of the leaf. In guardcells (GCs) MTs reappear around the pore at the junction ofthe periclinal and ventral walls. They subsequently appear throughoutmost of the cell cortex and the majority of stomatal complexesrecover fully organized MT arrays indistinguishable from thosein untreated cells. The results support and extend ultrastructuraland immunofluorescence observations that suggest that MTs inGCs of developing stomata are nucleated in the cell cortex. ²Present address: Department of Biology, The University of SouthwesternLouisiana, Lafayette, Louisiana 70504-2451, U.S.A. (Received April 24, 1990; Accepted July 7, 1990)