Motility in the siphonous green alga Bryopsis. II. Chloroplast movement requires organized arrays of both microtubules and actin filaments

The cortical cytoplasm of the giant cells of Bryopsis contains hundreds of interconnected microtubule (MT) bundles aligned along the cell's long axis. Actin fibers show an extensive but not exclusive superposition with these MT bundles. Chloroplasts move parallel to the bundles. Colchicine (0.5 mM), vinblastine (0.1 mM), and the herbicide ami-prophosmethyl (APM, 1-5 microM) strongly inhibit chloroplast movement and severely disrupt both the MT and the actin network. Additionally, APM leads to the appearance of large actin bundles up to 5 microns in diameter and several tens of micron in length. Erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA, 1 mM) does not block chloroplast movement, but affects chloroplast behavior by causing transient aggregations. The MT network is not significantly changed by EHNA, but actin fibers converge in large, radially symmetric complexes in regions of chloroplast aggregates. Cytochalasin D (CD, 1-10 micrograms/ml) leads to a significant but transient reduction of chloroplast speed within the first 60 min, as the actin network breaks down into small foci. Within the next 1 to 3 h of treatment, these foci segregate into massive clusters where chloroplasts remain immobilized. At the same time, chloroplast movement recovers in other areas of the cell. This recovery coincides with the reappearance of actin filament bundles in these cell regions. The MT cytoskeleton is not significantly affected by CD. These data are inconsistent with a mechanism of chloroplast movement in Bryopsis based solely on either MTs or actin, but instead they suggest an intimate interaction of both cytoskeletal networks in maintaining the spatial organization of the cytoplasm and in supporting chloroplast movement.     

Co-Localization of Particle Transport with Microtubules in Cytoplasmic Exudates of the Siphonous Green Alga Bryopsis

Organelles in the cortical cytoplasm of the siphonous green alga Bryopsis display various types of motile activities. One of them, saltatory movement along axially oriented linear tracks is typical for mitochondria and other small particles. A method is described which allows in vitro observation of such movements in thin layers of cytoplasm extruded from the alga and attached to a poly-l -lysine coated glass surface. By comparing video recordings of motile activities with the position of cytoskeletal elements visualized by immunofluorescence in the same area of a cytoplasmic exudate, it can be shown that tracks along which particles have moved in vitro are identical with microtubules (MTs). Depolymerization of MTs in the cytoplasmic exudates by MT-specific inhibitors stops particle movement, whereas depolymerization of actin filaments with cytochalasin D disrupts actin bundles but has little effect on particle motility. These data are consistent with the model of MT guided particle transport.     

Visualization of cytoskeletal changes through the life cycle inAcetabularia

Summary The cytoskeleton in the siphonous, marine green algaAcetabularia is visualized by immunocytochemistry using antibodies against plant alfa tubulin and animal smooth muscle actin. In the vegetative phase of the life cycle, when the cell grows a cylindrical stalk and until the reproductive cap is completed, actin forms continuous, parallel bundles that extend through the entire length of the stalk and cap rays respectively. Microtubules (MTs) cannot be detected until the primary nucleus, located in the rhizoid of the giant cell, divides to form thousands of secondary nuclei. MTs can then be seen radiating from each secondary nucleus that is encountered in the stalk on its migration upwards into the cap rays. They are oriented mostly parallel to the long axis of the cell. At arrival in the cap rays up to the white spot stage, when nuclei assume equidistant positions in the cap ray cytoplasm, a radiating system of MTs forms around each nucleus and dramatically increases until impressive radial arrays have developed. This phase coincides with a disappearance of actin bundles in the cap rays, but they are retained in the stalk cytoplasm. Shortly after that additional MTs appear around the disk like partitions of cap ray cytoplasm. Concomitantly, bundles of actin reappear colinearly with the circumferrential MTs eventually forming complete rings around each disk of cap ray cytoplasm. During this process the compartments of the future cysts are gradually bulging outwards and simultaneously the rings of actin sink inwards until domes are formed with the nuclei fixed in the top centers of the domes. At this stage the peripheral areas of the radiating MT systems around the nuclei start to break down, whereas the circumferrential MT systems remain intact. Subsequently, the rings of both actin and MTs decrease in diameter, and finally contract to a spot opposite the nucleus, while the cysts continue to develop their oval shape. After the cysts have become separated, they round up and enter several rounds of nuclear divisions. MTs form short radial arrays around each nucleus with minor changes due to a reduction of MTs during division followed by a reappearance after completion of each division. Actin is rearranged in the cysts to a cortical network of randomly oriented, short bundles, that is maintained until gamete formation sets in.These findings accentuate the involvement of Cytoskeletal elements in the key steps of morphogenesis inAcetabularia to an extent that is unknown in higher plants.     

Distribution and dynamics of the cytoskeleton in graviresponding protonemata and rhizoids of characean algae: exclusion of microtubules and a convergence of actin filaments in the apex suggest an actin-mediated gravitropism

The organization of the microtubule (MT) and actin microfilament (MF) cytoskeleton of tip-growing rhizoids and protonemata of characean green algae was examined by confocal laser scanning microscopy. This analysis included microinjection of fluorescent tubulin and phallotoxins into living cells, as well as immunofluorescence labeling of fixed material and fluorescent phallotoxin labeling of unfixed material. Although the morphologically very similar positively gravitropic (downward growing) rhizoids and negatively gravitropic (upward growing) protonemata show opposite gravitropic responses, no differences were detected in the extensive three-dimensional distribution of actin MFs and MTs in both cell types. Tubulin microinjection revealed that in contrast to internodal cells, fluorescent tubulin incorporated very slowly into the MT arrays of rhizoids, suggesting that MT dynamics are very different in tip-growing and diffusely expanding cells. Microtubules assembled from multiple sites at the plasma membrane in the basal zone, and a dense subapical array emerged from a diffuse nucleation centre on the basal side of the nuclear envelope. Immunofluorescence confirmed these distribution patterns but revealed more extensive MT arrays. In the basal zone, short branching clusters of MTs form two cortical hemicylinders. Subapical, axially oriented MTs are distributed in equal density throughout the peripheral and inner cytoplasm and are closely associated with subapical organelles. Microtubules, however, are completely absent from the apical zones of rhizoids and protonemata. Actin MFs were found in all zones of rhizoids and protonemata including the apex. Two files of axially oriented bundles of subcortical actin MFs and ring-like actin structures in the streaming endoplasm of rhizoids were detected in the basal zones by microinjection or rhodamine-phalloidin labeling. The subapical zone contains a dense array of mainly axially oriented actin MFs that co-distribute with the subapical MT array. In the apex, actin MFs form thicker bundles that converge into a remarkably distinct actin patch in the apical dome, whose position coincides with the position of the endoplasmic reticulum aggregate in the centre of the Spitzenk?rper. Actin MFs radiate from the actin patch towards the apical membrane. Together with results from previous inhibitor studies (Braun and Sievers, 1994, Eur J Cell Biol 63: 289–298), these results suggest that MTs have a stabilizing function in maintaining the polar cytoplasmic and cytoskeletal organization. The motile processes, however, are mediated by actin. In particular, the actin cytoskeleton appears to be involved in the structural and functional organization of the Spitzenk?rper and thus is responsible for controlling cell shape and growth direction. Despite the similar structural arrangements of the actin cytoskeleton, major differences in the function of actin MFs have been observed in rhizoids and protonemata. Since actin MFs are more directly involved in the gravitropic response of protonemata than of rhizoids, the opposite gravitropism in the two cell types seems to be based mainly on different properties and activities of the actin cytoskeleton. Received: 14 September 1997 / Accepted: 16 October 1997     

Microtubule anchoring by cortical actin bundles prevents streaming of the oocyte cytoplasm

The localisation of the determinants of the body axis during Drosophila oogenesis is dependent on the microtubule (MT) cytoskeleton. Mutations in the actin binding proteins Profilin, Cappuccino (Capu) and Spire result in premature streaming of the cytoplasm and a reorganisation of the oocyte MT network. As a consequence, the localisation of axis determinants is abolished in these mutants. It is unclear how actin regulates the organisation of the MTs, or what the spatial relationship between these two cytoskeletal elements is. Here, we report a careful analysis of the oocyte cytoskeleton. We identify thick actin bundles at the oocyte cortex, in which the minus ends of the MTs are embedded. Disruption of these bundles results in cortical release of the MT minus ends, and premature onset of cytoplasmic streaming. Thus, our data indicate that the actin bundles anchor the MTs minus ends at the oocyte cortex, and thereby prevent streaming of the cytoplasm. We further show that actin bundle formation requires Profilin but not Capu and Spire. Thus, our results support a model in which Profilin acts in actin bundle nucleation, while Capu and Spire link the bundles to MTs. Finally, our data indicate how cytoplasmic streaming contributes to the reorganisation of the MT cytoskeleton. We show that the release of the MT minus ends from the cortex occurs independently of streaming, while the formation of MT bundles is streaming dependent.     

Immobilisation of organelles and actin bundles in the cortical cytoplasm of the alga Chara corallina Klein ex. Wild

The mechanism by which sub-cortical actin bundles and membranous organelles are immobilised in the cortical cytoplasm of the alga Chara was studied by perfusing cells with a solution containing 1% Triton X-100. Light and scanning electron microscopy and the release of starch grains and chlorophyll-protein complexes indicated that the detergent extensively solubilised the chloroplasts. However, the sub-cortical actin bundles remained in situ even though they were originally separated from the plasma membrane by the chloroplasts. A fibrous layer between chloroplasts and plasma membrane became readily visible after detergent extraction of the cells and could be released by low-ionic-strength ethylenediaminetetraacetic acid, thioglycollate and trypsin. The same treatments applied to cells not subject to detergent extraction released the membrane-bound organelles and actin bundles and no fibrous meshwork was visible on subsequent extraction with Triton. It is, therefore, concluded that a detergent-insoluble cortical cytoskeleton exists and contributes to the immobility of the actin and cortical organelles in the cells.Abbreviation EDTA ethylenediaminetetraacetic acid     

Reorganization of the actin and microtubule cytoskeleton throughout blue-light-induced differentiation of characean protonemata into multicellular thalli

Summary The reorganization of the actin and microtubule (MT) cytoskeleton was immunocytochemically visualized by confocal laser scanning microscopy throughout the photomorphogenetic differentiation of tip-growing characean protonemata into multicellular green thalli. After irradiating dark-grown protonemata with blue or white light, decreasing rates of gravitropic tip-growth were accompanied by a series of events leading to the first cell division: the nucleus migrated towards the tip; MTs and plastids invaded the apical cytoplasm; the polar zonation of cytoplasmic organelles and the prominent actin patch at the cell tip disappeared and the tip-focused actin microfilaments (MFs) were reorganized into a homogeneous network. During prometaphase and metaphase, extranuclear spindle microtubules formed between the two spindle poles. Cytoplasmic MTs associated with the apical spindle pole decreased in number but did not disappear completely during mitosis. The basal cortical MTs represent a discrete MT population that is independent from the basal spindle poles and did not redistribute during mitosis and cytokinesis. Preprophase MT bands were never detected but cytokinesis was characterized by higher-plant-like phragmoplast MT arrays. Cytoplasmic actin MFs persisted as a dense network in the apical cytoplasm throughout the first cell division. They were not found in close contact with spindle MTs, but actin MFs were clearly coaligned along the MTs of the early phragmoplast. The later belt-like phragmoplast was completely depleted of MFs close to the time of cell plate fusion except for a few actin MF bundles that extended to the margin of the growing cell plate. The cell plate itself and young anticlinal cell walls showed strong actin immunofluorescence. After several anticlinal cell divisions, basal cells of the multicellular protonema produced nodal cell complexes by multiple periclinal divisions. The apical-dome cell of the new shoot which originated from a nodal cell becomes the meristem initial that regularly divides to produce a segment cell. The segment cell subsequently divides to produce a single file of alternating internodal cells and multicellular nodes which together form the complexly organized characean thallus. The actin and MT distribution of nodal cells resembles that of higherplant meristem cells, whereas the internodal cells exhibit a highly specialized cortical system of MTs and streaming-generating actin bundles, typical of highly vacuolated plant cells. The transformation from the asymmetric mitotic spindle of the polarized tip-growing protonema cell to the symmetric, higher-plant-like spindle of nodal thallus cells recapitulates the evolutionary steps from the more primitive organisms to higher plants.Abbreviations FITC fluorescein isothiocyanate - MF microfilament - MT microtubule - MSB microtubule-stabilizing buffer - PBS phosphate-buffered saline     

Dynein-dependent motility of microtubules and nucleation sites supports polarization of the tubulin array in the fungus Ustilago maydis

Microtubules (MTs) are often organized by a nucleus-associated MT organizing center (MTOC). In addition, in neurons and epithelial cells, motor-based transport of assembled MTs determines the polarity of the MT array. Here, we show that MT motility participates in MT organization in the fungus Ustilago maydis. In budding cells, most MTs are nucleated by three to six small and motile gamma-tubulin-containing MTOCs at the boundary of mother and daughter cell, which results in a polarized MT array. In addition, free MTs and MTOCs move rapidly throughout the cytoplasm. Disruption of MTs with benomyl and subsequent washout led to an equal distribution of the MTOC and random formation of highly motile and randomly oriented MTs throughout the cytoplasm. Within 3 min after washout, MTOCs returned to the neck region and the polarized MT array was reestablished. MT motility and polarity of the MT array was lost in dynein mutants, indicating that dynein-based transport of MTs and MTOCs polarizes the MT cytoskeleton. Observation of green fluorescent protein-tagged dynein indicated that this is achieved by off-loading dynein from the plus-ends of motile MTs. We propose that MT organization in U. maydis involves dynein-mediated motility of MTs and nucleation sites.     

Establishment and maintenance of nuclear position during zoospore formation in allomyces macrogynus: roles of the cytoskeleton

The involvement of the microtubule (MT) and actin microfilament (MF) cytoskeletons in establishing nuclear positions during zoosporogenesis in Allomyces macrogynus was assessed using selective cytoskeletal disrupting treatments and documented with light microscopy. These experiments were coupled with low-speed centrifugation studies to determine the degree to which cytoskeletal elements anchor nuclear position. At the onset of zoospore formation, nuclei were positioned only in cortical cytoplasmic regions of the zoosporangia (ZS). Immunofluorescence microscopy revealed that MTs primarily emanated from centrosomal regions into the surrounding cytoplasm at this stage. During delimitation of the cytoplasm into individual uninucleate zoospores, nuclei migrated from cortical regions to become distributed throughout the cytoplasm. Coincident with nuclear migrations, MTs were primarily organized at and emanated from nuclear surfaces, forming extensive perinuclear arrays. Nuclear migrations were suppressed in ZS induced to sporulate in the presence of cytochalasin D, an actin MF inhibiting compound. Disruption of MTs with nocodazole did not block nuclear migrations, although resultant nuclear spacing was irregular. Centrifugation treatments of control and drug-treated ZS demonstrated that nuclear positions were stabilized by perinuclear MT arrays. The results indicate that nuclear motility in ZS of A. macrogynus is the result of an actin-based system while perinuclear MTs arrays function to establish and fix nuclear position during zoospore formation. Copyright 1998 Academic Press.     

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     

Actin-organelle interaction: association with chloroplast in arabidopsis leaf mesophyll cells.

The role of the cytoskeleton in the regulation of chloroplast motility and positioning has been investigated by studying: (1) structural relationship of actin microfilaments, microtubules, and chloroplasts in cryofixed and freeze-substituted leaf cells of Arabidopsis; and (2) the effects of anti-actin (Latrunculin B; LAT-B) and anti-microtubule (Oryzalin) drugs on intracellular distribution of chloroplasts. Immunolabeling of leaf cells with two plant-actin specific antibodies, which react equivalently with all the expressed Arabidopsis actins, revealed two arrangements of actin microfilaments: longitudinal arrays of thick actin bundles and randomly oriented thin actin filaments that extended from the bundles. Chloroplasts were either aligned along the actin bundles or closely associated with the fine filaments. Baskets of actin microfilaments were also observed around the chloroplasts. The leaf cells labeled with an anti-tubulin antibody showed dense transverse arrays of cortical microtubules that exhibited no apparent association with chloroplasts. The application of LAT-B severely disrupted actin filaments and their association with chloroplasts. In addition, LAT-B induced aberrant aggregation of chloroplasts in the mesophyll and bundle sheath cells. Double labeling of LAT-B treated cells with anti-actin and anti-tubulin antibodies revealed that the microtubules in these cells were unaffected. Moreover, depolymerization of microtubules with Oryzalin did not affect the distribution of chloroplasts. These results provide evidence for the involvement of actin, but not tubulin, in the movement and positioning of chloroplasts in leaf cells. We propose that using motor molecules, some chloroplasts migrate along the actin cables directly, while others are pulled along the cables by the fine actin filaments. The baskets of microfilaments may anchor the chloroplasts during streaming and allow control over proper three-dimensional orientation to light.     

植物微管特效解聚剂APM对紫露草雄蕊毛细胞胞质环流的影响

采用体外渗透和显微注射的方法。将植物微管特效解聚剂甲基氨草磷(APM)引入紫露草雄蕊毛细胞后,发现原来沿着胞质束运动的胞质颗粒运动速度渐慢,进而胞质束消失,颗粒运动停止。显微注射后,还发现APM可通过胞间通道由被注射的细胞向两侧细胞扩散,从而也导致两侧细胞胞质束消失,颗粒运动停止。APM对胞质环流的抑制作用是可逆的。结果表明微管可能是胞质束的重要组份之一,植物胞质环流与微管的聚合与解聚状态有密切关系。     

Molecular motors are differentially distributed on Golgi membranes from polarized epithelial cells

Microtubules (MT) are required for the efficient transport of membranes from the trans-Golgi and for transcytosis of vesicles from the basolateral membrane to the apical cytoplasm in polarized epithelia. MTs in these cells are primarily oriented with their plus ends basally near the Golgi and their minus-ends in the apical cytoplasm. Here we report that isolated Golgi and Golgi-enriched membranes from intestinal epithelial cells possess the actin based motor myosin-I, the MT minus- end-directed motor cytoplasmic dynein and its in vitro motility activator dynactin (p150/Glued). The Golgi can be separated into stacks, possessing features of the Golgi cisternae, and small membranes enriched in the trans-Golgi network marker TGN 38/41. Whereas myosin-I is present on all membranes in the Golgi fraction, dynein is present only on the small membrane fraction. Dynein, like myosin-I, is associated with membranes as a cytoplasmic peripheral membrane protein. Dynein and myosin-I coassociate with membranes that bind to MTs and cross-link actin filaments and MTs in a nucleotide-dependent manner. We propose that cytoplasmic dynein moves Golgi membranes along MTs to the cell cortex where myosin-I provides local delivery through the actin- rich cytoskeleton to the apical membrane.     

Blue-light-induced reorganization of the actin cytoskeleton and the avoidance response of chloroplasts in epidermal cells of<Emphasis Type="Italic"> Vallisneria gigantea</Emphasis>

In leaf epidermal cells of the aquatic angiosperm Vallisneria gigantea Graebner, high-intensity blue light induces the actin-dependent avoidance response of chloroplasts. By semi-quantitative motion analysis and phalloidin staining, time courses of the blue-light-induced changes in the mode of movement of individual chloroplasts and in the configuration of actin filaments were examined in the presence and absence of a flavoprotein inhibitor, diphenylene iodonium. In dark-adapted cells, short, thick actin bundles seemed to surround each chloroplast, which was kept motionless in the outer periclinal cytoplasm of the cells. After 10 min of irradiation with high-intensity blue light, a rapid, unidirectional movement of chloroplasts was induced, concomitant with the appearance of aggregated, straight actin bundles stretched over the outer periclinal cytoplasm. Diphenylene iodonium inhibited the avoidance response of chloroplasts, apparently by delaying a change in the mode of chloroplast movement from random sway to unidirectional migration, by suppressing the appearance of aggregated, straight actin bundles. In partially irradiated individual cells, redistribution of chloroplasts and reorganization of actin filaments occurred only in the areas exposed to blue light. From the results, we propose that the short, thick actin bundles in the vicinity of chloroplasts function to anchor the chloroplasts in dark-adapted cells, and that the aggregated, straight actin bundles organized under blue-light irradiation provide tracks for unidirectional movement of chloroplasts.Preliminary results of part of the local irradiation study have already been reported in abstract form [N. Sakurai et al. (2002) J Photosci 9:326–328].     

Actin and tubulin cytoskeletons in germlings of the oomycete fungus Phytophthora infestans

Actin and tubulins of Phytophthora infestans germlings were detected with monoclonal antibodies on Western blots of crude extracts separated by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The Mr of actin was approximately 43,000, whereas alpha- and beta-tubulin, which migrated as a single band, had an Mr of 53,000. Rhodamine-phalloin revealed peripheral patches of actin in ungerminated cysts. In young germlings, actin fibers were visible in the conversion zone between cyst and germ tube and as connections between actin patches and the incipient germ tube. Actin patches also occurred throughout the peripheral cytoplasm of longer germ tubes, except for the hyphal apex, which commonly contained actin fibers, but actin patches only exceptionally. Associations between patches and fibers were frequent. A monoclonal antibody specific for actin also stained fibers, but in addition it revealed diffuse staining of the apex and fine granular structures, indicative of the presence of G-actin or of single actin filaments. Cysts incubated with a monoclonal antibody against tubulin contained an array of cytoplasmic microtubules (MTs) that arise from a nucleus-associated center. Some of these MTs circumflexed the nucleus, whereas others extended to the cyst periphery. In germ tubes, axially oriented MT bundles extended from the nucleus-associated center into the proximal and distal cytoplasm. Their density was highest near the nucleus, and their number decreased towards the tip, with only a few remaining at the extreme apex. Bundles of MTs were continuous from the nucleus to the subapical region, reaching lengths of up to 20 microns. Ultrastructurally the bundles consisted of as many as 10 MTs. The architecture of the actin and tubulin cytoskeletons in germ tubes of P. infestans bolsters the hypothesis that they maintain the spatial organization of the hyphal protoplast and support or accomplish intrahyphal movements.     

Spatial relationship between microtubules and plasma-membrane rosettes during the deposition of primary wall microfibrils in Closterium sp.

The mechanism by which cortical microtubules (MTs) control the orientation of cellulose microfibril deposition in elongating plant cells was investigated in cells of the green alga, Closterium sp., preserved by ultrarapid freezing. Cellulose microfibrils deposited during formation of the primary cell wall are oriented circumferentially, parallel to cortical MTs underlying the plasma membrane. Some of the microfibrils curve away from the prevailing circumferential orientation but then return to it. Freeze-fracture electron microscopy shows short rows of particle rosettes on the P-face of the plasma membrane, also oriented perpendicular to the long axis of the cell. Previous studies of algae and higher plants have provided evidence that such rosettes are involved in the deposition of cellulose microfibrils. The position of the rosettes relative to the underlying MTs was visualized by deep etching, which caused much of the plasma membrane to collapse. Membrane supported by the MTs and small areas around the rosettes resisted collapse. The rosettes were found between, or adjacent to, MTs, not directly on top of them. Rows of rosettes were often at a slight angle to the MTs. Some evidence of a periodic structure connecting the MTs to the plasma membrane was apparent in freeze-etch micrographs. We propose that rosettes are not actively or directly guided by MTs, but instead move within membrane channels delimited by cortical MTs attached to the plasma membrane, propelled by forces derived from the polymerization and crystallization of cellulose microfibrils. More widely spaced MTs presumably allow greater lateral freedom of movement of the rosette complexes and result in a more meandering pattern of deposition of the cellulose fibrils in the cell wall.Abbreviations E-face exoplasmic fracture face - MT microtubule - P-face protoplasmic fracture-face     

A Highlights from MBoC Selection: Persistent growth of microtubules at low density

Microtubules (MTs) often form a polarized array with minus ends anchored at the centrosome and plus ends extended toward the cell margins. Plus ends display behavior known as dynamic instability—transitions between rapid shortening and slow growth. It is known that dynamic instability is regulated locally to ensure entry of MTs into nascent areas of the cytoplasm, but details of this regulation remain largely unknown. Here, we test an alternative hypothesis for the local regulation of MT behavior. We used microsurgery to isolate a portion of peripheral cytoplasm from MTs growing from the centrosome, creating cytoplasmic areas locally depleted of MTs. We found that in sparsely populated areas MT plus ends persistently grew or paused but never shortened. In contrast, plus ends that entered regions of cytoplasm densely populated with MTs frequently transitioned to shortening. Persistent growth of MTs in sparsely populated areas could not be explained by a local increase in concentration of free tubulin subunits or elevation of Rac1 activity proposed to enhance MT growth at the cell leading edge during locomotion. These observations suggest the existence of a MT density–dependent mechanism regulating MT dynamics that determines dynamic instability of MTs in densely populated areas of the cytoplasm and persistent growth in sparsely populated areas.     

Dynamic Organization of Microtubules in Guard Cells of Viciafaba L. with Diurnal Cycle

Stomatal movement is regulated by changes in the volume of guardcells, thought to be mainly controlled by an osmo-regulatorysystem. In the present study, we examined the additional involvementof cytoskeletal events in the regulation of stomatal movement.Microtubules (MTs) in guard cells of Viciafaba L., grown undersunlight, were observed during the day and night by immunofluorescencemicroscopy. Cortical MTs began to be organized in a radial arrayat dawn and increased in numbers in the morning following theincrease in the stomatal aperture size. Thereafter, MTs becamelocalized near the nucleus and began to be destroyed from theevening to midnight, following the decrease in stomatal aperturesize. These diurnal changes in MT organization were observedeven two days after transfer from natural light condition tototal darkness, and were accompanied by corresponding changesin stomatal aperture. The increase in stomatal aperture sizein the early morning was inhibited by 50 µM propyzaraide,which destroys cortical MTs in guard cells, whereas the decreasein aperture size in the evening was suppressed by 10 µMtax-ol, which stabilizes cortical MTs. These results suggestthat radially-organized cortical MTs of guard cells may controldiurnal stomatal movement. (Received September 3, 1997; Accepted November 5, 1997)     

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