CELL DIVISION AND MORPHOGENESIS OF THE CENTRIC DIATOM CHAETOCEROS DECIPIENS (BACILLARIOPHYCEAE) II. ELECTRON MICROSCOPY AND A NEW PARADIGM FOR TIP GROWTH

Valve morphogenesis starts when the silica deposition vesicle (SDV) expands across a cleavage furrow covered by an unidentified layer, which may aid in its shaping. A labiate process (LP) is present only in the outer valve of terminal cells in the filament. Before these particular cells form setae, a layered "labiate process apparatus" (LPA) appears on the SDV in the exact center of the forming valve, near the microtubule center arising after cleavage. The LPA thereafter surmounts the lips of the LP as it forms. After the girdle bands separate slightly, two lateral protrusions develop in the corners of the cell. These nascent setae are lined internally by a cylindrical, fibrous band (sleeve), which assembles immediately ahead of the expanding edge of the SDV, very close to the plasmalemma. Then these protrusions, lined by the fibrous band, the SDV, and the forming silica wall, grow through two gaps in the girdle bands. The cytoplasm at the tip of the growing seta is naked. Immediately behind the tip, this fibrous band is adpressed to the plasmalemma and thereby apparently defines the diameter of the seta; it extends to internally ensheath the tipmost edge of the SDV for a short distance, like a tight-fitting inner sleeve. This structure is considered the major organelle involved in seta morphogenesis. Microtubules (MTs), while present, are variable in extent and disposition within the seta. Turgor pressure is considered irrelevant in driving seta growth. Instead, a new paradigm proposed for tip-growing cells generally, may apply to seta morphogenesis, as follows. If, as is suspected, the fibrous band contains actin, cycling of this actin (as in animal cells undergoing ruffling or filopodial extension) could drive seta extension via attachment of the band to the just-formed silica wall. The band is visualized as a molecular treadmill whose support base, the new wall, is being continually extended; extension is controlled and generated strictly at the tip.     

VALVE MORPHOGENESIS IN THE PENNATE DIATOM ACHNANTHES COARCTATA

Mitosis and valve morphogenesis in the pennate diatom Achnanthes coarctata (Bréb. in W. Sm.) Grun. are described. After cytokinesis, both daughter nuclei and their microtubule centers (MCs) are found near one side of the cell. Each new tubular silica deposition vesicle (SDV) arises centrally, forming a single rib running the length of the cell. Each MC then migrates around its nucleus and positions itself directly adjacent to the new SDV. The enlarging silicalemmas with their associated MCs, nuclei, microtubules (MTs) and microfilaments (MFs) appear in mirror image in the daughter cells. Both SDVs soon generate a second longitudinal rib alongside the first; the gap between the ribs ultimately becomes the future raphe fissure. The MC, MTs and nucleus are associated with each fissure. However, the subsequent behavior of the valve secreting machinery now becomes quite different in the daughter cells. In the cell that will form a raphid valve, the silicalemma, flanked by MFs, expands laterally in both directions over the cleavage furrow. Within the expanding SDV, silica secretion continues, eventually generating the structure of the mature valve, and during this phase the raphe fissure becomes delineated as in other raphid diatoms. In the other daughter cell, however, the MC and its MTs withdraw from the silicalemma, and the SDV moves laterally across the cleavage furrow until the double rib is at the corner of the cell. As silica is secreted into this expanding SDV, the raphe fissure completely fills in. This valve, therefore, lacks a raphe when mature and has a symmetry quite different from that of the valve formed in the other daughter cell. These events are compared with the course of morphogenesis described for other raphid diatoms.     

VALVE MORPHOGENESIS IN SURIRELLA (BACILLARIOPHYCEAE)1

Valve morphogenesis in two Surirellae (S. ovalis Brebisson and S. robusta Ehrenberg) is described. Mitosis takes place at the broad end of the cell. After cleavage, a new Microtubule Center (MC) arises near each spindle pole and moves to the adjacent plasmalemma. Soon, a specific group of microtubules (MTs) extends from very near the MC around the periphery of the cell. Concurrently, the new tubular Silica Deposition Vesicle (SDV) grows around the periphery of the cell close to these MTs. A double rib of silica is rapidly formed inside the SDV; the space between the ribs becomes the raphe. Mitochondria line up along the MTs, and the SDV may be molded around these to create the canal raphe. Soon, the SDV expands in two directions to create the face and the mantle of the new valve. Meanwhile, each daughter nucleus, accompanied by the MC, moves to its interphase position at the center of the cell; this movement is colchicine-sensitive. As in several other pennate diatoms, an interruption in the raphe of the mature valve coincides with the initial position of the MC. The canal raphe thickens rapidly around the mitochondria; a rudimentary raphe fiber may be associated with the creation of a tiny curvature at the inner raphe fissure. As the SDV expands in the large S. robusta, the daughter cell protoplasts slowly shrink by plasmolysis, thereby creating the complex curved surface of the new valve surmounted by the arching canal raphes which are now quite rigid. In S. ovalis, the daughter cell protoplasts remain appressed and therefore the new valve surface is basically flat. The symmetry of Surirella is quite different from that of other pennate diatoms. However, the cytoplasmic events accompanying valve morphogenesis are similar in all important respects to those described in other raphid pennate diatoms, and clearly supports a naviculoid origin for this genus.     

Feedback interactions between cell-cell adherens junctions and cytoskeletal dynamics in newt lung epithelial cells

To test how cell-cell contacts regulate microtubule (MT) and actin cytoskeletal dynamics, we examined dynamics in cells that were contacted on all sides with neighboring cells in an epithelial cell sheet that was undergoing migration as a wound-healing response. Dynamics were recorded using time-lapse digital fluorescence microscopy of microinjected, labeled tubulin and actin. In fully contacted cells, most MT plus ends were quiescent; exhibiting only brief excursions of growth and shortening and spending 87.4% of their time in pause. This contrasts MTs in the lamella of migrating cells at the noncontacted leading edge of the sheet in which MTs exhibit dynamic instability. In the contacted rear and side edges of these migrating cells, a majority of MTs were also quiescent, indicating that cell-cell contacts may locally regulate MT dynamics. Using photoactivation of fluorescence techniques to mark MTs, we found that MTs in fully contacted cells did not undergo retrograde flow toward the cell center, such as occurs at the leading edge of motile cells. Time-lapse fluorescent speckle microscopy of fluorescently labeled actin in fully contacted cells revealed that actin did not flow rearward as occurs in the leading edge lamella of migrating cells. To determine if MTs were required for the maintenance of cell-cell contacts, cells were treated with nocodazole to inhibit MTs. After 1-2 h in either 10 microM or 100 nM nocodazole, breakage of cell-cell contacts occurred, indicating that MT growth is required for maintenance of cell-cell contacts. Analysis of fixed cells indicated that during nocodazole treatment, actin became reduced in adherens junctions, and junction proteins alpha- and beta-catenin were lost from adherens junctions as cell-cell contacts were broken. These results indicate that a MT plus end capping protein is regulated by cell-cell contact, and in turn, that MT growth regulates the maintenance of adherens junctions contacts in epithelia.     

Accumulation of F-actin during cytokinesis inAllium. Correlation with microtubule distribution and the effects of drugs

Summary The distribution of F-actin in the phragmoplast/cell plate complex of formaldehyde-fixedAllium root cells was visualized with rhodaminephalloidin (RP). Increased RP fluorescence appears in late anaphase in a broad zone between separating chromosomes. The fluorescence is mostly amorphous in appearance and does not resemble the distinct actin fibers seen in interphase cells. The actin becomes more concentrated near the midplane by telophase and takes the form of a relatively bright layer of fluorescence adjacent to the forming cell plate. This distribution differs markedly from that of phragmoplast microtubules (MTs) which extend back from the plate toward the daughter nuclei. F-actin continues to accumulate in new parts of the expanding phragmoplast, while RP fluorescence gradually decreases near older portions of the plate. It disappears completely near the new wall in most interphase cells. Treatment of root tips with cytochalasin B or D before fixation markedly reduces RP fluorescence, but phragmoplast MTs remain. Colchicine or oryzalin treatment leads to the disappearance of both phragmoplast actin and MTs. The possible function of actin in the phragmoplast/cell plate complex is discussed.Abbreviations CB cytochalasin B - CD cytochalasin D - CIPC isopropyl N-(3-chlorophenyl-)carbamate - DIC differential interference contrast - MT microtubule - PBS phosphate buffered saline - PM plasmalemma - RP rhodamine-phalloidin     

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.     

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     

Cytoskeleton and morphogenesis in brown algae

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

LIM kinase 1 coordinates microtubule stability and actin polymerization in human endothelial cells

Microtubule (MT) destabilization promotes the formation of actin stress fibers and enhances the contractility of cells; however, the mechanism involved in the coordinated regulation of MTs and the actin cytoskeleton is poorly understood. LIM kinase 1 (LIMK1) regulates actin polymerization by phosphorylating the actin depolymerization factor, cofilin. Here we report that LIMK1 is also involved in the MT destabilization. In endothelial cells endogenous LIMK1 co-localizes with MTs and forms a complex with tubulin via the PDZ domain. MT destabilization induced by thrombin or nocodazole resulted in a decrease of LIMK1 colocalization with MTs. Overexpression of wild type LIMK1 resulted in MT destabilization, whereas the kinase-dead mutant of LIMK1 (KD) did not affect MT stability. Importantly, down-regulation of endogenous LIMK1 by small interference RNA resulted in abrogation of the thrombin-induced MTs destabilization and the inhibition of thrombin-induced actin polymerization. Expression of Rho kinase 2, which phosphorylates and activates LIMK1, dramatically decreases the interaction of LIMK1 with tubulin but increases its interaction with actin. Interestingly, expression of KD-LIMK1 or small interference RNA-LIMK1 prevents thrombin-induced microtubule destabilization and F-actin formation, suggesting that LIMK1 activity is required for thrombin-induced modulation of microtubule destabilization and actin polymerization. Our findings indicate that LIMK1 may coordinate microtubules and actin cytoskeleton.     

Rho Guanosine Triphosphatase Mediates the Selective Stabilization of Microtubules Induced by Lysophosphatidic Acid

The asymmetric distribution of stable, posttranslationally modified microtubules (MTs) contributes to the polarization of many cell types, yet the factors controlling the formation of these MTs are not known. We have found that lysophosphatidic acid (LPA) is a major serum factor responsible for rapidly generating stable, detyrosinated (Glu) MTs in serum-starved 3T3 cells. Using C3 toxin and val14 rho we showed that rho was both necessary and sufficient for the induction of Glu MTs by LPA and serum. Unlike previously described factors that induce MT stability, rho induced the stabilization of only a subset of the MTs and, in wound-edge cells, these stable MTs were appropriately oriented toward the leading edge of the cell. LPA had little effect on individual parameters of MT dynamics, but did induce long states of pause in a subset of MTs near the edge of the cell. Rho stimulation of MT stability was independent of actin stress fiber formation. These results identify rho as a novel regulator of the MT cytoskeleton that selectively stabilizes MTs during cell polarization by acting as a switch between dynamic and stable states of MTs rather than as a modulator of MT assembly and disassembly.     

Microtubule motor Ncd induces sliding of microtubules in vivo

The mitotic spindle is a microtubule (MT)-based molecular machine that serves for equal segregation of chromosomes during cell division. The formation of the mitotic spindle requires the activity of MT motors, including members of the kinesin-14 family. Although evidence suggests that kinesins-14 act by driving the sliding of MT bundles in different areas of the spindle, such sliding activity had never been demonstrated directly. To test the hypothesis that kinesins-14 can induce MT sliding in living cells, we developed an in vivo assay, which involves overexpression of the kinesin-14 family member Drosophila Ncd in interphase mammalian fibroblasts. We found that green fluorescent protein (GFP)-Ncd colocalized with cytoplasmic MTs, whose distribution was determined by microinjection of Cy3 tubulin into GFP-transfected cells. Ncd overexpression resulted in the formation of MT bundles that exhibited dynamic "looping" behavior never observed in control cells. Photobleaching studies and fluorescence speckle microscopy analysis demonstrated that neighboring MTs in bundles could slide against each other with velocities of 0.1 microm/s, corresponding to the velocities of movement of the recombinant Ncd in in vitro motility assays. Our data, for the first time, demonstrate generation of sliding forces between adjacent MTs by Ncd, and they confirm the proposed roles of kinesins-14 in the mitotic spindle morphogenesis.     

Regulation of leading edge microtubule and actin dynamics downstream of Rac1

Actin in migrating cells is regulated by Rho GTPases. However, Rho proteins might also affect microtubules (MTs). Here, we used time-lapse microscopy of PtK1 cells to examine MT regulation downstream of Rac1. In these cells, "pioneer" MTs growing into leading-edge protrusions exhibited a decreased catastrophe frequency and an increased time in growth as compared with MTs further from the leading edge. Constitutively active Rac1(Q61L) promoted pioneer behavior in most MTs, whereas dominant-negative Rac1(T17N) eliminated pioneer MTs, indicating that Rac1 is a regulator of MT dynamics in vivo. Rac1(Q61L) also enhanced MT turnover through stimulation of MT retrograde flow and breakage. Inhibition of p21-activated kinases (Paks), downstream effectors of Rac1, inhibited Rac1(Q61L)-induced MT growth and retrograde flow. In addition, Rac1(Q61L) promoted lamellipodial actin polymerization and Pak-dependent retrograde flow. Together, these results indicate coordinated regulation of the two cytoskeletal systems in the leading edge of migrating cells.     

The effect of drugs on diatom valve morphogenesis

Summary The effects of various drugs on cell wall (valve) morphogenesis was investigated in three species of diatoms (Pinnularia spp., Surirella robusta, andHantzschia amphioxys) using light microscopy (LM) and scanning electron microscopy (SEM). Treatment ofSurirella with the microtubule (MT) disrupting agent colchicine during early valve formation results in a characteristic malformation of the valve, whereby part of the normally circumferential raphe canal forms as an abnormal protruding lip on the valve surface, located up to 20 m from the edge of the valve. The position of this malformed lip coincides with the location of a microtubule center (MC) at the time of colchicine addition, suggesting that the MC may play a direct role in positioning the tip of the raphe canal during valve formation. The migration of this MC to the tip of the cell during early valve morphogenesis is reversibly inhibited by the metabolic inhibitor 2-4-dinitrophenol (DNP). The effect of colchicine onPinnularia valve formation is less severe, causing occasional malformation of the raphe, but little if any lateral displacement. InHantzschia, colchicine has no effect on the positioning of the raphe, but prolonged exposure causes fusion of the raphe canal with the valve face. Cochicine treatment also results in the absence of the normal curvature at the central interruption in the raphe, as well as abnormal pore formation in this central area. Addition of cytochalasin D during early valve formation inHantzschia causes the raphe canal to form in the center of the valve face, suggesting that the normal translocation of the raphe canal to the valve edge is actindependent. Comparison of valves from control and cytochalasintreatmentHantzschia suggest that the pore spacing within the valve is determined by the position relative to the raphe, and does not depend on whether to pores form on the side (mantle) or the face of the mature valve.Abbreviations DM diatom medium - DNP dinitrophenol - MT microtubule - MC microtubule center - PSS primary silicification site - SDV silica deposition vesicle     

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.     

A novel p21-activated kinase binds the actin and microtubule networks and induces microtubule stabilization.

Coordination of the different cytoskeleton networks in the cell is of central importance for morphogenesis, organelle transport, and motility. The Rho family proteins are well characterized for their effects on the actin cytoskeleton, but increasing evidence indicates that they may also control microtubule (MT) dynamics. Here, we demonstrate that a novel Cdc42/Rac effector, X-p21-activated kinase (PAK)5, colocalizes and binds to both the actin and MT networks and that its subcellular localization is regulated during cell cycle progression. In transfected cells, X-PAK5 promotes the formation of stabilized MTs that are associated in bundles and interferes with MTs dynamics, slowing both the elongation and shrinkage rates and inducing long paused periods. X-PAK5 subcellular localization is regulated tightly, since coexpression with active Rac or Cdc42 induces its shuttling to actin-rich structures. Thus, X-PAK5 is a novel MT-associated protein that may communicate between the actin and MT networks during cellular responses to environmental conditions.     

Converging populations of f-actin promote breakage of associated microtubules to spatially regulate microtubule turnover in migrating cells

BACKGROUND: In migrating cells, the retrograde flow of filamentous actin (f-actin) from the leading edge toward the cell body is accompanied by the synchronous motion of microtubules (MTs, ), whose plus ends undergo net growth. Thus, MTs must depolymerize elsewhere in the cell to maintain polymer mass over time. The source and location of depolymerized MTs is unknown. Here, we test the hypothesis that MT polymer loss occurs in central cell regions and is induced by the convergence of actin retrograde and anterograde flow, which buckles and breaks associated MTs and promotes minus-end depolymerization. RESULTS: We characterized the effects of calyculin A and ML-7 on the movement of f-actin and MTs by multi-spectral fluorescence recovery after photobleaching (FRAP) and fluorescent speckle microscopy (FSM). Our studies show that these drugs affect the rate of f-actin and MT convergence and MT buckling in a central cell region we call the "convergence zone." Increases in f-actin convergence are associated with faster MT turnover and an increase in both MT breakage and minus-end depolymerization, but they have no effect on MT plus end dynamic instability. CONCLUSIONS: We propose that f-actin movement into the convergence zone plays a major role in spatially modulating MT turnover during cell migration by regulating MT breakage, and thus minus-end dynamics, in central cell regions.     

Septin 6 localizes to microtubules in neuronal dendrites

In neuronal dendrites, septins localize to the base of the spine, a unique position which is sandwiched between the microtubule (MT)-rich dendritic shaft and the actin filament-rich spine. Here, we provide evidence for the association of SEPT6 with MTs in cultured rat hippocampal neurons. In normal cultures, SEPT6 clusters localized to MTs, but not to actin clusters. Only MT-disrupting agents (vincristine and nocodazole), but not microfilament-disrupting one (latrunculin A), induced the redistribution of SEPT6 to the disrupted MTs. The nascent MT fibers that were recovered from vincristine or nocodazole treatments also accompanied SEPT6. Blocking MT disruption by Taxol prevented such phenomena, proving that the redistribution of SEPT6 was due to the MT disruption. Our results indicate that SEPT6 complexes at the base of the dendritic spine are associated with MTs.     

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.     

Mitosis and mitotic wave propagation in the coenocytic alga,<Emphasis Type="Italic"> Vaucheria terrestris</Emphasis> sensu Goetz

Mitosis and cytoplasmic microtubule (MT) dynamics were observed for the first time in Vaucheria terrestris sensu Goetz. Mitosis could occasionally be seen in part of the cylindrical coenocytic cell. The frequency of encountering cells with dividing nuclei was highest (ca 12%) 4 h after the onset of light in 12 h light/12 h dark regimes; it decreased thereafter and approached zero during the dark period. From the anterior end of every interphase nucleus a unique, long MT bundle extended. Differential-interference optics reveals that there is a filamentous structure in front of the moving nucleus. In prophase, the interphase bundle disappeared and shorter MT bundles emanated from both ends of the nucleus. In metaphase, the cytoplasmic MTs completely disappeared, probably being recycled to spindles. Continuous MTs elongated in anaphase and developed into an interzonal spindle in telophase; this elongated up to as much as 10 m. The daughter nuclei were pushed away from each other by the interzonal spindle. Mitosis started synchronously in a relatively narrow region, and the mitotic stage propagated as a mitotic wave to adjacent regions, most frequently from tip to base. The role of the mitotic wave in tip growth and morphogenesis of a coenocytic cell is discussed.This paper is dedicated to the memory of Dr. Eiji Kamitsubo who passed away on 25 April 2003.     

Root contraction in hyacinth III. Orientation of cortical microtubules visualized by immunofluorescence microscopy

Summary Cortical microtubules (MTs) were visualized in root cortex cells ofHyacinthus orientalis L. using immunofluorescence techniques. Cellular MT orientation was determined adjacent to radial longitudinal and transverse walls of root tip, uncontracted, contracting, and fully contracted regions. As seen in longitudinal views, MTs formed parallel, apparently helical arrays which were oriented transversely, axially or obliquely depending upon the region. Transverse sectional views showed that MTs adjacent to transverse cell walls formed a variety of patterns which varied with developmental stage and cell location. Microtubules were oriented in crisscross or parallel arrays. The parallel arrays were oriented either parallel, perpendicular or oblique to the radius of the root. There was an apparent temporal progression in MT reorientation from outer cortical to inner cortical cell layers. A resultant progression of reoriented cell growth could account for root contraction. These findings corroborate earlier electron microscopic observations of changing MT orientation accompanying root contraction, and provide cytological evidence to test mathematical and biophysical models of the mechanics of cell expansion.Abbreviations MT microtubule - MF microfibril - MTSB microtubule stabilizing buffer - PBS phosphate buffered saline