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

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

Nuclear centering in Spirogyra: force integration by microfilaments along microtubules

The contribution of microtubules and microfilaments to the cytomechanics of transverse nuclear centering were investigated in the charophycean green alga Spirogyracrassa (Zygnematales). Cytoplasmic strands of enhanced rigidity and fasciate appearance radiate from the rim of the lenticular nucleus through the vacuole, frequently split once or twice and are attached to the helical chloroplast bands in the peripheral cytoplasm. The nucleus is encased in tubulin and a web of F-actin. Bundles of microtubules, emerging from the nuclear rim, are organized into dividing fascicles within the strands and reach to the inner surface of the chloroplast envelope. Organelles are translocated in both directions along similarly arranged fascicles of microfilament bundles which extend from the nucleus to the peripheral actin cytoskeleton. Application of microtubule- and/or microfilament-depolymerizing drugs affected the position of the nucleus only slowly, but in distinct ways. The differential effects suggest that nuclear centering depends on the tensional integrity of the perinuclear scaffold, with microfilaments conveying tension along stabilized microtubules and the actin cytoskeleton integrating the translocation forces generated within the scaffold. Received: 9 December 1996 / Accepted: 29 April 1997     

A primary role for microfilaments, but not microtubules, in hormone-induced cytoplasmic retraction

The distributions of microfilaments and microtubules were studied during transient hormone-induced changes in cell shape (retraction-respreading). Two cell types (fibroblasts and bone cells), differentially responsive to parathyroid hormone (PTH) and prostaglandin E₂ (PGE₂), were analysed. The cytoplasm of fibroblasts retracted in response to PGE₂ but not PTH, whereas bone cells could respond to both PGE₂ and PTH. Time-lapse photomicrography indicated that the retraction began within minutes of hormone addition, while respreading occurred over longer times, up to 8 h. Affinity-purified actin and tubulin antibodies were used to follow the appearance of microtubules and microfilaments during both the retraction and the respreading phases. Microtubules appeared not to reorganize noticeably, although they were squeezed closer together in cellular pseudopods; no extensive loss or growth was detectable. Microfilaments did alter drastically their appearance and distributions. Soon after hormone addition when earliest detectable cytoplasmic retraction was evident, microfilament bundles appeared to break down. Remaining microfilament bundles consisted of relatively short, non-aligned fragments or aggregates. During respreading, microfilament bundles regrew and realigned throughout the cytoplasm. These data suggest a primary role for microfilaments, but probably not microtubules, in these cell shape changes.     

Microtubules and tracheole migration in wing disks of Galleria mellonella

Tracheoles migrate into the lacunae of wing imaginal disks that are cultured in vitro in medium containing α-ecdysone. Colchicine and vinblastin prevented tracheole migration in vitro under conditions which did not affect the viability of the disks. Cytochalasin B also inhibited tracheole migration, but only at concentrations which were deleterious to the disks. Our ultrastructure findings were in accord with the hypothesis that microtubules, but perhaps not microfilaments, are required for tracheole migration.     

Caenorhabditis elegans morphogenesis: the role of the cytoskeleton in elongation of the embryo

During development Caenorhabditis elegans changes from an embryo that is relatively spherical in shape to a long thin worm. This paper provides evidence that the elongation of the body is caused by the outermost layer of embryonic cells, the hypodermis, squeezing the embryo circumferentially. The hypodermal cells surround the embryo and are linked together by cellular junctions. Numerous circumferentially oriented bundles of microfilaments are present at the outer surfaces of the hypodermal cells as the embryo elongates. Elongation is associated with an apparent pressure on the internal cells of the embryo, and cytochalasin D reversibly inhibits both elongation and the increase in pressure. Circumferentially oriented microtubules also are associated with the outer membranes of the hypodermal cells during elongation. Experiments with the microtubule inhibitors colcemid, griseofulvin, and nocodazole suggest that the microtubules function to distribute across the membrane stresses resulting from microfilament contraction, such that the embryo decreases in circumference uniformly during elongation. While the cytoskeletal organization of the hypodermal cells appears to determine the shape of the embryo during elongation, an extracellular cuticle appears to maintain the body shape after elongation.     

Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia

Previous studies (Holmes, K.V., and P.W. Choppin. J. Exp. Med. 124:501- 520; J. Cell Biol. 39:526-543) showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy. During cell fusion, micro tubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei. No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei. Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 microgram/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 microgram/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 microgram/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required. The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei.     

Association of actin filaments with axonal microtubule tracts

Axoplasmic organelles move on actin as well as microtubules in vitro and axons contain a large amount of actin, but little is known about the organization and distribution of actin filaments within the axon. Here we undertake to define the relationship of the microtubule bundles typically found in axons to actin filaments by applying three microscopic techniques: laser-scanning confocal microscopy of immuno-labeled squid axoplasm; electronmicroscopy of conventionally prepared thin sections; and electronmicroscopy of touch preparations-a thin layer of axoplasm transferred to a specimen grid and negatively stained. Light microscopy shows that longitudinal actin filaments are abundant and usually coincide with longitudinal microtubule bundles. Electron microscopy shows that microfilaments are interwoven with the longitudinal bundles of microtubules. These bundles maintain their integrity when neurofilaments are extracted. Some, though not all microfilaments decorate with the S1 fragment of myosin, and some also act as nucleation sites for polymerization of exogenous actin, and hence are definitively identified as actin filaments. These actin filaments range in minimum length from 0.5 to 1.5 µm with some at least as long as 3.5 µm. We conclude that the microtubule-based tracks for fast organelle transport also include actin filaments. These actin filaments are sufficiently long and abundant to be ancillary or supportive of fast transport along microtubules within bundles, or to extend transport outside of the bundle. These actin filaments could also be essential for maintaining the structural integrity of the microtubule bundles.     

The architecture of the accessory reproductive glands of the male desert locust 2: Microtubular structures

Cytoplasmic microtubules are distributed widely in the mature accessory reproductive glands of the male desert locust. They are not concentrated in any particular region of the cytoplasm, but they appear to be closely associated with mitochondria and the Golgi apparatus. The individual microtubules have a helical configuration, and they are often congregated in bundles. It is suggested that the microtubules arise from Golgi regions, and that they function as a scaffolding for the maintenance of the shape of the glandular cells.     

Distribution of actin and tubulin in cells and in glycerinated cell models after treatment with cytochalasin B (CB)

The organization of microfilaments and microtubules in cultured cells before and after the addition of cytochalasin B (CB) was studied both by electron microscopy and immunofluorescence microscopy using antibodies specific for actin, tubulin and tropomyosin. CB induces a rapid disorganization of normal microfilament bundles. Star-like patches of actin and tropomyosin are visualized in immunofluorescence microscopy and dense aggregates of condensed microfilaments are seen in electron microscopy. The integrity of the microtubules is not changed by CB treatment. Addition of CB to glycerinated cells, in contrast to normal cells, does not result in the disorganization of microfilament bundles. CB-treated glycerinated models can still contract upon addition of ATP. Thus the CB-induced rearrangement of microfilament bundles occurs only in vivo and not in glycerinated cell contractility models.     

Cortical microtubules and cortical microfilaments in the green alga,Micrasterias pinnatifida

Summary Cortical microtubules and cortical microfilaments were visualized in cells ofMicrasterias pinnatifida treated by freeze-substitution, and the pattern of their distribution was reconstructed from serial sections. Most cortical microtubules accompanied the long microfilaments that ran parallel to the microtubules. Cortical microfilaments not accompanied by the microtubules were also found. They were short and slightly curved. Both types of cortical microfilament were not grouped into bundles, and were 6–7 nm in diameter, a value that corresponds to the diameter of filaments of F-actin.     

Microtubules regulate aortic endothelial cell actin microfilament reorganization in intact and repairing monolayers

To understand the role of microtubules and microfilaments in regulating endothelial monolayer integrity and repair, and since microtubules and microfilaments show some co-alignment in endothelial cells, we tested the hypothesis that microtubules organize microfilament distribution. Disruption of microtubules with colchicine in resting confluent aortic endothelial monolayers resulted in disruption of microfilament distribution with a loss of dense peripheral bands, an increase in actin microfilament bundles, and an associated increase of focal adhesion proteins at the periphery of the cells. However, when microfilaments were disrupted with cytochalasin B, microtubule distribution did not change. During the early stages of wound repair of aortic endothelial monolayers, microtubules and microfilaments undergo a sequential series of changes in distribution prior to cell migration. They are initially distributed randomly relative to the wound edge, then align parallel to the wound edge and then elongate perpendicular to the wound edge. When microtubules in wounded cultures were disrupted, dense peripheral bands and lamellipodia formation were lost with increases in central stress fibers. However, following microfilament disruption, microtubule redistribution was not disrupted and the microtubules elongated perpendicular to the wound edge similar to non-treated cultures. Microtubules may organize independently of microfilaments while microfilaments require microtubules to maintain normal organization in confluent and repairing aortic endothelial monolayers.     

Stabilizing microtubules with taxol increases microfilament stability during freezing of rye root tips

We have used double fluorescence labelling to investigate the effect of freezing on microtubules and microfilaments in root-tip cells of rye (Secale cereale L. cv Rymin). Freezing to -5°C (which does not kill these cells) caused partial depolymerization of both, but microfilaments were more resistant than microtubules. When microtubules were stabilized against freeze-induced depolymerization by pre-treating seedlings with taxol, microfilaments exhibited enhanced stability as well. Almost all the frozen cells containing taxol-stabilized microtubules also contained microfilaments. When seedlings were treated with the microtubule-destabilizing drug APM prior to freezing, microfilaments became more susceptible to freeze-induced depolymerization than in controls. These data suggest a physical interaction between microtubules and microfilaments in these cells.     

Polarized cell growth, organelle motility, and cytoskeletal organization in conifer pollen tube tips are regulated by KCBP, the calmodulin-binding kinesin

Kinesin-like calmodulin-binding protein (KCBP), a member of the Kinesin 14 family, is a minus end directed C-terminal motor unique to plants and green algae. Its motor activity is negatively regulated by calcium/calmodulin binding, and its tail region contains a secondary microtubule-binding site. It has been identified but not functionally characterized in the conifer Picea abies. Conifer pollen tubes exhibit polarized growth as organelles move into the tip in an unusual fountain pattern directed by microfilaments but uniquely organized by microtubules. We demonstrate here that PaKCBP and calmodulin regulate elongation and motility. PaKCBP is a 140 kDa protein immunolocalized to the elongating tip, coincident with microtubules. This localization is lost when microtubules are disrupted with oryzalin, which also reorganizes microfilaments into bundles. Colocalization of PaKCBP along microtubules is enhanced when microfilaments are disrupted with latrunculin B, which also disrupts the fine network of microtubules throughout the tip while preserving thicker microtubule bundles. Calmodulin inhibition by W-12 perfusion reversibly slows pollen tube elongation, alters organelle motility, promotes microfilament bundling, and microtubule bundling coincident with increased PaKCBP localization. The constitutive activation of PaKCBP by microinjection of an antibody that displaces calcium/calmodulin and activates microtubule bundling repositions vacuoles in the tip before rapidly stopping organelle streaming and pollen tube elongation. We propose that PaKCBP is one of the target proteins in conifer pollen modulated by calmodulin inhibition leading to microtubule bundling, which alters microtubule and microfilament organization, repositions vacuoles and slows organelle motility and pollen tube elongation.     

Microtubules and microfilaments are both responsible for pollen tube elongation in the coniferPicea abies (Norway spruce)

Summary InPicea abies (Norway spruce), microtubules and actin microfllaments both form a dense matrix throughout the tube mainly parallel to the direction of elongation. In these conifer pollen tubes the organization of this matrix is different from that in angiosperms. This study tests our hypothesis that differences in cytoskeletal organization are responsible for differences in tube growth and physiology. Pollen grains were germinated in media containing cytoskeletal disrupters and analyzed for germination, tube length, tube branching, and tip swelling. Disruption of microtubules significantly inhibits tube elongation and induces tube branching and tip swelling. Tip swelling is probably caused by disruption of the microtubules in the tip that are perpendicular to the direction of elongation. Confocal microscopy indicates that colchicine and propyzamide cause fragmentation of microtubules throughout the tube. Oryzalin and amiprophosmethyl cause a complete loss of microtubules from the tip back toward the tube midpoint but leave microtubules intact from the midpoint back to the grain. Disruption of microfilaments by cytochalasins B and D and inhibition of myosin by N-ethylmaleimide or 2,3-butanedione monoxime stops tube growth and inhibits germination. Microfilament disruption induces short branches in tubes, probably originating from defective microfilament organization behind the tip. In addition, confocal microscopy coupled with microinjection of fluorescein-labeled phalloidin into actively growing pollen tubes indicates that microfllament bundles extend into the plastid-free zone at the tip but are specifically excluded from the growing tip. We conclude that microtubules and microfilaments coordinate to drive tip extension in conifer pollen tubes in a model that differs from angiosperms.     

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.     

Microtubule stability affects the unique motility of F-actin in Marchantia polymorpha

Actin microfilaments play crucial roles in diverse plant functions. Some specific cellular processes require interaction between F-actin and microtubules, and it is believed that there are direct or indirect connections between F-actin and microtubules. We previously reported that actin microfilaments exhibit unique dynamic motility in cells of the liverwort, Marchantia polymorpha; the relevance of this activity to microtubules has not been explored. To examine whether the dynamics of F-actin in M. polymorpha were somehow regulated by microtubules, we investigated the effects of stabilization or destabilization of microtubules on dynamics of actin bundles, which were visualized by Lifeact-Venus. To our surprise, both stabilization and destabilization of microtubules exerted similar effects on F-actin motility; apparent sliding movement of F-actin in M. polymorpha cells was accelerated by both oryzalin and paclitaxel, with the effect of paclitaxel more evident than that of oryzalin. Immunofluorescence staining revealed that some F-actin bundles were arrayed along with microtubules in M. polymorpha thallus cells. These results suggest that microtubules play regulatory roles in the unique F-actin dynamics in M. polymorpha.     

GFP-tagging of cell components reveals the dynamics of subcellular re-organization in response to infection of Arabidopsis by oomycete pathogens

Cytoplasmic aggregation, the rapid translocation of cytoplasm and subcellular components to the site of pathogen penetration, is one of the earliest reactions of plant cells against attack by microorganisms. We have investigated cytoplasmic aggregation during Arabidopsis-oomycete interactions. Infection by non-pathogenic Phytophthora sojae was prevented in the plant epidermal cell layer, whereas Peronospora parasitica isolates Cala2 (avirulent) and Noks1 (virulent) could both penetrate into the mesophyll cell layer. Epidermal cell responses to penetration by these oomycetes were examined cytologically with a range of transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-tagged cell components. These included plants containing GFP-TUA6 for visualizing microtubules, GFP-hTalin for actin microfilaments, GFP-tm-KKXX for endoplasmic reticulum (ER), and STtmd-GFP for the Golgi apparatus. In all interactions, actin microfilaments were actively re-arranged and formed large bundles in cytoplasmic strands focused on the penetration site. Aggregation of ER membrane and accumulation of Golgi bodies at the infection site were observed, suggesting that production and secretion of plant materials were activated around the penetration site. Microtubules did not become focused on the penetration site. No difference was evident between the responses of epidermal cells in the non-host, incompatible and compatible interactions. This result indicates that the induction of cytoplasmic aggregation in Arabidopsis epidermal cells was neither suppressed by the virulent strain of Peronospora, nor effective in stopping infection.     

Evidence for active interactions between microfilaments and microtubules in myxomycete flagellates

We have previously observed the apparent displacement of microfilaments over microtubules in the backbone structure of permeabilized flagellates of Physarum polycephalum upon addition of ATP (Uyeda, T. Q. P., and M. Furuya. 1987. Protoplasma. 140:190-192). We now report that disrupting the microtubular cytoskeleton by treatment with 0.2 mM Ca2+ for 3-30 s inhibits the movement of the microfilaments induced by subsequent treatment with 1 mM Mg-ATP and 10 mM EGTA. Stabilization of microtubules by pretreatment with 50 microM taxol retarded both the disintegrative effect of Ca2+ on the microtubules and the inhibitory effect of Ca2+ on the subsequent, ATP-induced movement of the microfilaments. These results suggest that the movement of the microfilaments depends on the integrity of the microtubular cytoskeleton. EM observation showed that the backbone structure in control permeabilized flagellates consists of two arrays of microtubules closely aligned with bundles of microfilaments of uniform polarity. The microtubular arrays after ATP treatment were no longer associated with microfilaments, yet their alignment was not affected by the ATP treatment. These results imply that the ATP treatment induces reciprocal sliding between the microfilaments and the microtubules, rather than between the microfilaments themselves or between the microtubules themselves. While sliding was best stimulated by ATP, the movement was partially induced by GTP or ATP gamma S, but not by ADP or adenylyl-imidodiphosphate (AMP-PNP). AMP-PNP added in excess to ATP, 50 microM vanadate, or 2 mM erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) inhibited the sliding. Thus, the pharmacological characteristics of this motility were partly similar to, although not the same as, those of the known microtubule-dependent motilities.     

Microtubular organization in flat epitheloid and stellate process-bearing astrocytes in culture

Microtubules and microfilament patterns in cultured astrocytes were revealed by using indirect immunofluorescent microscopy in conjunction with anti-tubulin immune serum and anti-actin immunoglobulins respectively. In flat epitheloid astroglial cells (either polygonal or elongated) colchicine-sensitive immunofluorescent fibres, which correspond to bundles of microtubules, extend from the perinuclear cytoplasm into the cell periphery by running for long distances through the different focal planes. These patterns of organization differ markedly from the patterns of organization of microfilaments which are arranged in fibres parallel to each other and often oriented along the cell boundary. In response to the combined treatments of serum withdrawal and administration of dBcAMP, flat epitheloid astrocytes adopt a morphology similar to that of the mature astrocytes in situ in the CNS, that is of stellate process-bearing cells. This is prevented or is reverted by the administration of colchicine at the appropriate times. There are strong suggestions indicating that during cell processes formation the microtubular network is reorganized and microtubules assembled into dense bundles which are oriented along the axis of the cell processes. In view of these results, we suggest that, in contrast to microfilaments, microtubules are not determinant for the maintenance of cellular shape in elongated or polygonal flat epitheloid astroglial cells but they are required for both the formation and maintenance of processes in stellate astrocytes.     

Polarity orientation and assembly process of microtubule bundles in nocodazole-treated, MAP2c-transfected COS cells.

Microtubule bundles reminiscent of those found in neuronal processes are formed in fibroblasts and Sf9 cells that are transfected with the microtubule-associated proteins tau, MAP2, or MAP2c. To analyze the assembly process of these bundles and its relation to the microtubule polarity, we depolymerized the bundles formed in MAP2c-transfected COS cells using nocodazole, and observed the process of assembly of microtubule bundles after removal of the drug in cells microinjected with rhodamine-labeled tubulin. Within minutes of its removal, numerous short microtubule fragments were observed throughout the cytoplasm. These short fragments were randomly oriented and were already bundled. Somewhat longer, but still short bundles, were then found in the peripheral cytoplasm. These bundles became the primordium of the larger bundles, and gradually grew in length and width. The polarity orientation of microtubules in the reformed bundle as determined by "hook" procedure using electron microscope was uniform with the plus end distal to the cell nucleus. The results suggest that some mechanism(s) exists to orient the polarity of microtubules, which are not in direct continuity with the centrosome, during the formation of large bundles. The observed process presents a useful model system for studying the organization of microtubules that are not directly associated with the centrosomes, such as those observed in axons.