Novel filamentous bundles in the cytoplasm of a unicellular eukaryote,Crithidia fasciculata

Summary Trypanosomes, an evolutionarily ancient group of unicellular eukaryotic parasites, appear to lack both microfilaments (actin) and intermediate filaments (IFs): the major cytoskeletal component common to all trypanosomes consists of a stable microtubular array intimately associated with the plasma membrane. We present here evidence of bundles of trans-cytoplasmic filaments ca. 10 nm in diameter, seen by transmission electron microscopy, that are formed in stationary cultures of an insect trypanosome,Crithidia fasciculata. Immunofluorescent labelling with an antibody raised against plant fibrillar bundles (AFB) and Western blotting with an antibody that cross-reacts with a broad range of IFs (anti-IFA) as well as with fibrillar bundles, indicates that these filaments appear to share antigenic determinants common to animal IFs and to fibrillar bundles of plant origin.Abbreviations AFB anti-fibrillar bundle antibody - anti-IFA anti-intermediate filament antibody - IF intermediate filament - SEM scanning electron microscope - TEM transmission electron microscope - YOL 1/34 anti--tubulin antibody     

Organization of the cytoskeleton in the coenocytic algaVaucheria longicaulis var.macounii: an experimental study

Summary The organization of the cytoskeleton of vegetative filaments ofVaucheria longicaulis Hoppaugh var.macounii Blum is investigated by fluorescence microscopy using monoclonal anti -tubulin antibodies and fluorescein (FITC)-labelled phalloidin. Confocal laser scanning microscopy observations give further information on the distribution of the cytoskeletal elements. Phalloidin labelling reveals F-actin bundles in the cortical cytoplasm of both fixed and unfixed vegetative filaments of this alga. In addition a more diffuse fluorescent component, seen at higher magnification to be made up of thinner F-actin bundles, can also be detected in unfixed cells. The distribution of the F-actin bundles resemble that of filamentous structures observed with differential interference contrast (DIC) microscopy in living cells. These structures seem to correspond to the microtubule associated reticulum (MAR) described in literature and overall the evidence suggests that actin and MAR elements are co-distributed. F-actin bundles are always found in association with focal masses (foci) of phalloidin-positive material. Foci are also observed by DIC microscopy associated with the cytoplasmic filamentous structures in living cells.Depolymerization of F-actin with cytochalasin D and the subsequent repolymerization that occurs on transfer ofVaucheria vegetative filaments to cytochalasin-free medium suggest that these foci are involved in the organization of the F-actin array. Immunofluorescence for -tubulin reveals microtubule bundles that are shorter in length and straighter in configuration than microfilament bundles. Microtubule bundles are associated with spot-like focal structures that, in many instances, show a close relationship with respect to nuclei. Oryzalin and cold temperature cause the depolymerization of the microtubule bundles and suggest, in conjunction with repolymerization studies, that these fluorescent spots associated with the ends of the microtubule bundles are involved in their organization; hence, they represent microtubule organizing centres or MTOCs. The importance of both microfilament and microtubule bundle focal regions is discussed with respect to the apical growth exhibited by the vegetative filaments of this alga.     

Monoclonal antibody to intermediate filament antigen cross-reacts with higher plant cells

The monoclonal antibody (anti-IFA) raised (Pruss et al., 1981, Cell 27:419-428) against an intermediate filament antigen, which is widespread throughout phylogeny, has been shown here to cross-react with higher plants. On immunoblotting, anti-IFA cross-reacted with proteins in homogenates of carrot suspension cells and of meristematic cells from onion root tips. A 50-kD cross-reactive protein was enriched in a fraction that consisted of detergent-insoluble bundles of 7-nm fibrils from carrot protoplasts (Powell et al., 1982, J. Cell Sci. 56:319-335). By use of indirect immunofluorescence, anti-IFA stained formaldehyde-fixed onion meristematic cells and carrot protoplasts in patterns approximating those obtained with monoclonal anti-tubulins. That anti-IFA was not recognizing plant tubulins was established by use of immunoblots of two-dimensional gels on which the proteins that comprised isolated fibrillar bundles and taxol-purified carrot tubulins had been separated. The two groups of proteins had different positional coordinates: anti-IFA recognized the fibrillar bundle proteins, and anti-tubulins recognized plant microtubule proteins with no cross-reaction to the heterologous proteins. Likewise, formaldehyde-fixed taxol microtubules from carrot cells could be stained with anti-tubulin but not with anti-IFA. It is concluded that an epitope common to intermediate filaments from animals co-distributes with microtubules in higher plant cells.     

The organization of F-actin in root tip cells ofAdiantum capillus veneris throughout the cell cycle

Summary The patterns of F-actin in relation to microtubule (Mt) organization in dividing root tip cells ofAdiantum capillus veneris were studied with rhodamine-phalloidin (RP) labelling and tubulin immunofluorescence. Interphase cells display a well organized network of cortical/subcortical, endoplasmic and perinuclear actin filaments (AFs), not particularly related to the interphase Mt arrays. The cortical AFs seem to persist during the cell cycle while the large subcortical AF bundles disappear by preprophase/prophase and reappear after cytokinesis is completed. In some but not all of the preprophase cells the cortical AFs tend to form a band (AF-PPB) coincident with the preprophase band of Mts (Mt-PPB). In metaphase and anaphase cells AFs are localized in the cell cortex, around the spindle and inside it coincidently with kinetochore Mt bundles. During cytokinesis AFs are consistently found in the phragmoplast. In oryzalin treated cells neither Mt-PPBs, spindles and phragmoplasts exist, nor such F-actin structures can be observed. In cells recovering from oryzalin, AF-PPBs, AF kinetochore bundles and AF phragmoplasts reform. They show the same pattern with the reinstating respective Mt arrays. In contrast, in cells treated with cytochalasin B (CB), AFs disappear but all categories of Mt arrays form normally.These observations show that F-actin organization in root tip cells ofA. capillus veneris differs from that of root tip cells of flowering plants examined so far. In addition, Mts seem to be crucial for F-actin organization as far as it concerns the PPB, the mitotic spindle, and the phragmoplast.Abbreviations AF actin filament - CB cytochalasin B - MBS m-male-imidobenzoyl-N-hydroxysuccinimide ester - MSB microtubule stabilizing buffer - Mt microtubule - PBS phosphate buffered saline - PPB preprophase band - RP rhodamine phalloidin     

Occurrence of two different intermediate filament proteins in the same filament in situ within a human glioma cell line : An immunoelectron microscopical study

Intermediate filament systems of an established glioma cell line have been characterized by double immunofluorescence microscopy and by immunoelectron microscopy using two antibodies, one of which recognizes glial fibrillary acid protein (GFA) but not vimentin, and the second which recognizes vimentin but not GFA. The results show that glioma cells express two immunologically distinct IF polypeptides which are found in the same 10-nm filaments. Juxtanuclear caps formed after exposure of the cells to colcemid consisted of intermediate filaments composed of both GFA and vimentin. In immunoelectron microscopy both untreated cells and cells treated with colcemid show discontinuous labelling when only a single antibody is used, but continuous labelling when both antibodies are used simultaneously.     

Immuno-gold localization of α-L-arabinofuranosyl residues in pollen tubes of Nicotiana alata Link et otto

Immuno-gold labelling using a monoclonal antibody (PCBC3) with a primary specificity for -L-arabinofuranosyl residues was used to locate these residues in pollen tubes of Nicotiana alata grown in vivo. The antibody bound to the outer fibrillar layer of the pollen-tube wall: the inner, non-fibrillar wall layer was not labelled. Cytoplasmic vesicles (0.2 m diameter) were also labelled. The antibody may bind to an arabinan in the pollen-tube wall.     

Intermediate filaments in the giant muscle cells of the nematode Ascaris lumbricoides; abundance and three-dimensional complexity of arrangements

The body muscle cells of the nematode Ascaris lumbricoides are characterized by massive amounts of intermediate filaments (IF). These occur in all three regions of this giant cell type. They traverse the cytoplasm of the balloon-like belly, which houses the nucleus, and occur as bundles in the arm-like extensions to the nerve. The organization of IF in the third region, the contractile fiber, was analyzed further by serial sections and three-dimensional reconstruction. IF bundles traverse the glycogen-rich lumina of the fiber and reach as baskets around the sarcomeres. Together with numerous dense bodies they form the Z-band-like arrangements. IF bundles reach the plasma membrane at hemidesmosome-like specializations often situated at deep membrane invaginations filled with a fibrillar component of the extracellular matrix. The ultrastructural appearance of IF bundles is connected to the contractional state of the sarcomeres. They appear straight in extended muscle but coil up upon contraction. In the pharynx massive IF bundles are oriented longitudinally. A second type of IF bundles follows the radially oriented sarcomeres. These reveal pronounced Z-band type structures with massive disks. IF surround the sarcomeres and seem to terminate at these disks. We discuss possible functions of the complex IF organization in body muscle and pharynx.     

An argyrophil fibrillar system and amitotic nuclear division in pars intermedia cells of the rainbow trout (Salmo irideus)

Summary Argyrophil fibrils were revealed in pars intermedia chromophobe cells of Salmo irideus. For the light microscopical demonstration of the fibrils a recently developed copper sulphate-silver protein technique for Bouin-fixed hypophyses was used.The fibrils, apparently belonging to one fibrillar system, are found only in limited regions of the cytoplasm. They occur in ring-, loop-, or cracknel shapes in the close vicinity of the rounded nucleus in the chromophobe cell, and continue in linear shape into the cell base or cell apex where they may end near the adjacent basement membrane.Electron microscopically, the fibrils are composed of filaments, 70–90 Å in diameter, which are arranged in parallel. Bundles of filaments are frequently found near the nucleus. In addition to perinuclear filaments, bundles of filaments with tiny offshoots occur in different areas of the cytoplasm, extending sometimes as far as the cell membrane which is located near the basement membrane of the pars intermedia epithelium.In cells with a bi- or trilobular, apparently amitotically dividing, nucleus a fibrillar loop or ring usually surrounds the interlobular part(s) of the nucleus. This relation of the fibrils to the dividing nucleus and their consisting of regularly arranged filaments indicates the significance of the fibrillar system in the chromophobe cell. It is thus suggested that the fibrillar system is involved in the constriction of the nucleus during amitosis.The number of chromophobe cells in which fibrils are light microscopically visible varied greatly among the rainbow trout used.Miss M. C. Wentzel is thanked for her technical assistance, Mr. M. Veenhuis for his advice and excellent help with the preparation of the electron micrographs, and Mr. L. Hoekstra for preparing the final drawings. Mr. N. J. Williams' corrections to the English are gratefully acknowledged.     

Monoclonal antibodies against plant proteins recognise animal intermediate filaments

Four monoclonal antibodies were raised against polypeptides present in a high-salt detergent-insoluble fraction from cells of Chlamydomonas reinhardtii. Indirect immunofluorescence microscopy of fibroblasts and epithelial cells grown in culture using these plant antibodies revealed staining arrays identical to those obtained with well characterised antibodies to animal intermediate filaments. Immunofluorescence microscopy of Chlamydomonas with these monoclonal antibodies and a monoclonal antibody that recognises all animal intermediate filaments (anti-IFA) gave a diffuse, patchy cytoplasmic staining pattern. Both the plant antibodies and anti-IFA stained interphase onion root tip cells in a diffuse perinuclear pattern. In metaphase through to telophase, the labelling patterns colocalised with those of microtubules. Labelling of the phragmoplast was also detected but not staining of the preprophase band. On Western blots of various animal cell lines and tissues, all the antibodies labelled known intermediate filament proteins. On Western blots of whole Chlamydomonas proteins, all the antibodies labelled a broad band in the 57,000 Mr range, and three antibodies labelled bands around 66,000 and 140,000 Mr but with variable intensities. On Western blots of whole onion root tip proteins, all the antibodies labelled 50,000 Mr (two to three bands) polypeptides and a diffuse band around 60,000 Mr and three of the antibodies also labelled several polypeptides in the 90,000-200,000 Mr range. The consistent labelling of these different bands by several different monoclonal antibodies recognising animal intermediate filaments makes these polypeptides putative plant intermediate filament proteins.     

γ-Tubulin has a conserved intrinsic property of self-polymerization into double stranded filaments and fibrillar networks

γ-Tubulin is essential for microtubule nucleation and also plays less understood roles in nuclear and cell-cycle-related functions. High abundancy of γ-tubulin in acentrosomal Arabidopsis cells facilitated purification and biochemical characterization of large molecular species of γ-tubulin. TEM, fluorescence, and atomic force microscopy of purified high molecular γ-tubulin forms revealed the presence of linear filaments with a double protofilament substructure, filament bundles and aggregates. Filament formation from highly purified γ-tubulin free of γ-tubulin complex proteins (GCPs) was demonstrated for both plant and human γ-tubulin. Moreover, γ-tubulin associated with porcine brain microtubules formed oligomers. Experimental evidence on the intrinsic ability of γ-tubulin to oligomerize/polymerize was supported by conservation of α- and β-tubulin interfaces for longitudinal and lateral interactions for γ-tubulins. STED (stimulated emission depletion) microscopy of Arabidopsis cells revealed fine, short γ-tubulin fibrillar structures enriched on mitotic microtubular arrays that accumulated at polar regions of acentrosomal spindles and the outer nuclear envelope before mitosis, and were also present in nuclei. Fine fibrillar structures of γ-tubulin representing assemblies of higher order were localized in cell-cycle-dependent manner at sites of dispersed γ-tubulin location in acentrosomal plant cells as well as at sites of local γ-tubulin enrichment after drug treatment. Our findings that γ-tubulin preserves the capability of prokaryotic tubulins to self-organize into filaments assembling by lateral interaction into bundles/clusters help understanding of the relationship between structure and multiple cellular functions of this protein species and suggest that besides microtubule nucleation and organization, γ-tubulin may also have scaffolding or sequestration functions.     

Oosome formation during in vitro oogenesis inBradysia tritici (syn.Sciara ocellaris)

Summary The development of follicles fromBradysia tritici (syn.Sciara ocellaris) during in vitro culture was studied. When follicles are isolated from 12-h-old females and placed in Robb's R-14 medium, their nurse cells regress with the same kinetics as in vivo and a histologically normal oosome forms at the posterior pole of the oocyte. Protein synthesis during in vitro development was studied by labelling follicles for 15 min and culturing them in vitro until the oosome had formed (28 h after eclosion of the donor). The time-course of protein labelling was defined by studying the incorporation kinetics of³H-amino acids into TCA-precipitable material; 50% of the radioactivity in the follicles was incorporated into TCA-precipitable material in less than 30 min. Autoradiographs of follicles labelled at different stages of oogenesis always showed a labelled oosome even if the labelling period was hours before oosome formation. These results indicate that the synthesis of oosome material starts long before the oosome forms at the end of vitellogenesis. Oosome formation can be inhibited by colchicine (20 g/ml) and is, therefore, likely to be dependent directly or indirectly on microtubule function.     

SB401, a pollen-specific protein from Solanum berthaultii, binds to and bundles microtubules and F-actin

We characterize a novel, pollen-specific, microtubule-associated protein, SB401, found in Solanum berthaultii. This protein binds to and bundles taxol-stabilized microtubules and enhances tubulin polymerization in a concentration-dependent manner, particularly at lower temperatures. Electron microscopy revealed that the protein decorates the entire length of microtubules. Cross-linking and electrophoresis studies showed that SB401 protein forms dimers, and suggest that dimerization could account for bundling. Double immunofluorescent staining of pollen tubes of S. berthaultii showed that SB401 protein co-localized with cortical microtubule bundles. SB401 protein also binds to and bundles actin filaments, and could connect actin filaments to microtubules. SB401 protein had a much higher affinity for microtubules than for actin filaments. In the presence of both cytoskeletal elements, the protein preferentially bound microtubules to form bundles. These results demonstrate that SB401 protein may have important roles in organizing the cytoskeleton in pollen tubes.     

Evidence for intermediate filaments in squirrelfish erythrophores of Holocentrus ascensionus (Rufus)

We have documented the presence of intermediate filaments (IF) in cultured erythrophores of the squirrelfish Holocentrus ascensionus (Rufus). SDS-PAGE and Western blots with monoclonal antibodies T11 and R12 demonstrated that isolated IF consisted of a pair of polypeptides of 54 and 52 kDa. Immunofluorescent studies revealed that the two proteins formed prominent radially oriented IF networks in erythrophores. Immunoelectron microscopic studies showed that the IF were distributed in a "spider-web"-like network of filaments which occasionally intersected with the microtubule surfaces. The IF proteins also were found in fish iridiphores but not in fish epithelial cells which cocultured with the chromatophores.     

Microtubules in pollen tube subprotoplasts: organization during protoplast formation and protoplast outgrowth

Summary Microtubules inNicotiana tabacum pollen tube subprotoplasts reassembled in wave-like to concentric cortical arrays. Crosslinks between microtubules were either 15 or 80 nm in length. Cortical actin filaments showed different distributions; no colocalization like that in pollen tubes was observed. Degradation of actin filaments by cytochalasin D had no influence on microtubule organization. Degradation of microtubules and/or actin filaments did not affect outgrowth of the subprotoplasts. Organization of the microtubules occurred independent of the presence of the generative cell and/or the vegetative nucleus. No relation of actin filament and microtubule organization with organelle distribution could be detected.Abbreviations AFs actin filaments - DAPI 4,6-diamidino-2-phenylindole - EGTA ethylene glycol bis (2-amino ethylether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MTs microtubules - SPPs subprotoplasts - TRITC tetramethyl rhodamine B isothiocyanate     

Effects of ATP and cyclic AMP on the in vitro assembly and stability of mammalian brain microtubules

The relevance of protein phosphorylation, transphosphorylation and binding phenomena in the kinetics of the ATP-induced assembly of cycle-purified microtubule protein from mammalian brain were studied. ATP was able to induce the polymerization of microtubules of normal appearance. However, the assembled structures, were unstable and microtubules depolymerized after achievement of a transitory maximum. Cyclic AMP reduced the amplitude of the polymerization maximum in a concentration-dependent manner, correlating with the stimulation of the endogenous phosphorylation reaction. When microtubule assembly was induced by GTP, in the presence of various concentrations of ATP, the slope of the depolymerization phase was found to depend on the concentration of ATP. Fluoride ion inhibited the endogenous phosphorylation reaction and reduced the disassembly rate, in a concentration-dependent manner. Evidence is also presented indicating that ATP did not bind to phosphocellulose-purified tubulin. These results further contribute to indicate that ATP and cyclic AMP, acting coordinately to control the phosphorylation extent of microtubule proteins are important factors to determine microtubule stability within the cell. Some implications of this mechanism for the regulation by cAMP of the initiation of DNA synthesis and mitosis are considered.Abbreviations MAPs microtubule-associated proteins - MAP2 microtubule-associated protein 2, Mes-4-morpholinoethanesulfonic acid - EGTA ethylene-glycol bis (-aminoethyl ether)N,N,N,N-tetraacetic acid - PMSF phenylmethylsulfonyl fluoride - PEI polyethyleneimine - PC phosphocellulose - DEAE Diethylaminoethyl - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate     

Extracellular coats on the surface of Strongylocentrotus purpuratus eggs: stereo electron microscopy of quick-frozen and deep-etched specimens

Summary Sea urchin (Strongylocentrotus purpuratus) eggs were fixed, quick-frozen, deep-etched, and rotary-replicated, and the three-dimensional structure of the external surface of the egg visualized using stereo electron microscopy. The cell surface is coated with three layers of filaments: the sheetlike vitelline layer adhering closely to the plasma membrane, a second layer of oblique fibrils extending from microvillar tips to the vitelline layer below, and a third, outermost layer of horizontal filaments coursing in bundles over the microvillar tips. After fertilization, the newly elevated vitelline envelope is transformed into a three-layered structure, the central layer being a tightly knit network of fine filaments decorated on each side with a loose network of thicker fibrils. Subsequently, the envelope becomes coated with paracrystalline protein released from the cortical granules, and microvillar casts are reshaped into angular, jagged peaks having two to five sides. The final structure of the fertilization envelope consists of a thick central layer of compact fibrillar material that is coated on each side with thin plates of paracrystalline protein.     

Microtubule assembly and oscillations induced by flash photolysis of caged-GTP

Microtubule assembly and oscillations have been induced using the rapid liberation of GTP by UV flash photolysis of caged-GTP and monitored by time-resolved X-ray scattering. The flash photolysis method of achieving assembly conditions is much faster than the temperature jump method used earlier (msec vs. s range). However, the structural transitions and their rates are similar to those described previously. This means that the rates of the transitions in microtubule assembly observed before are determined by the protein itself, and not by the rate at which assembly conditions are induced. The advantages and limitations of using the photolysis of caged-GTP in microtubule assembly studies are compared with temperature jump methods. Caged-GTP itself reduces the rate of microtubule assembly and oscillations at mM concentrations, consistent with a weak interaction between the nucleotide analogue and the protein. X-rays are capable of slowly liberating GTP and other breakdown products from caged-GTP, even in the absence of UV flash photolysis, thus causing an apparent X-ray-induced microtubule assembly. This effect depends on the X-ray dose but is independent of the caged-GTP concentrations used here (mM range), suggesting that the breakdown of caged-GTP is caused not by the direct absorption of X-rays by the compound but by another intermediate reaction such as the generation of radicals by the X-rays.Abbreviations DTT dithiothreitol - EGTA ethylene glycol-O,O-bis (2-amino ethyl ether)-N,N,N,N-tetraacetic acid - GDP guanosine-5-diphosphate - GTP guanosine-5-triphosphate - caged-GTP P³-1-(2-nitrophenyl) ethyl ester of GTP - HPLC high performance liquid chromatography - Mt-protein microtubule protein (=tubulin +MAPs) - MAP(s) microtubule-associated protein(s) - PC-tubulin phosphocellulose-purified tubulin - PIPES piperazine-1,4-bis(2-ethane sulfonic acid) - UV ultraviolet light Offprint requests to: E. Mandelkow     

Dynamics of the cytoskeleton of epidermal cells in situ and in culture

Summary The cytoskeleton of primary tissue-culture cells from the epidermis of Xenopus laevis tadpoles was investigated by phase-contrast, immunofluorescence, and electron microscopy. The connection between the arrangement of different types of filaments and the mechanical properties of the epidermis is discussed. The bilayered epidermis attains stability from thick bundles of tonofilaments interconnecting the basal desmosomes. Twisting of tonofilaments around each other can explain the occurrence of elastic filamentous curls forming a meshwork braced between rows of small desmosomes in the apical region of the epidermis. Actin is arranged as a diffuse meshwork and sometimes forms bundles intermingling with tonofilament bundles. Surface membranes and rows of small desmosomes are delineated by actin and contain -actinin. Actin raises the tension for rounding and spreading of cells. Microtubules stabilize already well-developed lamellae.     

Interrelation between the spatial disposition of actin filaments and microtubules during the differentiation of tracheary elements in culturedZinnia cells

Summary Changes in the spatial relationship between actin filaments and microtubules during the differentiation of tracheary elements (TEs) was investigated by a double staining technique in isolatedZinnia mesophyll cells. Before thickening of the secondary wall began to occur, the actin filaments and microtubules were oriented parallel to the long axis of the cell. Reticulate bundles of microtubules and aggregates of actin filaments emerged beneath the plasma membrane almost simultaneously, immediately before the start of the deposition of the secondary wall. The aggregates of actin filaments were observed exclusively between the microtubule bundles. Subsequently, the aggregates of actin filaments extended preferentially in the direction transverse to the long axis of the cell, and the arrays of bundles of microtubules which were still present between the aggregates of actin filaments became transversely aligned. The deposition of the secondary walls then took place along the transversely aligned bundles of microtubules.Disruption of actin filaments by cytochalasin B produced TEs with longitudinal bands of secondary wall, along which bundles of microtubules were seen, while TEs produced in the absence of cytochalasin B had transverse bands of secondary wall. These results indicate that actin filaments play an important role in the change in the orientation of arrays of microtubules from longitudinal to transverse. Disruption of microtubules by colchicine resulted in dispersal of the regularly arranged aggregates of actin filaments, but did not inhibit the formation of the aggregates itself, suggesting that microtubules are involved in maintaining the arrangement of actin filaments but are not involved in inducing the formation of the regularly arranged aggregates of actin filaments.These findings demonstrate that actin filaments cooperate with microtubules in controlling the site of deposition of the secondary wall in developing TEs.Abbreviations DMSO dimethylsulfoxide - EGTA ethyleneglycolbis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MSB microtubule-stabilizing buffer - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - TE tracheary element     

Localization of a kinesin-like protein in generative cells of tobacco

Summary We have obtained immunofluorescence and immunoblot evidence for the presence of kinesin-like protein (KLP) in pollen tubes of tobacco using an antibody generated against peptides encoded by theKATA gene ofArabidopsis. This antibody recognizes an M_r 140,000 polypeptide inArabidopsis seedlings, and stains the mitotic apparatus in this species as well as in tobacco suspension cells. In tobacco pollen tubes prepared for dual immunofluorescence localizations of KLP and -tubulin, the antibody binds transiently to microtubule (Mt) bundles and the nucleus in premitotic generative cells; it then stains the developing mitotic apparatus as the nuclear envelope breaks down. By metaphase, fluorescence is located over kinetochore fibers and associated Mts. Localization of KLP is concentrated in the midzone during anaphase, and by early cytokinesis, it closely brackets the cell plate. Phragmoplast fluorescence then spreads along the phragmoplast distal to the cell plate. Punctate staining is also detected along vegetative Mts. No KLP localization is seen in pollen tubes treated with antibody after it had been preadsorbed to the antigenic peptides. The antibody recognizes an M_r 110,000 polypeptide in extracts of tobacco pollen tubes, and a polypeptide of somewhat lower M_r inTradescantia pollen tubes. Our results show that KLP(s) related to KatAp are present in tobacco generative cells and may play roles in the organization and/or operation of the mitotic apparatus and phragmoplast.Abbreviations KLP kinesin-like protein - Mt microtubule - MA mitotic apparatus Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology