Binding of axonemal dynein to microtubules comprising the cytoplasmic transport system in insect ovarioles

Dynein isolated from ciliary axonemes of Tetrahymena is shown to bind in a characteristic fashion as arms to microtubules dissected from the nutritive tubes of insect ovarioles. The microtubules in nutritive tubes are associated with the transport of cytoplasmic components along their length, and the significance of their ability to bind axonemal dynein, to the possibility that microtubule/dynein interactions are involved in microtubule-associated movements, generally, is discussed.     

Alloaffinity filtration: a general approach to the purification of dynein and dynein-like molecules

Alloaffinity filtration simply and specifically separates certain axonemal dyneins and dynein arm components from crude mixtures on the basis of their ability to bind and decorate Tetrahymena axonemal microtubules on a filter in the absence of ATP and to detach and pass into the eluate when 0.5 mM ATP is added. The procedure, which may be performed repetitively, is successful in purifying a Tetrahymena dynein that has characteristics of 30 S dynein prepared by conventional methods, while other dyneins originally present in the mixture, e.g., 14 S Tetrahymena dynein, are not found in the ATP eluate. A relatively homogeneous population of dynein oligomers is obtained. Alloaffinity-purified 30 S Tetrahymena dynein consists of heavy-, intermediate-, and light-chain polypeptides that cosediment in a sucrose gradient in fixed molar ratios and that have structural features of in situ Tetrahymena arms. Dyneins from other species will bind to Tetrahymena microtubules and can be purified by this method. Alloaffinity-purified Chlamydomonas dynein is a set of polypeptides including the four heavy chains that characterize the outer arm.     

Studies on calmodulin-binding proteins (CaMBPs) in the cilia of Tetrahymena

As a first step to elucidate the involvement of calmodulin in Ca2+-dependent regulation of ciliary motility, molecular species and properties of calmodulin-binding proteins (CaMBPs) in Tetrahymena cilia were investigated by a modified [125I]calmodulin overlay method. At least 36 kinds of CaMBPs were detected. All the CaMBPs bound to calmodulin in Ca2+-dependent and calmodulin-specific manners, but they showed different Ca2+-dependencies. Several of CaMBPs bound to calmodulin in the presence of 100 microM trifluoperazine, several did in the presence of 8 M urea, and a few of them were highly sensitive to trypsin digestion. Among these CaMBPs, we noticed a 95 000-dalton (D) CaMBP present in the outerdoublet microtubule fraction, which possessed some attributes of the calmodulin counterpart suggested from the results of our previous paper [12]. We discussed a possibility that this protein might correspond to one of the protein components of the interdoublet link.     

Cloning,localization, and axonemal function of Tetrahymena centrin

Centrin, an EF hand Ca(2+) binding protein, has been cloned in Tetrahymena thermophila. It is a 167 amino acid protein of 19.4 kDa with a unique N-terminal region, coded by a single gene containing an 85-base pair intron. It has > 80% homology to other centrins and high homology to Tetrahymena EF hand proteins calmodulin, TCBP23, and TCBP25. Specific cellular localizations of the closely related Tetrahymena EF hand proteins are different from centrin. Centrin is localized to basal bodies, cortical fibers in oral apparatus and ciliary rootlets, the apical filament ring and to inner arm (14S) dynein (IAD) along the ciliary axoneme. The function of centrin in Ca(2+) control of IAD activity was explored using in vitro microtubule (MT) motility assays. Ca(2+) or the Ca(2+)-mimicking peptide CALP1, which binds EF hand proteins in the absence of Ca(2+), increased MT sliding velocity. Antibodies to centrin abrogated this increase. This is the first demonstration of a specific centrin function associated with axonemal dynein. It suggests that centrin is a key regulatory protein for Tetrahymena axonemal Ca(2+) responses, including ciliary reversal or chemotaxis.     

"Buttonin," a unique button-shaped microtubule-associated protein (75 kD) that decorates spindle microtubule surface hexagonally

A 75-kD protein was purified from sea urchin egg microtubule proteins through gel filtration. It enhanced the polymerization of porcine brain tubulin, but was not heat-stable and did not bind to calmodulin in the presence of calcium as demonstrated by calmodulin affinity column chromatography. Rotary shadowing of the freeze-etched 75-kD protein adsorbed on mica revealed the protein to be a spherical molecule (approximately 9 nm in diameter). Quick-freeze deep-etch electron microscopy revealed that the surface of microtubules polymerized with 75-kD protein was entirely covered with hexagonally packed, round, button-like structures that were quite uniform in shape and size (approximately 9 nm) and similar to the buttons observed on microtubules of mitotic spindles in vivo or microtubules isolated from mitotic spindles. Judging from calibration studies of molecular mass by gel filtration, the 75-kD protein probably exists in a dimeric form (approximately 150 kD) in its native condition. The stoichiometry of tubulin (dimer) versus 75-kD protein (dimer) in the polymerized pellet was 3-3.4:1. Hence, we concluded that the 75-kD protein was a unique microtubule-associated protein that formed the microtubule button in vivo and in vitro. We propose to name this protein "buttonin".     

The functional expression and motile properties of recombinant outer arm dynein from Tetrahymena

Cilia and flagella are motile organelles that play various roles in eukaryotic cells. Ciliary movement is driven by axonemal dyneins (outer arm and inner arm dyneins) that bind to peripheral microtubule doublets. Elucidating the molecular mechanism of ciliary movement requires the genetic engineering of axonemal dyneins; however, no expression system for axonemal dyneins has been previously established. This study is the first to purify recombinant axonemal dynein with motile activity. In the ciliated protozoan Tetrahymena, recombinant outer arm dynein purified from ciliary extract was able to slide microtubules in a gliding assay. Furthermore, the recombinant dynein moved processively along microtubules in a single-molecule motility assay. This expression system will be useful for investigating the unique properties of diverse axonemal dyneins and will enable future molecular studies on ciliary movement.     

Affinity-purified melittin antibody recognizes the calmodulin-binding domain on calmodulin target proteins

Melittin is a 26-amino acid amphipathic peptide which binds to calmodulin in a calcium-dependent manner. The utility of melittin as a peptide replica of the calmodulin-binding region of calmodulin acceptor proteins (CaMBPs) was investigated. Antibody against melittin was raised and purified by antigen affinity chromatography. Interaction of the antibody with CaMBPs was initially suggested by the ability of anti-melittin-Sepharose, but not nonimmune IgG-Sepharose, to bind calmodulin-dependent cyclic AMP phosphodiesterase. Direct interaction of melittin antibody with the calmodulin-binding domain of acceptor proteins was demonstrated by quantitative inhibition of calmodulin binding to the purified CaMBPs, myosin light chain kinase, and eel electric organ CaMBP55. These results indicate that melittin antibody identifies regions of structural similarity between calmodulin acceptor proteins, and this region includes a common calmodulin-binding domain.     

Structural and Functional Characterization of Paramecium Dynein: Initial Studies

ABSTRACT Dynein arms and isolated dynein from Paramecium tetraurelia ciliary axonemes are comparable in structure, direction of force generation, and microtubule translocation ability to other dyneins. In situ arms have dimensions and substructure similar to those of Tetrahymena. Based on spoke arrangement in intact axonemes, arms translocate axonemal microtubules in sliding such that active dynein arms are (-) end directed motors and the doublet to which the body and cape of the arms binds (N) translocates the adjacent doublet (N+1) upward. After salt extraction, based on ATPase activity, paramecium dynein is found as a 22S and a 14S species. the 22S dynein is a three-headed molecule that has unfolded from the in situ dimensions; the 14S dynein is single headed. Both dyneins can be photocleaved by UV light (350 nm) in the presence of Mg^2-, ATP and vanadate; the photocleavage pattern of 22S dynein differs from that seen with Tetrahymena. Both isolated dyneins translocate taxol-stabilized, bovine brain microtubules in vitro. Under standard conditions, 22S dynein, like comparable dyneins from other organisms, translocates at velocities that are about three times faster than 14S dynein.     

Structural and functional characterization of paramecium dynein: initial studies.

Dynein arms and isolated dynein from Paramecium tetraurelia ciliary axonemes are comparable in structure, direction of force generation, and microtubule translocation ability to other dyneins. In situ arms have dimensions and substructure similar to those of Tetrahymena. Based on spoke arrangement in intact axonemes, arms translocate axonemal microtubules in sliding such that active dynein arms are (-) end directed motors and the doublet to which the body and cape of the arms binds (N) translocates the adjacent doublet (N + 1) tipward. After salt extraction, based on ATPase activity, paramecium dynein is found as a 22S and a 14S species. The 22S dynein is a three-headed molecule that has unfolded from the in situ dimensions; the 14S dynein is single headed. Both dyneins can be photocleaved by UV light (350 nm) in the presence of Mg2+, ATP and vanadate; the photocleavage pattern of 22S dynein differs from that seen with Tetrahymena. Both isolated dyneins translocate taxol-stabilized, bovine brain microtubules in vitro. Under standard conditions, 22S dynein, like comparable dyneins from other organisms, translocates at velocities that are about three times faster than 14S dynein.     

Characterization and in vitro polymerization of Tetrahymena tubulin

Tetrahymena tubulin was purified from the cell extract using DEAE-Sephadex A-50 ion-exchanger and ammonium sulfate precipitation. About 2.2% of the total protein in the 20,000 X g supernatant was recovered as DEAE-Sephadex-purified tubulin fraction. Applying the temperature-dependent polymerization-depolymerization method to this fraction in the presence of Tetrahymena outer fibers as a seed, almost pure tubulin was obtained. Tetrahymena tubulin dimer showed different behavior on SDS-polyacrylamide gels from porcine brain tubulin, and showed very low affinity for colchicine, amounting to about one-twentieth of the binding to porcine brain tubulin. The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragment induced polymerization as demonstrated by viscometric measurements, but the reconstituted microtubules were very unstable in the absence of glycerol. Microtubule-depolymerizing agents such as Ca2+ ions, low temperature, or colchicine all inhibited in vitro polymerization. Although Tetrahymena tubulin purified by the polymerization-depolymerization method could copolymerize with porcine brain microtubules, the DEAE-Sephadex-purified tubulin fraction suppressed the initial rate of porcine brain microtubule assembly in vitro. There seemed to be no differences between cytoplasmic tubulin and outer fiber tubulin in colchicine binding activity or SDS-gel electrophoretic behavior, or between the fine structure of both reconstituted microtubules observed by electron microscopy.     

Calmodulin immunolocalization in outer segments of Xenopus laevis photoreceptors

Because adaptation of vertebrate photoreceptors to light is mediated by changes in the level of calcium in their outer segments (OS), proteins that bind calcium are important in phototransduction. This study has used immunofluorescence to investigate the distribution of the calcium-binding protein calmodulin within photoreceptor OS dissociated from amphibian ( Xenopus laevis) retinas. The OS of rods and cones had a streak of fluorescence to calmodulin at the ciliary axoneme. The OS of rods (but not cones) also displayed regularly spaced puncta of anti-calmodulin fluorescence along longitudinal lines coinciding with their multiple incisures. This location of calmodulin immunofluorescence closely matches the known location of microtubules within the OS of amphibian rods and cones. These findings provide evidence that calmodulin is closely associated with the microtubules of both the axonemal and incisural cytoskeletal systems in OS, and suggest that this association is important for calmodulin function in photoreceptors.     

Identification of elongation factor-1alpha as a Ca2+/calmodulin-binding protein in Tetrahymena cilia

Calmodulin (CaM) is known to be a ciliary component. However, the function of CaM in cilia or flagella has not been well understood. Immunoelectron microscopy using anti-CaM antibody showed that CaM was localized on the axonemal microtubules (MTs) and matrix of Tetrahymena cilia. To investigate the signal transduction of Ca(2+)/CaM in cilia, we performed Ca(2+)/CaM-affinity column chromatography in the membrane and matrix fraction. Elongation factor-1alpha (EF-1alpha) was identified as a Ca(2+)/CaM-binding protein in cilia. EF-1alpha is a highly conserved protein and functions in protein translation. In addition, EF-1alpha has been reported to interact with MTs and F-actin in several organisms. Immunoelectron microscopy showed that EF-1alpha was localized on the axonemal MTs. However, in immunoblot analysis, EF-1alpha was mainly extracted in the membrane and matrix fraction from the axonemal MTs by 1% Triton X-100 extraction. These results suggest that interaction between EF-1alpha and axonemal MTs is weak and sensitive to treatment with 1% Triton X-100 and that EF-1alpha mediates between axonemal MTs and CaM in the presence of Ca(2+). Moreover, EF-1alpha was also localized in cilia of Paramecium, suggesting that EF-1alpha functions as a target protein of Ca(2+)/CaM in ciliate cilia.     

Limited chymotryptic digestion of bovine adrenal 190,000-Mr microtubule-associated protein and preparation of a 27,000-Mr fragment which stimulates microtubule assembly

A heat stable microtubule-associated protein of Mr 190,000 (190-kDa MAP) has been purified from bovine adrenal cortex (Murofushi, H., Kotani, S., Aizawa, H., Hisanaga, S., Hirokawa, N., and Sakai, H. (1986) J. Cell Biol. 103, 1911-1919). Limited chymotryptic digestion of 190-kDa MAP produced a fragment of Mr 27,000 (27-kDa fragment), which bound to microtubules reconstituted in the presence of taxol. This fragment was purified with the aid of cosedimentation with microtubules. The purified 27-kDa fragment showed an ability to stimulate tubulin polymerization in the absence of taxol. Electron microscopic observation of microtubules reconstituted from purified 27-kDa fragment and tubulin revealed that the microtubules were in the form of thick bundles and that lateral projections which can be seen in microtubules reconstituted from intact 190-kDa MAP and tubulin were not observed. These results indicate that 27-kDa fragment includes or is a part of microtubule-binding domain of 190-kDa MAP and that this fragment is active in stimulating microtubule assembly. Amino acid analysis revealed that the 27-kDa fragment was rich in lysine, proline, and alanine, the sum of these three being about 45% of the total amino acids and that the contents of methionine, tyrosine, phenylalanine, and histidine were very low. These data suggest that the microtubule binding domain of the 190-kDa MAP comprises an unique structure.     

Activation of the dynein adenosinetriphosphatase by microtubules

Previous work has indicated that following the rapid adenosine 5'-triphosphate (ATP) induced dissociation of the microtubule-dynein complex, the rate-limiting step in the ATPase cycle is product release [Johnson, K. A. (1983) J. Biol. Chem. 258, 13825-13832], which occurs at a rate of approximately 2-6 s-1. In this report we complete the analysis of the ATPase cycle by examining the effect of microtubules on the rate of product release. For these studies we used repolymerized Tetrahymena axonemal microtubules and microtubule-associated protein (MAP) free bovine brain microtubules which were shown to be free of any measureable ATPase activity. Tetrahymena 22S dynein bound to these microtubules predominantly by the ATP-sensitive site and at a rate giving an apparent second-order rate constant of (0.2-1) X 10(6) M-1 s-1, which is 50-fold greater than the rate observed with brain microtubules containing MAPs. ATP induced the rapid dissociation of the microtubule-dynein complex with an apparent second-order rate constant vs. ATP concentration equal to 1.6 X 10(6) M-1 s-1; this value is only slightly lower than that observed in the presence of MAPs. After the ATP-induced dissociation, the dynein reassociated with the microtubules following a lag period due to the time required to hydrolyze the ATP. The duration of the lag time for reassociation decreased with increasing microtubule concentration, suggesting that microtubules increased the rate of ATP turnover. Direct measurements at steady state showed that the specific activity of the dynein increased with increasing microtubule concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microtubule-membrane interactions in cilia. II. Photochemical cross-linking of bridge structures and the identification of a membrane-associated dynein-like ATPase

Photochemical cross-linking of both Tetrahymena and Aequipecten ciliary membrane proteins with the lipophilic reagent 4,4'-dithiobisphenylazide links together a high molecular weight dynein-like ATPase, membrane tubulin, and at least two other proteins. Electron microscopy of detergent-extracted cilia reveals that the cross-linked complex remains attached to the outer-doublet microtubules by a microtubule-membrane bridge. Cleavage of the reagent's disulfide bond releases the bridge- membrane complex and the dynein-like membrane-associated ATPase. Electron microscopy was used to ensure that the dynein-like protein did not result from the solubilization of the dynein arms attached to the outer-doublet microtubules. The dynein-like protein has been isolated using sucrose gradients and is similar to axonemal dynein with respect to its sedimentation characteristics nucleotide specificity, and divalent cation requirements. Photochemical cross-linking of ciliary membrane porteins in vivo results initially in the modification of ciliary beat and, eventually, in the cessation of ciliary movement. These results suggest that a dynein-like ATPase comprises the bridge which links the ciliary membrane to the outer-doublet microtubules and that this bridge is involved in the modulation of normal ciliary movement.     

Immune localization of calmodulin in the ciliated cells of hamster tracheal epithelium

Melachronous beating of cilia of epithelial surfaces of most respiratory airways moves the overlying mucous layer in a caudal direction. The molecular mechanisms controlling ciliary beat remain largely unknown. Calcium, an element in its cationic form, is ubiquitous in biological functions and its concentration is critical for ciliary beating. Calmodulin, a calcium-binding protein which regulates the activity of many enzymes and cellular processes, may regulate ciliary beating by controlling enzymes responsible for mechanochemical movement between adjacent peripheral microtubule doublets composing the ciliary axoneme. As a first step in describing a calmodulin-related controlling mechanism for ciliary beating, calmodulin was localized in the ciliated cells lining the respiratory tracts of hamsters by electron microscopy, using an indirect immunoperoxidase technique with anticalmodulin antibodies as the molecular probe. Thin-sections revealed calmodulin located on microtubules and dynein arms of the ciliary shaft, basal body, apical cytoskeletal microtubules, and plasma membranes in specimens fixed with 1 mM Ca+2. Specimens fixed with less Ca+2 (1 microM), Mn+2, Mg+2, and EGTA showed a diffuse pattern of calmodulin with loci of greatest densities on basal body microtubule triplets. Demembranated specimens showed a less specific localization on axonemal microtubules but only on cells fixed with Ca+2. Calmodulin, by binding calcium, may function in ciliary beating in the respiratory tract of mammals either directly or indirectly through its effects on the energy-producing enzymes and by control of Ca+2 flux through plasma membranes.     

Vinblastine-induced aggregation of brine shrimp (Artemia) tubulin

Tubulin from the brine shrimp Artemia readily assembles in vitro in the absence of microtubule-associated proteins under conditions which do not permit assembly of tubulin from brain. Heated microtubule-associated protein preparations from bovine brain do, however, interact with Artemia tubulin, resulting in stimulation of tubulin assembly and formation of morphologically normal cold-sensitive microtubules. Addition of vinblastine to mixtures containing microtubules assembled in the presence of neural microtubule-associated proteins caused a drop and then a rise in turbidity of the solution. The turbidity changes were accompanied by the appearance of coils, presumably derived from the microtubules which disappeared upon addition of vinblastine. Coils also resulted when microtubule-associated proteins and vinblastine were added to tubulin before polymerization was initiated. Vinblastine prevented normal assembly and caused disruption of Artemia microtubules polymerized in the absence of microtubule-associated proteins. Under these conditions clumped or compact coils, different in appearance from those formed in the presence of the microtubule-associated proteins, were observed. The data confirm that tubulin from Artemia, an organism that is phylogenetically far removed from mammals, has retained binding sites for vinblastine and microtubule-associated proteins and that the interrelationship of these sites has been at least partially preserved. The incomplete depolymerization of Artemia microtubules in response to vinblastine when microtubule-associated proteins are absent suggests that the longitudinal tubulin-tubulin interactions involved in microtubule formation are more stable for Artemia than for neural tubulin.     

Acetylation of lysine 40 in alpha-tubulin is not essential in Tetrahymena thermophila

In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of alpha- and beta- tubulin heterodimer, precluding distinctions among microtubular systems based on tubulin primary sequence isotypes. Tetrahymena tubulins also are modified by several types of posttranslational reactions including acetylation of alpha-tubulin at lysine 40, a modification found in most eukaryotes. In Tetrahymena, axonemal alpha-tubulin and numerous other microtubules are acetylated. We completely replaced the single type of alpha-tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position. No acetylated tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40. Surprisingly, mutants lacking detectable acetylated tubulin are indistinguishable from wild-type cells. Thus, acetylation of alpha- tubulin at lysine 40 is non-essential in Tetrahymena. In addition, isoelectric focusing gel analysis of axonemal tubulin from cells unable to acetylate alpha-tubulin leads us to conclude that: (a) most or all ciliary alpha-tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated alpha-tubulin molecules in wild-type cells contain one or more additional charge-altering modifications.     

The third calmodulin family protein in Tetrahymena. Cloning of the cDNA for Tetrahymena calcium-binding protein of 23 kDa (TCBP-23)

Recently, we proved the existence of the second calmodulin family protein in Tetrahymena (Tetrahymena calcium-binding protein of 25 kDa, TCBP-25) by analyzing its cDNA (Takemasa, T., Ohnishi, K., Kobayashi, T., Takagi, T., Konishi, K., and Watanabe, Y. (1989) J. Biol. Chem. 264, 19293-19301). During the amino acid sequence determination of TCBP-25, we became aware of the fact that another polypeptide carrying calcium-binding domains of EF-hand type existed in addition to Tetrahymena calmodulin and TCBP-25. This third calmodulin family protein from Tetrahymena was confirmed by isolating its cDNA clones. One of the cloned cDNAs contains 763 nucleotides and encodes a protein that is composed of 207 amino acid residues and has a molecular mass of 23,413 daltons. This predicted protein possesses four EF-hand type calcium-binding domains, so we have designated it as Tetrahymena calcium-binding protein of 23 kDa (TCBP-23). TCBP-23 is similar (35% homology) but clearly different from TCBP-25. The TCBP-23 gene is actively transcribed in vivo as a 0.84-kilobase RNA. Thus, it follows that Tetrahymena cells have three different calmodulin family proteins: calmodulin, TCBP-25 and TCBP-23. These proteins are expected to provide important clues for solving the mechanisms of calcium-dependent phenomena, such as ciliary reversal.     

Isolation and characterization of a unique 15 kilodalton trypanosome subpellicular microtubule-associated protein.

A protein of 15 kDa (p15) was isolated from Trypanosoma brucei subpellicular microtubules by tubulin affinity chromatography. The protein bound tubulin specifically both in its native form and after SDS-PAGE in tubulin overlay experiments. p15 promoted both the in vitro polymerization of purified calf brain tubulin and the bundling of preformed mammalian microtubules. Immunolabeling identified p15 at multiple sites along microtubule polymers comprising calf brain tubulin and p15 as well as on the subpellicular microtubules of cryosectioned trypanosomes. Antibodies directed against p15 did not cross react with mammalian microtubules. It is suggested that p15 is a trypanosome-specific microtubule-associated protein (MAP) that contributes to the unique organization of the subpellicular microtubules.