Structure and mass analysis of 14S dynein obtained from Tetrahymena cilia.

Scanning transmission electron microscopic analysis revealed that the 14S fraction of Tetrahymena dynein was of a mixture of two types of particles in approximately equal proportions. The 14S dynein molecules were roughly ellipsoid in shape with approximate axes of 9.5 and 14.5 nm. About half of the particles had tails 20-24-nm long. By the integration of electron scattering intensities, particles with tails had an average mass of 510 kD with a SD of 90 kD. The globular heads of both types of particles had an average mass of 330 kD with a SD of 60 kD. The mass of the tail structure was about 180 kD. By SDS-PAGE, the 14S dynein consisted of two high molecular mass polypeptides above 300 kD that could be distinguished by immunoblot analysis.

Aggregation and thermo-inactivation of glutamine synthetase from an extreme thermophile, Bacillus caldolyticus.

The extreme thermophile, Bacillus caldolyticus, contains two regulatory isoforms of glutamine synthetase (glutamate-ammonia ligase, EC 6.3.1.2), E-I and E-II, produced as separate gene products. Light scattering and electron microscopy data indicate that these thermophilic enzymes aggregate to higher molecular weight species in two stages: initial polymerization of native dodecamers, followed by 'melting' of the aggregated species to produce amorphous denatured protein. The initial stages of the aggregation occurred at temperatures below those for time-dependent denaturation, especially for E-II. In contrast, mesophilic (B. subtilis) enzyme showed no evidence of temperature-dependent aggregation. Thus, aggregation may be a stabilizing mechanism for the thermophilic systems. Bound metal ions and substrates caused dramatic increases in the temperatures at which aggregation and loss of activity occurred for thermophilic enzymes. Certain combinations of ligands (e.g., MnATP + L-glutamate) acted synergistically, so that these complexes denatured only above 90 degrees C. Various models were considered for heat-driven aggregation followed by denaturation, plus ligand stabilization. Taken together, the data are most consistent with unfolding of subunits within the dodecameric unit, rather than unfolding to monomers prior to aggregation.

Ethanol, an efficient coolant for rapid freezing of biological material.

Supercooled ethanol was used for freeze-quenching protozoan samples and gave reasonable ultrastructural preservation. The use of ethanol as a coolant has considerable advantages in terms of its relative safety, high cooling efficiency and low cost when compared with other coolants currently used for freeze-quenching.

Activation of the dynein adenosinetriphosphatase by cross-linking to microtubules.

The microtubule-dynein complex consisting of 22S dynein from Tetrahymena cilia and MAP-free microtubules was subjected to treatment with various concentrations of 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide (EDC), a zero-length cross-linker, at 28 degrees C for 1 h. Following cross-linking of the microtubule-dynein complex, nearly all of the ATPase activity cosedimented with the microtubules in the presence of ATP. Electron microscopic observation by negative staining revealed that, following treatment with 1 mM EDC, the complex did not dissociate in the presence of ATP, although the dynein decoration pattern was disordered. The complex treated with 3 mM EDC exhibited normal microtubule-dynein patterns even after the addition of ATP. The ATPase activity of the microtubule-dynein complex was enhanced about 30-fold by the treatment with 1-3 mM EDC. These results indicate that the ATPase activation was caused by the close proximity of the dynein ATPase sites to the microtubules and provide further support for the functional interaction of all three dynein heads with the microtubule. The maximal specific activity was 12 mumol min-1 (mg of dynein)-1, corresponding to a turnover rate of 150 s-1, which may be the rate-limiting step at infinite microtubule concentration and may represent the maximum rate of force production in the axoneme.

Immunological comparison of 22S, 19S, and 12S dyneins from Paramecium cilia.

Three forms of dynein (22S, 19S, and 12S) were purified from Paramecium cilia. Two classes of monoclonal antibodies against purified 22S dynein were generated. One class reacted on immunoblots with the heavy chains of 22S, 19S, and 12S dyneins; the second class reacted with an 88 kD intermediate chain of 22S dynein. Polyclonal antiserum to the heavy chains of 22S dynein reacted with the alpha-heavy chain of 22S and 19S dyneins. A previously described antiserum raised against 22S dynein [Travis et al.: Biochim. Biophys. Acta 966:73-83, 1988] recognized the gamma-heavy chain of 22S dynein which was also present in 19S and 12S dyneins, along with the 88 and 76 kD intermediate chains of 22S dynein. This antiserum was also able to immunoprecipitate dynein from crude extracts of cilia. Electron microscopy revealed that the 22S dynein consisted mainly of two-headed particles with some three-headed particles present. The 12S dynein was mainly one-headed particles. The 19S dynein was a mixture of three-, two-, and one-headed particles. The immunological and electron microscopic studies showed that 19S dynein arises from 22S dynein, and that 12S dynein is heterogeneous, composed of the gamma-heavy chain of 22S dynein and a unique dynein ATPase. The polyclonal antibodies were also used to detect cross-reactive proteins in other organisms. Both the anti-heavy chain and the anti-22S dynein sera reacted strongly with 22S outer arm dynein of Tetrahymena, but not with the 14S dynein of this organism.

Inner and outer arm axonemal dyneins from the Antarctic rockcod Notothenia coriiceps.

Adaptive compensation of enzymatic activities is common among cold-living poikilotherms. Their enzymes often demonstrate higher activities at low temperatures than do homologs from temperate or thermophilic species. To understand the molecular features necessary for cold adaptation of microtubule motor proteins, we have initiated studies of the flagellar dynein ATPases of Antarctic fishes (body temperature range = -1.8 to +2 degrees C). Dyneins were isolated by high-salt extraction of demembranated sperm axonemes from the Antarctic yellowbelly rockcod, Notothenia coriiceps. Although solubilization of inner arms was incomplete, an inner arm dynein was recognized as a discrete complex containing one major dynein heavy chain (DHC) and sedimenting through sucrose gradients at approximately 12 S. Like inner arm dyneins from Chlamydomonas, the fish complex contained an actin-immunoreactive protein of 43 kDa and a 30-kDa protein. One isoform of the inner arm DHC gene family of N. coriiceps was detected by the polymerase chain reaction, and Southern analysis established that this DHC gene is present at one copy per haploid genome. Outer arm dynein was extracted quantitatively by high-salt treatment, contained two DHCs (one major, one minor), and sedimented through sucrose gradients as a polydisperse, aggregating system. Associated with the outer arm DHCs were five presumptive intermediate chains (ICs) of 66-91 kDa, immunologically defined by their cross-reactivity to four monoclonal antibodies specific for ICs from other organisms. The basal (non-microtubule-stimulated) specific ATPase activities of the N. coriiceps inner and outer arm dyneins were approximately 0.07 and approximately 0.04 micromol of P(i) min(-1) mg(-1), respectively, at 0 degrees C, attained their maxima (approximately 0.1 micromol of P(i) min(-1) mg(-1)) at 9 and 19 degrees C, respectively, and at higher temperatures declined substantially. Furthermore, the activities of the fish dyneins at temperatures < or = 15 degrees C were significantly larger than that of outer arm dynein from the mesophile Tetrahymena. These results suggest that the greater catalytic efficiencies of N. coriiceps inner and outer arm dyneins at low temperatures are due to enhanced polypeptide flexibility in the active sites of their protein subunits. We conclude that temperature adaptation of flagellar dyneins from Antarctic fishes is compatible with substantial conservation of primary and quaternary structure.

Membrane deformation of living glial cells using atomic force microscopy.

Using atomic force microscopy (AFM) it has been possible to detect actin filaments that are beneath the cell membrane of living cells despite the fact that the AFM tip is applied to the surface of the cell. To determine whether the AFM tip actually penetrates or deforms the cell membrane we determined whether an intracellularly trapped fluorescent indicator was lost from cells during AFM. Using epifluorescence illumination to monitor the presence of fluo-3 in the cell, we found that AFM did not cause dye leakage from the cell. Further, force-distance curves indicated that standard tips did not penetrate the membrane while sharper Supertips TM did. In addition, the physiology of cells was found to be unaffected by AFM with standard tips since volume regulatory signal transduction mechanisms were intact in such studies. Thus, traditional AFM tips deform the cell membrane in order to reveal the presence of subcellular structures.

Structure and mass of mammalian respiratory ciliary outer arm 19S dynein.

Mammalian respiratory ciliary outer arm dyneins isolated as the major ATPase peak migrating at 19S on sucrose density gradients were examined by transmission electron microscopy of negatively stained samples and scanning transmission electron microscopy of unstained samples. The predominant discrete particle structure observed was composed of two globular heads apparently connected by amorphous or indistinct material. The heads were either circular or slightly elliptical of mean 13 +/- 1 X 10 +/- 2 nm dimensions. The mass of this structure averaged 1.22 +/- 0.34 million daltons with the individual globular heads averaging 310 +/- 77 kilodaltons (kD). Negative staining revealed that one or both of the globular heads often contained a central accumulation of stain measuring 2.5 +/- 1 nm across. A second type of structure, appearing with lesser frequency in the 19S fraction than in the unfractionated dynein preparation loaded onto the sucrose gradient, was a single globular head of 13 +/- 1 X 10 +/- 2 nm often with 2 +/- 1 nm centrally accumulated stain and with or without an appendage. This one-headed particle thus resembled one-half of the two-headed particle. Mass measurements were lower, however, for isolated, single globular heads, averaging 220 +/- 111 kD. A third type of particle observed was a ring-like structure with 4 +/- 1 nm centrally accumulated stain and without appendages. The ring structure was slightly larger in diameter, 14 +/- 1 nm, and had a greater peripheral accumulation of negative stain than either of the one- or two-headed particles, suggesting that it was not derived therefrom.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and mass analysis of 12S and 19S dynein obtained from bull sperm flagella.

The Brookhaven scanning transmission electron microscope (STEM) was used to elucidate the structures and masses of 12S and 19S dynein extracted from bull sperm flagella. The 12S particle was a single globular particle with an average mass of 311 +/- 10 kdaltons. The 19S dynein particles consisted of two globular heads joined to a common base. The average mass of the 19S particle was 1.6 +/- 0.04 X 10(6) daltons. Thus, with the exception of the larger mass, the bull sperm 19S dynein molecule resembles the two-headed 21S dynein obtained from sea urchin sperm flagella and the 18S dynein obtained from Chlamydomonas with the possibility of a third head giving rise to the 12S particle. The structure, mass and polypeptide composition of bull sperm flagella dynein is compared with outer arm dyneins previously obtained from Chlamydomonas, Tetrahymena, and sea urchin sperm flagella.

Structure of the alpha-, beta-, and gamma-heavy chains of 22 S outer arm dynein obtained from Tetrahymena cilia.

Here we document the UV-induced, vanadate-dependent cleavage of the alpha-, beta-, and gamma-heavy chains of 22 S outer arm dynein obtained from Tetrahymena cilia. All three polypeptides have a single site of photocleavage in the presence of Mg2+, ATP, and vanadate (termed V1 cleavage). The alpha-chain yields complementary fragments with masses of 232 and 185 kDa, the beta-chain has complementary fragments with masses of 225 and 195 kDa, and the gamma-chain has complementary fragments with masses of 242 and 161 kDa. In the absence of ATP, only the beta-chain undergoes V1 cleavage. All three polypeptides have one single site of V2 cleavage, which are unaffected by the presence of nucleotide and only require the presence of Mn2+ and vanadate. V2 cleavage always occurs on the larger V1 fragments and is separated from the V1 site by 52, 48, and 57 kDa for the alpha-, beta-, and gamma-heavy chains, respectively. We have also found a third type of UV-induced vanadate-dependent cleavage which we have termed VMT cleavage. VMT cleavage occurs when dynein is bound to microtubules in an ATP-sensitive manner under V1 solution conditions that should only support cleavage of the beta-chain (i.e. vanadate, Mg2+, and absence of ATP). Under these conditions V1 cleavage of the beta-chain and V2 cleavage of all three chains occur. This is the first documented evidence of V2 cleavage occurring under V1 solution conditions and implies a change in dynein structure when it binds to a microtubule. Using a combination of polyclonal and monoclonal antibodies, we have been able to construct linear polypeptide maps of all three heavy chains. Their relationship to the polypeptide maps previously obtained for heavy chains obtained from the dynein of Chlamydomonas and sea urchin axonemes is discussed.

Adenosine 5'-O-(3-thiotriphosphate) hydrolysis by dynein.

The interaction of dynein with ATP gamma S, a phosphorothioate analogue of ATP, has been investigated in depth. The hydrolyses of ATP gamma S and of ATP were shown to be mutually competitive. ATP gamma S induced complete dissociation of the microtubule-dynein complex such that the time course of dissociation monitored by stopped-flow light-scattering methods followed a single exponential. The ATP gamma S concentration dependence of the rate of dissociation was hyperbolic, indicating that the dissociation is at least a two-step process: M.D + ATP gamma S in equilibrium M.D.ATP gamma S----M + D.ATP gamma S. The fit to the hyperbola gives an apparent Kd = 0.5 mM for the binding of ATP gamma S to the microtubule-dynein complex, and the maximal rate of 45 s-1 defines the rate of dissociation of the ternary M.D.ATP gamma S complex. Rapid quench-flow experiments demonstrated that the hydrolysis of ATP gamma S by dynein exhibited an initial burst of product formation. The size of the burst was 1.2 mol/10(6) g of dynein, comparable to that in the case of ATP hydrolysis. The steady-state rate of ATP gamma S turnover by dynein was activated by MAP-free microtubules. Because the rate of ATP gamma S turnover is severalfold (4-8) slower than ATP turnover, the rate-limiting step must be release of thiophosphate, not ADP. Thus, microtubules can activate the rate of thiophosphate release. The stereochemical course of phosphoric residue transfer was determined by using ATP gamma S stereospecifically labeled in the gamma position with 18O.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymerization of Antarctic fish tubulins at low temperatures: energetic aspects.

Tubulins were purified from the brain tissues of three Antarctic fishes, Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus, by ion-exchange chromatography and one cycle of temperature-dependent microtubule assembly and disassembly in vitro, and the functional properties of the protein were examined. The preparations contained the alpha- and beta-tubulins and were free of microtubule-associated proteins. At temperatures between 0 and 24 degrees C, the purified tubulins polymerized readily and reversibly to yield both microtubules and microtubule polymorphs (e.g., "hooked" microtubules and protofilament sheets). Critical concentrations for polymerization of the tubulins ranged from 0.87 mg/mL at 0 degrees C to 0.02 mg/mL at 18 degrees C. The van't Hoff plot of the apparent equilibrium constant for microtubule elongation at temperatures between 0 and 18 degrees C was linear and gave a standard enthalpy change (delta H degree) of +26.9 kcal/mol and a standard entropy change (delta S degree) of +123 eu. At 10 degrees C, tubulin from N. gibberifrons polymerized efficiently at high ionic strength; the critical concentration increased monotonically from 0.041 to 0.34 mg/mL as the concentration of NaCl added to the assembly buffer was increased from 0 to 0.4 M. Together, the results indicate that the polymerization of tubulins from the Antarctic fishes is entropically driven and suggest that an increased reliance on hydrophobic interactions underlies the energetics of microtubule formation at low temperatures. Thus, evolutionary modification to increase the proportion of hydrophobic interactions (relative to other bond types) at sites of interdimer contact may be one adaptive mechanism that enables the tubulins of cold-living poikilotherms to polymerize efficiently at low temperatures.

Phosphorothioate analogs of ATP as the substrates of dynein and ciliary or flagellar movement.

The phosphorothioate analog of ATP has a sulfur atom replacing a non-bridging oxygen atom of the triphosphate moiety of ATP. Due to the tetrahedral nature of the phosphorus atom, stereoisomers are known to exist, designated as the Sp and Rp isomers. We have reported [Shimizu & Furusawa (1986) Biochemistry 25, 5787] on the hydrolytic activity of the 22S dynein from Tetrahymena cilia towards the phosphorothioate analogs of ATP. In this paper, we extend our study and report on the microtubule-dynein dissociation by these analogs and on their ability to support sea urchin flagellar dynein enzymatic activity as well as ciliary or flagellar motility. It has been shown that the microtubule--22S-dynein complex is dissociated by the binding of ATP to the dynein enzymatic sites [Porter & Johnson (1983) J. Biol. Chem. 258, 6575]. We studied the dissociation by adenosine 5'-[alpha-thio]triphosphate (ATP[alpha S]), Sp or Rp, by light-scattering stopped-flow methods. The dissociation by (Sp)ATP[alpha S] was rapid and the rate of the light-scattering change by (Sp)ATP[alpha S] was a hyperbolic function of the nucleotide concentration, indicating that dissociation was a two-step process. On the other hand, (Rp)ATP[alpha S] up to 2 mM induced only slow and partial dissociation of the complex, while, in the presence of vanadate, it induced complete dissociation with a slightly higher rate (0.5 s-1). The adenosine 5'-[beta-thio]triphosphate (ATP[beta S]) isomers did not induce dissociation. The hydrolytic activity of the outer arm dynein from sea urchin sperm flagella towards these analogs was similar to that of 22S dynein. The ratios of Vmax (nmol.mg protein-1.min-1)/apparent Km (microM) of this dynein were 400-720, 53, 9.7, 0.62 and 0.028 for ATP, ATP[alpha S] (Sp or Rp), ATP[beta S] (Sp or Rp), respectively, in the presence of Mg2+ as the supporting cation. This dynein exhibited the same stereospecificity at beta phosphate as the 22S dynein or myosin. The detergent models of Tetrahymena or sea urchin spermatozoa were reactivated only by ATP or (Sp)ATP[alpha S] while other analogs were ineffective. The maximal beat frequency of the cilia or flagella reactivated by (Sp)ATP[alpha S] was one-quarter to one-half of that produced by ATP reactivation.

Mechanism of polynucleotide phosphorylase.

The de novo polymerization of RNA initiated by polynucleotide phosphorylase from nucleoside diphosphates was examined. End group analysis performed under conditions designed to specifically end label the polymer revealed no evidence for a 5'-pyrophosphate-terminated polymer. However, we observed preferential incorporation of the ADP alpha S(RP) diastereomer into the 5' end (Marlier & Benkovic, 1982) in chain initiation, suggesting that the enzyme incorporates a nucleoside diphosphate specifically into the 5' end of the product, with subsequent enzymatic removal of the polyphosphate linkage. No evidence could be obtained for a covalent adduct between the enzyme and the 5' end of the polymer chain, despite the high processivity of the polymerization reaction. Gel electrophoretic analysis showed the polymer to be highly disperse, varying from 1 to 30 kb. Scanning transmission electron microscopy supported this product analysis and further suggested that (i) each subunit can produce an RNA polymer and (ii) both 5' and 3' ends of the RNA can be bound simultaneously to the same or differing enzyme molecules.

Brain and egg tubulins from antarctic fishes are functionally and structurally distinct.

The multitubulin hypothesis proposes that chemically distinct tubulins may possess different polymerization properties or may form functionally different microtubules. To test this hypothesis, we have examined the functional properties and the structures of singlet-specific nonneural and neural tubulins from Antarctic fishes. Tubulins were purified from eggs of Notothenia coriiceps neglecta, and from brain tissues of N. coriiceps neglecta or N. gibberifrons, by DEAE ion-exchange chromatography and cycles of microtubule assembly/disassembly. At temperatures between 0 and 20 degrees C, each of these tubulins polymerized efficiently in vitro to yield microtubules of normal morphology. Critical concentrations for polymerization of egg tubulin ranged from 0.057 mg/ml at 3 degrees C to 0.002 mg/ml at 18 degrees C, whereas those for brain tubulin at like temperatures were 4-10-fold larger. Polymerization of both tubulins was entropically driven, but the apparent standard enthalpy and entropy changes for microtubule elongation by egg tubulin (delta Happ0 = +33.9 kcal/mol, delta Sapp0 = +151 entropy units) were significantly greater than values observed for brain tubulin (delta Happ0 = +26.5 kcal/mol, delta Sapp0 = +121 entropy units). Egg tubulin was composed of approximately six alpha and two beta chains and lacked the beta III isotype, whereas brain tubulin was more complex (greater than or equal to 10 of each chain type). Furthermore, egg alpha tubulins were more basic, and their carboxyl termini more resistant to cleavage by subtilisin, than were the alpha chains of brain. We conclude that brain and egg tubulins from the Antarctic fishes are functionally distinct in vitro, due either to qualitative or quantitative differences in isotypic composition, to differential posttranslational modification of shared isotypes, or to both.

Activation of ATPase activity of 14S dynein from Tetrahymena cilia by microtubules.

The ATPase activity of 14S dynein was activated by the presence of microtubule-associated-protein-free microtubules. The activation was 2.5-3.5 fold at 10 mg microtubule/ml, and the activity increased further with increasing microtubule concentration. The microtubule-14S-dynein complex, microtubule bundles with 14S dynein, was treated with a zero-length chemical cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). The ATPase activity of the complex responded to EDC in a biphasic, concentration-dependent manner and, at most, it was enhanced 5-10 fold. The complex treated with EDC was no longer unbundled by addition of ATP, as revealed by electron-microscopic observation. Several ATP analogues, which support in vitro microtubule translocation mediated by 14S dynein, were turned over faster by this mechanochemical enzyme in the presence of microtubules than in their absence. However, some ATP analogues which do not support the translocation were also turned over faster in the presence of microtubules. Thus, microtubule-dynein motility and substrate-turnover activation are not tightly coupled, which indicates that all three major motor systems, actin- heavy-meromyosin, microtubule-kinesin [Shimizu, T., Furusawa, K., Ohashi, S., Toyoshima, Y. Y., Okuno, M., Malik, F. & Vale, R. D. (1991) J. Cell Biol. 112, 1189-1197] and microtubule-dynein, have this characteristic property in common.

Dynein structure and function.

The structure of dynein isolated from several sources follows the pattern first observed with Tetrahymena 22S dynein, which has three globular heads attached by three flexible strands to a root-like base. Recent biochemical data indicate that there is one ATPase site on each dynein head and that all three heads interact with microtubules in an ATP-sensitive manner. Accordingly, images of dynein in situ can be interpreted in terms of a model for crossbridge action where the roots of the bouquet anchor the dynein to the A-tubule and all three heads reach out to interact with the B-tubule in an ATP-dependent reaction to produce a force for sliding.

Decoration of the microtubule surface by one kinesin head per tubulin heterodimer.

Kinesin, a microtubule-dependent ATPase, is believed to be involved in anterograde axonal transport. The kinesin head, which contains both microtubule and ATP binding sites, has the necessary components for the generation of force and motility. We have used saturation binding and electron microscopy to examine the interaction of the kinesin motor domain with the microtubule surface and found that binding saturated at one kinesin head per tubulin heterodimer. Both negative staining and cryo-electron microscopy revealed a regular pattern of kinesin bound to the microtubule surface, with an axial repeat of 8 nm. Optical diffraction analysis of decorated microtubules showed a strong layer-line at this spacing, confirming that one kinesin head binds per tubulin heterodimer. The addition of Mg-ATP to the microtubule-kinesin complex resulted in the complete dissociation of kinesin from the microtubule surface.