Mechanochemical aspects of axonemal dynein activity studied by in vitro microtubule translocation.

We have determined the relationship between microtubule length and translocation velocity from recordings of bovine brain microtubules translocating over a Paramecium 22S dynein substratum in an in vitro assay chamber. For comparison with untreated samples, the 22S dynein has been subjected to detergent and/or to pretreatments that induce phosphorylation of an associated 29 kDa light chain. Control and treated dyneins have been used at the same densities in the translocation assays. In any given condition, translocation velocity (v) shows an initial increase with microtubule length (L) and then reaches a plateau. This situation may be represented by a hyperbola of the general form v = aL/(L+b), which is formally analogous to the Briggs-Haldane relationship, which we have used to interpret our data. The results indicate that the maximum translocation velocity Vo(= a) is increased by pretreatment, whereas the length constant KL(= b), which corresponds to Km, does not change with pretreatment, implying that the mechanochemical properties of the pretreated dyneins differ from those of control dyneins. The conclusion that KL is constant for defined in vitro assays rules out the possibility that the velocity changes seen are caused by changes in geometry in the translocation assays or by the numbers of dyneins or dynein heads needed to produce maximal translocational velocity. From our analysis, we determine that f, the fraction of cycle time during which the dynein is in the force-generating state, is small--roughly 0.01, comparable to the f determined previously for heavy meromyosin. The practical limits of these mechanochemical changes imply that the maximum possible ciliary beat frequency is about 120 Hz, and that in the physiological range of 5-60 Hz, beat frequency could be controlled by varying the numbers of phosphorylated outer arm dyneins along an axonemal microtubule.     

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.     

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.     

The dynein heavy chain family

Dynein is the large molecular motor that translocates to the (-) ends of microtubules. Dynein was first isolated from Tetrahymena cilia four decades ago. The analysis of the primary structure of the dynein heavy chain and the discovery that many organisms express multiple dynein heavy chains have led to two insights. One, dynein, whose motor domain comprises six AAA modules and two potential mechanical levers, generates movement by a mechanism that is fundamentally different than that which underlies the motion of myosin and kinesin. And two, organisms with cilia or flagella express approximately 14 different dynein heavy chain genes, each gene encodes a distinct dynein protein isoform, and each isoform appears to be functionally specialized. Sequence comparisons demonstrate that functionally equivalent isoforms of dynein heavy chains are well conserved across species. Alignments of portions of the motor domain result in seven clusters: (i) cytoplasmic dynein Dyhl; (ii) cytoplasmic dynein Dyh2; (iii) axonemal outer arm dynein alpha; (iv) outer arm dyneins beta and gamma; (v) inner arm dynein 1alpha; (vi) inner arm dynein 1beta; and (vii) a group of apparently single-headed inner arm dyneins. Some of the dynein groups contained more than one representative from a single organism, suggesting that these may be tissue-specific variants.     

Modulation of cytoplasmic dynein ATPase activity by the accessory subunits

The microtubule-based motor molecule cytoplasmic dynein has been proposed to be regulated by a variety of mechanisms, including phosphorylation and specific interaction with the organelle-associated complex, dynactin. In this study, we examined whether the intermediate chain subunits of cytoplasmic dynein are involved in modulation of ATP hydrolysis, and thereby affect motility. Treatment of testis cytoplasmic dynein under hypertonic salt conditions resulted in separation of the intermediate chains from the remainder of the dynein molecule, and led to a 4-fold enhancement of ATP hydrolysis. This result suggests that the accessory subunits act as negative regulators of dynein heavy chain activity. Comparison of ATPase activities of dyneins with differing intermediate chain isoforms showed significant differences in basal ATP hydrolysis rates, with testis dynein 7-fold more active than dynein from brain. Removal of the intermediate chain subunits led to an equalization of ATPase activity between brain and testis dyneins, suggesting that the accessory subunits are responsible for the observed differences in tissue activity. Finally, our preparative procedures have allowed for the identification and purification of a 1:1 complex of dynein with dynactin. As this interaction is presumed to be mediated by the dynein intermediate chain subunits, we now have defined experimental conditions for further exploration of dynein enzymatic and motility regulation.     

Kinesin and cytoplasmic dynein binding to brain microsomes.

Movement of cellular organelles in a directional manner along polar microtubules is driven by the motor proteins, kinesin and cytoplasmic dynein. The binding of these proteins to a microsomal fraction from embryonic chicken brain is investigated here. Both motors exhibit saturation binding to the vesicles, and proteolysis of vesicle membrane proteins abolishes binding. The maximal binding for kinesin is 12 +/- 1.7 and 43 +/- 2 pmol per mg of vesicle protein with or without 1 mM ATP, respectively. The maximal binding for cytoplasmic dynein is 55 +/- 3.8 and 73 +/- 3.7 pmol per mg of vesicle protein with or without ATP, respectively. These values correspond to 1-6 sites per vesicle of 100-nm diameter. The nonhydrolyzable ATP analog, adenyl-5'-yl imidodiphosphate (AMP-PNP), inhibited kinesin binding to vesicles but increased kinesin binding to microtubules. An antibody to the kinesin light chain also inhibited vesicle binding to kinesin. In the absence but not presence of ATP, competition between the two motors for binding was observed. We suggest that there are two distinguishable binding sites for kinesin and cytoplasmic dynein on these organelles in the presence of ATP and a shared site in the absence of ATP.     

Structural characterization of myosin from bovine brain

Myosins isolated from bovine brain, rabbit skeletal muscle, and chicken gizzard smooth muscle and their heavy meromyosin and light meromyosin fractions were studied in the electron microscope by negative staining with uranyl acetate. Under similar conditions of preparation and polymerization, the three myosins formed paracrystals of different structures. The light meromyosin portion of the skeletal muscle myosin also assembled in a different fashion than the brain or smooth muscle light meromyosins; the latter two assembled similarly. The heavy meromyosin portion from each of the three myosins was shown to interact with the actins isolated from each of the three tissue sources by the formation of the characteristic arrowhead patterns with similar periodicities. The brain heavy meromyosin attachment to both skeletal and brain actins was dissociated by ATP. It is suggested that differences in the light meromyosin portions of the three myosins may account in part for their differences in assembly in vivo.     

Calmodulin confers calcium sensitivity on ciliary dynein ATPase

Extraction of demembranated cilia of Tetrahymena by Tris-EDTA (denoted by the suffix E) yields 14S-E and 30S-E dyneins with ATPase activities that are slightly increased by Ca++. This effect is moderately potentiated when bovine brain calmodulin is added to the assay mixture. Extraction with 0.5 M KCl (denoted by the suffix K) yeilds a 14S-K dynein with a low basal ATPase activity in the presence of Ca++. Subsequent addition of calmodulin causes marked activation (up to 10- fold) of ATPase activity. Although 14S-K and 14S-E dyneins have Ca++- dependent ATPase activities that differ markedly in the degree of activation, the concentration of calmodulin required for half-maximal saturation is similar for both, approximately 0.1 microM. Both 30S-K and 30S-E dyneins, however, require approximately 0.7 microM bovine brain calmodulin to reach half-maximal activation of their Ca++- dependent ATPase activities. Tetrahymena calmodulin is as effective as bovine brain calmodulin in activating 30S dynein , but may be slightly less effective than the brain calmodulin in activating 14S dynein. Rabbit skeletal muscle troponin C also activates the Ca++-dependent ATPase activity of 30S dynein and, to a lesser extent, that of 14S dynein, but in both cases is less effective than calmodulin. The interaction of calmodulin with dynein that results in ATPase activation is largely complete in less than 1 min, and is prevented by the presence of low concentrations of ATP. Adenylyl imidodiphosphate can partially prevent activation of dynein ATPase by calmodulin plus Ca++, but at much higher concentrations than required for prevention by ATP. beta, gamma-methyl-adenosine triphosphate appears not to prevent this activation. The presence of Ca++-dependent calmodulin-binding sites on 14S and 30S dyneins was demonstrated by the Ca++-dependent retention of the dyneins on a calmodulin-Sepharose-4B column. Gel electrophoresis of 14S dynein that had been purified by the affinity-chromatography procedure showed that presence of two major and one minor high molecular weight components. Similar analysis of 30S dynein purified by this procedure also revealed on major and one minor high molecular weight components that were different from the major components of 14S dynein. Ca++-dependent binding sites for calmodulin were shown to be present on axonemes that had been extracted twice with Tris-EDTA or with 0.5 M KCl by the use of 35S-labeled Tetrahymena calmodulin. It is concluded that the 14S and 30S dyneins of Tetrahymena contain Ca++- dependent binding sites for calmodulin and the calmodulin mediates the Ca++-regulation of the dynein ATPases of Tetrahymena cilia.     

Depolymerization of F-actin by deoxyribonuclease I.

Deoxyribonuclease I causes depolymerization of filamentous muscle actin to form a stable complex of 1 mole DNAase I:1 mole actin. The regulatory proteins tropomyosin and troponin bind to filamentous actin and slow down but do not prevent the depolymerization. In the absense of ATP, heavy meromyosin binds tightly to actin filaments and blocks completely the DNAase I: actin filament interaction. Addition of ATP releases heavy meromyosin; DNAase I is then rapidly inhibited and the actin filaments are depolymerized.     

Evidence suggesting the presence of common antigenic determinant between dynein and tubulin.

The present experiments showed that the guinea pig antiserum prepared against the main polypeptides of 14 S dynein from Tetrahymena cilia reacted with sea urchin sperm flagellar dynein and with bovine brain high molecular weight protein to give rise to a precipitin line confluent with that formed between the antiserum and Tetrahymena dynein. Furthermore, it was found that this antiserum also reacted with tubulins from Tetrahymena cilia, sea urchin sperm flagella and bovine brain to give rise to the confluent precipitin line. Among muscle proteins, only actin preparation from rabbit skeletal muscle reacted with the anti-Tetrahymena dynein serum, whereas neither rabbit skeletal muscle myosin, chicken skeletal muscle tropomyosin nor chicken skeletal muscle troponin reacted with the antiserum. These results suggest that dynein and tubulin and probably actin share an antigenic determinant regardless of different protein species and of different animal species. The common antigenic determinant was detected only when the proteins denatured with urea/sodium dodecyl sulfate/beta-mercaptoethanol/N-ethylmaleimide were used, but it was not detected at all when the native proteins were used. This implies that a certain common antigenic determinant which is involved in the precipitin line formation exists in the primary structures of dyneins and tubulins and probably actin, and is hidden inside the tertiary structures of the native protein molecules.     

Directional instability of microtubule transport in the presence of kinesin and dynein, two opposite polarity motor proteins

Kinesin and dynein are motor proteins that move in opposite directions along microtubules. In this study, we examine the consequences of having kinesin and dynein (ciliary outer arm or cytoplasmic) bound to glass surfaces interacting with the same microtubule in vitro. Although one might expect a balance of opposing forces to produce little or no net movement, we find instead that microtubules move unidirectionally for several microns (corresponding to hundreds of ATPase cycles by a motor) but continually switch between kinesin-directed and dynein-directed transport. The velocities in the plus-end (0.2-0.3 microns/s) and minus-end (3.5-4 microns/s) directions were approximately half those produced by kinesin (0.5 microns/s) and ciliary dynein (6.7 microns/s) alone, indicating that the motors not contributing to movement can interact with and impose a drag upon the microtubule. By comparing two dyneins with different duty ratios (percentage of time spent in a strongly bound state during the ATPase cycle) and varying the nucleotide conditions, we show that the microtubule attachment times of the two opposing motors as well as their relative numbers determine which motor predominates in this assay. Together, these findings are consistent with a model in which kinesin-induced movement of a microtubule induces a negative strain in attached dyneins which causes them to dissociate before entering a force-generating state (and vice versa); reversals in the direction of transport may require the temporary dissociation of the transporting motor from the microtubule. The bidirectional movements described here are also remarkably similar to the back-and-forth movements of chromosomes during mitosis and membrane vesicles in fibroblasts. These results suggest that the underlying mechanical properties of motor proteins, at least in part, may be responsible for reversals in microtubule-based transport observed in cells.     

Cytoplasmic dynein is a vesicle protein.

Microtubule-based organelle transport is thought to be mediated by the force-generating proteins cytoplasmic dynein and kinesin. These motor proteins have been characterized based on their ability to associate with and translocate microtubules. We show here that cytoplasmic dynein is also present as a peripheral membrane protein of purified synaptic vesicles. The vesicle-associated cytoplasmic dynein is identified by its photo-induced cleavage in the presence of ATP and vanadate. Purified, soluble cytoplasmic dynein is competent to bind to vesicle membranes stripped of endogenous peripheral membrane proteins by alkaline pH. Dynein binding to membranes is saturable at a concentration of 1.00 +/- 0.15 pmol/micrograms vesicle protein and has a dissociation constant of 22.3 +/- 2.4 nM. The association of cytoplasmic dynein with the membrane cannot be reversed by incubation with ATP. Furthermore, following binding to membranes, dynein retains its ability to bind ATP and to be photo-cleaved in the presence of vanadate. The presence of cytoplasmic dynein on synaptic vesicles and its ability to bind to extracted membranes supports current models of microtubule-based organelle translocation.     

Preparation of microtubules from rat liver and testis: cytoplasmic dynein is a major microtubule associated protein

A microtubule associated protein from brain tissue (MAP 1C), has been found to possess many properties in common with ciliary and flagellar dyneins (Paschal et al.:J. Cell Biol. 105:1273-1282, 1987). However, this protein, now designated as cytoplasmic dynein, exhibited several properties which distinguish it from axonemal forms of the enzyme. We have investigated these characteristics further in a study of cytoplasmic dyneins from non-neuronal tissues. Rat liver and testis in particular were found to contain high levels of cytoplasmic dynein. The yield of dynein from testis was over 70 micrograms/g of tissue, making this the best source of cytoplasmic dynein of all tissues so far examined. The characterization of dynein from these sources has confirmed and extended our previous observations concerning the unique properties of cytoplasmic dynein. Activation of liver and testis dynein occurred at low (less than 1 mg/ml) tubulin concentration. Polypeptides identified as subunits of brain cytoplasmic dynein (74, 59, 57, 55, and 53 kDa) were present in liver and testis preparations. In addition, polypeptides at 150 and 45 kDa were found to copurify with the non-neuronal dyneins. The liver and testis enzyme hydrolyzed pyrimidine nucleotides at rates up to 12.5 times faster than ATP, though the relative affinity of cytoplasmic dynein for CTP was much lower (Km = 1.0 mM) than that for ATP. The properties of the testis enzyme were consistent with its identification as a cytoplasmic dynein rather than a sperm axonemal precursor. These data indicate that cytoplasmic dyneins may be widespread in distribution and that they share certain biochemical properties unique from those of axonemal dyneins. These characteristics are consistent with the proposal that cytoplasmic dynein plays a universal role in retrograde organelle motility.     

Dissociation of double-headed cytoplasmic dynein into single-headed species and its motile properties

Cytoplasmic dynein is a minus-end directed microtubule motor and plays important roles in the transport of various intracellular cargoes. Cytoplasmic dynein comprises two identical heavy chains and forms a dimer (double-headed dynein); the total molecular weight of the cytoplasmic dynein complex is about 1.5 million. The dynein motor domain is structurally very different from those of kinesin and myosin, and our understanding of the mechanisms of dynein energy transduction is limited mainly because of the difficulty in obtaining a sufficient quantity of purified and active cytoplasmic dynein. We purified cytoplasmic dynein, which was free from dynactin and other dynein-associated proteins. The purified cytoplasmic dynein was active in an in vitro motility assay. The controlled dialysis of the purified dynein against 4 M urea resulted in its complete dissociation into monomeric species (single-headed dynein). The separation of the dynein heads by the treatment was reversible. The MgATPase activities of the single-headed and reconstituted double-headed dynein were comparable to that of intact dynein. The double-headed dynein bundled microtubules in the absence of ATP; the single-headed dynein did not. The single-headed dynein produced in vitro microtubule-gliding motility at velocities very similar to those of double-headed dynein at various ATP concentrations. These results indicate that a single cytoplasmic dynein heavy chain is sufficient to produce robust microtubule motility. Application of the double- and single-headed dynein molecules in various assay systems will elucidate the mechanism of action of the cytoplasmic dynein.     

Amino acid composition of dynein and comparison with myosin.

A comparison is made between dynein [flagellar ATPase; EC 3.6.1.3], purified from sea urchin sperm flagella, and muscle myosin. The amino acid composition of dynein was found to be statistically different from that of myosin. The same was true of their tryptic fragments retaining ATPase activity, i.e., Fragment A of dynein and heavy meromyosin. At low ionic strength, no superprecipitation took place when ATP was added to a mixture of dynein and actin, and stimulation of the Mg2+-ATPase activity of dynein remained below 50% even when a one-hundred-fold excess of actin was present. No viscosity drop was caused by adding ATP to a solution containing dynein and actin. Anti-myosin antiserum did not react with dynein, while anti-Fragment A antiserum formed no precipit-n line against myosin. Furthermore, the amount of dynein that combined with F-actin was less than one-fifth of the amount of dynein that fully combined with microtubules. These results are consistent with the dissimilarity in enzymatic and other physiocochemical properties of these two proteins.     

Calcium regulates ATP-sensitive microtubule binding by Chlamydomonas outer arm dynein

The Chlamydomonas outer dynein arm contains three distinct heavy chains (alpha, beta, and gamma) that exhibit different motor properties. The LC4 protein, which binds 1-2 Ca2+ with KCa = 3 x 10-5 m, is associated with the gamma heavy chain and has been proposed to act as a sensor to regulate dynein motor function in response to alterations in intraflagellar Ca2+ levels. Here we genetically dissect the outer arm to yield subparticles containing different motor unit combinations and assess the microtubule-binding properties of these complexes both prior to and following preincubation with tubulin and ATP, which was used to inhibit ATP-insensitive (structural) microtubule binding. We observed that the alpha heavy chain exhibits a dominant Ca2+-independent ATP-sensitive MT binding activity in vitro that is inhibited by attachment of tubulin to the structural microtubule-binding domain. Furthermore, we show that ATP-sensitive microtubule binding by a dynein subparticle containing only the beta and gamma heavy chains does not occur at Ca2+ concentrations below pCa 6 but is maximally activated above pCa 5. This activity was not observed in mutant dyneins containing small deletions in the microtubule-binding region of the beta heavy chain or in dyneins that lack both the alpha heavy chain and the motor domain of the beta heavy chain. These findings strongly suggest that Ca2+ binding directly to a component of the dynein complex regulates ATP-sensitive interactions between the beta heavy chain and microtubules and lead to a model for how individual motor units are controlled within the outer dynein arm.     

Characterization of outer arm dynein in sea anemone, Anthopleura midori.

Outer arm dynein was purified from sperm flagella of a sea anemone, Anthopleura midori, and its biochemical and biophysical properties were characterized. The dynein, obtained at a 20S ATPase peak by sucrose density gradient centrifugation, consisted of two heavy chains, three intermediate chains, and seven light chains. The specific ATPase activity of dynein was 1.3 micromol Pi/mg/min. Four polypeptides (296, 296, 225, and 206 kDa) were formed by UV cleavage at 365 nm of dynein in the presence of vanadate and ATP. In addition, negatively stained images of dynein molecules and the hook-shaped image of the outer arm of the flagella indicated that sea anemone outer arm dynein is two-headed. In contrast to protist dyneins, which are three-headed, outer arm dyneins of flagella and cilia in multicellular animals are two-headed molecules corresponding to the two heavy chains. Phylogenetic considerations were made concerning the diversity of outer arm dyneins.     

Autoinhibitory and other autoregulatory elements within the dynein motor domain

The dyneins are a family of microtubule motor proteins. The motor domain, which represents the C-terminal 2/3 of the dynein heavy chain, exhibits homology to the AAA family of ATPases. It consists of a ring of six related but divergent AAA+ units, with two substantial sized protruding projections, the stem, or tail, which anchors the protein to diverse subcellular sites, and the stalk, which binds microtubules. This article reviews recent efforts to probe the mechanism by which the dyneins produce force, and work from the authors' lab regarding long-range conformational regulation of dynein enzymatic activity.     

Identification of dynein heavy chain genes expressed in human and mouse testis: chromosomal localization of an axonemal dynein gene

Dynein heavy chains are involved in microtubule-dependent transport processes. While cytoplasmic dyneins are involved in chromosome or vesicle movement, axonemal dyneins are essential for motility of cilia and flagella. Here we report the isolation of dynein heavy chain (DHC)-like sequences in man and mouse. Using polymerase chain reaction and reverse-transcribed human and mouse testis RNA cDNA fragments encoding the conserved ATP binding region of dynein heavy chains were amplified. We identified 11 different mouse and eight human dynein-like sequences in testis which show high similarity to known dyneins of different species such as rat, sea urchin or green algae. Sequence similarities suggest that two of the mouse clones and one human clone encode putative cytoplasmic dynein heavy chains, whereas the other sequences show higher similarity to axonemal dyneins. Two of nine axonemal dynein isoforms identified in the mouse testis are more closely related to known outer arm dyneins, while seven clones seem to belong to the inner arm dynein group. Of the isolated human isoforms three clones were classified as outer arm and four clones as inner arm dynein heavy chains. Each of the DHC cDNAs corresponds to an individual gene as determined by Southern blot experiments. The alignment of the deduced protein sequences between human (HDHC) and mouse (MDHC) dynein fragments reveals higher similarity between single human and mouse sequences than between two sequences of the same species. Human and mouse cDNA fragments were used to isolate genomic clones. Two of these clones, gHDHC7 and gMDHC7, are homologous genes encoding axonemal inner arm dyneins. While the human clone is assigned to 3p21, the mouse gene maps to chromosome 14.     

Cytoplasmic dynein, the dynactin complex, and kinesin are interdependent and essential for fast axonal transport

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150(Glued) (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150(Glued) were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.