Identification and immunolocalization of cytoplasmic dynein in Dictyostelium

A high molecular weight microtubule binding protein has been isolated from homogenates of Dictyostelium. Because of its sedimentation velocity (20s), ATP-sensitive binding to microtubules, UV-vanadate-ATP mediated fragmentation, prominent CTPase activity, and its ability to produce limited microtubule movement in vitro, we consider this protein to be a form of cytoplasmic dynein. A polyclonal antibody monospecific to this protein was produced, and dynein's intracellular distribution in ameboid cells was examined by immunofluorescence. The antibody labels a punctate cytoplasmic pattern, localizes to a spherical region adjacent to the nucleus, and also appears to label the nuclei. The punctate staining pattern is consistent with cytoplasmic dynein's proposed function in organelle transport. The spherical juxtanuclear object stained is coincident with this cell's microtubule organizing center, an obvious termination point for minus-end directed microtubule motors. By immunofluorescence, there does not appear to be a substantial amount of dynein in the intranuclear mitotic spindles of Dictyostelium. These data provide evidence for localization of cytoplasmic dynein in cells, and suggest that Dictyostelium will be a useful system in which to study the molecular biology of microtubule-associated motor enzymes.     

C-sequence of the Dictyostelium cytoplasmic dynein participates in processivity modulation

We examined the functional roles of C-sequence, a 47-kDa non-AAA+ module at the C-terminal end of the 380-kDa Dictyostelium dynein motor domain. When the distal segment of the C-sequence was deleted from the motor domain, the single-molecule processivity of the dimerized motor domain was selectively impaired without its ensemble motile ability and ATPase activity being severely affected. When the hinge-like sequence between the distal and proximal C-sequence segments was made more or less flexible, the dimeric motor showed lower or higher processivity, respectively. These results suggest a potential function of the distal C-sequence segment as a modulator of processivity.     

ATP hydrolysis cycle-dependent tail motions in cytoplasmic dynein

The motor protein dynein is predicted to move the tail domain, a slender rod-like structure, relative to the catalytic head domain to carry out its power stroke. Here, we investigated ATP hydrolysis cycle-dependent conformational dynamics of dynein using fluorescence resonance energy transfer analysis of the dynein motor domain labeled with two fluorescent proteins. We show that dynein adopts at least two conformational states (states I and II), and the tail undergoes ATP-induced motions relative to the head domain during transitions between the two states. Our measurements also suggest that in the course of the ATP hydrolysis cycle of dynein, the tail motion from state I to state II takes place in the ATP-bound state, whereas the motion from state II to state I occurs in the ADP-bound state. The latter tail motion may correspond to the predicted power stroke of dynein.     

Kinetic evidence for multiple dynein ATPase sites

We have examined the kinetics of ATP-induced dissociation of the microtubule-dynein complex at low ATP concentrations in the presence of vanadate, which inhibits the enzyme after the binding and hydrolysis of a single ATP per site (Shimizu, T., and Johnson, K. A. (1983) J. Biol. Chem. 258, 13833-13840). Four aspects of the dissociation reaction could not be explained by a model of dynein with a single ATP-sensitive microtubule binding site. First, titration of the light-scattering amplitude versus ATP concentration in the presence of vanadate gave Mr = 720,000/ATP binding site, indicating approximately 2.8 sites/2 million molecular weight particle. Second, the dissociation reaction was incomplete at concentrations of less than 2 microM ATP in the absence of vanadate, while the addition of vanadate led to complete dissociation at an increased rate. Third, the time course of dissociation induced by less than or equal to 1 microM ATP in the presence of vanadate was biphasic, with a small but distinct lag. Fourth, the ATP concentration dependence of the rate of dissociation in the absence of vanadate was concave upward at concentrations of ATP less than 5 microM, whereas the plot was linear in the presence of vanadate. These data suggest that dynein has three ATP-sensitive microtubule binding sites and each site must bind ATP for dynein to detach from the microtubule.     

A cytoplasmic dynein tail mutation impairs motor processivity

Mutations in the tail of the cytoplasmic dynein molecule have been reported to cause neurodegenerative disease in mice. The mutant mouse strain Legs at odd angles (Loa) has impaired retrograde axonal transport, but the molecular deficiencies in the mutant dynein molecule, and how they contribute to neurodegeneration, are unknown. To address these questions, we purified dynein from wild-type mice and the Legs at odd angles mutant mice. Using biochemical, single-molecule, and live-cell-imaging techniques, we find a marked inhibition of motor run-length in vitro and in vivo, and significantly altered motor domain coordination in the dynein from mutant mice. These results suggest a potential role for the dynein tail in motor function, and provide direct evidence for a link between single-motor processivity and disease.     

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.     

Two approaches to isolate cytoplasmic dynein ATPase from Neurospora crassa

Cytoplasmic dynein is a force-producing enzyme that, in association with dynactin, conducts minus-end directed transport of various organelles along microtubules. Biochemical analyses of cytoplasmic dynein and dynactin have been conducted primarily in vertebrate systems, whereas genetic analyses have been explored mainly in yeast and the filamentous fungi. To provide a complementary biochemical approach for the study of fungal dynein, we isolated/partially purified cytoplasmic dynein ATPase from the filamentous fungus Neurospora crassa. N. crassa dynein was partially purified by slightly modifying the existing procedures, described for mammalian cytoplasmic dynein that uses dynein-microtubule binding, followed by release with ATP and sucrose gradient fractionation. A novel approach was also used to isolate dynein-specific ATPase by gel filtration (Sepharose CL-4B). The K(m), ATP obtained by isolating dynein ATPase using gel filtration was similar to that obtained by using conventional method, suggests that contaminant proteins do not interfere with the dynein ATPase activity. Like vertebrate dynein, N. crassa dynein is a general NTPase with highest activity toward ATP, and only the ATPase activity is stimulated by microtubules. The K(m), ATP for N. crassa cytoplasmic dynein is 10- to 15-fold higher than that of the vertebrate enzyme.     

Regulatory ATPase sites of cytoplasmic dynein affect processivity and force generation

The heavy chain of cytoplasmic dynein contains four nucleotide-binding domains referred to as AAA1-AAA4, with the first domain (AAA1) being the main ATP hydrolytic site. Although previous studies have proposed regulatory roles for AAA3 and AAA4, the role of ATP hydrolysis at these sites remains elusive. Here, we have analyzed the single molecule motility properties of yeast cytoplasmic dynein mutants bearing mutations that prevent ATP hydrolysis at AAA3 or AAA4. Both mutants remain processive, but the AAA4 mutant exhibits a surprising increase in processivity due to its tighter affinity for microtubules. In addition to changes in motility characteristics, AAA3 and AAA4 mutants produce less maximal force than wild-type dynein. These results indicate that the nucleotide binding state at AAA3 and AAA4 can allosterically modulate microtubule binding affinity and affect dynein processivity and force production.     

Rhodopsin's carboxy-terminal cytoplasmic tail acts as a membrane receptor for cytoplasmic dynein by binding to the dynein light chain Tctex-1.

The interaction of cytoplasmic dynein with its cargoes is thought to be indirectly mediated by dynactin, a complex that binds to the dynein intermediate chain. However, the roles of other dynein subunits in cargo binding have been unknown. Here we demonstrate that dynein translocates rhodopsin-bearing vesicles along microtubules. This interaction occurs directly between the C-terminal cytoplasmic tail of rhodopsin and Tctex-1, a dynein light chain. C-terminal rhodopsin mutations responsible for retinitis pigmentosa inhibit this interaction. Our results point to an alternative docking mechanism for cytoplasmic dynein, provide novel insights into the role of motor proteins in the polarized transport of post-Golgi vesicles, and shed light on the molecular basis of retinitis pigmentosa.     

Kinetic analysis of axoneme and dynein ATPase from sea urchin sperm

The behavior of the ATPase of axoneme (detergent-treated flagellum) and dynein from sea urchin sperm was investigated. The activation of the ATPase by divalent cations was attributed to formation of a complex of ATP and the divalent cation; the metal-ATP complex is an effective substrate. However, free ATP is a modifier of the ATPase. Free ATP markedly changes the affinity of the metal-ATP complex to the enzymes. Calcium-activated ATPase activity of axoneme decreased at high concentration of CaCl₂, but that of dynein did not decrease.     

Kinetic characterization of the ATPase cycle of the molecular chaperone Hsc66 from Escherichia coli

Hsc66 from Escherichia coli is a constitutively expressed hsp70 class molecular chaperone whose activity is coupled to ATP binding and hydrolysis. To better understand the mechanism and regulation of Hsc66, we investigated the kinetics of ATP hydrolysis and the interactions of Hsc66 with nucleotides. Steady-state experiments revealed that Hsc66 has a low affinity for ATP (K(m)(ATP) = 12.7 microM) compared with other hsp70 chaperones. The kinetics of nucleotide binding were determined by analyzing changes in the Hsc66 absorbance spectrum using stopped-flow methods at 23 degrees C. ATP binding results in a rapid, biphasic increase of Hsc66 absorbance at 280 nm; this is interpreted as arising from a two-step process in which ATP binding (k(a)(ATP) = 4.2 x 10(4) M(-1) s(-1), k(d)(ATP) = 1.1 s(-1)) is followed by a slow conformational change (k(conf) = 0. 1 s(-1)). Under single turnover conditions, the ATP-induced transition decays exponentially with a rate (k(decay) = 0.0013 s(-1)) similar to that observed in both steady-state and single turnover ATP hydrolysis experiments (k(hyd) = 0.0014 s(-1)). ADP binding to Hsc66 results in a monophasic transition in the absence (k(a)(ADP) = 7 x 10(5) M(-1) s(-1), k(d)(ADP) = 60 s(-1)) and presence of physiological levels of inorganic phosphate (k(a)(ADP(P(i)) = 0.28 x 10(5) M(-1) s(-1), k(d)(ADP(P(i)) = 9.1 s(-1)). These results indicate that ATP hydrolysis is the rate-limiting step under steady-state conditions and is >10(3)-fold slower than the rate of ADP/ATP exchange. Thus, in contrast to DnaK and eukaryotic forms of hsp70 that have been characterized to date, the R if T equilibrium balance for Hsc66 is shifted in favor of the low peptide affinity T state, and regulation of the reaction cycle is expected to occur at the ATP hydrolysis step rather than at nucleotide exchange.     

Characterization of the microtubule-activated ATPase of brain cytoplasmic dynein (MAP 1C)

We recently found that the brain cytosolic microtubule-associated protein 1C (MAP 1C) is a microtubule-activated ATPase, capable of translocating microtubules in vitro in the direction corresponding to retrograde transport. (Paschal, B. M., H. S. Shpetner, and R. B. Vallee. 1987b. J. Cell Biol. 105:1273-1282; Paschal, B. M., and R. B. Vallee. 1987. Nature [Lond.]. 330:181-183.). Biochemical analysis of this protein (op. cit.) as well as scanning transmission electron microscopy revealed that MAP 1C is a brain cytoplasmic form of the ciliary and flagellar ATPase dynein (Vallee, R. B., J. S. Wall, B. M. Paschal, and H. S. Shpetner. 1988. Nature [Lond.]. 332:561-563). We have now characterized the ATPase activity of the brain enzyme in detail. We found that microtubule activation required polymeric tubulin and saturated with increasing tubulin concentration. The maximum activity at saturating tubulin (Vmax) varied from 186 to 239 nmol/min per mg. At low ionic strength, the Km for microtubules was 0.16 mg/ml tubulin, substantially lower than that previously reported for axonemal dynein. The microtubule-stimulated activity was extremely sensitive to changes in ionic strength and sulfhydryl oxidation state, both of which primarily affected the microtubule concentrations required for half-maximal activation. In a number of respects the brain dynein was enzymatically similar to both axonemal and egg dyneins. Thus, the ATPase required divalent cations, calcium stimulating activity less effectively than magnesium. The MgATPase was inhibited by metavandate (Ki = 5-10 microM for the microtubule-stimulated activity), 1 mM NEM, and 1 mM EHNA. In contrast to other dyneins, the brain enzyme hydrolyzed CTP, TTP, and GTP at higher rates than ATP. Thus, the enzymological properties of the brain cytoplasmic dynein are clearly related to those of other dyneins, though the brain enzyme is unique in its substrate specificity and in its high sensitivity to stimulation by microtubules.     

A single-headed recombinant fragment of Dictyostelium cytoplasmic dynein can drive the robust sliding of microtubules

A cytoplasmic dynein is a microtubule-based motor protein involved in diverse cellular functions, such as organelle transport and chromosome segregation. The dynein has two ring-shaped heads that contain six repeats of the AAA domain responsible for ATP hydrolysis. It has been proposed that the ATPase-dependent swing of a stalk and a stem emerging from each of the heads generates the power stroke (Burgess, S.A. (2003) Nature 421, 715-718). To understand the molecular mechanism of the dynein power stroke, it is essential to establish an easy and reproducible method to express and purify the recombinant dynein with full motor activities. Here we report the expression and purification of the C-terminal 380-kDa fragment of the Dictyostelium cytoplasmic dynein heavy-chain fused with an affinity tag and green fluorescent protein. The purified single-headed recombinant protein drove the robust minus-end-directed sliding of microtubules at a velocity of 1.2 microm/s. This recombinant protein had a high basal ATPase activity (approximately 4s(-1)), which was further activated by >15-fold on the addition of 40 microM microtubules. These results show that the 380-kDa recombinant fragment retains all the structures required for motor functions, i.e. the ATPase activity highly stimulated by microtubules and the robust motility.     

Kinetic properties of dynein ATPase from Tetrahymena pyriformis. The initial phosphate burst of dynein ATPase and its interaction with ATP analogs.

1. Dynein was extracted with 0.5 M KCl from Tetrahymena axonemes. SDS-gel electrophoresis of the extract indicated that about 50% of the extracted protein had a molecular weight of about 3.5 X 10(5), and that 90% of the proteins with this weight had been extracted. 2. The ATPase [EC 3.6.1.3] reaction of the KCl-extracted dynein fraction was enhanced by 60-80% by addition of the outer doublet fraction. It showed an initial burst of Pi liberation of about 1 mol per mol of proteins with a molecular weight of 3.5 X 10(5). 3. We examined the interaction of the dynein-tubulin system from Tetrahymena cilia with ten ATP analogs [2'-dATP, 3'-dATP, epsilonATP, FTP, 8-NH(CH3)-ATP, 8,3'-S-cyclo-ATP, 8-Br-ATP, 8-OCH3-ATP, 8-SCH3-ATP, and AMPPNP]. Among them, 2'-dATP and 3'-dATP were good substrates for dynein ATPase, as they induced the dissociation of dynein arms from the B-tubule of outer doublets, the sliding movement between outer doublets, and the bending movement of axonemes. The other analogs did not induce the dissociation or the sliding movement. 4. Among the ATP analogs tested, only 2'-dATP and 3'-dATP induced the reorientation of cilia on the Triton model of Tetrahymena; the reorientation rates were smaller than that induced by ATP.     

Isolation and characterization of dynein ATPase from bull spermatozoa.

To determine whether the abnormal insulin-secretory activity encountered in obese mice is due to an anomaly in the production of cyclic AMP, islets of lean and obese mice were incubated with forskolin under various conditions. Our data show that, in addition to the well-known quantitative differences in insulin-secretory activity between islets of lean and obese mice, there are important qualitative differences. The islets of obese mice accumulated less cyclic AMP than did those of lean mice in response to given doses of forskolin, yet their insulin secretion was enhanced to much higher values. In the islets of obese mice, but not in those of lean mice, the stimulatory effect of forskolin on insulin secretion was evident even at non-stimulatory concentrations of glucose or in Ca2+-deprived incubation media, showing that the islet of the obese mouse is less dependent on a primary stimulus (glucose) and on the provision of normal concentrations of Ca2+ in the bathing medium than is the islet of the lean mouse.     

Dictyostelium myosin II mutations that uncouple the converter swing and ATP hydrolysis cycle

During the ATP hydrolysis cycle of the Dictyostelium myosin II motor domain, two conserved alpha-helices, the SH1/SH2 helix and the relay helix, rotate in a coordinated way to induce the swing motion of the converter domain. A network of hydrophobic and ionic interactions in these two helices and the converter may ensure that the motions of these helices are effectively transmitted to the converter. To examine the roles of these interactions in the ATPase-dependent converter swing, we disrupted two conserved hydrophobic linkages among them by means of a point mutation (I499A or F692A). The resulting mutations induced only limited changes in the kinetic parameters of ATP hydrolysis, except for a marked increase of basal MgATPase activity. However, the mutant myosins completely lost their in vitro and in vivo motor functions. Measurements of the intrinsic tryptophan fluorescence and the GFP-based FRET revealed that the converter domain of these mutants did not swing during steady-state ATP hydrolysis or in the presence of tightly trapped Mg.ADP.V(i), which shows that the point mutations induced the uncoupling of the converter swing and ATP hydrolysis cycle. These results highlight the importance of these hydrophobic linkages for transmitting the coordinated twist motions of the helices to the converter as well as the requirement of this converter swing for force generation.     

Long range allosteric control of cytoplasmic dynein ATPase activity by the stalk and C-terminal domains

The dynein motor domain consists of a ring of six AAA domains with a protruding microtubule-binding stalk and a C-terminal domain of unknown function. To understand how conformational information is communicated within this complex structure, we produced a series of recombinant and proteolytic rat motor domain fragments, which we analyzed enzymatically. A recombinant 210-kDa half-motor domain fragment surprisingly exhibited a 6-fold higher steady state ATPase activity than a 380-kDa complete motor domain fragment. The increased ATPase activity was associated with a complete loss of sensitivity to inhibition by vanadate and an approximately 100-fold increase in the rate of ADP release. The time course of product release was discovered to be biphasic, and each phase was stimulated approximately 1000-fold by microtubule binding to the 380-kDa motor domain. Both the half-motor and full motor domain fragments were remarkably resistant to tryptic proteolysis, exhibiting either two or three major cleavage sites. Cleavage near the C terminus of the 380-kDa motor domain released a 32-kDa fragment and abolished sensitivity to vanadate. Cleavage at this site was insensitive to ATP or 5'-adenylyl-beta,gamma-imidodiphosphate but was blocked by ADP-AlF3 or ADP-vanadate. Based on these data, we proposed a model for long range allosteric control of product release at AAA1 and AAA3 through the microtubule-binding stalk and the C-terminal domain, the latter of which may interact with AAA1 to close the motor domain ring in a cross-bridge cycle-dependent manner.     

Inhibition of dynein ATPase by vanadate, and its possible use as a probe for the role of dynein in cytoplasmic motility.

Vanadate selectively inhibited dynein ATPase, 10^?7$\text{M}$ causing 50% inhibition under favorable conditions. Actomyosin ATPase was inhibited only by up to a thousand times higher concentration. In both cases vanadate inhibition was not competitive with ATP. Reversal by catecholamines was correlated with reduction of vanadate. The motility of demembranated sea urchin or mammalian sperm was arrested by vanadate concentrations similar to those which inhibited dynein ATPase; a thousand times higher concentration was needed to paralyze live sperm. The possible utility of vanadate sensitivity as a probe for dynein involvement in non-axonemal motile systems was explored with respect to brain ATPase associated with tubulin obtained by cycles of assembly, and ATPases associated with mitotic apparatus isolated from sea urchin embryos.     

Dynein from Dictyostelium: primary structure comparisons between a cytoplasmic motor enzyme and flagellar dynein

We report here the cloning and sequencing of a cytoplasmic dynein heavy chain gene from the cellular slime mold Dictyostelium discoideum. Using a combination of approaches, we have isolated 14,318 bp of DNA sequence which contains an open-reading frame of 4,725 amino acids. The deduced molecular weight of the polypeptide predicted by this reading frame is 538,482 D. Overall, the polypeptide sequence is 51% similar and 28% identical to the recently published sequences of the beta-dynein heavy chain from sea urchin flagella (Gibbons, I. R., B. H. Gibbons, G. Mocz, and D. J. Asai. 1991. Nature (Lond.). 352: 640-643; Ogawa, K. 1991. Nature (Lond.). 352:643-645). It contains four GXXXXGKT/S motifs that form part of a consensus sequence for ATP-binding domains; these motifs are clustered near the middle of the polypeptide. The distribution of the regions sharing sequence similarity between the Dictyostelium and sea urchin heavy chain polypeptides suggests that the amino termini of dyneins may contain domains that specify axonemal or cytoplasmic functions.