Molecular structure of dynein and motility of a giant sperm axoneme provided with only the outer dynein arm.

The peculiar sperm axoneme of the dipteran Asphondylia ruebsaameni is characterized by an extraordinarily high number of microtubule doublets (up to 2,500) arranged in double parallel spirals. Doublets of the inner row of each spiral are tilted, so that their outer arms point towards the B-tubule of the next doublet in the outer row. Doublets are provided with only the outer arm, and no structure related to the central pair/radial spoke complex is present. When analyzed by quick-freeze, deep-etch electron microscopy, the structure of the dynein arms was shown to share the same organization described in other organisms; however, it appears to be somewhat more complex than that previously found in a related dipteran species, Monarthropalpus flavus, since the foot region of the arms displays a globular extra-domain that is intercalated between adjacent arms. Treatment of demembranated sperm with ATP and vanadate induced conformational changes in the dynein arms. SDS-page suggested the presence of a single dynein high molecular weight band or, in the gels with the best electrophoretic resolution, of two very closely spaced bands. This polypeptide positively reacted with a polyclonal antibody raised against a specific amino acid sequence located in the phosphate-binding loop of the dynein catalytic site. Dynein heavy chain-related DNA sequences corresponding to the catalytic phosphate-binding region were amplified by RT-PCR. Two distinct fragments (Asph-ax1 and Asph-ax2) encoding axonemal dynein sequences were identified. Southern blot analysis performed on genomic DNA using these sequences as a probe showed that they are part of different genes. An intron was identified in the Asph-ax1 fragment at a position corresponding to the site of a nucleotide deletion in the putative pseudogene of Monarthropalpus. Asphondylia spermatozoa exhibited in vivo a whirling movement both in the deferent duct and in the spermatheca, but they were unable to undergo processive movement in vitro. They propagated a three-dimensional wave only when constrained in a bent configuration by some mechanical means. The phylogenetic relationships between the two dipteran species, Monarthopalpus and Asphondylia, based on these biochemical and molecular data are also discussed.     

Splitting the ciliary axoneme: implications for a "switch-point" model of dynein arm activity in ciliary motion

In the presence of specific inhibitors of beat. 20 microM VO4(3-) or pCa 4, mussel gill lateral (L) cilia can be arrested in two positions--"hands down" or "hands up"--at opposite ends of the stroke cycle. Cilia move to these positions by doublet microtubule sliding. Axonemes of arrested cilia, still tethered to the cell, are intact after demembranation and protease treatment. When reactivated by 4 mM ATP with inhibitors present, about 40% split apart. Splits are not random but occur preferentially between different specific doublets in the two opposite arrest positions. Several different related patterns of splitting are observed; for every pattern in "hands down" axonemes, there is a corresponding complementary split pattern in "hands up" axonemes. In some split patterns two doublets remain firmly attached to the central pair; these also differ depending on axonemal position. Although some of the patterns seen may be artifactual or difficult to explain, the complementary splitting patterns are predictable with simple assumptions by a "switch point" hypothesis of ciliary activity where, during each recovery stroke, doublets 6-8 have active dynein arms, while during each effective stroke, arms on doublets 1-4 become active, and arms 6-8 are turned off. Because of a difference between the patterns seen and the predictions, the status of the arms on doublet 9 is unresolved. The patterns also suggest that a spoke-central sheath attachment cycle may correlate with switching of arm activity during the generation of an asymmetric beat.     

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.     

Polarity and asymmetry in the arrangement of dynein and related structures in the Chlamydomonas axoneme

Understanding the molecular architecture of the flagellum is crucial to elucidate the bending mechanism produced by this complex organelle. The current known structure of the flagellum has not yet been fully correlated with the complex composition and localization of flagellar components. Using cryoelectron tomography and subtomogram averaging while distinguishing each one of the nine outer doublet microtubules, we systematically collected and reconstructed the three-dimensional structures in different regions of the Chlamydomonas flagellum. We visualized the radial and longitudinal differences in the flagellum. One doublet showed a distinct structure, whereas the other eight were similar but not identical to each other. In the proximal region, some dyneins were missing or replaced by minor dyneins, and outer-inner arm dynein links were variable among different microtubule doublets. These findings shed light on the intricate organization of Chlamydomonas flagella, provide clues to the mechanism that produces asymmetric flagellar beating, and pose a new challenge for the functional study of the flagella.     

Solution structure of a dynein motor domain associated light chain

Dyneins are molecular motors that translocate towards the minus ends of microtubules. In Chlamydomonas flagellar outer arm dynein, light chain 1 (LC1) associates with the nucleotide binding region within the gamma heavy chain motor domain and consists of a central leucine-rich repeat section that folds as a cylindrical right handed spiral formed from six beta-beta-alpha motifs. This central cylinder is flanked by terminal helical subdomains. The C-terminal helical domain juts out from the cylinder and is adjacent to a hydrophobic surface within the repeat region that is proposed to interact with the dynein heavy chain. The position of the C-terminal domain on LC1 and the unexpected structural similarity between LC1 and U2A' from the human spliceosome suggest that this domain interacts with the dynein motor domain.     

A model for the coordinated stepping of cytoplasmic dynein

Cytoplasmic dynein play an important role in transporting various intracellular cargos by coupling their ATP hydrolysis cycle with their conformational changes. Recent experimental results showed that the cytoplasmic dynein had a highly variable stepping pattern including “hand-over-hand”, “inchworm” and “nonalternating-inchworm”. Here, we developed a model to describe the coordinated stepping patterns of cytoplasmic dynein, based on its working cycle, construction and the interaction between its leading head and tailing head. The kinetic model showed how change in the distance between the two heads influences the rate of cytoplasmic dynein under different stepping patterns. Numerical simulations of the distribution of step size and striding rate are in good quantitative agreement with experimental observations. Hence, our coordinated stepping model for cytoplasmic dynein successfully explained its diverse stepping patterns as a molecular motor. The cooperative mechanism carried out by the two heads of cytoplasmic dynein shed light on the strategies adopted by the cytoplasmic dynein in executing various functions.     

Inhibition of axoneme and dynein ATPase from sea urchin sperm by free ATP

Sea urchin sperm flagellar ATPase (EC 3.6.1.3) has magnesium-ATP as an effective substrate and is inhibited by free ATP. The inhibition is prevented by high concentration of KCl or NaCl. 0.4 M KCl extracts 48% of ATPase activity from axoneme. The 0.4 M KCl extract and 0.4 M KCl-treated axoneme are also inhibited by free ATP and this inhibition is reversed by KCl. Dynein purified twice by sucrose density gradient centrifugation is alsof inhibited by free ATP; this inhibition is also reversed by KCl.     

A novel dynein light intermediate chain colocalizes with the retrograde motor for intraflagellar transport at sites of axoneme assembly in chlamydomonas and Mammalian cells

The assembly of cilia and flagella depends on bidirectional intraflagellar transport (IFT). Anterograde IFT is driven by kinesin II, whereas retrograde IFT requires cytoplasmic dynein 1b (cDHC1b). Little is known about how cDHC1b interacts with its cargoes or how it is regulated. Recent work identified a novel dynein light intermediate chain (D2LIC) that colocalized with the mammalian cDHC1b homolog DHC2 in the centrosomal region of cultured cells. To see whether the LIC might play a role in IFT, we characterized the gene encoding the Chlamydomonas homolog of D2LIC and found its expression is up-regulated in response to deflagellation. We show that the LIC subunit copurifies with cDHC1b during flagellar isolation, dynein extraction, sucrose density centrifugation, and immunoprecipitation. Immunocytochemistry reveals that the LIC colocalizes with cDHC1b in the basal body region and along the length of flagella in wild-type cells. Localization of the complex is altered in a collection of retrograde IFT and length control mutants, which suggests that the affected gene products directly or indirectly regulate cDHC1b activity. The mammalian DHC2 and D2LIC also colocalize in the apical cytoplasm and axonemes of ciliated epithelia in the lung, brain, and efferent duct. These studies, together with the identification of an LIC mutation, xbx-1(ok279), which disrupts retrograde IFT in Caenorhabditis elegans, indicate that the novel LIC is a component of the cDHC1b/DHC2 retrograde IFT motor in a variety of organisms.     

The motor activity of mammalian axonemal dynein studied in situ on doublet microtubules

Flagellar dynein generates forces that produce relative shearing between doublet microtubules in the axoneme; this drives propagated bending of flagella and cilia. To better understand dynein's role in coordinated flagellar and ciliary motion, we have developed an in situ assay in which polymerized single microtubules glide along doublet microtubules extruded from disintegrated bovine sperm flagella at a pH of 7.8. The exposed, active dynein remain attached to their respective doublet microtubules, allowing gliding of individual microtubules to be observed in an environment that allows direct control of chemical conditions. In the presence of ATP, translocation of microtubules by dynein exhibits Michaelis-Menten type kinetics, with V(max) = 4.7 +/- 0.2 microm/s and K(m) = 124 +/- 11 microM. The character of microtubule translocation is variable, including smooth gliding, stuttered motility, oscillations, buckling, complete dissociation from the doublet microtubule, and occasionally movements reversed from the physiologic direction. The gliding velocity is independent of the number of dynein motors present along the doublet microtubule, and shows no indication of increased activity due to ADP regulation. These results reveal fundamental properties underlying cooperative dynein activity in flagella, differences between mammalian and non-mammalian flagellar dynein, and establish the use of natural tracks of dynein arranged in situ on the doublet microtubules of bovine sperm as a system to explore the mechanics of the dynein-microtubule interactions in mammalian flagella.     

Relaxation-based structure refinement and backbone molecular dynamics of the dynein motor domain-associated light chain

The light chain 1 (LC1) polypeptide is a member of the leucine-rich repeat protein family and binds at or near the ATP hydrolytic site within the motor domain of the gamma heavy chain from Chlamydomonas outer arm dynein. It consists of an N-terminal helix, a central barrel formed from six leucine-rich repeats that fold as beta beta alpha units, and a C-terminal helical domain that protrudes from the main axis defined by the leucine-rich repeats. Interaction with the gamma heavy chain is likely mediated through a hydrophobic patch on the larger beta sheet face, and the C-terminal region is predicted to insert into the dynein ATP hydrolytic site. Here we have used 1H-15N heteronuclear relaxation measurements obtained at 500 and 600 MHz to refine and validate the LC1 solution structure. In this refined structure, the C-terminal helix is significantly reoriented by more than 20 degrees as compared to the control and provides a more precise understanding of the potential regulatory role of this domain. We also employed the refined structure to perform a dynamic analysis of LC1 using the 600 MHz data set. These results, which were cross validated using the 500 MHz data set, strongly support identification of the predicted LC1 binding surfaces and provide additional insight into the interaction mechanisms of leucine-rich repeat proteins.     

Inhibition of axoneme and dynein ATPase from sea urchin sperm by free ATP.

Sea urchin sperm flagellar ATPase (EC 3.6.1.3) has magnesium-ATP as an effective substrate and is inhibited by free ATP. The inhibition is prevented by high concentration of KCl or NaCl. 0.4 M KCl extracts 48% of ATPase activity from axoneme. The 0.4 M KCl extract and 0.4 M KCl-treated axoneme are also inhibited by free ATP and this inhibition is reversed by KCl. Dynein purified twice by sucrose density gradient centrifugation is also inhibited by free ATP; this inhibition is also reversed by KCl.     

Dynactin increases the processivity of the cytoplasmic dynein motor

Cytoplasmic dynein supports long-range intracellular movements of cargo in vivo but does not appear to be a processive motor protein by itself. We show here that the dynein activator, dynactin, binds microtubules and increases the average length of cytoplasmic-dynein-driven movements without affecting the velocity or microtubule-stimulated ATPase kinetics of cytoplasmic dynein. Enhancement of microtubule binding and motility by dynactin are both inhibited by an antibody to dynactin's microtubule-binding domain. These results indicate that dynactin acts as a processivity factor for cytoplasmic-dynein-based motility and provide the first evidence that cytoskeletal motor processivity can be affected by extrinsic factors.     

A direct interaction between cytoplasmic dynein and kinesin I may coordinate motor activity

Cytoplasmic dynein and kinesin I are both unidirectional intracellular motors. Dynein moves cargo toward the cell center, and kinesin moves cargo toward the cell periphery. There is growing evidence that bi-directional motility is regulated in the cell, potentially through direct interactions between oppositely oriented motors. We have identified a direct interaction between cytoplasmic dynein and kinesin I. Using the yeast two-hybrid assay and affinity chromatography, we demonstrate that the intermediate chain of dynein binds to kinesin light chains 1 and 2. The interaction is both direct and specific. Co-immunoprecipitation experiments demonstrate an interaction between endogenous proteins in rat brain cytosol. Double-label immunocytochemistry reveals a partial co-localization of vesicle-associated motor proteins. Together these observations suggest that soluble motors can interact, potentially allowing kinesin I to actively localize dynein to cellular sites of function. There is also a vesicle population with both dynein and kinesin I bound that may be capable of bi-directional motility along cellular microtubules.     

25 Angstrom resolution structure of a cytoplasmic dynein motor reveals a seven-member planar ring

Dyneins form one of the three major families of cytoskeleton-based motor proteins that together drive most of the visible forms of cell and organelle movement. We present here a 3D reconstruction of a cytoplasmic dynein motor domain obtained by electron microscopy, at 25 Angstrom resolution. This work demonstrates a basic motor architecture of a flat, slightly elliptical ring composed of seven densities arranged around a partially enclosed central cavity. We have used specific Fab tags to localize the microtubule-binding domain; the connecting stalk emerges at one end of the motor's long axis. Through proposed fitting of representative AAA domain structures, we show that the nucleotide catalytic P-1 domain is likely located at the opposite end of the motor. Thus mechanisms that couple nucleotide hydrolysis with microtubule binding must be propagated around a ring structure, in a manner clearly distinct from kinesin or myosin-mediated movements. Analysis of the Fab tagged datasets reveals classes of particles with stalks protruding at distinct angles from the motor. There is a approximately 40 degrees variation in microtubule-binding stalk angle that may reflect linkage to dynein's mechanochemical cycle. Overall, the work provides sufficient resolution to begin the mapping of landmark features onto a dynein motor, and provides a foundation for understanding the mechanics of dynein movement.     

A split motor domain in a cytoplasmic dynein

The heavy chain of dynein forms a globular motor domain that tightly couples the ATP-cleavage region and the microtubule-binding site to transform chemical energy into motion along the cytoskeleton. Here we show that, in the fungus Ustilago maydis, two genes, dyn1 and dyn2, encode the dynein heavy chain. The putative ATPase region is provided by dyn1, while dyn2 includes the predicted microtubule-binding site. Both genes are located on different chromosomes, are transcribed into independent mRNAs and are translated into separate polypeptides. Both Dyn1 and Dyn2 co-immunoprecipitated and co-localized within growing cells, and Dyn1-Dyn2 fusion proteins partially rescued mutant phenotypes, suggesting that both polypeptides interact to form a complex. In cell extracts the Dyn1-Dyn2 complex dissociated, and microtubule affinity purification indicated that Dyn1 or associated polypeptides bind microtubules independently of Dyn2. Both Dyn1 and Dyn2 were essential for cell survival, and conditional mutants revealed a common role in nuclear migration, cell morphogenesis and microtubule organization, indicating that the Dyn1-Dyn2 complex serves multiple cellular functions.     

Endocytic internalization in budding yeast requires coordinated actin nucleation and myosin motor activity

Actin polymerization essential for endocytic internalization in budding yeast is controlled by four nucleation promoting factors (NPFs) that each exhibits a unique dynamic behavior at endocytic sites. How each NPF functions and is regulated to restrict actin assembly to late stages of endocytic internalization is not known. Quantitative analysis of NPF biochemical activities, and genetic analysis of recruitment and regulatory mechanisms, defined a linear pathway in which protein composition changes at endocytic sites control actin assembly and function. We show that yeast WASP initiates actin assembly at endocytic sites and that this assembly and the recruitment of a yeast WIP-like protein by WASP recruit a type I myosin with both NPF and motor activities. Importantly, type I myosin motor and NPF activities are separable, and both contribute to endocytic coat inward movement, which likely represents membrane invagination. These results reveal a mechanism in which actin nucleation and myosin motor activity cooperate to promote endocytic internalization.     

Design and regulation of the AAA+ microtubule motor dynein

Dyneins are highly complex molecular motors that transport their attached cargo towards the minus end of microtubules. These enzymes are required for many essential motile activities within the cytoplasm and also power eukaryotic cilia and flagella. Each dynein contains one or more heavy chain motor units that consist of an N-terminal stem domain that is involved in cargo attachment, and six AAA+ domains (AAA1-6) plus a C-terminal globular segment that are arranged in a heptameric ring. At least one AAA+ domain (AAA1) is capable of ATP binding and hydrolysis, and the available data suggest that one or more additional domains also may bind nucleotide. The ATP-sensitive microtubule binding site is located at the tip of a 10nm coiled coil stalk that emanates from between AAA4 and AAA5. The function of this motor both in the cytoplasm and the flagellum must be tightly regulated in order to result in useful work. Consequently, dyneins also contain a series of additional components that serve to define the cargo-binding properties of the enzyme and which act as sensors to transmit regulatory inputs to the motor units. Here we describe the two basic dynein designs and detail the various regulatory systems that impinge on this motor within the eukaryotic flagellum.