Cytoplasmic dynein

The organization and function of eukaryotic cells rely on the action of many different molecular motor proteins. Cytoplasmic dynein drives the movement of a wide range of cargoes towards the minus ends of microtubules, and these events are needed, not just at the single-cell level, but are vital for correct development. In the present paper, I review recent progress on understanding dynein's mechanochemistry, how it is regulated and how it binds to such a plethora of cargoes. The importance of a number of accessory factors in these processes is discussed.     

Cytoplasmic dynein nomenclature

A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms.     

Cytoplasmic dynein: a key player in neurodegenerative and neurodevelopmental diseases

Cytoplasmic dynein is the most important molecular motor driving the movement of a wide range of cargoes towards the minus ends of microtubules.As a molecular motor protein,dynein performs a variety of basic cellular functions including organelle transport and centrosome assembly.In the nervous system,dynein has been demonstrated to be responsible for axonal retrograde transport.Many studies have revealed direct or indirect evidence of dynein in neurodegenerative diseases such as amyotrophic lateral sclerosis,Charcot-Marie-Tooth disease,Alzheimer’s disease,Parkinson’s disease and Huntington’s disease.Among them,a number of mutant proteins involved in various neurodegenerative diseases interact with dynein.Axonal transport disruption is presented as a common feature occurring in neurodegenerative diseases.Dynein heavy chain mutant mice also show features of neurodegenerative diseases.Moreover,defects of dynein-dependent processes such as autophagy or clearance of aggregation-prone proteins are found in most of these diseases.Lines of evidence have also shown that dynein is associated with neurodevelopmental diseases.In this review,we focus on dynein involvement in different neurological diseases and discuss potential underlying mechanisms.     

Cytoplasmic dynein mediates adenovirus binding to microtubules

During infection, adenovirus (Ad) capsids undergo microtubule-dependent retrograde transport as part of a program of vectorial transport of the viral genome to the nucleus. The microtubule-associated molecular motor, cytoplasmic dynein, has been implicated in the retrograde movement of Ad. We hypothesized that cytoplasmic dynein constituted the primary mode of association of Ad with microtubules. To evaluate this hypothesis, an Ad-microtubule binding assay was established in which microtubules were polymerized with taxol, combined with Ad in the presence or absence of microtubule-associated proteins (MAPs), and centrifuged through a glycerol cushion. The addition of purified bovine brain MAPs increased the fraction of Ad in the microtubule pellet from 17.3% +/- 3.5% to 80.7% +/- 3.8% (P < 0.01). In the absence of tubulin polymerization or in the presence of high salt, no Ad was found in the pellet. Ad binding to microtubules was not enhanced by bovine brain MAPs enriched for tau protein or by the addition of bovine serum albumin. Enhanced Ad-microtubule binding was also observed by using a fraction of MAPs purified from lung A549 epithelial cell lysate which contained cytoplasmic dynein. Ad-microtubule interaction was sensitive to the addition of ATP, a hallmark of cytoplasmic dynein-dependent microtubule interactions. Immunodepletion of cytoplasmic dynein from the A549 cell lysate abolished the MAP-enhanced Ad-microtubule binding. The interaction of Ad with both dynein and dynactin complexes was demonstrated by coimmunoprecipitation. Partially uncoated capsids isolated from cells 40 min after infection also exhibited microtubule binding. In summary, the primary mode of Ad attachment to microtubules occurs though cytoplasmic dynein-mediated binding.     

Cytoplasmic dynein intermediate chain phosphorylation regulates binding to dynactin

Previously, we identified dynactin as a cargo receptor or adaptor for cytoplasmic dynein, mediated by an interaction between the dynein intermediate chain and p150(Glued). To test phosphorylation as a potential regulatory mechanism for this interaction, we analyzed cytoplasmic dynein by two-dimensional gel analysis and detected two intermediate chain variants, one of which was eliminated by phosphatase treatment. Overlay assays demonstrated that p150(Glued) bound dephosphorylated but not phosphorylated intermediate chains. We then subjected the purified cytoplasmic dynein intermediate chain to mass spectrometry and identified a single phosphorylated tryptic fragment corresponding to the p150(Glued)-binding domain. Fragmentation and retention time analysis mapped the phosphorylation site to serine 84. Site-directed mutants designed to mimic the dephosphorylated or phosphorylated intermediate chain disrupted both in vitro phosphorylation and in vivo phosphorylation of transfected proteins. Mutants mimicking the dephosphorylated form bound p150(Glued) in vitro and overexpression perturbed transport of dynein-dependent membranes. Mutants mimicking the phosphorylated form displayed diminished p150(Glued) binding in vitro and did not disrupt dynein-mediated transport when expressed in vivo. These findings represent the first mapping of an intermediate chain phosphorylation site and suggest that this phosphorylation plays an important role in regulating the binding of cytoplasmic dynein to dynactin.     

Mitochondria could be a potential key mediator linking the intestinal microbiota to depression

The intestinal microbiota has been reported to affect depression, a common mental condition with severe health-related consequences. However, what mediates the effect of the intestinal microbiota on depression has not been well elucidated. We summarize the roles of the mitochondria in eliciting beneficial effects on the gut microbiota to ameliorate symptoms of depression. It is well known that mitochondria play a key role in depression. An important pathogenic factor, namely inflammatory response, may adversely impact mitochondrial functionality to maintain cellular homeostasis. Dysfunction of mitochondria not only affects neuronal function but also reduces neuron cell numbers. We posit that the intestinal microbiota could affect neuronal mitochondrial function through short-chain fatty acids such as butyrate. Brain inflammatory processes could also be affected through the modulation of gut permeability and blood lipopolysaccharide levels. Aberrant mitochondria functionality coupled to adverse cellular homeostasis could be a key mediator for the effect of the intestinal microbiota on the progression of depression.     

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.     

Big steps toward understanding dynein

Dynein is a microtubule-based molecular motor that is involved in various biological functions, such as axonal transport, mitosis, and cilia/flagella movement. Although dynein was discovered 50 years ago, the progress of dynein research has been slow due to its large size and flexible structure. Recent progress in understanding the force-generating mechanism of dynein using x-ray crystallography, cryo-electron microscopy, and single molecule studies has provided key insight into the structure and mechanism of action of this complex motor protein.It has been 50 years since dynein was discovered and named by Ian Gibbons as a motor protein that drives cilia/flagella bending (Gibbons, 1963; Gibbons and Rowe, 1965). In the mid-1980s, dynein was also found to power retrograde transport in neurons (Paschal and Vallee, 1987). Subsequently, the primary amino acid sequence of the cytoplasmic dynein heavy chain, which contains the motor domain, was determined from the cDNA sequence (Mikami et al., 1993; Zhang et al., 1993). Like other biological motors, such as kinesins and myosins, the amino acid sequence of the dynein motor domain is well conserved. There are 16 putative genes that encode dynein heavy chains in the human genome (Yagi, 2009). Among these is one gene encoding cytoplasmic dynein heavy chain and one encoding retrograde intraflagellar transport dynein heavy chain, while the rest encode for heavy chains of axonemal dyneins. Most of the genes encoding the human dynein heavy chain have a counterpart in Chlamydomonas reinhardtii, which suggests that their functions are conserved from algae to humans.Dynein is unique compared with kinesin and myosin because dynein molecules form large molecular complexes. For example, one axonemal outer arm dynein molecule of C. reinhardtii is composed of three dynein heavy chains, two intermediate chains, and more than ten light chains (King, 2012). Mammalian cytoplasmic dynein consists of two heavy chains and several smaller subunits (Fig. 1 A; Vallee et al., 1988; Allan, 2011). The cargoes of cytoplasmic dynein are various membranous organelles, including lysosomes, endosomes, phagosomes, and the Golgi complex (Hirokawa, 1998). It is likely that one cytoplasmic dynein heavy chain can adapt to diverse cargos and functions by changing its composition.Open in a separate windowFigure 1.Atomic structures of cytoplasmic dynein. (A) Schematic structure of cytoplasmic dynein complex, adapted from Allan (2011). (B) The primary structure of cytoplasmic dynein. (C and D) The atomic model of D. discoideum cytoplasmic dynein motor domain (PDB accession no. 3VKG) overlaid on a microtubule (EMDB-5193; Sui and Downing, 2010) according to the orientation determined by Mizuno et al. (2007) (C) Side view. (D) View from the plus end of microtubule. (E) Schematic domain structure of dynein.Dynein must have a distinct motor mechanism from kinesin and myosin, because it belongs to the AAA+ family of proteins and does not have the conserved amino acid motifs, called the switch regions, present in kinesins, myosins, and guanine nucleotide-binding proteins (Vale, 1996). Therefore, studying dynein is of great interest because it will reveal new design principles of motor proteins. This review will focus on the mechanism of force generation by cytoplasmic and axonemal dynein heavy chains revealed by recent structural and biophysical studies.

Anatomy of dynein

To understand the chemomechanical cycle of dynein based on its molecular structure, it is important to obtain well-diffracting crystals and build accurate atomic models. Recently, Kon and colleagues determined the crystal structures of Dictyostelium discoideum cytoplasmic dynein motor domain, first at 4.5-Å resolution (Kon et al., 2011), and subsequently at 2.8 Å (without the microtubule binding domain) and 3.8-Å (wild type) resolution (Kon et al., 2012). Carter and colleagues also determined the crystal structures of the Saccharomyces cerevisiae (yeast) cytoplasmic dynein motor domain, first at 6-Å resolution (Carter et al., 2011), and later at 3.3–3.7-Å resolution (Schmidt et al., 2012). According to these crystal structures as well as previous EM studies, the overall structure of the dynein heavy chain is divided into four domains: tail, linker, head, and stalk (Fig. 1, B–E). Simply put, each domain carries out one essential function of a motor protein: the tail is the cargo binding domain, the head is the site of ATP hydrolysis, the linker is the mechanical amplifier, and the stalk is the track-binding domain.The tail, which is not part of the motor domain and is absent from crystal structures, is located at the N-terminal ∼1,400 amino acid residues and involved in cargo binding (gray in Fig. 1, B and E). The next ∼550 residues comprise the “linker” (pink in Fig. 1, B–E), which changes its conformation depending on the nucleotide state (Burgess et al., 2003; Kon et al., 2005). This linker domain was first observed by negative staining EM in combination with single particle analysis of dynein c, an isoform of inner arm dynein from C. reinhardtii flagella (Burgess et al., 2003). According to the crystal structures, the linker is made of bundles of α-helices and lies across the AAA+ head domain, forming a 10-nm-long rod-like structure (Fig. 1, C and D). Recent class averaged images of D. discoideum cytoplasmic dynein show that the linker domain is stiff along its entire length when undocked from the head (Roberts et al., 2012). The head (motor) domain of dynein is composed of six AAA+ (ATPase associated with diverse cellular activities) modules (Neuwald et al., 1999; color-coded in Fig. 1, B–E). Although many AAA+ family proteins are a symmetric homohexamer (Ammelburg et al., 2006), the AAA+ domains of dynein are encoded by a single heavy chain gene and form an asymmetric heterohexamer. Among the six AAA+ domains, hydrolysis at the first AAA domain mainly provides the energy for dynein motility (Imamula et al., 2007; Kon et al., 2012). The hexameric ring has two distinct faces: the linker face and the C-terminal face. The linker face is slightly convex and the linker domain lies across this side (Fig. 1 D, left side). The other side of the ring has the C-terminal domain (Fig. 1 D, right side).The stalk domain of dynein was identified as the microtubule-binding domain (MTBD; Gee et al., 1997). It emanates from the C-terminal face of AAA4 and is composed of antiparallel α-helical coiled-coil domain (yellow in Fig. 1, B–E). The tip of the stalk is the actual MTBD. Interestingly, the crystal structures revealed another antiparallel α-helical coiled coil that emerges from AAA5 (orange in Fig. 1, B–E), and this region is called the buttress (Carter et al., 2011) or strut (Kon et al., 2011), which was also observed as the bifurcation of the stalk by negative-staining EM (Burgess et al., 2003; Roberts et al., 2009). The tip of the buttress/strut is in contact with the middle of the stalk and probably works as a mechanical reinforcement of the stalk.

The chemomechanical cycle of dynein

Based on structural and biochemical data, a putative chemomechanical cycle of dynein is outlined in Fig. 2 (A–E). In the no-nucleotide state, dynein is bound to a microtubule through its stalk domain, and its tail region is bound to cargoes (Fig. 2 A). The crystal structures of yeast dynein are considered to be in this no-nucleotide state. When ATP is bound to the AAA+ head, the MTBD quickly detaches from the microtubule (Fig. 2 B; Porter and Johnson, 1983). The ATP binding also induces “hinging” of the linker from the head (Fig. 2 C). According to the biochemical analysis of recombinant D. discoideum dynein (Imamula et al., 2007), the detachment from the microtubule (Fig. 2, A and B) is faster than the later hinging (Fig. 2, B and C). As a result of these two reactions, the head rotates or shifts toward the minus end of the microtubule (for more discussion about “rotate” versus “shift” see the “Dyneins in the axoneme” section) and the MTBD steps forward. The directionality of stepping seems to be mainly determined by the MTBD, because the direction of dynein movement does not change even if the head domain is rotated relative to the microtubule by insertion or deletion of the stalk (Carter et al., 2008). In the presence of ADP and vanadate, dynein is considered to be in this state (Fig. 2 C).Open in a separate windowFigure 2.Presumed chemomechanical cycle and stepping of dynein. (A–E) Chemomechanical cycle of dynein. The pre- and post-power stroke states are also called the primed and unprimed states, respectively. The registries of the stalk coiled coil are denoted as α and β according to Gibbons et al. (2005). (F and G) Processive movement of kinesin (F) and dynein (G). (F) Hand-over-hand movement of kinesin. A step by one head (red) is always followed by the step of another head (green). The stepping of kinesin is on one protofilament of microtubule. (G) Presumed stepping of dynein. The step size varies and the interhead separation can be large. A step by one head (red) is not always flowed by the step of another head (green). (H) A model of strain-based dynein ATPase activation. (G, top) Without strain, the gap between the AAA1 and AAA2 is open and the motor domain cannot hydrolyze ATP. (G, bottom) Under a strain imposed between MTBD and tail (thin black arrows), the gap becomes smaller (thick black arrows) and turns on ATP hydrolysis by dynein.After the MTBD rebinds to the microtubule at the forward site (Fig. 2 D), release of hydrolysis products from the AAA+ head is activated (Holzbaur and Johnson, 1989) and the hinged linker goes back to the straight conformation (Fig. 2 E; Kon et al., 2005). The crystal structure of D. discoideum dynein is considered to be in the state after phosphate release and before ADP release. This straightening of the linker is considered to be the power-generating step and brings the cargo forward relative to the microtubule.

The MTBD of dynein

As outlined in Fig. 2, the nucleotide state of the head domain may control the affinity of the MTBD to the microtubule. Conversely, the binding of the MTBD to the microtubule should activate the ATPase activity of the head domain. This two-way communication is transmitted through the simple ∼17-nm-long α-helical coiled-coil stalk and the buttress/strut, and its structural basis has been a puzzling question.Currently there are three independent MTBD atomic structures in the Protein Data Bank (PDB): One of the crystal structures of the D. discoideum dynein motor domain contains the MTBD (Fig. 3 A), and Carter et al. (2008) crystallized the MTBD of mouse cytoplasmic dynein fused with a seryl tRNA-synthetase domain (Fig. 3 C). The MTBD structure of C. reinhardtii axonemal dynein was solved using nuclear magnetic resonance (PDB accession no. 2RR7; Fig. 3 B). The MTBD is mostly composed of α-helices and the three structures are quite similar to each other within the globular MTBD (Fig. 3). Note that dynein c has an additional insert at the MTBD–microtubule interface (Fig. 3 B, inset), whose function is not yet clear. The three structures start to deviate from the junction between the MTBD and the coiled-coil region of the stalk (Fig. 3, A–C, blue arrowheads). Particularly, one of the stalk α-helix (CC2) in D. discoideum dynein motor domain appears to melt at the junction with the MTBD (Fig. 3 A, red arrowhead). This structural deviation suggests that the stalk coiled coil at the junction is flexible, which is consistent with the observation by EM (Roberts et al., 2009).Open in a separate windowFigure 3.Atomic models of the MTBD of dynein. (A) D. discoideum cytoplasmic dynein (PDB accession no. 3VKH). (B) C. reinhardtii dynein c (PDB accession no. 2RR7). The inset shows the side view, highlighting the dynein c–specific insert. (C) Mouse cytoplasmic dynein (PDB accession no. 3ERR). (D) Mouse cytoplasmic dynein fit to the MTBD–microtubule complex derived from cryo-EM (PDB accession no. 3J1T). All the MTBD structures were aligned using least square fits and color-coded with a gradient from the N to C terminus. CC1, coiled coil helix 1; CC2, coiled coil helix 2. The blue arrowheads points to the junction between MTBD and the stalk, where a well-conserved proline residue (colored pink) is located. In C and D, two residues (isoleucine 3269 and leucine 3417) are shown as spheres. The two residues form hydrophobic contacts in the β-registry (C), whereas they are separated in the α-registry (D) because of the sliding between the two α-helices (blue and red arrows). Conformational changes observed in the mouse dynein MTBD in complex with a microtubule by cryo-EM are shown by black arrows. Note that the cryo-EM density map does not have enough resolution to observe sliding between CC1 and CC2. The sliding was modeled based on targeted molecular dynamics (Redwine et al., 2012).Various mechanisms have been proposed to explain how the affinity between the MTBD and a microtubule is controlled. Gibbons et al. (2005) proposed “the helix-sliding hypothesis” (for review see Cho and Vale, 2012). In brief, this hypothesis proposes that the sliding between two α-helices CC1 and CC2 (Fig. 3, C and D; blue and red arrows) may control the affinity of this domain to a microtubule. When Gibbons’s classification (Gibbons et al., 2005) of the sliding state is applied to the three MTBD structures, the stalk in the D. discoideum dynein motor domain is in the “α-registry” state (not visible in Fig. 3 A because of the melting of CC2), which corresponds to the strong binding state. However, the mouse cytoplasmic and C. reinhardtii axonemal MTBDs have the “β-registry” stalk (Fig. 3 C), which corresponds to the weak binding state.To observe conformational changes induced by the α-registry and/or microtubule binding, Redwine et al. (2012) solved the structure of mouse dynein MTBD in complex with a microtubule at 9.7-Å resolution using cryo-EM and single particle analysis. The MTBD was coupled with seryl tRNA-synthetase to fix the stalk helix in the α-registry. At this resolution, α-helices are visible, and they used molecular dynamics to fit the crystal structure of mouse MTBD (β-registry) to the cryo-EM density map. According to this result, the first helix H1 moves ∼10 Å to a position that avoids a clash with the microtubule (Fig. 3 D, black arrows). This also induces opening of the stalk helix (CC1). Together with mutagenesis and single-molecule motility assays, Redwine et al. (2012) proposed that this new structure represents the strong binding state. Currently, it is not clear why the MTBD structure of D. discoideum dynein motor domain (α-registry, Fig. 3 A) is not similar to the new α-registry mouse dynein MTBD, and this problem needs to be addressed by further studies.

Structures around the first ATP binding site

Another central question about motor proteins is how Ångstrom-scale changes around the nucleotide are amplified to generate steps >8 nm. For dynein, the interface between the first nucleotide-binding pocket and the linker seem to be the key force-generating element (Fig. 4). The crystal structures of dynein give us clues about how nucleotide-induced conformational changes may be transmitted to and amplified by the linker domain.Open in a separate windowFigure 4.Structures around the first ATP binding site. (A) Schematic domain structure of the head domain. Regions contacting the linker domain are colored purple. (B) AAA submodules surrounding the first nucleotide-binding pocket (PDB accession no. 3VKG, chain A). The linker is connected to AAA1 domain by the “N-loop.” To highlight that the two finger-like structures are protruding, the shadow of the atomic structure has been cast on the plane parallel to the head domain. (C) Interaction between the linker and the two finger-like structures. The pink arrowhead points to the hinge-like structure of the linker. The pink numbers indicates the subdomain of the linker.The main ATP catalytic site is located between AAA1 and AAA2 (Fig. 4, A and B). There are four ADP molecules in the D. discoideum dynein crystal structures, but the first ATP binding site alone drives the microtubule-activated ATPase activity, based on biochemical experiments on dyneins whose ATP binding sites were mutated (Kon et al., 2012).One AAA+ module is composed of a large submodule and a small α submodule (Fig. 4 B). The large α/β submodule is located inside of the ring and the small α submodule is located outside. The large submodule bulges toward the linker face, and the overall ring forms a dome-like shape (Fig. 1 D).The main ATP catalytic site is surrounded by three submodules: AAA1 large α/β, AAA1 small α, and AAA2 large α/β (Fig. 4, A and B). Based on the structural changes of other AAA+ proteins (Gai et al., 2004; Suno et al., 2006; Wendler et al., 2012), the gap between AAA1 and AAA2 modules is expected to open and close during the ATPase cycle.In fact, the size of the gap varies among the dynein crystal structures. The crystal structures of yeast dyneins show a larger gap between AAA1 and AAA2, which might be the reason why no nucleotide was found in the binding pocket. Although Schmidt et al. (2012) soaked the crystals in a high concentration of various nucleotides (up to 25 mM of ATP), no electron densities corresponding to the nucleotide were observed at the first ATP binding site. Among dynein crystal structures, one of D. discoideum dynein (PDB accession no. 3VKH, chain A) has the smallest gap, but it is still considered to be in an “open state” because the arginine finger in the AAA2 module (Fig. 4 B, red) is far from the phosphates of ADP. Because the arginine finger is essential for ATP hydrolysis in other AAA+ proteins (Ogura et al., 2004), the gap is expected to close and the arginine finger would stabilize the negative charge during the transition state of ATP hydrolysis.The presumed open/close conformational change between AAA1 and AAA2 would result in the movement of two “finger-like” structures protruding from the AAA2 large α/β submodule (Fig. 4 B). The two finger-like structures are composed of the H2 insert β-hairpin and preSensor I (PS-I) insert. In D. discoideum dynein crystal structure, the two finger-like structures are in contact with the “hinge-like cleft” of the linker (Fig. 4 C, pink arrowhead). The hinge-like cleft is one of the thinnest parts of the linker, where only one α-helix is connecting between the linker subdomains 2 and 3.In the yeast crystal structures, which have wider gaps between AAA1 and AAA2, the two finger-like structures are not in direct contact with the linker and separated by 18 Å. Instead, the N-terminal region of the linker is in contact with the AAA5 domain (Fig. 4 A). To test the functional role of the linker–AAA5 interaction, Schmidt et al. (2012) mutated a residue involved in the interaction (Phe3446) and found that the mutation resulted in severe motility defects, showing strong microtubule binding and impaired ATPase activities. In D. discoideum dynein crystals, there is no direct interaction between AAA5 and the linker, which suggests that the gap between AAA1 and AAA2 may influence the interaction between the head and linker domain. The contact between the linker and AAA5 may also influence the gap around AAA5, because the gap between AAA5 and AAA6 is large in yeast dynein crystal, whereas the one between AAA4 and AAA5 is large in D. discoideum dynein.The movement of two finger-like structures would induce remodeling of the linker. According to the recent cryo-EM 3D reconstructions of cytoplasmic dynein and axonemal dynein c (Roberts et al., 2012), the linker is visible across the head and there is a large gap between AAA1 and AAA2 in the no-nucleotide state. This linker structure is considered to be the “straight” state (Fig. 2, A and E). In the presence of ADP vanadate, the gap between AAA1 and AAA2 is closed and the N-terminal region of linker is near AAA3, which corresponds to the pre-power stroke “hinged” state (Fig. 2, C and D). The transition from the hinged state to the straight state of the linker is considered to be the force-generating step of dynein.

Processivity of dynein

As the structure of dynein is different from other motor proteins, dynein’s stepping mechanism is also distinct. Both dynein and kinesin are microtubule-based motors and move processively. Based on the single molecule tracking experiment with nanometer accuracy (Yildiz et al., 2004), it is widely accepted that kinesin moves processively by using its two motor domain alternately, called the “hand-over-hand” mechanism. To test whether dynein uses a similar mechanism to kinesin or not, recently Qiu et al. (2012) and DeWitt et al. (2012) applied similar single-molecule approaches to dynein.To observe the stepping, the two head domains of yeast recombinant cytoplasmic dynein were labeled with different colors and the movement of two head domains was tracked simultaneously. If dynein walks by the hand-over-hand mechanism, the step size would be 16 nm and the stepping of one head domain would always be followed by the stepping of another head domain (alternating pattern), and the trailing head would always take a step (Fig. 2 F). Contrary to this prediction, both groups found that the stepping of the head domains is not coordinated when the two head domains are close together. These observations indicated that the chances of a leading or trailing head domain stepping are not significantly different (Fig. 2 G; DeWitt et al., 2012; Qiu et al., 2012).This stepping pattern predicts that the distance between the head domains can be long. In fact, the distance between the two head domains is on average ∼18 ± 11 nm (Qiu et al., 2012) or 28.4 ± 10.7 nm (DeWitt et al., 2012), and as large as ∼50 nm (DeWitt et al., 2012). When the two head domains are separated, there is a tendency where stepping of the trailing head is preferred over that of the forward head.In addition, even though the recombinant cytoplasmic dynein is a homodimer, the two heavy chains do not function equally. While walking along the microtubule, the leading head tends to walk on the right side, whereas the trailing head walks on the left side (DeWitt et al., 2012; Qiu et al., 2012). This arrangement suggests that the stepping mechanism is different between the two heads. In fact, when one of the two dynein heavy chains is mutated to abolish the ATPase activity at AAA1, the heterodimeric dynein still moves processively (DeWitt et al., 2012), with the AAA1-mutated dynein heavy chain remaining mostly in the trailing position. This result clearly demonstrates that allosteric communication between the two AAA1 domains is not required for processivity of dynein. It is likely that the mutated head acts as a tether to the microtubule, as it is known that wild-type dynein can step processively along microtubules under external load even in the absence of ATP (Gennerich et al., 2007).These results collectively show that dynein moves by a different mechanism from kinesin. It is likely that the long stalk and tail allow dynein to move in a more flexible manner.

Dyneins in the axoneme

As mentioned in the introduction, >10 dyneins work in motile flagella and cilia. The core of flagella and cilia is the axoneme, which is typically made of nine outer doublet microtubules and two central pair microtubules (“9 + 2,” Fig. 5 A). The axonemes are found in various eukaryotic cells ranging from the single-cell algae C. reinhardtii to human. Recent extensive cryo-electron tomography (cryo-ET) in combination with genetics revealed the highly organized and complex structures of axonemes that are potentially important for regulating dynein activities (Fig. 5, C and D; Nicastro et al., 2006; Bui et al., 2008, 2009, 2012; Heuser et al., 2009, 2012; Movassagh et al., 2010; Lin et al., 2012; Carbajal-González et al., 2013; Yamamoto et al., 2013).Open in a separate windowFigure 5.Arrangement of axonemal dyneins. (A) The schematic structure of the motile 9 + 2 axoneme, viewed from the base of flagella. (B) Quasi-planar asymmetric movement of the 9 + 2 axoneme typically observed in trachea cilia or in C. reinhardtii flagella. (C and D) 3D structure of a 96-nm repeat of doublet microtubules in the distal/central region of C. reinhardtii flagella (EMDB-2132; Bui et al., 2012). N-DRC, the nexin-dynein regulatory complex; ICLC, intermediate chain/light chain complex. Inner arm dynein subspecies are labeled according to Bui et al. (2012) and Lin et al. (2012). To avoid the confusion with the linker domain of dynein, the structures connecting between outer and inner arm dyneins are labeled as “connecters,” which are normally called “linkers.” Putative ATP binding sites of outer arm dynein determined by biotin-ADP (Oda et al., 2013) are indicated by orange circles. The atomic structure of cytoplasmic dynein is placed into the β-heavy chain of outer arm dynein and its enlarged view is shown in the inset. (D) Two doublet microtubules, viewed from the base of flagella.The basic mechanochemical cycles of axonemal dyneins are believed to be shared with cytoplasmic dynein. Dynein c is an inner arm dynein of C. reinhardtii and used extensively to investigate the conformational changes of dynein, as shown in Fig. 2 (A–E), by combining EM and single-particle analysis (Burgess et al., 2003; Roberts et al., 2012). Structural changes of axonemal dyneins complexed with microtubules are also observed by quick-freeze and deep-etch EM (Goodenough and Heuser, 1982; Burgess, 1995), cryo-EM (Oda et al., 2007), negative-staining EM (Ueno et al., 2008), and cryo-ET (Movassagh et al., 2010). According to these studies, the AAA+ head domains are constrained near the A-tubule in the no-nucleotide state. In the presence of nucleotide, the head domains move closer to the B-tubule and/or the minus end of microtubule, and their appearance becomes heterogeneous, which is consistent with the observation of isolated dynein c that shows greater flexibility between tail and stalk in the ADP/vanadate state (Burgess et al., 2003).One of the controversies about the structural changes of axonemal dyneins is whether their stepping involves “rotation” or “shift” of the head (Fig. 2, B to D). The stalk angle relative to the microtubule seems to be a constant ∼60° irrespective of the nucleotide state (Ueno et al., 2008; Movassagh et al., 2010). This angle is similar to the angle obtained from cryo-EM study of the MTBD–microtubule complex (Redwine et al., 2012). Based on these observations, Ueno et al. (2008) and Movassagh et al. (2010) hypothesize that the “shift” of the head pulls the B-microtubule toward the distal end. However, Roberts et al. (2012) propose that the “rotation” of head and stalk is involved in the stepping based on the docking of dynein c head into an averaged flagella tomogram obtained by Movassagh et al. (2010). This issue needs to be resolved by more reliable and high-resolution data, but these two models may not be mutually exclusive. For example, averaged tomograms may be biased toward the microtubule-bound stalk because tomograms are aligned using microtubules.To interpret these structural changes of axonemal dyneins, docking atomic models of dynein is necessary. According to Roberts et al. (2012), the linker face of inner arm dynein c is oriented outside of axoneme (Fig. 5 D). For outer arm dyneins, we used cryo-EM in combination with biotin-ADP-streptavidin labeling and showed that the ATP binding site, most likely AAA1, is on the left side of the AAA+ head (Fig. 5 C; Oda et al. (2013)). Assuming that the stalks extend out of the plane toward the viewer, the linker face of outer arm dynein is oriented outside of axoneme (Fig. 5 C, inset; and Fig. 5 D). If it were the opposite, the AAA1 would be located on the right side of the AAA+ head. In summary, both inner and outer arm dynein seem to have the same arrangement, with their linker face oriented outside of the axoneme (Fig. 5 D).A unique characteristic of axonemal dyneins is that these dyneins are under precise temporal and spatial control. To generate a planer beating motion (Fig. 5 B), dyneins should be asymmetrically controlled, because the dyneins located on doublets 2–4 drive the effective stroke, whereas the ones on doublets 6–8 drive the recovery stroke (Fig. 5 A). Based on the cryo-ET observation of axonemes, Nicastro et al. (2006) proposed that “linkers” between dyneins provide hard-wiring to coordinate motor activities. Because the linkers in axonemes are distinct structures from the linker domain of dynein, for clarity, here we call them “connecters.” According to the recent cryo-ET of proximal region of C. reinhardtii flagella (Bui et al., 2012), there are in fact asymmetries among nine doublets that are localized to the connecters between outer and inner arm dynein, called the outer-inner dynein (OID) connecters (Fig. 5, A and C). Recently we identified that the intermediate chain 2 (IC2) of outer arm dynein is a part of the OID connecters, and a mutation of the N-terminal region of IC2 affects functions of both outer and inner arm dyneins (Oda et al., 2013), which supports the idea that the connecters between dyneins are involved in axonemal dynein regulation.

Closing remarks

Thanks to the crystal structures, we can now design and interpret experiments such as single molecule assays and EM based on the atomic models of dynein. Our understanding of the molecular mechanism and cellular functions of dyneins will be significantly advanced by these experiments in the near future.One important direction of dynein research is to understand the motor mechanisms closer to the in vivo state. For example, the step sizes of cytoplasmic dynein purified from porcine brain is ∼8 nm independent of load (Toba et al., 2006). This result suggests that intermediate and light chain bound to the dynein heavy chain may modulate the motor activity of dynein. To address such questions, Trokter et al. (2012) reconstituted human cytoplasmic dynein complex from recombinant proteins, although the reconstituted dynein did not show robust processive movement. Further studies are required to understand the movement of cytoplasmic dynein. Similarly, axonemal dyneins should also be studied using mutations in a specific gene that does not affect the overall flagella structure, rather than depending on null mutants that cause the loss of large protein complexes.Detailed full chemomechanical cycle of dynein and its regulation are of great importance. Currently, open/closed states of the gap between AAA1 and AAA2 are not clearly correlated with the chemomechanical cycle of dynein. Soaking dynein crystal with nucleotides showed that the presence of ATP alone is not sufficient to close the gap, at least in the crystal (Schmidt et al., 2012). This result suggests that other factors such as a conformational change of the linker are required. For other motors, ATP hydrolysis is an irreversible chemical step, which is often “gated” by strain. In the case of kinesin, ATP is hydrolyzed by a motor domain only when a forward strain is applied by the other motor domain through the neck linker (Cross, 2004; Kikkawa, 2008). A similar strain-based gating mechanism may play important roles in controlling the dynein ATPase. Upon MTBD binding to the forward binding site, a strain between MTBD and tail would be applied to the dynein molecule. The Y-shaped stalk and strut/buttress under the strain would force the head domain to close the gap between AAA4 and AAA5 (Fig. 2 H). Similarly, the linker under the strain would be hooked onto the two finger-like structures and close the gap between AAA1 and AAA2 (Fig. 2 H). The gap closure then triggers ATP hydrolysis by dynein. This strain-based gating of dynein is consistent with the observation that the rate of nonadvancing backward steps, which would depend on ATP hydrolysis, is increased by load applied to dynein (Gennerich et al., 2007). To explain cilia and flagella movement, the geometric clutch hypothesis has been proposed (Lindemann, 2007), which contends that the forces transverse (t-force) to the axonemal axis act on the dynein to regulate dynein activities. In the axoneme, dynein itself can be the sensor of the t-force by the strain-based gating mechanism. Further experiments are required to test this idea, but the strain-based gating could be a shared property of biological motors.     

Cytoplasmic dynein: advances in microtubule-based motility

It is four years since the discovery that a cytoplasmic form of dynein was able to produce force along microtubules in the opposite direction to kinesin. Recent evidence has supported a role for this cytoplasmic dynein in retrograde organelle transport, as well as other forms of intracellular motility.     

Computational approaches to understanding protein aggregation in neurodegeneration

The generation of toxic non-native protein conformers has emerged as a unifying thread among disorders such as Alzheimer＇s disease, Parkinson＇s disease, and amyotrophic lateral sclerosis. Atomic-level detail regarding dynamical changes that facilitate protein aggre- gation, as well as the structural features of large-scale ordered aggregates and soluble non-native oligomers, would contribute signifi- cantly to current understanding of these complex phenomena and offer potential strategies for inhibiting formation of cytotoxic species. However, experimental limitations often preclude the acquisition of high-resolution structural and mechanistic information for aggregating systems. Computational methods, particularly those combine both aU-atom and coarse-grained simulations to cover a wide range of time and length scales, have thus emerged as crucial tools for investigating protein aggregation. Here we review the current state of computational methodology for the study of protein self-assembly, with a focus on the application of these methods toward understanding of protein aggregates in human neurodegenerative disorders.     

Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein

Using a yeast two-hybrid human brain cDNA library screen, the cytoplasmic dynein light chain (LC8), a 10-kDa protein, was found to interact strongly with the phosphoprotein (P) of two lyssaviruses: rabies virus (genotype 1) and Mokola virus (genotype 3). The high degree of sequence divergence between these P proteins (only 46% amino acid identity) favors the hypothesis that this interaction is a common property shared by all lyssaviruses. The P protein-dynein LC8 interaction was confirmed by colocalization with laser confocal microscopy in infected cells and by coimmunoprecipitation. The dynein-interacting P protein domain was mapped to the 186 amino acid residues of the N-terminal half of the protein. Dynein LC8 is a component of both cytoplasmic dynein and myosin V, which are involved in a wide range of intracellular motile events, such as microtubule minus-end directed organelle transport in axon "retrograde transport" and actin-based vesicle transport, respectively. Our results provide support for a model of viral nucleocapsid axoplasmic transport. Furthermore, the role of LC8 in cellular mechanisms other than transport, e.g., inhibition of neuronal nitric oxide synthase, suggests that the P protein interactions could be involved in physiopathological mechanisms of rabies virus-induced pathogenesis.     

Cytoplasmic dynein nucleates microtubules to organize them into radial arrays in vivo

Numerous evidence demonstrates that dynein is crucial for organization of microtubules (MTs) into radial arrays, but its exact function in this process is unclear. Here, we studied the role of cytoplasmic dynein in MT radial array formation in the absence of the centrosome. We found that dynein is a potent MT nucleator in vitro and that stimulation of dynein activity in cytoplasmic fragments of melanophores induces nucleation-dependent formation of MT radial array in the absence of the centrosome. This new property of dynein, in combination with its known role as an MT motor that is essential for MT array organization in the absence and presence of the centrosome, makes it a unique molecule whose activity is necessary and sufficient for the formation and maintenance of MT radial arrays in cells.     

Cytoplasmic dynein ATPase activity is regulated by dynactin-dependent phosphorylation

Cytoplasmic dynein is a microtubule-associated motor that utilizes ATP hydrolysis to conduct minus-end directed transport of various organelles. Dynactin is a multisubunit complex that has been proposed to both link dynein with cargo and activate dynein motor function. The mechanisms by which dynactin regulates dynein activity are not clear. In this study, we examine the role of dynactin in regulating dynein ATPase activity. We show that dynein-microtubule binding and ATP-dependent release of dynein from microtubules are reduced in dynactin null mutants, Deltaro-3 (p150(Glued)) and Deltaro-4 (Arp1), relative to wild-type. The dynein-microtubule binding activity, but not the ATP-dependent release of dynein from microtubules, is restored by in vitro mixing of extracts from dynein and dynactin mutants. Dynein produced in a Deltaro-3 mutant has approximately 8-fold reduced ATPase activity relative to dynein isolated from wild-type. However, dynein ATPase activity from wild-type is not reduced when dynactin is separated from dynein, suggesting that dynein produced in a dynactin mutant is inactivated. Treatment of dynein isolated from the Deltaro-3 mutant with lambda protein phosphatase restores the ATPase activity to near wild-type levels. The reduced dynein ATPase activity observed in dynactin null mutants is mainly due to altered affinity for ATP. Radiolabeling experiments revealed that low molecular mass proteins, particularly 20- and 8-kDa proteins, that immunoprecipitate with dynein heavy chain are hyperphosphorylated in the dynactin mutant and dephosphorylated upon lambda protein phosphatase treatment. The results suggest that cytoplasmic dynein ATPase activity is regulated by dynactin-dependent phosphorylation of dynein light chains.     

Cytoplasmic dynein subunit heterogeneity: implications for axonal transport

The formation and maintenance of neuronal synapses is dependent on the active transport of material between the cell body and the axon terminal. Cytoplasmic dynein is one motor for microtubule-based axonal transport. Two pools of cytoplasmic dynein have been identified in the axon. They are distinguished by their intermediate and light intermediate chain subunits. Each pool is transported at different rates down the axon in association with different proteins or organelles. This review presents several models to discuss the potential functional roles of these different pools of cytoplasmic dynein during axonal transport.     

Cytoplasmic dynein/dynactin mediates the assembly of aggresomes

Aggresomes are pericentrosomal cytoplasmic structures into which aggregated, ubiquitinated, misfolded proteins are sequestered. Misfolded proteins accumulate in aggresomes when the capacity of the intracellular protein degradation machinery is exceeded. Previously, we demonstrated that an intact microtubule cytoskeleton is required for the aggresome formation [Johnston et al., 1998: J. Cell Biol. 143:1883-1898]. In this study, we have investigated the involvement of microtubules (MT) and MT motors in this process. Induction of aggresomes containing misfolded DeltaF508 CFTR is accompanied by a redistribution of the retrograde motor cytoplasmic dynein that colocalizes with aggresomal markers. Coexpression of the p50 (dynamitin) subunit of the dynein/dynactin complex prevents the formation of aggresomes, even in the presence of proteasome inhibitors. Using in vitro microtubule binding assays in conjunction with immunogold electron microscopy, our data demonstrate that misfolded DeltaF508 CFTR associate with microtubules. We conclude that cytoplasmic dynein/dynactin is responsible for the directed transport of misfolded protein into aggresomes. The implications of these findings with respect to the pathogenesis of neurodegenerative disease are discussed.     

Cytoplasmic dynein and dynactin in cell division and intracellular transport

Since the initial discovery of cytoplasmic dynein, it has become apparent that this microtubule-based motor is involved in several cellular functions including cell division and intracellular transport. Another multisubunit complex, dynactin, may be required for most, if not all, cytoplasmic dynein-driven activities and may provide clues to dynein's functional diversity. Recent genetic and biochemical findings have illuminated the cellular roles of dynein and dynactin and provided insight into the functional mechanism of this complex motor.     

Cytoplasmic dynein plays a role in mammalian mitotic spindle formation

The formation and functioning of a mitotic spindle depends not only on the assembly/disassembly of microtubules but also on the action of motor enzymes. Cytoplasmic dynein has been localized to spindles, but whether or how it functions in mitotic processes is not yet known. We have cloned and expressed DNA fragments that encode the putative ATP- hydrolytic sites of the cytoplasmic dynein heavy chain from HeLa cells and from Dictyostelium. Monospecific antibodies have been raised to the resulting polypeptides, and these inhibit dynein motor activity in vitro. Their injection into mitotic mammalian cells blocks the formation of spindles in prophase or during recovery from nocodazole treatment at later stages of mitosis. Cells become arrested with unseparated centrosomes and form monopolar spindles. The injected antibodies have no detectable effect on chromosome attachment to a bipolar spindle or on motions during anaphase. These data suggest that cytoplasmic dynein plays a unique and important role in the initial events of bipolar spindle formation, while any later roles that it may play are redundant. Possible mechanisms of dynein's involvement in mitosis are discussed.     

Cytoplasmic dynein as a facilitator of nuclear envelope breakdown.

During prophase in higher cells, centrosomes localize to deep invaginations in the nuclear envelope in a microtubule-dependent process. Loss of nuclear membranes in prometaphase commences in regions of the nuclear envelope that lie outside of these invaginations. Dynein and dynactin complex components concentrate on the nuclear envelope prior to any changes in nuclear envelope organization. These observations suggest a model in which dynein facilitates nuclear envelope breakdown by pulling nuclear membranes and associated proteins poleward along astral microtubules leading to nuclear membrane detachment. Support for this model is provided by the finding that interference with dynein function drastically alters nuclear membrane dynamics in prophase and prometaphase.