Dynein-based Motility Research Articles

Composition, organization and mechanisms of the transition zone, a gate for the cilium.

The cilium evolved to provide the ancestral eukaryote with the ability to move and sense its environment. Acquiring these functions required the compartmentalization of a dynein-based motility apparatus and signaling proteins within a discrete subcellular organelle contiguous with the cytosol. Here, we explore the potential molecular mechanisms for how the proximal-most region of the cilium, termed transition zone (TZ), acts as a diffusion barrier for both membrane and soluble proteins and helps to ensure ciliary autonomy and homeostasis. These include a unique complement and spatial organization of proteins that span from the microtubule-based axoneme to the ciliary membrane; a protein picket fence; a specialized lipid microdomain; differential membrane curvature and thickness; and lastly, a size-selective molecular sieve. In addition, the TZ must be permissive for, and functionally integrates with, ciliary trafficking systems (including intraflagellar transport) that cross the barrier and make the ciliary compartment dynamic. The quest to understand the TZ continues and promises to not only illuminate essential aspects of human cell signaling, physiology, and development, but also to unravel how TZ dysfunction contributes to ciliopathies that affect multiple organ systems, including eyes, kidney, and brain.

Bicaudal D Family Adaptor Proteins Control the Velocity of Dynein-Based Movements

Cargo transport along microtubules is driven by the collective function of microtubule plus- and minus-end-directed motors (kinesins and dyneins). How the velocity of cargo transport is driven by opposing teams of motors is still poorly understood. Here, we combined inducible recruitment of motors and adaptors to Rab6 secretory vesicles with detailed tracking of vesicle movements to investigate how changes in the transport machinery affect vesicle motility. We find that the velocities of kinesin-based vesicle movements are slower and more homogeneous than those of dynein-based movements. We also find that Bicaudal D (BICD) adaptor proteins can regulate dynein-based vesicle motility. BICD-related protein 1 (BICDR-1) accelerates minus-end-directed vesicle movements and affects Rab6 vesicle distribution. These changes are accompanied by reduced axonal outgrowth in neurons, supporting their physiological importance. Our study suggests that adaptor proteins can modulate the velocity of dynein-based motility and thereby control the distribution of transport carriers.

Involvement of microtubular network and its motors in productive endocytic trafficking of mouse polyomavirus.

Infection of non-enveloped polyomaviruses depends on an intact microtubular network. Here we focus on mouse polyomavirus (MPyV). We show that the dynamics of MPyV cytoplasmic transport reflects the characteristics of microtubular motor-driven transport with bi-directional saltatory movements. In cells treated with microtubule-disrupting agents, localization of MPyV was significantly perturbed, the virus was retained at the cell periphery, mostly within membrane structures resembling multicaveolar complexes, and at later times post-infection, only a fraction of the virus was found in Rab7-positive endosomes and multivesicular bodies. Inhibition of cytoplasmic dynein-based motility by overexpression of dynamitin affected perinuclear translocation of the virus, delivery of virions to the ER and substantially reduced the numbers of infected cells, while overexpression of dominant-negative form of kinesin-1 or kinesin-2 had no significant impact on virus localization and infectivity. We also found that transport along microtubules was important for MPyV-containing endosome sequential acquisition of Rab5, Rab7 and Rab11 GTPases. However, in contrast to dominant-negative mutant of Rab7 (T22N), overexpression of dominant-negative mutant Rab11 (S25N) did not affect the virus infectivity. Altogether, our study revealed that MPyV cytoplasmic trafficking leading to productive infection bypasses recycling endosomes, does not require the function of kinesin-1 and kinesin-2, but depends on functional dynein-mediated transport along microtubules for translocation of the virions from peripheral, often caveolin-positive compartments to late endosomes and ER – a prerequisite for efficient delivery of the viral genome to the nucleus.

The 2.8 Å crystal structure of the dynein motor domain

Dyneins are microtubule-based AAA(+) motor complexes that power ciliary beating, cell division, cell migration and intracellular transport. Here we report the most complete structure obtained so far, to our knowledge, of the 380-kDa motor domain of Dictyostelium discoideum cytoplasmic dynein at 2.8 Å resolution; the data are reliable enough to discuss the structure and mechanism at the level of individual amino acid residues. Features that can be clearly visualized at this resolution include the coordination of ADP in each of four distinct nucleotide-binding sites in the ring-shaped AAA(+) ATPase unit, a newly identified interaction interface between the ring and mechanical linker, and junctional structures between the ring and microtubule-binding stalk, all of which should be critical for the mechanism of dynein motility. We also identify a long-range allosteric communication pathway between the primary ATPase and the microtubule-binding sites. Our work provides a framework for understanding the mechanism of dynein-based motility.

Structural Basis for the Regulation of Microtubule-Binding Affinity by Cytoplasmic Dynein

The molecular motor cytoplasmic dynein is responsible for most minus-end directed microtubule-based transport in eukaryotic cells. Dynein is especially important in neurons, where defects in dynein-based motility have been linked to neurodegenerative and neurodevelopmental diseases in humans. Due to the complexity and size of dynein our understanding of it has lagged behind that of other cytoskeletal motor proteins. A major goal in the field is to determine how ATP hydrolysis events in dynein's AAA+ motor domain “ring” are communicated to the microtubule-binding domain (MTBD), which is located 250A away. Information between these two sites is proposed to be conveyed via changes in the register of a long coiled-coil “stalk” that connects the MTBD to the AAA+ ring. Both low and high affinity registers have been identified and a crystal structure of the MTBD with the stalk locked into the low affinity register has been solved. To understand the structural changes that occur upon MT binding and how they might be regulated we have obtained a ∼10A resolution cryo-EM reconstruction of microtubules decorated with the MTBD of cytoplasmic dynein locked into the “high affinity” state. We used our density to perform molecular dynamics flexible fitting with the available crystal structure and observe significant rearrangements in three of the six alpha helices that make up the MTBD. Our pseudo-atomic model accounts for several mutations that were previously identified for their effects on microtubule-binding affinity. Based on our results we propose a model for the coordination between changes in the register of the coiled-coil and dynein's affinity for its track.

Tuning Microtubule‐Based Transport Through Filamentous MAPs: The Problem of Dynein

We recently proposed that regulating the single-to-multiple motor transition was a likely strategy for regulating kinesin-based transport in vivo. In this study, we use an in vitro bead assay coupled with an optical trap to investigate how this proposed regulatory mechanism affects dynein-based transport. We show that tau's regulation of kinesin function can proceed without interfering with dynein-based transport. Surprisingly, at extremely high tau levels--where kinesin cannot bind microtubules (MTs)--dynein can still contact MTs. The difference between tau's effects on kinesin- and dynein-based motility suggests that tau can be used to tune relative amounts of plus-end and minus-end-directed transport. As in the case of kinesin, we find that the 3RS isoform of tau is a more potent inhibitor of dynein binding to MTs. We show that this isoform-specific effect is not because of steric interference of tau's projection domains but rather because of tau's interactions with the motor at the MT surface. Nonetheless, we do observe a modest steric interference effect of tau away from the MT and discuss the potential implications of this for molecular motor structure.

Force-Induced Bidirectional Stepping of Cytoplasmic Dynein

Cytoplasmic dynein is a minus-end-directed microtubule motor whose mechanism of movement remains poorly understood. Here, we use optical tweezers to examine the force-dependent stepping behavior of yeast cytoplasmic dynein. We find that dynein primarily advances in 8 nm increments but takes other sized steps (4-24 nm) as well. An opposing force induces more frequent backward stepping by dynein, and the motor walks backward toward the microtubule plus end at loads above its stall force of 7 pN. Remarkably, in the absence of ATP, dynein steps processively along microtubules under an external load, with less force required for minus-end- than for plus-end-directed movement. This nucleotide-independent walking reveals that force alone can drive repetitive microtubule detachment-attachment cycles of dynein's motor domains. These results suggest a model for how dynein's two motor domains coordinate their activities during normal processive motility and provide new clues for understanding dynein-based motility in living cells.

Mechanism of Dynamitin-mediated Disruption of Dynactin

Dynamitin is a commonly used inhibitor of cytoplasmic dynein-based motility in living cells. Dynamitin does not inhibit dynein directly but instead acts by causing disassembly of dynactin, a multiprotein complex required for dynein-based movement. In dynactin, dynamitin is closely associated with the subunits p150(Glued) and p24, which together form the shoulder and projecting arm structures of the dynactin molecule. In this study, we explore the way in which exogenous dynamitin effects dynactin disruption. We find that pure, recombinant dynamitin is an elongated protein with a strong propensity for self-assembly. Titration experiments reveal that free dynamitin binds dynactin before it causes release of subunits. When dynamitin is added to dynactin at an equimolar ratio of exogenous dynamitin subunits to endogenous dynamitin subunits (1x= 4 mol of exogenous dynamitin per mole of dynactin), exogenous dynamitin exchanges with endogenous dynamitin, and partial release of p150(Glued) is observed. When added in vast excess (> or =25x; 100 mol of exogenous dynamitin per mole of dynactin), recombinant dynamitin causes complete release of both p150(Glued) subunits, two dynamitins and one p24, but not other dynactin subunits. Our data suggest that dynamitin mediates disruption of dynactin by binding to endogenous dynamitin subunits. This binding destabilizes the shoulder structure that links the p150(Glued) arm to the Arp1 filament and leads to subunit release.

The golgin Lava lamp mediates dynein-based Golgi movements during Drosophila cellularization

Drosophila melanogaster cellularization is a dramatic form of cytokinesis in which a membrane furrow simultaneously encapsulates thousands of cortical nuclei of the syncytial embryo to generate a polarized cell layer. Formation of this cleavage furrow depends on Golgi-based secretion and microtubules. During cellularization, specific Golgi move along microtubules, first to sites of furrow formation and later to accumulate within the apical cytoplasm of the newly forming cells. Here we show that Golgi movements and furrow formation depend on cytoplasmic dynein. Furthermore, we demonstrate that Lava lamp (Lva), a golgin protein that is required for cellularization, specifically associates with dynein, dynactin, cytoplasmic linker protein-190 (CLIP-190) and Golgi spectrin, and is required for the dynein-dependent targeting of the secretory machinery. The Lva domains that bind these microtubule-dependent motility factors inhibit Golgi movement and cellularization in a live embryo injection assay. Our results provide new evidence that golgins promote dynein-based motility of Golgi membranes.

Dynactin polices two-way organelle traffic.

How is the bidirectional motion of organelles controlled? In this issue, Deacon et al. (2003) reveal the unexpected finding that dynactin (previously known to control dynein-based motility) binds to kinesin II and regulates anterograde movement of Xenopus melanosomes. This result suggests that dynactin may be a key player in coordinating vesicle traffic in this system.

Distinct cell cycle-dependent roles for dynactin and dynein at centrosomes.

Centrosomal dynactin is required for normal microtubule anchoring and/or focusing independently of dynein. Dynactin is present at centrosomes throughout interphase, but dynein accumulates only during S and G2 phases. Blocking dynein-based motility prevents recruitment of dynactin and dynein to centrosomes and destabilizes both centrosomes and the microtubule array, interfering with cell cycle progression during mitosis. Destabilization of the centrosomal pool of dynactin does not inhibit dynein-based motility or dynein recruitment to centrosomes, but instead causes abnormal G1 centriole separation and delayed entry into S phase. The correct balance of centrosome-associated dynactin subunits is apparently important for satisfaction of the cell cycle mechanism that monitors centrosome integrity before centrosome duplication and ultimately governs the G1 to S transition. Our results suggest that, in addition to functioning as a microtubule anchor, dynactin contributes to the recruitment of important cell cycle regulators to centrosomes.

Dynactin is required for microtubule anchoring at centrosomes.

The multiprotein complex, dynactin, is an integral part of the cytoplasmic dynein motor and is required for dynein-based motility in vitro and in vivo. In living cells, perturbation of the dynein-dynactin interaction profoundly blocks mitotic spindle assembly, and inhibition or depletion of dynein or dynactin from meiotic or mitotic cell extracts prevents microtubules from focusing into spindles. In interphase cells, perturbation of the dynein-dynactin complex is correlated with an inhibition of ER-to-Golgi movement and reorganization of the Golgi apparatus and the endosome-lysosome system, but the effects on microtubule organization have not previously been defined. To explore this question, we overexpressed a variety of dynactin subunits in cultured fibroblasts. Subunits implicated in dynein binding have effects on both microtubule organization and centrosome integrity. Microtubules are reorganized into unfocused arrays. The pericentriolar components, gamma tubulin and dynactin, are lost from centrosomes, but pericentrin localization persists. Microtubule nucleation from centrosomes proceeds relatively normally, but microtubules become disorganized soon thereafter. Overexpression of some, but not all, dynactin subunits also affects endomembrane localization. These data indicate that dynein and dynactin play important roles in microtubule organization at centrosomes in fibroblastic cells and provide new insights into dynactin-cargo interactions.

Characterization of a Chlamydomonas insertional mutant that disrupts flagellar central pair microtubule-associated structures.

Two alleles at a new locus, central pair-associated complex 1 (CPC1), were selected in a screen for Chlamydomonas flagellar motility mutations. These mutations disrupt structures associated with central pair microtubules and reduce flagellar beat frequency, but do not prevent changes in flagellar activity associated with either photophobic responses or phototactic accumulation of live cells. Comparison of cpc1 and pf6 axonemes shows that cpc1 affects a row of projections along C1 microtubules distinct from those missing in pf6, and a row of thin fibers that form an arc between the two central pair microtubules. Electron microscopic images of the central pair in axonemes from radial spoke-defective strains reveal previously undescribed central pair structures, including projections extending laterally toward radial spoke heads, and a diagonal link between the C2 microtubule and the cpc1 projection. By SDS-PAGE, cpc1 axonemes show reductions of 350-, 265-, and 79-kD proteins. When extracted from wild-type axonemes, these three proteins cosediment on sucrose gradients with three other central pair proteins (135, 125, and 56 kD) in a 16S complex. Characterization of cpc1 provides new insights into the structure and biochemistry of the central pair apparatus, and into its function as a regulator of dynein-based motility.

Actin-related protein 1 and cytoplasmic dynein-based motility - what's the connection?

The actin-related protein Arp1 works in conjunction with the microtubule-based motor cytoplasmic dynein to drive many types of intracellular motility. In vertebrate cells, Arp1 is present exclusively in the form of a 37-nm filament that constitutes the backbone of dynactin, a 1.2-MDa macromolecular complex containing nine other polypeptides. Dynactin has been proposed to function as the link between dynein and its cargo. Recent work indicates that the dynactin subunit p150 Glued mediates the interaction of the dynactin molecule with dynein and microtubules, leaving the Arp1 filament as a possible cargo-binding domain. Mechanisms for binding of F-actin to membranes are discussed as models of the Arp1-membrane interaction.

Chapter 70 Inhibitors of the Dynein ATPase and Ciliary or Flagellar Motility

Publisher Summary When a substance inhibits a single event without affecting other events, it is called a specific inhibitor. As dynein ATPase and dynein-based motility, are thought to be homologous, if an inhibitor inhibits a group (or a family) of homologous events without affecting others, it also is regarded as specific. With regard to inhibitors of the dynein ATPase, their specificity for dynein and ciliary or flagellar motility relative to their inhibitory effects on other motile systems and motor enzymes should be taken into consideration. In discussing the inhibitors of interest, it is important to keep in mind certain aspects of inhibition kinetics. These parameters are the half-maximal concentration of the inhibitor for the inhibition and the mode of inhibition. In some cases, these parameters are significant for diagnosis of an effect. With enzymes, inhibition usually is classified into three categories: competitive, noncompetitive, and uncompetitive inhibition. These are based on the effects of the inhibitor on the Michaelis constant of an enzyme. Ciliary or flagellar motility or in vitro motility by dynein or other motors are not true enzyme reactions, but usually they can be analyzed in an analogous manner. The parameters are also applicable to the inhibitors that interact noncovalently with their targets. Chemical modifiers also are sometimes fairly specific and effective inhibitors of physiological events, especially for enzyme reactions. In this case, the second parameter does not exist. In addition, the inhibition depends on the progression of chemical modification, so that it is influenced by the concentration of the inhibitor, as well as on the duration of modification. This chapter discusses some of such inhibitor in detail.

Two activators of microtubule-based vesicle transport.

Cytoplasmic dynein purified by nucleotide dependent microtubule affinity has significant minus end-directed vesicle motor activity that decreases with each further purification step. Highly purified dynein causes membrane vesicles to bind but not move on microtubules. We exploited these observations to develop an assay for factors that, in combination with dynein, would permit minus end-directed vesicle motility. At each step of the purification, non-dynein fractions were recombined with dynein and assayed for vesicle motility. Two activating fractions were identified by this method. One, called Activator I, copurified with 20S dynein by velocity sedimentation but could be separated from it by ion exchange chromatography. Activator I increased only the frequency of dynein-driven vesicle movements. Activator II, sedimenting at 9S, increased both the frequency and velocity of vesicle transport and also supported plus end movements. Our results suggest that dynein-based motility is controlled at multiple levels and provide a preliminary characterization of two regulatory factors.