BICD2, dynactin,and LIS1 cooperate in regulating dynein recruitment to cellular structures

Cytoplasmic dynein is the major microtubule minus-end–directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein–dynactin interaction are poorly understood. In this study, we focus on dynein–dynactin recruitment to cargo by the conserved motor adaptor Bicaudal D2 (BICD2). We show that dynein and dynactin depend on each other for BICD2-mediated targeting to cargo and that BICD2 N-terminus (BICD2-N) strongly promotes stable interaction between dynein and dynactin both in vitro and in vivo. Direct visualization of dynein in live cells indicates that by itself the triple BICD2-N–dynein–dynactin complex is unable to interact with either cargo or microtubules. However, tethering of BICD2-N to different membranes promotes their microtubule minus-end–directed motility. We further show that LIS1 is required for dynein-mediated transport induced by membrane tethering of BICD2-N and that LIS1 contributes to dynein accumulation at microtubule plus ends and BICD2-positive cellular structures. Our results demonstrate that dynein recruitment to cargo requires concerted action of multiple dynein cofactors.     

Regulation of dynein‐dynactin‐driven vesicular transport

Most of the long‐range intracellular movements of vesicles, organelles and other cargoes are driven by microtubule (MT)‐based molecular motors. Cytoplasmic dynein, a multisubunit protein complex, with the aid of dynactin, drives transport of a wide variety of cargoes towards the minus end of MTs. In this article, I review our current understanding of the mechanisms underlying spatiotemporal regulation of dynein‐dynactin‐driven vesicular transport with a special emphasis on the many steps of directional movement along MT tracks. These include the recruitment of dynein to MT plus ends, the activation and processivity of dynein, and cargo recognition and release by the motor complex at the target membrane. Furthermore, I summarize the most recent findings about the fine control mechanisms for intracellular transport via the interaction between the dynein‐dynactin motor complex and its vesicular cargoes.      

A Neurospora crassa Arp1 mutation affecting cytoplasmic dynein and dynactin localization

Of the actin-related proteins, Arp1 is the most similar to conventional actin, and functions solely as a component of the multisubunit complex dynactin. Dynactin has been identified as an activator of the microtubule-associated motor cytoplasmic dynein. The role of Arp1 within dynactin is two-fold: (1) it serves as a structural scaffold protein for other dynactin subunits; and (2) it has been proposed to link dynactin, and thereby dynein, with membranous cargo via interaction with spectrin. Using the filamentous fungus Neurospora crassa, we have identified genes encoding subunits of cytoplasmic dynein and dynactin. In this study, we describe a genetic screen for N. crassa Arp1 (ro-4) mutants that are defective for dynactin function. We report that the ro-4(E8) mutant is unusual in that it shows alterations in the localization of cytoplasmic dynein and dynactin and in microtubule organization. In the mutant, dynein/dynactin complexes co-localize with bundled microtubules at hyphal tips. Given that dynein transports membranous cargo from hyphal tips to distal regions, the cytoplasmic dynein and dynactin complexes that accumulate along microtubule tracts at hyphal tips in the ro-4(E8) mutant may have either reduced motor activity or be delayed for activation of motor activity following cargo binding.     

Apoptotic cleavage of cytoplasmic dynein intermediate chain and p150(Glued) stops dynein-dependent membrane motility.

Cytoplasmic dynein is the major minus end-directed microtubule motor in animal cells, and associates with many of its cargoes in conjunction with the dynactin complex. Interaction between cytoplasmic dynein and dynactin is mediated by the binding of cytoplasmic dynein intermediate chains (CD-IC) to the dynactin subunit, p150(Glued). We have found that both CD-IC and p150(Glued) are cleaved by caspases during apoptosis in cultured mammalian cells and in Xenopus egg extracts. Xenopus CD-IC is rapidly cleaved at a conserved aspartic acid residue adjacent to its NH(2)-terminal p150(Glued) binding domain, resulting in loss of the otherwise intact cytoplasmic dynein complex from membranes. Cleavage of CD-IC and p150(Glued) in apoptotic Xenopus egg extracts causes the cessation of cytoplasmic dynein--driven endoplasmic reticulum movement. Motility of apoptotic membranes is restored by recruitment of intact cytoplasmic dynein and dynactin from control cytosol, or from apoptotic cytosol supplemented with purified cytoplasmic dynein--dynactin, demonstrating the dynamic nature of the association of cytoplasmic dynein and dynactin with their membrane cargo.     

Backseat drivers: Regulation of dynein motility

Control of the activity of the microtubule motor cytoplasmic dynein 1 is essential for its function in intracellular transport. A recent paper by McKenney et al. published in Science shows that activation of processive dynein motility requires the formation of cargo adaptor-dynein-dynactin complexes.Cells rely on their intracellular components being in the right place at the right time. In eukaryotic cells, microtubule-based transport by motor proteins belonging to the dynein and kinesin families plays a crucial role in regulating the spatial-temporal distribution of a multitude of membrane bound organelles, protein complexes, and ribonucleoprotein complexes. Disruption of these transport functions can play a key role in pathological processes and the activities of microtubule motors are frequently usurped by both viral and bacterial pathogens to aid their replication¹. Cytoplasmic dynein 1 is the predominant motor protein complex mediating transport towards the minus end of microtubules and it transports many different cargoes². The dynein holoenzyme is composed of a dimeric heavy chain that contains the microtubule-binding and AAA-ATPase motor domains associated with a series of smaller accessory proteins implicated in regulation and cargo binding. Targeting of the motor to a specific cargo is often mediated by so-called ''adaptor proteins'' that can associate with both the cargo (e.g., endosomes) and the motor complex itself.Diverse functions and diverse cargoes necessitate a high level of cytoplasmic dynein regulation. Such regulation must limit motor activity to prevent wasteful ATP hydrolysis in the absence of cargo transport and prevent inappropriate movement of cargo-free motors on microtubules. Regulation must allow exquisite responses to dynamic spatial and temporal cues for cargo transport and allow for the selective recognition of a wide range of cargoes/adaptors that, ostensibly at least, may be quite different. It must also support bidirectional transport processes.A second multiprotein complex, dynactin³, helps to regulate cytoplasmic dynein 1. Indeed, dynactin is required for almost all known functions of cytoplasmic dynein. It is thought that dynactin plays a key role in attachment to cargo and promotes dynein activity. Despite this, its precise mechanism of action and the role of cargo attachment itself has remained unclear.Recently, a study by McKenney et al.⁴ in Science and a complimentary study by Schlager et al.⁵ in EMBO J, have taken crucial steps forward, uncovering a role for tripartite cargo adaptor-dynein-dynactin complexes in directly promoting dynein activity (Figure 1). Both of these studies utilize elegant biochemical purification coupled with the technical feat of high-resolution, single-molecule, multicolor TIRF microscopy to examine the properties of these assemblies as they move on labelled microtubules in vitro.Open in a separate windowFigure 1Schematic showing proposed organization of an active dynein-dynactin-cargo complex. Cargo (e.g., an endosome) couples to the motor complex via surface receptors (e.g., a Rab GTPases) that recruit specific adaptor proteins (e.g., BiCD2, Hook). These in turn recruit dynein/dynactin and stabilize their association, supporting the microtubule binding and processivity of the dynein-dynactin complex.McKenney et al.⁴ begin by showing that cytoplasmic dynein purified from rat brain (that is free from both dynactin and cargo proteins) binds to microtubules but does not engage in the processive long distance movements characteristic of motility in vivo. This implies that dynein requires activation. To isolate transport-active complexes, the authors use an alternative approach — affinity purification (from RPE-1 cells) via the cargo adaptor BicD2 (that couples dynein to Rab6-containing organelles). This yields stable associations of BicD2, dynein and dynactin, which when examined in TIRF motility assays, exhibit speeds and run lengths approaching those observed in vivo. Importantly, the authors reveal that these complexes consist of a single copy of dynein, dynactin and BicD2 (a dimer), demonstrating that the intrinsic processivity of the holoenzyme is directly enhanced and ruling out effects from cooperation between motor complexes in this system.The authors then ask which components of this tripartite complex are needed for dynein activation — is BicD2 required or is dynactin sufficient? They show that removal of BicD2 results in a loss of processive motility and dissociation of dynein from dynactin, demonstrating the importance of the cargo adaptor itself in formation of stable dynein-dynactin complexes and motility. Schlager et al.⁵ come to similar conclusions in their study using recombinant human dynein produced in baculovirus (an achievement in its own right), showing that purified dynactin is unable to activate motility in the absence of BicD2.To determine whether this mechanism is unique to BicD2 or whether it holds for other cargo adaptors, McKenney et al. expand their study to include three other adaptors — Rab11-FIP3 (for recycling endosomes), Spindly (kinetochores) and Hook (early endosomes). They show that all three can be used to purify dynein-dynactin and that those complexes are capable of processive motility in a manner comparable to those derived from BicD2 affinity purification.These studies thus highlight a crucial role for the cargo adaptor, coupled with dynactin, in dynein activation. This is somewhat reminiscent of several kinesin family proteins which exist in an inactive state in the absence of cargo⁶. In the future it will be important to understand why dynein is inactive in the absence of dynactin/cargo and what changes occur within the complex upon cargo adaptor/dynactin binding to cause its conversion to a processive motor. Clues may come from comparison with S. Cerevisiae dynein which appears constitutively active and may associate with dynactin in the absence of cargo⁷. It will also be important to determine the regulatory signals that control formation and dissociation of these active complexes, how they interact with other dynein regulators such as the Lis1-NudEL complex and how they are affected by the action of plus-end-directed motors associated with the same cargo.Further progress should also come from understanding of the structural and biophysical characteristics of the motor-cargo interfaces that we now know must ultimately drive dynein activation. Indeed, the fact that four distinct cargo adaptors can promote formation of transport-active complexes may imply the existence of common features in dynein-cargo adaptor recognition mechanisms that support activation. Importantly, the establishment of these elegant in vitro systems that recapitulate many of the properties of cytoplasmic dynein in vivo will now allow for a full molecular dissection of this ubiquitous and fascinating process.     

Mammalian Golgi-associated Bicaudal-D2 functions in the dynein-dynactin pathway by interacting with these complexes

Genetic analysis in Drosophila suggests that Bicaudal-D functions in an essential microtubule-based transport pathway, together with cytoplasmic dynein and dynactin. However, the molecular mechanism underlying interactions of these proteins has remained elusive. We show here that a mammalian homologue of Bicaudal-D, BICD2, binds to the dynamitin subunit of dynactin. This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co-localizes with dynactin at microtubule plus ends. Overexpression studies using green fluorescent protein-tagged forms of BICD2 verify its intracellular distribution and co-localization with dynactin, and indicate that the C-terminus of BICD2 is responsible for Golgi targeting. Overexpression of the N-terminal domain of BICD2 disrupts minus-end-directed organelle distribution and this portion of BICD2 co-precipitates with cytoplasmic dynein. Nocodazole treatment of cells results in an extensive BICD2-dynactin-dynein co-localization. Taken together, these data suggest that mammalian BICD2 plays a role in the dynein- dynactin interaction on the surface of membranous organelles, by associating with these complexes.     

Assessing the Impact of Electrostatic Drag on Processive Molecular Motor Transport

The bidirectional movement of intracellular cargo is usually described as a tug-of-war among opposite-directed families of molecular motors. While tug-of-war models have enjoyed some success, recent evidence suggests underlying motor interactions are more complex than previously understood. For example, these tug-of-war models fail to predict the counterintuitive phenomenon that inhibiting one family of motors can decrease the functionality of opposite-directed transport. In this paper, we use a stochastic differential equations modeling framework to explore one proposed physical mechanism, called microtubule tethering, that could play a role in this “co-dependence” among antagonistic motors. This hypothesis includes the possibility of a trade-off: weakly bound trailing molecular motors can serve as tethers for cargoes and processing motors, thereby enhancing motor–cargo run lengths along microtubules; however, this introduces a cost of processing at a lower mean velocity. By computing the small- and large-time mean-squared displacement of our theoretical model and comparing our results to experimental observations of dynein and its “helper protein” dynactin, we find some supporting evidence for microtubule tethering interactions. We extrapolate these findings to predict how dynein–dynactin might interact with the opposite-directed kinesin motors and introduce a criterion for when the trade-off is beneficial in simple systems.     

Dynactin's pointed-end complex is a cargo-targeting module

Dynactin is an essential part of the cytoplasmic dynein motor that enhances motor processivity and serves as an adaptor that allows dynein to bind cargoes. Much is known about dynactin''s interaction with dynein and microtubules, but how it associates with its diverse complement of subcellular binding partners remains mysterious. It has been suggested that cargo specification involves a group of subunits referred to as the “pointed-end complex.” We used chemical cross-linking, RNA interference, and protein overexpression to characterize interactions within the pointed-end complex and explore how it contributes to dynactin''s interactions with endomembranes. The Arp11 subunit, which caps one end of dynactin''s Arp1 filament, and p62, which binds Arp11 and Arp1, are necessary for dynactin stability. These subunits also allow dynactin to bind the nuclear envelope prior to mitosis. p27 and p25, by contrast, are peripheral components that can be removed without any obvious impact on dynactin integrity. Dynactin lacking these subunits shows reduced membrane binding. Depletion of p27 and p25 results in impaired early and recycling endosome movement, but late endosome movement is unaffected, and mitotic spindles appear normal. We conclude that the pointed-end complex is a bipartite structural domain that stabilizes dynactin and supports its binding to different subcellular structures.     

mNUDC is required for plus‐end‐directed transport of cytoplasmic dynein and dynactins by kinesin‐1

Lissencephaly is a devastating neurological disorder caused by defective neuronal migration. The LIS1 (or PAFAH1B1) gene was identified as the gene mutated in lissencephaly patients, and was found to regulate cytoplasmic dynein function and localization. In particular, LIS1 is essential for anterograde transport of cytoplasmic dynein as a part of the cytoplasmic dynein–LIS1–microtubule complex in a kinesin‐1‐dependent manner. However, the underlying mechanism by which a cytoplasmic dynein–LIS1–microtubule complex binds kinesin‐1 is unknown. Here, we report that mNUDC (mammalian NUDC) interacts with kinesin‐1 and is required for the anterograde transport of a cytoplasmic dynein complex by kinesin‐1. mNUDC is also required for anterograde transport of a dynactin‐containing complex. Inhibition of mNUDC severely suppressed anterograde transport of distinct cytoplasmic dynein and dynactin complexes, whereas motility of kinesin‐1 remained intact. Reconstruction experiments clearly demonstrated that mNUDC mediates the interaction of the dynein or dynactin complex with kinesin‐1 and supports their transport by kinesin‐1. Our findings have uncovered an essential role of mNUDC for anterograde transport of dynein and dynactin by kinesin‐1.     

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

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

Bicaudal D induces selective dynein-mediated microtubule minus end-directed transport

Bicaudal D is an evolutionarily conserved protein, which is involved in dynein-mediated motility both in Drosophila and in mammals. Here we report that the N-terminal portion of human Bicaudal D2 (BICD2) is capable of inducing microtubule minus end-directed movement independently of the molecular context. This characteristic offers a new tool to exploit the relocalization of different cellular components by using appropriate targeting motifs. Here, we use the BICD2 N-terminal domain as a chimera with mitochondria and peroxisome-anchoring sequences to demonstrate the rapid dynein-mediated transport of selected organelles. Surprisingly, unlike other cytoplasmic dynein-mediated processes, this transport shows very low sensitivity to overexpression of the dynactin subunit dynamitin. The dynein-recruiting activity of the BICD2 N-terminal domain is reduced within the full-length molecule, indicating that the C-terminal part of the protein might regulate the interaction between BICD2 and the motor complex. Our findings provide a novel model system for dissection of the molecular mechanism of dynein motility.     

A Role for Spectrin in Dynactin‐dependent Melanosome Transport in Xenopus laevis Melanophores

The bi‐directional movement of pigment granules in frog melanophores involves the microtubule‐based motors cytoplasmic dynein, which is responsible for aggregation, and kinesin  II and myosin  V, which are required for dispersion of pigment. It was recently shown that dynactin acts as a link between dynein and kinesin  II and melanosomes, but it is not fully understood how this is regulated and if more proteins are involved. Here, we suggest that spectrin, which is known to be associated with Golgi vesicles as well as synaptic vesicles in a number of cells, is of importance for melanosome movements in Xenopus laevis melanophores. Large amounts of spectrin were found on melanosomes isolated from both aggregated and dispersed melanophores. Spectrin and two components of the oligomeric dynactin complex, p150^glued and Arp1/centractin, co‐localized with melanosomes during aggregation and dispersion, and the proteins were found to interact as determined by co‐immunoprecipitation. Spectrin has been suggested as an important link between cargoes and motor proteins in other cell types, and our new data indicate that spectrin has a role in the specialized melanosome transport processes in frog melanophores, in addition to a more general vesicle transport.     

Characterization of the p22 Subunit of Dynactin Reveals the Localization of Cytoplasmic Dynein and Dynactin to the Midbody of Dividing Cells

Dynactin, a multisubunit complex that binds to the microtubule motor cytoplasmic dynein, may provide a link between dynein and its cargo. Many subunits of dynactin have been characterized, elucidating the multifunctional nature of this complex. Using a dynein affinity column, p22, the smallest dynactin subunit, was isolated and microsequenced. The peptide sequences were used to clone a full-length human cDNA. Database searches with the predicted amino acid sequence of p22 indicate that this polypeptide is novel. We have characterized p22 as an integral component of dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments indicate that p22 binds directly to the p150^Glued subunit of dynactin. Immunocytochemistry with antibodies to p22 demonstrates that this polypeptide localizes to punctate cytoplasmic structures and to the centrosome during interphase, and to kinetochores and to spindle poles throughout mitosis. Antibodies to p22, as well as to other dynactin subunits, also revealed a novel localization for dynactin to the cleavage furrow and to the midbodies of dividing cells; cytoplasmic dynein was also localized to these structures. We therefore propose that dynein/dynactin complexes may have a novel function during cytokinesis.     

Load‐dependent detachment kinetics plays a key role in bidirectional cargo transport by kinesin and dynein

Bidirectional cargo transport along microtubules is carried out by opposing teams of kinesin and dynein motors. Despite considerable study, the factors that determine whether these competing teams achieve net anterograde or retrograde transport in cells remain unclear. The goal of this work is to use stochastic simulations of bidirectional transport to determine the motor properties that most strongly determine overall cargo velocity and directionality. Simulations were carried out based on published optical tweezer characterization of kinesin‐1 and kinesin‐2, and for available data for cytoplasmic dynein and the dynein‐dynactin‐BicD2 (DDB) complex. By varying dynein parameters and analyzing cargo trajectories, we find that net cargo transport is predicted to depend minimally on the dynein stall force, but strongly on dynein load‐dependent detachment kinetics. In simulations, dynein is dominated by kinesin‐1, but DDB and kinesin‐1 are evenly matched, recapitulating recent experimental work. Kinesin‐2 competes less well against dynein and DDB, and overall, load‐dependent motor detachment is the property that most determines a motor's ability to compete in bidirectional transport. It follows that the most effective intracellular regulators of bidirectional transport are predicted to be those that alter motor detachment kinetics rather than motor velocity or stall force.      

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.     

Dynactins p25 and p27 are predicted to adopt the LbetaH fold

Dynactin is a multimeric protein essential for the minus-end-directed transport driven by microtubule-based motor dynein. The pointed-end subcomplex in dynactin contains p62, p27, p25, and Arp11 subunits, and is thought to participate in interactions with membranous cargoes. We used sequence and structure prediction analysis to study dynactins p25 and p27. Here we present evidence that strongly supports that dynactins p27 and p25 contain the isoleucine-patch motif and adopt the left-handed parallel beta-helix fold. The structural models we obtained could contribute to the understanding of the complex interactions that dynactins are able to establish with cargo particles, microtubules or other dynactin subunits.     

Structural dynamics and multiregion interactions in dynein-dynactin recognition

Cytoplasmic dynein is a 1.2-MDa multisubunit motor protein complex that, together with its activator dynactin, is responsible for the majority of minus end microtubule-based motility. Dynactin targets dynein to specific cellular locations, links dynein to cargo, and increases dynein processivity. These two macromolecular complexes are connected by a direct interaction between dynactin's largest subunit, p150(Glued), and dynein intermediate chain (IC) subunit. Here, we demonstrate using NMR spectroscopy and isothermal titration calorimetry that the binding footprint of p150(Glued) on IC involves two noncontiguous recognition regions, and both are required for full binding affinity. In apo-IC, the helical structure of region 1, the nascent helix of region 2, and the disorder in the rest of the chain are determined from coupling constants, amide-amide sequential NOEs, secondary chemical shifts, and various dynamics measurements. When bound to p150(Glued), different patterns of spectral exchange broadening suggest that region 1 forms a coiled-coil and region 2 a packed stable helix, with the intervening residues remaining disordered. In the 150-kDa complex of p150(Glued), IC, and two light chains, the noninterface segments remain disordered. The multiregion IC binding interface, the partial disorder of region 2 and its potential for post-translational modification, and the modulation of the length of the longer linker by alternative splicing may provide a basis for elegant and multifaceted regulation of binding between IC and p150(Glued). The long disordered linker between the p150(Glued) binding segments and the dynein light chain consensus sequences could also provide an attractive recognition platform for diverse cargoes.