Rapid Actin-Dependent Viral Motility in Live Cells

During the course of an infection, viruses take advantage of a variety of mechanisms to travel in cells, ranging from diffusion within the cytosol to active transport along cytoskeletal filaments. To study viral motility within the intrinsically heterogeneous environment of the cell, we have developed a motility assay that allows for the global and unbiased analysis of tens of thousands of virus trajectories in live cells. Using this assay, we discovered that poliovirus exhibits anomalously rapid intracellular movement that was independent of microtubules, a common track for fast and directed cargo transport. Such rapid motion, with speeds of up to 5 μm/s, allows the virus particles to quickly explore all regions of the cell with the exception of the nucleus. The rapid, microtubule-independent movement of poliovirus was observed in multiple human-derived cell lines, but appeared to be cargo-specific. Other cargo, including a closely related picornavirus, did not exhibit similar motility. Furthermore, the motility is energy-dependent and requires an intact actin cytoskeleton, suggesting an active transport mechanism. The speed of this microtubule-independent but actin-dependent movement is nearly an order of magnitude faster than the fastest speeds reported for actin-dependent transport in animal cells, either by actin polymerization or by myosin motor proteins.     

Transport of African swine fever virus from assembly sites to the plasma membrane is dependent on microtubules and conventional kinesin

African swine fever virus (ASFV) is a large DNA virus that assembles in perinuclear viral factories located close to the microtubule organizing center. In this study, we have investigated the mechanism by which ASFV reaches the cell surface from the site of assembly. Immunofluorescence microscopy revealed that at 16 h postinfection, mature virions were aligned along microtubules. Furthermore, virus movement to the cell periphery was inhibited when microtubules were depolymerized by nocodazole. In addition, ASFV infection resulted in the increased acetylation of microtubules as well as their protection against depolymerization by nocodazole. Immunofluorescence microscopy showed that conventional kinesin was recruited to virus factories and to a large fraction of virus particles in the cytoplasm. Consistent with a role for conventional kinesin during ASFV egress to the cell periphery, overexpression of the cargo-binding domain of the kinesin light chain severely inhibited the movement of particles to the plasma membrane. Based on our observations, we propose that ASFV is recognized as cargo by conventional kinesin and uses this plus-end microtubule motor to move from perinuclear assembly sites to the plasma membrane.     

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate (“map”) the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. “Cargo mapping” consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to “map” them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.     

Intracellular transport based on actin polymerization

In addition to the intracellular transport of particles (cargo) along microtubules, there are in the cell two actin-based transport systems. In the actomyosin system the transport is driven by myosin, which moves the cargo along actin microfilaments. This transport requires the hydrolysis of ATP in the myosin molecule motor domain that induces conformational changes in the molecule resulting in the myosin movement along the actin filament. The other actin-based transport system of the cell does not involve myosin or other motor proteins. This system is based on a unidirectional actin polymerization, which depends on ATP hydrolysis in actin polymers and is initiated by proteins bound to the surface of transported particles. Obligatory components of the actin-based transport are proteins of the WASP/Scar family and a complex of Arp2/3 proteins. Moreover, the actin-based systems often contain dynamin and cortactin. It is known that a system of actin filaments formed on the surface of particles, the so-called “comet-like tail”, is responsible for intracellular movements of pathogenic bacteria, micropinocytotic vesicles, clathrin-coated vesicles, and phagosomes. This movement is reproduced in a cell-free system containing extract of Xenopus oocytes. The formation of a comet-like structure capable of transporting vesicles from the plasma membrane into the cell depth has been studied in detail by high performance electron microscopy combined with electron tomography. A similar mechanism provides the movement of vesicles containing membrane rafts enriched with sphingolipids and cholesterol, changes in position of the nuclear spindle at meiosis, and other processes. This review will consider current ideas about actin polymerization and its regulation by actin-binding proteins and show how these mechanisms are realized in the intracellular actin-based vesicular transport system.     

More Than Just a Cargo Adapter,Melanophilin Prolongs and Slows Processive Runs of Myosin Va

Myosin Va (myoVa) is a molecular motor that processively transports cargo along actin tracks. One well studied cargo in vivo is the melanosome, a pigment organelle that is moved first by kinesin on microtubules and then handed off to myoVa for transport in the actin-rich dendritic periphery of melanocytes. Melanophilin (Mlph) is the adapter protein that links Rab27a-melanosomes to myoVa. Using total internal reflection fluorescence microscopy and quantum dot-labeled full-length myoVa, we show at the single-molecule level that Mlph increases the number of processively moving myoVa motors by 17-fold. Surprisingly, myoVa-Mlph moves ∼4-fold slower than myoVa alone and with twice the run length. These two changes greatly increase the time spent on actin, a property likely to enhance the transfer of melanosomes to the adjacent keratinocyte. In contrast to the variable stepping pattern of full-length myoVa, the myoVa-Mlph complex shows a normal gating pattern between the heads typical of a fully active motor and consistent with a cargo-dependent activation mechanism. The Mlph-dependent changes in myoVa depend on a positively charged cluster of amino acids in the actin binding domain of Mlph, suggesting that Mlph acts as a “tether” that links the motor to the track. Our results provide a molecular explanation for the uncharacteristically slow speed of melanosome movement by myoVa in vivo. More generally, these data show that proteins that link motors to cargo can modify motor properties to enhance their biological role.     

Intracellular Vesicle Acidification Promotes Maturation of Infectious Poliovirus Particles

The autophagic pathway acts as part of the immune response against a variety of pathogens. However, several pathogens subvert autophagic signaling to promote their own replication. In many cases it has been demonstrated that these pathogens inhibit or delay the degradative aspect of autophagy. Here, using poliovirus as a model virus, we report for the first time bona fide autophagic degradation occurring during infection with a virus whose replication is promoted by autophagy. We found that this degradation is not required to promote poliovirus replication. However, vesicular acidification, which in the case of autophagy precedes delivery of cargo to lysosomes, is required for normal levels of virus production. We show that blocking autophagosome formation inhibits viral RNA synthesis and subsequent steps in the virus cycle, while inhibiting vesicle acidification only inhibits the final maturation cleavage of virus particles. We suggest that particle assembly, genome encapsidation, and virion maturation may occur in a cellular compartment, and we propose the acidic mature autophagosome as a candidate vesicle. We discuss the implications of our findings in understanding the late stages of poliovirus replication, including the formation and maturation of virions and egress of infectious virus from cells.     

JIP3 Activates Kinesin-1 Motility to Promote Axon Elongation

Kinesin-1 is a molecular motor responsible for cargo transport along microtubules and plays critical roles in polarized cells, such as neurons. Kinesin-1 can function as a dimer of two kinesin heavy chains (KHC), which harbor the motor domain, or as a tetramer in combination with two accessory light chains (KLC). To ensure proper cargo distribution, kinesin-1 activity is precisely regulated. Both KLC and KHC subunits bind cargoes or regulatory proteins to engage the motor for movement along microtubules. We previously showed that the scaffolding protein JIP3 interacts directly with KHC in addition to its interaction with KLC and positively regulates dimeric KHC motility. Here we determined the stoichiometry of JIP3-KHC complexes and observed approximately four JIP3 molecules binding per KHC dimer. We then determined whether JIP3 activates tetrameric kinesin-1 motility. Using an in vitro motility assay, we show that JIP3 binding to KLC engages kinesin-1 with microtubules and that JIP3 binding to KHC promotes kinesin-1 motility along microtubules. We tested the in vivo relevance of these findings using axon elongation as a model for kinesin-1-dependent cellular function. We demonstrate that JIP3 binding to KHC, but not KLC, is essential for axon elongation in hippocampal neurons as well as axon regeneration in sensory neurons. These findings reveal that JIP3 regulation of kinesin-1 motility is critical for axon elongation and regeneration.     

Extensibility of the Extended Tail Domain of Processive and Nonprocessive Myosin V Molecules

Myosin V is a single-molecule motor that moves organelles along actin. When myosin V pulls loads inside the cell in a highly viscous environment, the force on the motor is unlikely to be constant. We propose that the tether between the single-molecule motor and the cargo (i.e., the extended tail domain of the molecule) must be able to absorb the sudden mechanical motions of the motor and allow smooth relaxation of the motion of the cargo to a new position. To test this hypothesis, we compared the elastic properties of the extended tail domains of processive (mouse myosin Va) and nonprocessive (Drosophila myosin V) molecular motors. The extended tail domain of these myosins consists of mechanically strong coiled-coil regions interspersed with flexible loops. In this work we explored the mechanical properties of coiled-coil regions using atomic force microscopy. We found that the processive and nonprocessive coiled-coil fragments display different unfolding patterns. The unfolding of coiled-coil structures occurs much later during the atomic force microscopy stretch cycle for processive myosin Va than for nonprocessive Drosophila myosin V, suggesting that this elastic tether between the cargo and motor may play an important role in sustaining the processive motions of this single-molecule motor.     

Filament-Filament Switching Can Be Regulated by Separation Between Filaments Together with Cargo Motor Number

How intracellular transport controls the probability that cargos switch at intersections between filaments is not well understood. In one hypothesis some motors on the cargo attach to one filament while others attach to the intersecting filament, and the ensuing tug-of-war determines which filament is chosen. We investigate this hypothesis using 3D computer simulations, and discover that switching at intersections increases with the number of motors on the cargo, but is not strongly dependent on motor number when the filaments touch. Thus, simply controlling the number of active motors on the cargo cannot account for in vivo observations that found reduced switching with increasing motor number, suggesting additional mechanisms of regulation. We use simulations to show that one possible way to regulate switching is by simultaneously adjusting the separation between planes containing the crossing filaments and the total number of active motors on the cargo. Heretofore, the effect of filament-filament separation on switching has been unexplored. We find that the switching probability decreases with increasing filament separation. This effect is particularly strong for cargos with only a modest number of motors. As the filament separation increases past the maximum head-to-head distance of the motor, individual motors walking along a filament will be unable to reach the intersecting filament. Thus, any switching requires that other motors on the cargo attach to the intersecting filament and haul the cargo along it, while motor(s) engaged on the original filament detach. Further, if the filament separation is large enough, the cargo can have difficulty proceeding along the initial filament because the engaged motors can walk underneath the intersecting filament, but the cargo itself cannot fit between the filaments. Thus, the cargo either detaches entirely from the original filament, or must dip to the side of the initial filament and then pass below the crossing filament.     

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.     

Actin cytoskeleton is involved in targeting of a viral Hsp70 homolog to the cell periphery

The cell-to-cell movement of plant viruses involves translocation of virus particles or nucleoproteins to and through the plasmodesmata (PDs). As we have shown previously, the movement of the Beet yellows virus requires the concerted action of five viral proteins including a homolog of cellular approximately 70-kDa heat shock proteins (Hsp70h). Hsp70h is an integral component of the virus particles and is also found in PDs of the infected cells. Here we investigate subcellular distribution of Hsp70h using transient expression of Hsp70h fused to three spectrally distinct fluorescent proteins. We found that fluorophore-tagged Hsp70h forms motile granules that are associated with actin microfilaments, but not with microtubules. In addition, immobile granules were observed at the cell periphery. A pairwise appearance of these granules at the opposite sides of cell walls and their colocalization with the movement protein of Tobacco mosaic virus indicated an association of Hsp70h with PDs. Treatment with various cytoskeleton-specific drugs revealed that the intact actomyosin motility system is required for trafficking of Hsp70h in cytosol and its targeting to PDs. In contrast, none of the drugs interfered with the PD localization of Tobacco mosaic virus movement protein. Collectively, these findings suggest that Hsp70h is translocated and anchored to PDs in association with the actin cytoskeleton.     

Relation of beet yellows virus to the phloem and to movement in the sieve tube

In minor veins of leaves of Beta vulgaris L. (sugar beet) yellows virus particles were found both in parenchyma cells and in mature sieve elements. In parenchyma cells the particles were usually confined to the cytoplasm, that is, they were absent from the vacuoles. In the sieve elements, which at maturity have no vacuoles, the particles were scattered throughout the cell. In dense aggregations the particles tended to assume an orderly arrangement in both parenchyma cells and sieve elements. Most of the sieve elements containing virus particles had mitochondria, plastids, endoplasmic reticulum, and plasma membrane normal for mature sieve elements. Some sieve elements, however, showed evidence of degeneration. Virus particles were present also in the pores of the sieve plates, the plasmodesmata connecting the sieve elements with parenchyma cells, and the plasmodesmata between parenchyma cells. The distribution of the virus particles in the phloem of Beta is compatible with the concept that plant viruses move through the phloem in the sieve tubes and that this movement is a passive transport by mass flow. The observations also indicate that the beet yellows virus moves from cell to cell and in the sieve tube in the form of complete particles, and that this movement may occur through sieve-plate pores in the sieve tube and through plasmodesmata elsewhere.     

Chemophoresis engine: A general mechanism of ATPase-driven cargo transport

Cell polarity regulates the orientation of the cytoskeleton members that directs intracellular transport for cargo-like organelles, using chemical gradients sustained by ATP or GTP hydrolysis. However, how cargo transports are directly mediated by chemical gradients remains unknown. We previously proposed a physical mechanism that enables directed movement of cargos, referred to as chemophoresis. According to the mechanism, a cargo with reaction sites is subjected to a chemophoresis force in the direction of the increased concentration. Based on this, we introduce an extended model, the chemophoresis engine, as a general mechanism of cargo motion, which transforms chemical free energy into directed motion through the catalytic ATP hydrolysis. We applied the engine to plasmid motion in a ParABS system to demonstrate the self-organization system for directed plasmid movement and pattern dynamics of ParA-ATP concentration, thereby explaining plasmid equi-positioning and pole-to-pole oscillation observed in bacterial cells and in vitro experiments. We mathematically show the existence and stability of the plasmid-surfing pattern, which allows the cargo-directed motion through the symmetry-breaking transition of the ParA-ATP spatiotemporal pattern. We also quantitatively demonstrate that the chemophoresis engine can work even under in vivo conditions. Finally, we discuss the chemophoresis engine as one of the general mechanisms of hydrolysis-driven intracellular transport.     

dnc-1/dynactin 1 Knockdown Disrupts Transport of Autophagosomes and Induces Motor Neuron Degeneration

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. We previously showed that the expression of dynactin 1, an axon motor protein regulating retrograde transport, is markedly reduced in spinal motor neurons of sporadic ALS patients, although the mechanisms by which decreased dynactin 1 levels cause neurodegeneration have yet to be elucidated. The accumulation of autophagosomes in degenerated motor neurons is another key pathological feature of sporadic ALS. Since autophagosomes are cargo of dynein/dynactin complexes and play a crucial role in the turnover of several organelles and proteins, we hypothesized that the quantitative loss of dynactin 1 disrupts the transport of autophagosomes and induces the degeneration of motor neuron. In the present study, we generated a Caenorhabditis elegans model in which the expression of DNC-1, the homolog of dynactin 1, is specifically knocked down in motor neurons. This model exhibited severe motor defects together with axonal and neuronal degeneration. We also observed impaired movement and increased number of autophagosomes in the degenerated neurons. Furthermore, the combination of rapamycin, an activator of autophagy, and trichostatin which facilitates axonal transport dramatically ameliorated the motor phenotype and axonal degeneration of this model. Thus, our results suggest that decreased expression of dynactin 1 induces motor neuron degeneration and that the transport of autophagosomes is a novel and substantial therapeutic target for motor neuron degeneration.     

In vitro reconstitution of an mRNA-transport complex reveals mechanisms of assembly and motor activation

The assembly and composition of ribonucleic acid (RNA)–transporting particles for asymmetric messenger RNA (mRNA) localization is not well understood. During mitosis of budding yeast, the Swi5p-dependent HO expression (SHE) complex transports a set of mRNAs into the daughter cell. We recombinantly reconstituted the core SHE complex and assessed its properties. The cytoplasmic precomplex contains only one motor and is unable to support continuous transport. However, a defined interaction with a second, RNA-bound precomplex after its nuclear export dimerizes the motor and activates processive RNA transport. The run length observed in vitro is compatible with long-distance transport in vivo. Surprisingly, SHE complexes that either contain or lack RNA cargo show similar motility properties, demonstrating that the RNA-binding protein and not its cargo activates motility. We further show that SHE complexes have a defined size but multimerize into variable particles upon binding of RNAs with multiple localization elements. Based on these findings, we provide an estimate of number, size, and composition of such multimeric SHE particles in the cell.     

Statistics of active transport in Xenopus melanophores cells

The transport of cell cargo, such as organelles and protein complexes in the cytoplasm, is determined by cooperative action of molecular motors stepping along polar cytoskeletal elements. Analysis of transport of individual organelles generated useful information about the properties of the motor proteins and underlying cytoskeletal elements. In this work, for the first time (to our knowledge), we study collective movement of multiple organelles using Xenopus melanophores, pigment cells that translocate several thousand of pigment granules (melanosomes), spherical organelles of a diameter of ∼1 μm. These cells disperse melanosomes in the cytoplasm in response to high cytoplasmic cAMP, while at low cAMP melanosomes cluster at the cell center. Obtained results suggest spatial and temporal organization, characterized by strong correlations between movement of neighboring organelles, with correlation length of ∼4 μm and pair lifetime ∼5 s. Furthermore, velocity statistics revealed strongly non-Gaussian velocity distribution with high velocity tails demonstrating exponential behavior suggestive of strong velocity correlations. Depolymerization of vimentin intermediate filaments using a dominant-negative vimentin mutant or actin with cytochalasin B reduced correlation of behavior of individual particles. Based on our analysis, we concluded that steric repulsion is dominant, but both intermediate filaments and actin microfilaments are involved in dynamic cross-linking organelles in the cytoplasm.     

Cargos Rotate at Microtubule Intersections during Intracellular Trafficking

Intracellular cargos are transported by molecular motors along actin and microtubules, but how their dynamics depends on the complex structure of the cytoskeletal network remains unclear. In this study, we investigated this longstanding question by measuring simultaneously the rotational and translational dynamics of cargos at microtubule intersections in living cells. We engineered two-faced particles that are fluorescent on one hemisphere and opaque on the other and used their optical anisotropy to report the rotation of cargos. We show that cargos undergo brief episodes of unidirectional and rapid rotation while pausing at microtubule intersections. Probability and amplitude of the cargo rotation depend on the geometry of the intersecting filaments. The cargo rotation is not random motion due to detachment from microtubules, as revealed by statistical analyses of the translational and rotational dynamics. Instead, it is an active rotation driven by motor proteins. Although cargos are known to pause at microtubule intersections, this study reveals a different dimension of dynamics at this seemingly static state and, more importantly, provides direct evidence showing the correlation between cargo rotation and the geometry of underlying microtubule intersections.     

Two anterograde intraflagellar transport motors cooperate to build sensory cilia on C. elegans neurons

Cilia have diverse roles in motility and sensory reception and their dysfunction contributes to cilia-related diseases. Assembly and maintenance of cilia depends on the intraflagellar transport (IFT) of axoneme, membrane, matrix and signalling proteins to appropriate destinations within the organelle. In the current model, these diverse cargo proteins bind to multiple sites on macromolecular IFT particles, which are moved by a single anterograde IFT motor, kinesin-II, from the ciliary base to its distal tip, where cargo-unloading occurs. Here, we describe the observation of fluorescent IFT motors and IFT particles moving along distinct domains within sensory cilia of wild-type and IFT-motor-mutant Caenorhabditis elegans. We show that two anterograde IFT motor holoenzymes, kinesin-II and Osm-3-kinesin, cooperate in a surprising way to control two pathways of IFT that build distinct parts of cilia. Instead of each motor independently moving its own specific cargo to a distinct destination, the two motors function redundantly to transport IFT particles along doublet microtubules adjacent to the transition zone to form the axoneme middle segment. Next, Osm-3-kinesin alone transports IFT particles along the distal singlet microtubules to stabilize the distal segment. Thus, the subtle coordinate activity of these IFT motors creates two sequential transport pathways.     

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.     

Localization of EB1, IFT polypeptides, and kinesin-2 in Chlamydomonas flagellar axonemes via immunogold scanning electron microscopy

Intraflagellar transport (IFT) refers to the bi-directional movement of particles and associated cargo along the axonemes of eukaryotic flagella and cilia. To provide a new perspective on the morphology of IFT particles, their association with the axoneme, and their composition, we have used immunogold localization coupled to detection via scanning electron microscopy. Here we co-localize in the Chlamydomonas flagellar axoneme polypeptides labeled with specific antibodies. Chlamydomonas EB1 localizes to the distal tip of the axoneme, as expected from previous immunofluorescent data (Pedersen et al. Curr Biol2003;13(22):1969-1974), thus demonstrating the utility of this approach. Using antibodies to IFT-related polypeptides, particles can be identified associated with the axoneme that fall into one of two classes: The first class is composed of IFT particles labeled with polyclonal antibodies to kinesin-2 and monoclonal antibodies to either IFT139 (an IFT complex A polypeptide) or IFT172 (a complex B polypeptide). The second class is comprised of particles that label with antibodies to IFT139 alone; thus, discrete particles are present associated with the axoneme that are composed only of complex A polypeptides. When IFT particles were purified by sucrose gradient ultracentrifugation, they appeared as more or less spherical aggregates of varying dimensions labeled with antibodies to IFT139 and to the motor protein kinesin-2. By contrast, isolated IFT particles that were labeled with IFT172 antibodies were not labeled with kinesin-2 antibodies. The data are discussed in terms of the total polypeptide composition of an IFT particle and the interaction of the particles with the motors that power IFT.