Dynein is the motor for retrograde axonal transport of organelles.

Vesicular organelles in axons of nerve cells are transported along microtubules either toward their plus ends (fast anterograde transport) or toward their minus ends (retrograde transport). Two microtubule-based motors were previously identified by examining plastic beads induced to move along microtubules by cytosol fractions from the squid giant axon: (i) an anterograde motor, kinesin, and (ii) a retrograde motor, which is characterized here. The retrograde motor, a cytosolic protein previously termed HMW1, was purified from optic lobes and extruded axoplasm by nucleotide-dependent microtubule affinity and release; microtubule gliding was used as the assay of motor activity. The following properties of the retrograde motor suggest that it is cytoplasmic dynein: (i) sedimentation at 20-22 S with a heavy chain of Mr greater than 200,000 that coelectrophoreses with the alpha and beta subunits of axonemal dynein, (ii) cleavage by UV irradiation in the presence of ATP and vanadate, and (iii) a molecular structure resembling two-headed dynein from axonemes. Furthermore, bead movement toward the minus end of microtubules was blocked when axoplasmic supernatants were treated with UV/vanadate. Treatment of axoplasmic supernatant with UV/vanadate also blocks the retrograde movement of purified organelles in vitro without changing the number of anterograde moving organelles, indicating that dynein interacts specifically with a subgroup of organelles programmed to move toward the cell body. However, purified optic lobe dynein, like purified kinesin, does not by itself promote the movement of purified organelles along microtubules, suggesting that additional axoplasmic factors are necessary for retrograde as well as anterograde transport.     

Unconventional myosin traffic in cells reveals a selective actin cytoskeleton

Eukaryotic cells have a self-organizing cytoskeleton where motors transport cargoes along cytoskeletal tracks. To understand the sorting process, we developed a system to observe single-molecule motility in a cellular context. We followed myosin classes V, VI, and X on triton-extracted actin cytoskeletons from Drosophila S2, mammalian COS-7, and mammalian U2OS cells. We find that these cells vary considerably in their global traffic patterns. The S2 and U2OS cells have regions of actin that either enhance or inhibit specific myosin classes. U2OS cells allow for 1 motor class, myosin VI, to move along stress fiber bundles, while motility of myosin V and X are suppressed. Myosin X motors are recruited to filopodia and the lamellar edge in S2 cells, whereas myosin VI motility is excluded from the same regions. Furthermore, we also see different velocities of myosin V motors in central regions of S2 cells, suggesting regional control of motor motility by the actin cytoskeleton. We also find unexpected features of the actin cytoskeletal network, including a population of reversed filaments with the barbed-end toward the cell center. This myosin motor regulation demonstrates that native actin cytoskeletons are more than just a collection of filaments.     

Tracking melanosomes inside a cell to study molecular motors and their interaction

Cells known as melanophores contain melanosomes, which are membrane organelles filled with melanin, a dark, nonfluorescent pigment. Melanophores aggregate or disperse their melanosomes when the host needs to change its color in response to the environment (e.g., camouflage or social interactions). Melanosome transport in cultured Xenopus melanophores is mediated by myosin V, heterotrimeric kinesin-2, and cytoplasmic dynein. Here, we describe a technique for tracking individual motors of each type, both individually and in their interaction, with high spatial (approximately 2 nm) and temporal (approximately 1 msec) localization accuracy. This method enabled us to observe (i) stepwise movement of kinesin-2 with an average step size of 8 nm; (ii) smoother melanosome transport (with fewer pauses), in the absence of intermediate filaments (IFs); and (iii) motors of actin filaments and microtubules working on the same cargo nearly simultaneously, indicating that a diffusive step is not needed between the two systems of transport. In concert with our previous report, our results also show that dynein-driven retrograde movement occurs in 8-nm steps. Furthermore, previous studies have shown that melanosomes carried by myosin V move 35 nm in a stepwise fashion in which the step rise-times can be as long as 80 msec. We observed 35-nm myosin V steps in melanophores containing no IFs. We find that myosin V steps occur faster in the absence of IFs, indicating that the IF network physically hinders organelle transport.     

A myosin motor that selects bundled actin for motility

Eukaryotic cells organize their contents through trafficking along cytoskeletal filaments. The leading edge of a typical metazoan cytoskeleton consists of a dense and complex arrangement of cortical actin. A dendritic mesh is found across the broad lamellopodium, with long parallel bundles at microspikes and filopodia. It is currently unclear whether and how myosin motors identify the few actin filaments that lead to the correct destination, when presented with many similar alternatives within the cortex. Here we show that myosin X, an actin-based motor that concentrates at the distal tips of filopodia, selects the fascin-actin bundle at the filopodial core for motility. Myosin X moves individual actin filaments poorly in vitro, often supercoiling actin into plectonemes. However, single myosin X motors move robustly and processively along fascin-actin bundles. This selection requires only parallel, closely spaced filaments, as myosin X is also processive on artificial actin bundles formed by molecular crowding. Myosin X filopodial localization is perturbed in fascin-depleted HeLa cells, demonstrating that fascin bundles also direct motility in vivo. Our results demonstrate that myosin X recognizes the local structural arrangement of filaments in long bundles, providing a mechanism for sorting cargo to distant target sites.     

Myosin Va maneuvers through actin intersections and diffuses along microtubules

Certain types of intracellular organelle transport to the cell periphery are thought to involve long-range movement on microtubules by kinesin with subsequent handoff to vertebrate myosin Va (myoVa) for local delivery on actin tracks. This process may involve direct interactions between these two processive motors. Here we demonstrate using single molecule in vitro techniques that myoVa is flexible enough to effectively maneuver its way through actin filament intersections and Arp2/3 branches. In addition, myoVa surprisingly undergoes a one-dimensional diffusive search along microtubules, which may allow it to scan efficiently for kinesin and/or its cargo. These features of myoVa may help ensure efficient cargo delivery from the cell center to the periphery.     

The interaction between cytoplasmic dynein and dynactin is required for fast axonal transport

Fast axonal transport is characterized by the bidirectional, microtubule-based movement of membranous organelles. Cytoplasmic dynein is necessary but not sufficient for retrograde transport directed from the synapse to the cell body. Dynactin is a heteromultimeric protein complex, enriched in neurons, that binds to both microtubules and cytoplasmic dynein. To determine whether dynactin is required for retrograde axonal transport, we examined the effects of anti-dynactin antibodies on organelle transport in extruded axoplasm. Treatment of axoplasm with antibodies to the p150^Glued subunit of dynactin resulted in a significant decrease in the velocity of microtubule-based organelle transport, with many organelles bound along microtubules. We examined the molecular mechanism of the observed inhibition of motility, and we demonstrated that antibodies to p150^Glued disrupted the binding of cytoplasmic dynein to dynactin and also inhibited the association of cytoplasmic dynein with organelles. In contrast, the anti-p150^Glued antibodies had no effect on the binding of dynactin to microtubules nor on cytoplasmic dynein-driven microtubule gliding. These results indicate that the interaction between cytoplasmic dynein and the dynactin complex is required for the axonal transport of membrane-bound vesicles and support the hypothesis that dynactin may function as a link between the organelle, the microtubule, and cytoplasmic dynein during vesicle transport.     

Ultrastructural studies of hepatocyte cytoskeleton in experimental cholestasis by quick-freezing and deep-etching method

The ultrastructural association between the cytoskeleton and other organelles was studied by the quick-freezing and deep-etching method in rats treated with alpha-naphthylisothiocyanate (ANIT), or phalloidin, and in rats with obstructive jaundice. Cytoplasmic filaments were classified by measuring their diameters, and actin filaments were identified by specific decoration with myosin subfragment 1 (S1). S1-positive actin filaments and S1-negative intermediate filaments (12-14 nm in diameter) were observed to form a three-dimensional network around bile canaliculi, and were more numerous than in controls, not only in phalloidin-treated rats and rats with obstructive jaundice, but also in ANIT-administered rats. In all cholestatic rats, vesicular structures were also more numerous than in controls in the pericanalicular regions, and were closely associated with the microfilaments and the intermediate filaments. Filaments of a new type were localized between the lamellae of rough-surfaced endoplasmic reticulum and mitochondria, and between the lamellae of Golgi sacs and vesicles. Other thin filaments were also observed within the network of actin filaments. These filaments were 4-6 nm in diameter on replica membranes and were never decorated with S1. They were also directly connected with the canalicular membranes. Cytoskeletal components associated with membrane-bound organelles, including these new filaments, were suggested to be involved in the localization and migration of organelles.     

A monoclonal antibody against kinesin inhibits both anterograde and retrograde fast axonal transport in squid axoplasm.

One of our monoclonal antibodies against the heavy chain of bovine kinesin (H2) also recognized the heavy chain of squid kinesin. The immunofluorescence pattern of H2 in axoplasm was similar to that seen in mammalian cells with antibodies specific for kinesin light and heavy chains, indicating that squid kinesin is also concentrated on membrane-bounded organelles. Although kinesin is assumed to be a motor for translocation of membrane-bounded organelles in fast axonal transport, direct evidence has been lacking. Perfusion of axoplasm with purified H2 at 0.1-0.4 mg/ml resulted in a profound inhibition of both the rates and number of organelles moving in anterograde and retrograde directions in the interior of the axoplasm, and comparable inhibition was noted in bidirectional movement along individual microtubules at the periphery. Maximal inhibition developed over 30-60 min. Perfusion with higher concentrations of H2 (greater than 1 mg of IgG per ml) were less effective, whereas perfusion with 0.04 mg of H2 per ml resulted in minimal inhibition. Movement of membrane-bounded organelles after perfusion with comparable levels of irrelevant mouse IgG (0.04 to greater than 1 mg/ml) were not distinguishable from perfusion with buffer controls. Inhibition of fast axonal transport by an antibody specific for kinesin provides direct evidence that kinesin is involved in the translocation of membrane-bounded organelles in axons. Moreover, the inhibition of bidirectional axonal transport by H2 raises the possibility that kinesin may play some role in both anterograde and retrograde axonal transport.     

Microinjection into an identified axon to study the mechanism of fast axonal transport.

Microinjection into an axon of an identified invertebrate neuron is shown to be a useful technique for analyzing the mechanisms of fast axonal transport. It permits direct assessment of the effect of agents that cannot permeate the plasma membrane on the translocation of material in the axon. The actin filament depolymerizer DNase I, when injected into the axon of the Aplysia neuron R2, caused a local block of fast transport of [3H]glycoprotein. Two agents that should interfere with the functioning of actin filaments without causing extensive depolymerization, tne N-ethylmaleimide-modified nuclease S1 fragment of myosin (injected) and dihydrocytochalasin B (applied externally). had no effect. Together these results suggest that actin plays a structural role in the axonal cytoskeleton rather than a role in transport force generation, the effect of DNase I being mediated by structural disordering of the axoplasm. Experiments were also done with inhibitors of dynein, the microtubule-associated ATPase. erythro-9-[3-(2-Hydroxynonyl)]adenine blocked transport but vanadate was ineffective.     

Coatomer, but not P200/myosin II, is required for the in vitro formation of trans-Golgi network-derived vesicles containing the envelope glycoprotein of vesicular stomatitis virus

Using a cytosol and nucleotide dependent assay that we previously developed, we have investigated the requirement for coat proteins in the in vitro production of trans-Golgi network (TGN)-derived vesicles from a Madin-Darby canine kidney (MDCK) cell Golgi fraction that contains the ³⁵S-labeled, terminally glycosylated, envelope glycoprotein of vesicular stomatitis virus (VSV-G) accumulated in the TGN. We found that the TGN-derived vesicles, like those involved in intra-Golgi transport and in retrograde transport to the endoplasmic reticulum, contain a coatomer coat and that coatomer is required for their formation. Thus, after they are produced with GTPγS, the coated vesicles could be captured on beads containing anticoatomer antibody. Moreover, a cytosolic protein fraction depleted of coatomer could not support vesicle formation but it did so after purified coatomer was added. We also determined that P200/myosin II does not play an essential role in the in vitro generation of TGN-derived vesicles. Thus, removal of this protein from the cytosol, by differential salt precipitation or binding to phalloidin-induced actin filaments, had no effect on vesicle generation. Nevertheless, immunodepletion of cytosol using the anti-P200/myosin II AD7 antibody abolished vesicle generation and that antibody was capable of effectively immunocapturing coated vesicles, even when these were generated in the absence of P200/myosin II. These effects, however, are explained by the unexpected finding that the AD7 antibody interacts with undenatured coatomer.     

Actin cores of hair-cell stereocilia support myosin motility.

The actin cores of hair-cell stereocilia were tested as a substrate for the movement of myosin-coated beads in an in vitro assay. Large numbers of stereocilia from bullfrog sacculi and semicircular canals were isolated by blotting onto coverglasses and were demembranated to expose the polar actin tracks of their cytoskeletal cores. Silica or polystyrene beads, coated with thick filaments of chicken skeletal muscle myosin, were added to this core preparation in the presence of ATP. Myosin-coated beads could reach some of the cores by diffusion alone, but the efficiency and precision of the assay were improved considerably by the use of "optical tweezers" (a gradient-force optical trap) to deposit the beads directly on the cores. Beads applied in this fashion bound and moved unidirectionally at 1-2 microns/s, escaping the retarding force of the trap. Actin filaments within the stereocilia are cross-linked by fimbrin, but this did not appear to interfere with the motility of myosin. Beads coated with optic-lobe kinesin were also tested for movement; these bound and moved unidirectionally at 0.1-0.2 microns/s when applied to microtubule-based kinociliary cores, but not when applied to actin-based stereociliary cores. Our results are consistent with, and lend support to, a model for hair cell adaptation in which a molecular motor such as myosin maintains tension on the mechanically gated transduction channels. Optical tweezers and video-enhanced differential interference contrast optics provide high efficiency and improved optical resolution for the in vitro analysis of myosin motility.     

Myosin VI is a processive motor with a large step size

Myosin VI is a molecular motor involved in intracellular vesicle and organelle transport. To carry out its cellular functions myosin VI moves toward the pointed end of actin, backward in relation to all other characterized myosins. Myosin V, a motor that moves toward the barbed end of actin, is processive, undergoing multiple catalytic cycles and mechanical advances before it releases from actin. Here we show that myosin VI is also processive by using single molecule motility and optical trapping experiments. Remarkably, myosin VI takes much larger steps than expected, based on a simple lever-arm mechanism, for a myosin with only one light chain in the lever-arm domain. Unlike other characterized myosins, myosin VI stepping is highly irregular with a broad distribution of step sizes.     

Movement of scallop myosin on Nitella actin filaments: regulation by calcium.

In order to determine if Ca2+ regulates scallop myosin movement on actin, we have measured motility of scallop myosin along actin filaments using a direct visual assay. This procedure consists of covalently linking myosin to 1-micron beads and pipetting them onto a parallel array of actin filaments located on the cytoplasmic face of a Nitella internodal cell. In the absence of Ca2+, scallop myosin-coated beads exhibit no directed motion; however, in the presence of pCa2+ of greater than 5.84, these beads undergo linear translocations with average velocities of 2.0 micron/s. This Ca2+ -sensitive motility requires the presence of regulatory light chains on the scallop myosin. Removal of regulatory light chains with 10 mM EDTA produces a "desensitized" myosin, no longer sensitive to Ca2+, which moves at rates of 0.09-0.3 micron in the presence or absence of Ca2+. Readdition of regulatory light chains to preparations of desensitized myosin once again confers Ca2+-sensitive motility. The Ca2+ dependence of scallop-myosin motility shows a sharp transition, consistent with the Ca2+ activation sensitivity of the actin-activated ATPase. Furthermore, relative rates of movement of calcium-regulated myosins from various molluscan species are consistent with their respective rates of ATP hydrolysis. Thus, myosin motility along actin filaments provides a sensitive and direct assay of myosin activity and is suitable for studying myosin regulation.     

Regulated bidirectional motility of melanophore pigment granules along microtubules in vitro

Although many types of membrane-bound organelles rely upon microtubule-based transport for their proper placement within the cytoplasm, the molecular mechanisms that regulate intracellular motility remain largely unknown. To address this problem, we have studied the microtubule-dependent dispersion and aggregation of pigment granules from an immortalized Xenopus melanophore cell line. We have reconstituted pigment granule motility along bovine brain microtubules in vitro using a microscope-based motility assay. Pigment granules, or melanosomes, move along single microtubules bidirectionally; however, analysis of the polarities of this movement shows that melanosomes that have been purified from dispersed cells exhibit mostly plus end-directed motility, while movement of organelles from aggregating cells is biased toward the minus end. Removal of all soluble proteins from the melanosome fractions by density gradient centrifugation does not diminish organelle motility, demonstrating that all the components required for transport have a stable association with the melanosome membranes. Western blotting shows the presence of the plus end-directed motor, kinesin-II, and the minus end-directed motor, cytoplasmic dynein in highly purified melanosomes. Therefore, purified melanosomes retain their ability to move along microtubules as well as their regulated state. Direct biochemical comparison of melanosomes from aggregated and dispersed cells may elucidate the molecular mechanisms that regulate organelle transport in melanophores.     

Motion of myosin head domains during activation and force development in skeletal muscle

Muscle contraction is driven by a change in the structure of the head domain of myosin, the "working stroke" that pulls the actin filaments toward the midpoint of the myosin filaments. This movement of the myosin heads can be measured very precisely in intact muscle cells by X-ray interference, but until now this technique has not been applied to physiological activation and force generation following electrical stimulation of muscle cells. By using this approach, we show that the long axes of the myosin head domains are roughly parallel to the filaments in resting muscle, with their center of mass offset by approximately 7 nm from the C terminus of the head domain. The observed mass distribution matches that seen in electron micrographs of isolated myosin filaments in which the heads are folded back toward the filament midpoint. Following electrical stimulation, the heads move by approximately 10 nm away from the filament midpoint, in the opposite direction to the working stroke. The time course of this motion matches that of force generation, but is slower than the other structural changes in the myosin filaments on activation, including the loss of helical and axial order of the myosin heads and the change in periodicity of the filament backbone. The rate of force development is limited by that of attachment of myosin heads to actin in a conformation that is the same as that during steady-state isometric contraction; force generation in the actin-attached head is fast compared with the attachment step.     

Dimerized Drosophila myosin VIIa: a processive motor

The molecular mechanism of processive movement of single myosin molecules from classes V and VI along their actin tracks has recently attracted extraordinary attention. Another member of the myosin superfamily, myosin VII, plays vital roles in the sensory function of Drosophila and mammals. We studied the molecular mechanism of Drosophila myosin VIIa, using transient kinetics and single-molecule motility assays. Myosin VIIa moves along actin filaments as a processive, double-headed single molecule when dimerized by the inclusion of a leucine zipper at the C terminus of the coiled-coil domain. Its motility is approximately 8-10 times slower than that of myosin V, and its step size is 30 nm, which is consistent with the presence of five IQ motifs in its neck region. The kinetic basis for the processive motility of myosin VIIa is the relative magnitude of the release rate constants of phosphate (fast) and ADP (slow) as in myosins V and VI. The ATPase pathway is rate-limited by a reversible interconversion between two distinct ADP-bound actomyosin states, which results in high steady-state occupancy of a strongly actin-bound myosin species. The distinctive features of myosin VIIa (long run lengths, slow motility) will be very useful in video-based single-molecule applications. In cells, this kinetic behavior would allow myosin VIIa to exert and hold tension on actin filaments and, if dimerized, to function as a processive cargo transporter.     

Particles move along actin filament bundles in nerve growth cones.

Organelle movement along actin filaments has been demonstrated in dissociated squid axoplasm [Kurznetsov, S. A., Langford, G.M. & Weiss, D. G. (1992) Nature (London) 356, 722-725 and Bearer, E.L., DeGiorgis, J.A., Bodner, R.A., Kao, A.W. & Reese, T.S. (1993) Proc. Natl. Acad. Sci. USA 90, 11252-11256] but has not been shown to occur in intact neurons. Here we demonstrate that intracellular transport occurs along actin filament bundles in intact neuronal growth cones. We used video-enhanced differential interference contrast microscopy to observe intracellular transport in superior cervical ganglion neurons cultured under conditions that enhance the visibility of actin bundles within growth cone lamellipodia. Intracellular particles, ranging in size from < 0.5-1.5 microns, moved along linear structures (termed transport bundles) at an average maximum rate of 0.48 micron/sec. After particle movement had been viewed, cultures were preserved by rapid perfusion with chemical fixative. To determine whether particle transport occurred along actin, we then used fluorescence microscopy to correlate this movement with actin and microtubule distributions in the same growth cones. The observed transport bundles colocalized with actin but not with microtubules. The rates of particle movement and the association of moving particles with actin filament bundles suggest that myosins may participate in the transport of organelles (or other materials) in intact neurons.     

Arabidopsis myosin XI mutant is defective in organelle movement and polar auxin transport

Myosins are eukaryotic molecular motors moving along actin filaments. Only a small set of myosin classes is present in plants, in which myosins have been found to play a role in cytoplasmic streaming and chloroplast movement. Whereas most studies have been done on green algae, more recent data suggest a role of higher plant myosin at the postcytokinetic cell wall. Here we characterize a loss-of-function mutation for a myosin of plant-specific class XI and demonstrate myosin functions during plant development in Arabidopsis. T-DNA insertion in MYA2 caused pleiotropic effects, including flower sterility and dwarf growth. Elongation of epidermal cells, such as in hypocotyls and anther filaments, was reduced by up to 50% of normal length. This effect on anther filaments is responsible for flower sterility. In the meristems of root tips, it was evident that cell division was delayed and that cell plates were mislocated. Like zwichel, a kinesin-related mutation causing two-branched trichomes, the mya2 knockout causes branching defects, but here the trichomes remained unbranched. Growth was also impaired in pollen tubes and root hairs, cells that are highly dependent on vesicle transport. A failure in vesicle flow could be directly confirmed, because cytoplasmic streaming of vesicles and, more so, of large endoplasmic reticulum-based organelles was slowed. The defect in vesicle trafficking was accompanied by failures in basipetal auxin transport, measured in stem segments of inflorescences. This result strongly suggests a causal link between auxin-dependent processes and the distribution of vesicles and membrane-bound molecules by plant myosin.     

Myosin V and Kinesin act as tethers to enhance each others' processivity

Organelle transport to the periphery of the cell involves coordinated transport between the processive motors kinesin and myosin V. Long-range transport takes place on microtubule tracks, whereas final delivery involves shorter actin-based movements. The concept that motors only function on their appropriate track required further investigation with the recent observation that myosin V undergoes a diffusional search on microtubules. Here we show, using single-molecule techniques, that a functional consequence of myosin V's diffusion on microtubules is a significant enhancement of the processive run length of kinesin when both motors are present on the same cargo. The degree of run length enhancement correlated with the net positive charge in loop 2 of myosin V. On actin, myosin V also undergoes longer processive runs when kinesin is present on the same cargo. The process that causes run length enhancement on both cytoskeletal tracks is electrostatic. We propose that one motor acts as a tether for the other and prevents its diffusion away from the track, thus allowing more steps to be taken before dissociation. The resulting run length enhancement likely contributes to the successful delivery of cargo in the cell.