Short-range axonal/dendritic transport by myosin-V: A model for vesicle delivery to the synapse

Myosin-V is a versatile motor involved in short-range axonal/dendritic transport of vesicles in the actin-rich cortex and synaptic regions of nerve cells. It binds to several different kinds of neuronal vesicles by its globular tail domain but the mechanism by which it is recruited to these vesicles is not known. In this study, we used an in vitro motility assay derived from axoplasm of the squid giant axon to study the effects of the globular tail domain on the transport of neuronal vesicles. We found that the globular tail fragment of myosin-V inhibited actin-based vesicle transport by displacing native myosin-V and binding to vesicles. The globular tail domain pulled down kinesin, a known binding partner of myosin-V, in affinity isolation experiments. These data confirmed earlier evidence that kinesin and myosin-V interact to form a hetero-motor complex. The formation of a kinesin/myosin-V hetero-motor complex on vesicles is thought to facilitate the coordination of long-range movement on microtubules and short-range movement on actin filaments. The direct interaction of motors from both filament systems may represent the mechanism by which the transition of vesicles from microtubules to actin filaments is regulated. These results are the first demonstration that the recombinant tail of myosin-V inhibits vesicle transport in an in vitro motility assay. Future experiments are designed to determine the functional significance of the interaction between myosin-V and kinesin and to identify other proteins that bind to the globular tail domain of myosin-V.     

Alignment, zippering and splaying of microtubules during axogenesis

Neurodegenerative diseases may result in part from defects in motor-driven vesicle transport in neuronal cells. Myosin-V, an actin-based motor that is highly enriched in the brain, mediates the movement of vesicles on cortical actin filaments. Recent evidence suggests that the globular tail of myosin-V interacts with the microtubule-based motor, kinesin, to form a 'hetero-motor' complex on vesicles. The complex of these two motors, one microtubule-based and the other actin-based, facilitates the movement of vesicles from microtubules to actin filaments. Based on our studies of vesicle transport by these two motors in extracts of squid neurons, we hypothesize that one of the functions of the tail–tail interaction is to provide feedback between the two proteins to allow seamless transition of vesicles from microtubules to actin filaments. To study the interactions of the globular tail domain of myosin-V to kinesin and to neuronal vesicles, we used a GST-tagged globular tail fragment in motility assays. The MyoV tail fragment inhibited vesicle transport by 81–91% and thereby exhibited a dominant negative effect. These data show that the recombinant protein blocked the activity of native myosin-V presumably by binding to vesicles and competing away the native myosin-V motors. The GST-MyoV-tail fragment pulled down kinesin by immunoprecipitation from squid brain homogenates and therefore it exhibited binding properties of native myosin-V. These data show that the headless myosin-V fragment is an effective inhibitor of vesicle transport in cell extracts. These studies support the hypothesis that tail–tail interactions may be a mechanism for feedback between myosin-V and kinesin to allow transition of vesicles from microtubules to actin filaments. Acknowledgements: Supported by NSF grant MCB9974709.     

Symposium 4: Dynamics of the Neuronal Cytoskeleton. Actin‐based vesicle transport in nerve cell extracts: implications for neurodegenerative diseases

Neurodegenerative diseases may result in part from defects in motor‐driven vesicle transport in neuronal cells. Myosin‐V, an actin‐based motor that is highly enriched in the brain, mediates the movement of vesicles on cortical actin filaments. Recent evidence suggests that the globular tail of myosin‐V interacts with the microtubule‐based motor, kinesin, to form a ‘hetero‐motor’ complex on vesicles. The complex of these two motors, one microtubule‐based and the other actin‐based, facilitates the movement of vesicles from microtubules to actin filaments. Based on our studies of vesicle transport by these two motors in extracts of squid neurons, we hypothesize that one of the functions of the tail–tail interaction is to provide feedback between the two proteins to allow seamless transition of vesicles from microtubules to actin filaments. To study the interactions of the globular tail domain of myosin‐V to kinesin and to neuronal vesicles, we used a GST‐tagged globular tail fragment in motility assays. The MyoV tail fragment inhibited vesicle transport by 81–91% and thereby exhibited a dominant negative effect. These data show that the recombinant protein blocked the activity of native myosin‐V presumably by binding to vesicles and competing away the native myosin‐V motors. The GST‐MyoV‐tail fragment pulled down kinesin by immunoprecipitation from squid brain homogenates and therefore it exhibited binding properties of native myosin‐V. These data show that the headless myosin‐V fragment is an effective inhibitor of vesicle transport in cell extracts. These studies support the hypothesis that tail–tail interactions may be a mechanism for feedback between myosin‐V and kinesin to allow transition of vesicles from microtubules to actin filaments. Acknowledgements: Supported by NSF grant MCB9974709.     

A simple kinetic model describes the processivity of myosin-v

Myosin-V is a motor protein responsible for organelle and vesicle transport in cells. Recent single-molecule experiments have shown that it is an efficient processive motor that walks along actin filaments taking steps of mean size close to 36 nm. A theoretical study of myosin-V motility is presented following an approach used successfully to analyze the dynamics of conventional kinesin but also taking some account of step-size variations. Much of the present experimental data for myosin-V can be well described by a two-state chemical kinetic model with three load-dependent rates. In addition, the analysis predicts the variation of the mean velocity and of the randomness-a quantitative measure of the stochastic deviations from uniform, constant-speed motion-with ATP concentration under both resisting and assisting loads, and indicates a substep of size d(0) approximately 13-14 nm (from the ATP-binding state) that appears to accord with independent observations.     

Kinesin motors as molecular machines

Molecular motor proteins, fueled by energy from ATP hydrolysis, move along actin filaments or microtubules, performing work in the cell. The kinesin microtubule motors transport vesicles or organelles, assemble bipolar spindles or depolymerize microtubules, functioning in basic cellular processes. The mechanism by which motor proteins convert energy from ATP hydrolysis into work is likely to differ in basic ways from man-made machines. Several mechanical elements of the kinesin motors have now been tentatively identified, permitting researchers to begin to decipher the mechanism of motor function. The force-producing conformational changes of the motor and the means by which they are amplified are probably different for the plus- and minus-end kinesin motors.     

In vitro assays demonstrate that pollen tube organelles use kinesin-related motor proteins to move along microtubules

The movement of pollen tube organelles relies on cytoskeletal elements. Although the movement of organelles along actin filaments in the pollen tube has been studied widely and is becoming progressively clear, it remains unclear what role microtubules play. Many uncertainties about the role of microtubules in the active transport of pollen tube organelles and/or in the control of this process remain to be resolved. In an effort to determine if organelles are capable of moving along microtubules in the absence of actin, we extracted organelles from tobacco pollen tubes and analyzed their ability to move along in vitro-polymerized microtubules under different experimental conditions. Regardless of their size, the organelles moved at different rates along microtubules in the presence of ATP. Cytochalasin D did not inhibit organelle movement, indicating that actin filaments are not required for organelle transport in our assay. The movement of organelles was cytosol independent, which suggests that soluble factors are not necessary for the organelle movement to occur and that microtubule-based motor proteins are present on the organelle surface. By washing organelles with KI, it was possible to release proteins capable of gliding carboxylated beads along microtubules. Several membrane fractions, which were separated by Suc density gradient centrifugation, showed microtubule-based movement. Proteins were extracted by KI treatment from the most active organelle fraction and then analyzed with an ATP-sensitive microtubule binding assay. Proteins isolated by the selective binding to microtubules were tested for the ability to glide microtubules in the in vitro motility assay, for the presence of microtubule-stimulated ATPase activity, and for cross-reactivity with anti-kinesin antibodies. We identified and characterized a 105-kD organelle-associated motor protein that is functionally, biochemically, and immunologically related to kinesin. This work provides clear evidence that the movement of pollen tube organelles is not just actin based; rather, they show a microtubule-based motion as well. This unexpected finding suggests new insights into the use of pollen tube microtubules, which could be used for short-range transport, as actin filaments are in animal cells.     

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.     

A molecular network for the transport of the TI-VAMP/VAMP7 vesicles from cell center to periphery

The compartmental organization of eukaryotic cells is?maintained dynamically by vesicular trafficking. SNARE proteins play a crucial role in intracellular membrane fusion and need to be targeted to their proper donor or acceptor membrane. The molecular mechanisms that allow for the secretory vesicles carrying the v-SNARE TI-VAMP/VAMP7 to leave the?cell center, load onto microtubules, and reach the periphery to mediate exocytosis are largely unknown. Here, we show that the TI-VAMP/VAMP7 partner Varp, a Rab21 guanine nucleotide exchange factor, interacts with GolginA4 and the kinesin 1 Kif5A. Activated Rab21-GTP in turn binds to MACF1, an actin and microtubule regulator, which is itself a partner of GolginA4. These components are required for directed movement of TI-VAMP/VAMP7 vesicles from the cell center to the cell periphery. The molecular mechanisms uncovered here suggest an integrated view of the transport of vesicles carrying a specific v-SNARE toward the cell surface.     

Force-dependent stepping kinetics of myosin-V

Myosin-V is a processive two-headed actin-based motor protein involved in many intracellular transport processes. A key question for understanding myosin-V function and the communication between its two heads is its behavior under load. Since in vivo myosin-V colocalizes with other much stronger motors like kinesins, its behavior under superstall forces is especially relevant. We used optical tweezers with a long-range force feedback to study myosin-V motion under controlled external forward and backward loads over its full run length. We find the mean step size remains constant at approximately 36 nm over a wide range of forces from 5 pN forward to 1.5 pN backward load. We also find two force-dependent transitions in the chemomechanical cycle. The slower ADP-release is rate limiting at low loads and depends only weakly on force. The faster rate depends more strongly on force. The stronger force dependence suggests this rate represents the diffusive search of the leading head for its binding site. In contrast to kinesin motors, myosin-V's run length is essentially independent of force between 5 pN of forward to 1.5 pN of backward load. At superstall forces of 5 pN, we observe continuous backward stepping of myosin-V, indicating that a force-driven reversal of the power stroke is possible.     

Molecular motors in axonal transport

Neurons require a large amount of intracellular transport. Cytoplasmic polypeptides and membrane-bounded organelles move from the perikaryon, down the length of the axon, and to the synaptic terminals. This movement occurs at distinct rates and is termed axonal transport. Axonal transport is divided into the slow transport of cytoplasmic proteins including glycolytic enzymes and cytoskeletal structures and the fast transport of membrane-bounded organelles along linear arrays of microtubules. The polypeptide compositions of the rate classes of axonal transport have been well characterized, but the underlying molecular mechanisms of this movement are less clear. Progress has been particularly slow toward understanding force-generation in slow transport, but recent developments have provided insight into the molecular motors involved in fast axonal transport. Recent advances in the cellular and molecular biology of one fast axonal transport motor, kinesin, have provided a clearer understanding of organelle movement along microtubules. The availability of cellular and molecular probes for kinesin and other putative axonal transport motors have led to a reevaluation of our understanding of intracellular motility.     

Myosin-V makes two brownian 90 degrees rotations per 36-nm step

Myosin-V processively walks on actin filaments in a hand-over-hand fashion. The identical structures of the heads predict a symmetric hand-over-hand mechanism where regular, unidirectional rotation occurs during a 36-nm step. We investigated this by observing how fixed myosin-V rotates actin filaments. Actin filaments randomly rotated 90 degrees both clockwise and counter-clockwise during each step. Furthermore, ATP-dependent rotations were regularly followed by ATP-independent ones. Kinetic analysis indicated that the two 90 degrees rotations relate to the coordinated unbinding and rebinding of the heads with actin. We propose a 'brownian rotation hand-over-hand' model, in which myosin-V randomly rotates by thermally twisting its elastic neck domains during the 36-nm step. The brownian rotation may be advantageous for cargo transport through a crowded actin meshwork and for carrying cargoes reliably via multiple myosin-V molecules in the cell.     

Novel dendritic kinesin sorting identified by different process targeting of two related kinesins: KIF21A and KIF21B

Neurons use kinesin and dynein microtubule-dependent motor proteins to transport essential cellular components along axonal and dendritic microtubules. In a search for new kinesin-like proteins, we identified two neuronally enriched mouse kinesins that provide insight into a unique intracellular kinesin targeting mechanism in neurons. KIF21A and KIF21B share colinear amino acid similarity to each other, but not to any previously identified kinesins outside of the motor domain. Each protein also contains a domain of seven WD-40 repeats, which may be involved in binding to cargoes. Despite the amino acid sequence similarity between KIF21A and KIF21B, these proteins localize differently to dendrites and axons. KIF21A protein is localized throughout neurons, while KIF21B protein is highly enriched in dendrites. The plus end-directed motor activity of KIF21B and its enrichment in dendrites indicate that models suggesting that minus end-directed motor activity is sufficient for dendrite specific motor localization are inadequate. We suggest that a novel kinesin sorting mechanism is used by neurons to localize KIF21B protein to dendrites since its mRNA is restricted to the cell body.     

Cytoplasmic dynein and microtubule transport in the axon: The action connection

The neuron uses two families of microtubule-based motors for fast axonal transport, kinesin, and cytoplasmic dynein. Cytoplasmic dynein moves membranous organelles from the distal regions of the axon to the cell body. Because dynein is synthesized in the cell body, it must first be delivered to the axon tip. It has recently been shown that cytoplasmic dynein is moved from the cell body along the axon by two different mechanisms. A small amount is associated with fast anterograde transport, the membranous organelles moved by kinesin. Most of the dynein is transported in slow component b, the actin-based transport compartment. Dynactin, a protein complex that binds dynein, is also transported in slow component b. The dynein in slow component b binds to microtubules in an ATP-dependent manner in vitro, suggesting that this dynein is enzymatically active. The finding that functionally active dynein, and dynactin, are associated with the actin-based transport compartment suggests a mechanism whereby dynein anchored to the actin cytoskeleton via dynactin provides the motive force for microtubule movement in the axon.     

Complex formation with kinesin motor domains affects the structure of microtubules

Microtubules are highly dynamic components of the cytoskeleton. They are important for cell movement and they are involved in a variety of transport processes together with motor proteins, such as kinesin. The exact mechanism of these transport processes is not known and so far the focus has been on structural changes within the motor domains, but not within the underlying microtubule structure.Here we investigated the interaction between kinesin and tubulin and our experimental data show that microtubules themselves are changing structure during that process. We studied unstained, vitrified samples of microtubules composed of 15 protofilaments using cryo electron microscopy and helical image analysis. 3D maps of plain microtubules and microtubules decorated with kinesin have been reconstructed to approximately 17A resolution. The alphabeta-tubulin dimer could be identified and, according to our data, alpha- and beta-tubulin adopt different conformations in plain microtubules. Significant differences were detected between maps of plain microtubules and microtubule-kinesin complexes. Most pronounced is the continuous axial inter-dimer contact in the microtubule-kinesin complex, suggesting stabilized protofilaments along the microtubule axis. It seems, that mainly structural changes within alpha-tubulin are responsible for this observation. Lateral effects are less pronounced. Following our data, we believe, that microtubules play an active role in intracellular transport processes through modulations of their core structure.     

The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors.

Many intracellular compartments, including MHC class II-containing lysosomes, melanosomes, and phagosomes, move along microtubules in a bidirectional manner and in a stop-and-go fashion due to the alternating activities of a plus-end directed kinesin motor and a minus-end directed dynein-dynactin motor. It is largely unclear how motor proteins are targeted specifically to different compartments. Rab GTPases recruit and/or activate several proteins involved in membrane fusion and vesicular transport. They associate with specific compartments after activation, which makes Rab GTPases ideal candidates for controlling motor protein binding to specific membranes. We and others [7] have identified a protein, called RILP (for Rab7-interacting lysosomal protein), that interacts with active Rab7 on late endosomes and lysosomes. Here we show that RILP prevents further cycling of Rab7. RILP expression induces the recruitment of functional dynein-dynactin motor complexes to Rab7-containing late endosomes and lysosomes. Consequently, these compartments are transported by these motors toward the minus end of microtubules, effectively inhibiting their transport toward the cell periphery. This signaling cascade may be responsible for timed and selective dynein motor recruitment onto late endosomes and lysosomes.     

Models of motor-assisted transport of intracellular particles

One-dimensional models are presented for the macroscopic intracellular transport of vesicles and organelles by molecular motors on a network of aligned intracellular filaments. A motor-coated vesicle or organelle is described as a diffusing particle binding intermittently to filaments, when it is transported at the motor velocity. Two models are treated in detail: 1) a unidirectional model, where only one kind of motor is operative and all filaments have the same polarity; and 2) a bidirectional model, in which filaments of both polarities exist (for example, a randomly polarized actin network for myosin motors) and/or particles have plus-end and minus-end motors operating on unipolar filaments (kinesin and dynein on microtubules). The unidirectional model provides net particle transport in the absence of a concentration gradient. A symmetric bidirectional model, with equal mixtures of filament polarities or plus-end and minus-end motors of the same characteristics, provides rapid transport down a concentration gradient and enhanced dispersion of particles from a point source by motor-assisted diffusion. Both models are studied in detail as a function of the diffusion constant and motor velocity of bound particles, and their rates of binding to and detachment from filaments. These models can form the basis of more realistic models for particle transport in axons, melanophores, and the dendritic arms of melanocytes, in which networks of actin filaments and microtubules coexist and motors for both types of filament are implicated.     

Microtubule-dependent movement of late endocytic vesicles in vitro: requirements for Dynein and Kinesin

Our previous studies demonstrated that fluorescent early endocytic vesicles prepared from rat liver after injection of Texas red asialoorosomucoid contain asialoglycoprotein and its receptor and move and undergo fission along microtubules using kinesin I and KIFC2, with Rab4 regulating KIFC2 activity (J. Cell Sci. 116, 2749, 2003). In the current study, procedures to prepare fluorescent late endocytic vesicles were devised. In addition, flow cytometry was utilized to prepare highly purified fluorescent endocytic vesicles, permitting validation of microscopy-based experiments as well as direct biochemical analysis. These studies revealed that late vesicles bound to and moved along microtubules, but in contrast to early vesicles, did not undergo fission. As compared with early vesicles, late vesicles had reduced association with receptor, Rab4, and kinesin I but were highly associated with dynein, Rab7, dynactin, and KIF3A. Dynein and KIF3A antibodies inhibited late vesicle motility, whereas kinesin I and KIFC2 antibodies had no effect. Dynamitin antibodies prevented the association of late vesicles with microtubules. These results indicate that acquisition and exchange of specific motor and regulatory proteins characterizes and may regulate the transition of early to late endocytic vesicles. Flow cytometric purification should ultimately facilitate detailed proteomic analysis and mapping of endocytic vesicle-associated proteins.     

Aspergillus myosin-V supports polarized growth in the absence of microtubule-based transport

In the filamentous fungus Aspergillus nidulans, both microtubules and actin filaments are important for polarized growth at the hyphal tip. Less clear is how different microtubule-based and actin-based motors work together to support this growth. Here we examined the role of myosin-V (MYOV) in hyphal growth. MYOV-depleted cells form elongated hyphae, but the rate of hyphal elongation is significantly reduced. In addition, although wild type cells without microtubules still undergo polarized growth, microtubule disassembly abolishes polarized growth in MYOV-depleted cells. Thus, MYOV is essential for polarized growth in the absence of microtubules. Moreover, while a triple kinesin null mutant lacking kinesin-1 (KINA) and two kinesin-3s (UNCA and UNCB) undergoes hyphal elongation and forms a colony, depleting MYOV in this triple mutant results in lethality due to a severe defect in polarized growth. These results argue that MYOV, through its ability to transport secretory cargo, can support a significant amount of polarized hyphal tip growth in the absence of any microtubule-based transport. Finally, our genetic analyses also indicate that KINA (kinesin-1) rather than UNCA (kinesin-3) is the major kinesin motor that supports polarized growth in the absence of MYOV.     

Insulin-induced GLUT4 translocation involves protein kinase C-lambda-mediated functional coupling between Rab4 and the motor protein kinesin

Insulin stimulates glucose transport by promoting translocation of GLUT4 proteins from the perinuclear compartment to the cell surface. It has been previously suggested that the microtubule-associated motor protein kinesin, which transports cargo toward the plus end of microtubules, plays a role in translocating GLUT4 vesicles to the cell surface. In this study, we investigated the role of Rab4, a small GTPase-binding protein, and the motor protein KIF3 (kinesin II in mice) in insulin-induced GLUT4 exocytosis in 3T3-L1 adipocytes. Photoaffinity labeling of Rab4 with [gamma-(32)P]GTP-azidoanilide showed that insulin stimulated Rab4 GTP loading and that this insulin effect was inhibited by pretreatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 or expression of dominant-negative protein kinase C-lambda (PKC-lambda). Consistent with previous reports, expression of dominant-negative Rab4 (N121I) decreased insulin-induced GLUT4 translocation by 45%. Microinjection of an anti-KIF3 antibody into 3T3-L1 adipocytes decreased insulin-induced GLUT4 exocytosis by 65% but had no effect on endocytosis. Coimmunoprecipitation experiments showed that Rab4, but not Rab5, physically associated with KIF3, and this was confirmed by showing in vitro association using glutathione S-transferase-Rab4. A microtubule capture assay demonstrated that insulin stimulation increased the activity for the binding of KIF3 to microtubules and that this activation was inhibited by pretreatment with the PI3-kinase inhibitor LY294002 or expression of dominant-negative PKC-lambda. Taken together, these data indicate that (i) insulin signaling stimulates Rab4 activity, the association of Rab4 with kinesin, and the interaction of KIF3 with microtubules and (ii) this process is mediated by insulin-induced PI3-kinase-dependent PKC-lambda activation and participates in GLUT4 exocytosis in 3T3-L1 adipocytes.     

Tctex-1, a novel interaction partner of Rab3D, is required for osteoclastic bone resorption

Vesicular transport along microtubules must be strictly regulated to sustain the unique structural and functional polarization of bone-resorbing osteoclasts. However, the molecular mechanisms bridging these vesicle-microtubule interactions remain largely obscure. Rab3D, a member of the Rab3 subfamily (Rab3A/B/C/D) of small exocytotic GTPases, represents a core component of the osteoclastic vesicle transport machinery. Here, we identify a new Rab3D-interacting partner, Tctex-1, a light chain of the cytoplasmic dynein microtubule motor complex, by a yeast two-hybrid screen. We demonstrate that Tctex-1 binds specifically to Rab3D in a GTP-dependent manner and co-occupies Rab3D-bearing vesicles in bone-resorbing osteoclasts. Furthermore, we provide evidence that Tctex-1 and Rab3D intimately associate with the dynein motor complex and microtubules in osteoclasts. Finally, targeted disruption of Tctex-1 by RNA interference significantly impairs bone resorption capacity and mislocalizes Rab3D vesicles in osteoclasts, attesting to the notion that components of the Rab3D-trafficking pathway contribute to the maintenance of osteoclastic resorptive function.