Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport.These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles ¹. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available.Building on the classic inverted motility assay ², the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo ³. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface.The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA⁴, quantum dots ⁵ or a wide variety of antigens via biotinylated antibodies ^4-6.Download video file.^{(57M, mov)}     

Traffic control: adaptor proteins guide dynein–cargo takeoff

The precise movement of intracellular components requires active transport by molecular motors along the filamentous tracks of the cytoskeleton. While yeast cytoplasmic dynein can walk for some distance along microtubules, mammalian dynein is non‐processive. This has raised the question of how this motor can transport cargo. In two recent papers by the Carter, Bullock and Vale labs, mammalian dynein processivity has now been successfully reconstituted in vitro in the presence of adaptor proteins.     

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.     

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.     

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.     

Kinesin and dynein-dynactin at intersecting microtubules: motor density affects dynein function

Kinesin and cytoplasmic dynein are microtubule-based motor proteins that actively transport material throughout the cell. Microtubules can intersect at a variety of angles both near the nucleus and at the cell periphery, and the behavior of molecular motors at these intersections has implications for long-range transport efficiency and accuracy. To test motor function at microtubule intersections, crossovers were arranged in vitro using flow to orient successive layers of filaments. Single kinesin and cytoplasmic dynein-dynactin molecules fused with green-fluorescent protein, and artificial bead cargos decorated with multiple motors, were observed while they encountered intersections. Single kinesins tend to cross intersecting microtubules, whereas single dynein-dynactins have a more varied response. For bead cargos, kinesin motion is independent of motor number. Dynein beads with high motor numbers pause, but their actions become more varied as the motor number decreases. These results suggest that regulating the number of active dynein molecules could change a motile cargo into one that is anchored at an intersection, consistent with dynein's proposed transport and tethering functions in the cell.     

MAPping out distribution routes for kinesin couriers

In the crowded environment of eukaryotic cells, diffusion is an inefficient distribution mechanism for cellular components. Long‐distance active transport is required and is performed by molecular motors including kinesins. Furthermore, in highly polarised, compartmentalised and plastic cells such as neurons, regulatory mechanisms are required to ensure appropriate spatio‐temporal delivery of neuronal components. The kinesin machinery has diversified into a large number of kinesin motor proteins as well as adaptor proteins that are associated with subsets of cargo. However, many mechanisms contribute to the correct delivery of these cargos to their target domains. One mechanism is through motor recognition of sub‐domain‐specific microtubule (MT) tracks, sign‐posted by different tubulin isoforms, tubulin post‐translational modifications, tubulin GTPase activity and MT‐associated proteins (MAPs). With neurons as a model system, a critical review of these regulatory mechanisms is presented here, with a particular focus on the emerging contribution of compartmentalised MAPs. Overall, we conclude that – especially for axonal cargo – alterations to the MT track can influence transport, although in vivo, it is likely that multiple track‐based effects act synergistically to ensure accurate cargo distribution.     

Regulating cytoskeleton-based vesicle motility

During vesicular transport, the assembly of the coat complexes and the selection of cargo proteins must be coordinated with the subsequent translocation of vesicles from the donor to an acceptor compartment. Here, we review recent progress toward uncovering the molecular mechanisms that connect transport vesicles to the protein machinery responsible for cytoskeleton-mediated motility. An emerging theme is that vesicle cargo proteins, either directly or through binding interactions with coat proteins, are able to influence cytoskeletal dynamics and motor protein function. Hence, a vesicle's cargo composition may help direct its intracellular motility and targeting.     

Dynein anchors its mRNA cargo after apical transport in the Drosophila blastoderm embryo

Molecular motors actively transport many types of cargo along the cytoskeleton in a wide range of organisms. One class of cargo is localized mRNAs, which are transported by myosin on actin filaments or by kinesin and dynein on microtubules. How the cargo is kept at its final intracellular destination and whether the motors are recycled after completion of transport are poorly understood. Here, we use a new RNA anchoring assay in living Drosophila blastoderm embryos to show that apical anchoring of mRNA after completion of dynein transport does not depend on actin or on continuous active transport by the motor. Instead, apical anchoring of RNA requires microtubules and involves dynein as a static anchor that remains with the cargo at its final destination. We propose a general principle that could also apply to other dynein cargo and to some other molecular motors, whereby cargo transport and anchoring reside in the same molecule.     

The role of selective transport in neuronal polarization

Neurons are functionally and morphologically polarized and possess two distinct types of neurites: axons and dendrites. Key molecules for axon formation are transported along microtubules and accumulated at the distal end of the nascent axons. In this review, we summarize recent advances in the understanding of the mechanisms involved in selective transport in neurons. In addition, we focus on motor proteins, cargo, cargo adaptors, and the loading and unloading of cargo.     

Targeted movement of cell end factors in fission yeast

Kinesins are microtubule-based motor proteins that transport cargo to specific locations within the cell. However, the mechanisms by which cargoes are directed to specific cellular locations have remained elusive. Here, we investigated the in vivo movement of the Schizosaccharomyces pombe kinesin Tea2 to establish how it is targeted to microtubule tips and cell ends. Tea2 is loaded onto microtubules in the middle of the cell, in close proximity to the nucleus, and then travels using its intrinsic motor activity primarily at the tips of polymerizing microtubules. The microtubule-associated protein Mal3, an EB1 homologue, is required for loading and/or processivity of Tea2 and this function can be substituted by human EB1. In addition, the cell-end marker Tea1 is required to anchor Tea2 to cell ends. Movement of Tea1 and the CLIP170 homologue Tip1 to cell ends is abolished in Tea2 rigor (ATPase) mutants. We propose that microtubule-based transport from the vicinity of the nucleus to cell ends can be precisely regulated, with Mal3 required for loading/processivity, Tea2 for movement and Tea1 for cell-end anchoring.     

Melanosomes on the move: a model to understand organelle dynamics

Advances in live-cell microscopy have revealed the extraordinarily dynamic nature of intracellular organelles. Moreover, movement appears to be critical in establishing and maintaining intracellular organization and organellar and cellular function. Motility is regulated by the activity of organelle-associated motor proteins, kinesins, dyneins and myosins, which move cargo along polar MT (microtubule) and actin tracks. However, in most instances, the motors that move specific organelles remain mysterious. Over recent years, pigment granules, or melanosomes, within pigment cells have provided an excellent model for understanding the molecular mechanisms by which motor proteins associate with and move intracellular organelles. In the present paper, we discuss recent discoveries that shed light on the mechanisms of melanosome transport and highlight future prospects for the use of pigment cells in unravelling general molecular mechanisms of intracellular transport.     

Directed attachment of antibodies to kinesin‐powered molecular shuttles

Biomolecular motors, such as kinesin, have been used to shuttle a range of biological and synthetic cargo in microfluidic architectures. A critical gap in this technology is the ability to controllably link macromolecular cargo on microtubule (MT) shuttles without forming extraneous byproducts that may potentially limit their application. Here we present a generalized approach for functionalizing MTs with antibodies in which covalent bonds are formed between the carbohydrate in F_c region of polyclonal antibodies and the positively charged amino acids on the MT surface using the crosslinker succinimidyl 4‐hydrazidoterephthalate hydrochloride (SHTH). Antibody‐functionalized MTs (Ab‐MTs) produced through this approach maintained motility characteristics and antigenic selectivity, and did not produce undesirable byproducts common to other approaches. We also demonstrate and characterize the application of these Ab‐MTs for capturing and transporting bacterial and viral antigens. While this approach cannot be applied to monoclonal antibodies, which lack a carbohydrate moiety, it may be used for selectively functionalizing MT shuttles with a variety of carbohydrate‐containing cargoes. Biotechnol. Bioeng. 2009; 104: 1182–1188. © 2009 Wiley Periodicals, Inc.     

Characterization of shuttle mechanisms for the transport of reducing equivalents into mitochondria

The malate-aspartate, fatty acid, and α-glycerophosphate shuttles for the transport of reducing equivalents into mitochondria were reconstituted, using isolated hepatic mitochondria and the extramitochondrial components of the shuttles. Clofibrate and thyroxin increased, while propylthiouracil treatment decreased, the activity of mitochondrial α-glycerophosphate dehydrogenase. Despite these changes, the activity of the reconstituted α-glycerophosphate shuttle was similar in mitochondria from control rats and those from rats treated with clofibrate and propylthiouracil. There was an increase in the activity of the shuttle using mitochondria from thyroxin-treated rats. Rotenone caused 60–90% inhibition of this shuttle, suggesting that rotenone-sensitive NADH dehydrogenase participates in the pathway of oxidation of extramitochondrial hydrogen. Palmitate, oleate, and octanoate were equally effective in reconstituting a cyclic fatty acid shuttle. The shuttle was inhibited by various compounds affecting mitochondrial metabolism, including oligomycin, dinitrophenol, cyanide, rotenone, atractyloside, and α-bromopalmitate. Carnitine and several dicarboxylic and tricarboxylic acids which stimulate fatty acid elongation, augmented fatty acid shuttle activity. The malate-aspartate shuttle was inhibited by cycloserine, amino-oxyacetic acid, and hydrazine, and stimulated by pyridoxal phosphate, at the same concentrations which affected the activities of cytoplasmic and mitochondrial glutamic oxalacetic transaminase. This shuttle was inhibited by uncouplers, antimycin, azide, cyanide, rotenone, amobarbital, oligomycin, and several inhibitors of anion transport including iodobenzylmalonate and avenaciolide. The reconstituted shuttle is sufficiently active to provide about 70–80% of the oxalacetate required for maximal rates of gluconeogenesis. Extrapolations based on the rates of mitochondrial oxidation of acetaldehyde and the activity of the microsomal ethanol oxidizing system suggest that any one of the shuttles could account for the rate of ethanol metabolism in vitro by the alcohol dehydrogenase pathway.     

Bidirectional transport of organelles: unity and struggle of opposing motors

Bidirectional transport along microtubules is ensured by opposing motor proteins: cytoplasmic dynein that drives cargo to the minus-ends and various kinesins that generally move to the plus-ends of microtubules. Regulation of motor proteins that are simultaneously bound to the same organelle is required to maintain directional transport and prevent pausing of cargo pulled away by motors of opposite polarity. Debates of the recent decade have been focused on two possible mechanisms of such regulation: (i) coordination, which implies that only one type of motors is active at a given time, and (ii) tug-of-war, which assumes that both motors are active at the same time and that direction of transport depends on the outcome of motor's confrontation. The initial idea of coordination has been challenged by observations of simultaneous activity of plus- and minus-end-directed motors applied to the same cargo. Analysis of the available data indicates that coordination and tug-of-war theories rather complement than contradict each other: cargo interacts with two teams of active motors, the resulting direction and the winner team are determined by coordination complexes, but the activity of the loser team is never completely inhibited and remains at some background level. Such persisting activity might enhance the overall efficiency of transport by increasing processivity or helping to overcome the obstacles on microtubule track.     

The mechanochemistry of integrated motor protein complexes

The assembly of molecular motor proteins into multi-unit protein complexes plays an important role in determining the intracellular transport and trafficking properties of many subcellular commodities. Yet, it is not known how proteins within these complexes interact and function collectively. Considering the established ties between motor transport and diseases, it has become increasingly important to investigate the functional properties of these essential transport ‘motifs’. Doing so requires that the composite motile and force-generating properties of multi-unit motor assemblies are characterized. However, such analyses are typically confounded by a lack of understanding of the links between the structural and mechanical properties of many motor complexes. New experimental challenges also emerge when one examines motor cooperation. Distributions in the mechanical microstates available to motor ensembles must be examined in order to fully understand the transport behavior of multi-motor complexes. Furthermore, mechanisms by which motors communicate must be explored to determine whether motor groups can move cargo together in a truly cooperative fashion. Resolving these issues requires the development of experimental methods that allow the dynamics of complex systems of transport proteins to be monitored with the same precision available to single-molecule biophysical assays. Herein, we discuss key fundamental principles governing the function of motor complexes and their relation to mechanisms that regulate intracellular cargo transport. We also outline new experimental strategies to resolve these essential features of intracellular transport.     

Nuclear localization signal peptides induce molecular delivery along microtubules

Many essential processes in eukaryotic cells depend on regulated molecular exchange between its two major compartments, the cytoplasm and the nucleus. In general, nuclear import of macromolecular complexes is dependent on specific peptide signals and their recognition by receptors that mediate translocation through the nuclear pores. Here we address the question of how protein products bearing such nuclear localization signals arrive at the nuclear membrane before import, i.e., by simple diffusion or perhaps with assistance of cytoskeletal elements or cytoskeleton-associated motor proteins. Using direct single-particle tracking and detailed statistical analysis, we show that the presence of nuclear localization signals invokes active transport along microtubules in a cell-free Xenopus egg extract. Chemical and antibody inhibition of minus-end directed cytoplasmic dynein blocks this active movement. In the intact cell, where microtubules project radially from the centrosome, such an interaction would effectively deliver nuclear-targeted cargo to the nuclear envelope in preparation for import.