Two kinesins transport cargo primarily via the action of one motor: implications for intracellular transport

The number of microtubule motors attached to vesicles, organelles, and other subcellular commodities is widely believed to influence their motile properties. There is also evidence that cells regulate intracellular transport by tuning the number and/or ratio of motor types on cargos. Yet, the number of motors responsible for cargo motion is not easily characterized, and the extent to which motor copy number affects intracellular transport remains controversial. Here, we examined the load-dependent properties of structurally defined motor assemblies composed of two kinesin-1 molecules. We found that a group of kinesins can produce forces and move with velocities beyond the abilities of single kinesin molecules. However, such capabilities are not typically harnessed by the system. Instead, two-kinesin assemblies adopt a range of microtubule-bound configurations while transporting cargos against an applied load. The binding arrangement of motors on their filament dictates how loads are distributed within the two-motor system, which in turn influences motor-microtubule affinities. Most configurations promote microtubule detachment and prevent both kinesins from contributing to force production. These results imply that cargos will tend to be carried by only a fraction of the total number of kinesins that are available for transport at any given time, and provide an alternative explanation for observations that intracellular transport depends weakly on kinesin number in vivo.     

CRMP/UNC-33 organizes microtubule bundles for KIF5-mediated mitochondrial distribution to axon

Neurons are highly specialized cells with polarized cellular processes and subcellular domains. As vital organelles for neuronal functions, mitochondria are distributed by microtubule-based transport systems. Although the essential components of mitochondrial transport including motors and cargo adaptors are identified, it is less clear how mitochondrial distribution among somato-dendritic and axonal compartment is regulated. Here, we systematically study mitochondrial motors, including four kinesins, KIF5, KIF17, KIF1, KLP-6, and dynein, and transport regulators in C. elegans PVD neurons. Among all these motors, we found that mitochondrial export from soma to neurites is mainly mediated by KIF5/UNC-116. Interestingly, UNC-116 is especially important for axonal mitochondria, while dynein removes mitochondria from all plus-end dendrites and the axon. We surprisingly found one mitochondrial transport regulator for minus-end dendritic compartment, TRAK-1, and two mitochondrial transport regulators for axonal compartment, CRMP/UNC-33 and JIP3/UNC-16. While JIP3/UNC-16 suppresses axonal mitochondria, CRMP/UNC-33 is critical for axonal mitochondria; nearly no axonal mitochondria present in unc-33 mutants. We showed that UNC-33 is essential for organizing the population of UNC-116-associated microtubule bundles, which are tracks for mitochondrial trafficking. Disarrangement of these tracks impedes mitochondrial transport to the axon. In summary, we identified a compartment-specific transport regulation of mitochondria by UNC-33 through organizing microtubule tracks for different kinesin motors other than microtubule polarity.     

Highly Loaded Behavior of Kinesins Increases the Robustness of Transport Under High Resisting Loads

Kinesins are nano-sized biological motors which walk by repeating a mechanochemical cycle. A single kinesin molecule is able to transport its cargo about 1 μm in the absence of external loads. However, kinesins perform much longer range transport in cells by working collectively. This long range of transport by a team of kinesins is surprising because the motion of the cargo in cells can be hindered by other particles. To reveal how the kinesins are able to accomplish their tasks of transport in harsh intracellular circumstances, stochastic studies on the kinesin motion are performed by considering the binding and unbinding of kinesins to microtubules and their dependence on the force acting on kinesin molecules. The unbinding probabilities corresponding to each mechanochemical state of kinesin are modeled. The statistical characterization of the instants and locations of binding are captured by computing the probability of unbound kinesin being at given locations. It is predicted that a group of kinesins has a more efficient transport than a single kinesin from the perspective of velocity and run length. Particularly, when large loads are applied, the leading kinesin remains bound to the microtubule for long time which increases the chances of the other kinesins to bind to the microtubule. To predict effects of this behavior of the leading kinesin under large loads on the collective transport, the motion of the cargo is studied when the cargo confronts obstacles. The result suggests that the behavior of kinesins under large loads prevents the early termination of the transport which can be caused by the interference with the static or moving obstacles.     

Intracellular cargo transport by kinesin-3 motors

Intracellular transport along microtubules enables cellular cargoes to efficiently reach the extremities of large, eukaryotic cells. While it would take more than 200 years for a small vesicle to diffuse from the cell body to the growing tip of a one-meter long axon, transport by a kinesin allows delivery in one week. It is clear from this example that the evolution of intracellular transport was tightly linked to the development of complex and macroscopic life forms. The human genome encodes 45 kinesins, 8 of those belonging to the family of kinesin-3 organelle transporters that are known to transport a variety of cargoes towards the plus end of microtubules. However, their mode of action, their tertiary structure, and regulation are controversial. In this review, we summarize the latest developments in our understanding of these fascinating molecular motors.     

Diffusion of kinesin motors on cargo can enhance binding and run lengths during intracellular transport

Cellular cargoes, including lipid droplets and mitochondria, are transported along microtubules using molecular motors such as kinesins. Many experimental and computational studies focused on cargoes with rigidly attached motors, in contrast to many biological cargoes that have lipid surfaces that may allow surface mobility of motors. We extend a mechanochemical three-dimensional computational model by adding coupled-viscosity effects to compare different motor arrangements and mobilities. We show that organizational changes can optimize for different objectives: Cargoes with clustered motors are transported efficiently but are slow to bind to microtubules, whereas those with motors dispersed rigidly on their surface bind microtubules quickly but are transported inefficiently. Finally, cargoes with freely diffusing motors have both fast binding and efficient transport, although less efficient than clustered motors. These results suggest that experimentally observed changes in motor organization may be a control point for transport.     

Role of Unc104/KIF1-related motor proteins in mitochondrial transport in Neurospora crassa

Eukaryotic cells use diverse cytoskeleton-dependent machineries to control inheritance and intracellular positioning of mitochondria. In particular, microtubules play a major role in mitochondrial motility in the filamentous fungus Neurospora crassa and in mammalian cells. We examined the role of two novel Unc104/KIF1-related members of the kinesin family, Nkin2 and Nkin3, in mitochondrial motility in Neurospora. The Nkin2 protein is required for mitochondrial interactions with microtubules in vitro. Mutant hyphae lacking Nkin2 show mitochondrial motility defects in vivo early after germination of conidiospores. Nkin3, a member of a unique fungal-specific subgroup of small Unc104/KIF1-related proteins, is not associated with mitochondria in wild-type cells. However, it is highly expressed and recruited to mitochondria in Deltankin-2 mutants. Mitochondria lacking Nkin2 require Nkin3 for binding to microtubules in vitro, and mitochondrial motility defects in Deltankin-2 mutants disappear with up-regulation of Nkin3 in vivo. We propose that mitochondrial transport is mediated by Nkin2 in Neurospora, and organelle motility defects in Deltankin-2 mutants are rescued by Nkin3. Apparently, a highly versatile complement of organelle motors allows the cell to efficiently respond to exogenous challenges, a process that might also account for the great variety of different mitochondrial transport systems that have evolved in eukaryotic cells.     

Kinesin Recycling in Stationary Membrane Tubes

Collections of motors dynamically organize to extract membrane tubes. These tubes grow but often pause or change direction as they traverse an underlying microtubule (MT) network. In vitro, membrane tubes also stall: they stop growing in length despite a large group of motors available at the tip to pull them forward. In these stationary membrane tubes in vitro, we find that clusters of processive kinesin motors form and reach the tip of the tube at regular time intervals. The average times between cluster arrivals depends on the time over which motors depart from the tip, suggesting that motors are recycled toward the tip. Numerical simulations of the motor dynamics in the membrane tube and on the MTs show that the presence of cooperative binding between motors quantitatively accounts for the clustering observed experimentally. Cooperative binding along the length of the MT and a nucleation point at a distance behind the tip define the recycling period. Based on comparison of the numerical results and experimental data, we estimate a cooperative binding probability and concentration regime where the recycling phenomenon occurs.     

Peroxisomes,lipid droplets,and endoplasmic reticulum “hitchhike” on motile early endosomes

Intracellular transport is mediated by molecular motors that bind cargo to be transported along the cytoskeleton. Here, we report, for the first time, that peroxisomes (POs), lipid droplets (LDs), and the endoplasmic reticulum (ER) rely on early endosomes (EEs) for intracellular movement in a fungal model system. We show that POs undergo kinesin-3– and dynein-dependent transport along microtubules. Surprisingly, kinesin-3 does not colocalize with POs. Instead, the motor moves EEs that drag the POs through the cell. PO motility is abolished when EE motility is blocked in various mutants. Most LD and ER motility also depends on EE motility, whereas mitochondria move independently of EEs. Covisualization studies show that EE-mediated ER motility is not required for PO or LD movement, suggesting that the organelles interact with EEs independently. In the absence of EE motility, POs and LDs cluster at the growing tip, whereas ER is partially retracted to subapical regions. Collectively, our results show that moving EEs interact transiently with other organelles, thereby mediating their directed transport and distribution in the cell.     

Alphaherpesvirus infection disrupts mitochondrial transport in neurons

Mitochondria are dynamic organelles that are essential for cellular metabolism but can be functionally disrupted during pathogen infection. In neurons, mitochondria are transported on microtubules via the molecular motors kinesin-1 and dynein and recruited to energy-requiring regions such as synapses. Previous studies showed that proteins from pseudorabies virus (PRV), an alphaherpesvirus, localize to mitochondria and affect mitochondrial function. We show that PRV and herpes simplex virus type 1 (HSV-1) infection of rodent superior cervical ganglion (SCG) neurons disrupts mitochondrial motility and morphology. During PRV infection, glycoprotein B (gB)-dependent fusion events result in electrical coupling of neurons and increased action potential firing rates. Consequently, intracellular [Ca(2+)] increases and alters mitochondrial dynamics through a mechanism involving the Ca(2+)-sensitive cellular protein Miro and reduced recruitment of kinesin-1 to mitochondria. This disruption in mitochondrial dynamics is required for efficient growth and spread of PRV, indicating that altered mitochondrial transport enhances alphaherpesvirus pathogenesis and infection.     

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.     

Metazoan motor models: kinesin superfamily in C. elegans

In eukaryotic cells members of the kinesin family mediate intracellular transport by carrying cellular cargo on microtubule tracks. The nematode Caenorhabditis elegans genome encodes 21 members of the kinesin family, which show significant homology to their mammalian orthologs. Based on motor domain sequence homology and placement of the motor domain in the protein, the C. elegans kinesins have been placed in eight distinct groups; members of which participate in embryonic development, protein transport, synaptic membrane vesicles movement and in the axonal growth. Among 21 kinesins, at least 11 play a central role in spindle movement and chromosomal segregation. Understanding the function of C. elegans kinesins and related proteins may help navigate through the intricacies of intracellular traffic in a simple animal.     

Roles of Na(+)-Ca2+ exchange and of mitochondria in the regulation of presynaptic Ca2+ and spontaneous glutamate release

The release of neurotransmitter from presynaptic terminals depends on an increase in the intracellular Ca2+ concentration ([Ca2+]i). In addition to the opening of presynaptic Ca2+ channels during excitation, other Ca2+ transport systems may be involved in changes in [Ca2+]i. We have studied the regulation of [Ca2+]i in nerve terminals of hippocampal cells in culture by the Na(+)-Ca2+ exchanger and by mitochondria. In addition, we have measured changes in the frequency of spontaneous excitatory postsynaptic currents (sEPSC) before and after the inhibition of the exchanger and of mitochondrial metabolism. We found rather heterogeneous [Ca2+]i responses of individual presynaptic terminals after inhibition of Na(+)-Ca2+ exchange. The increase in [Ca2+]i became more uniform and much larger after additional treatment of the cells with mitochondrial inhibitors. Correspondingly, sEPSC frequencies changed very little when only Na(+)-Ca2+ exchange was inhibited, but increased dramatically after additional inhibition of mitochondria. Our results provide evidence for prominent roles of Na(+)-Ca2+ exchange and mitochondria in presynaptic Ca2+ regulation and spontaneous glutamate release.     

Stepping and Crowding of Molecular Motors: Statistical Kinetics from an Exclusion Process Perspective

Motor enzymes are remarkable molecular machines that use the energy derived from the hydrolysis of a nucleoside triphosphate to generate mechanical movement, achieved through different steps that constitute their kinetic cycle. These macromolecules, nowadays investigated with advanced experimental techniques to unveil their molecular mechanisms and the properties of their kinetic cycles, are implicated in many biological processes, ranging from biopolymerization (e.g., RNA polymerases and ribosomes) to intracellular transport (motor proteins such as kinesins or dyneins). Although the kinetics of individual motors is well studied on both theoretical and experimental grounds, the repercussions of their stepping cycle on the collective dynamics still remains unclear. Advances in this direction will improve our comprehension of transport process in the natural intracellular medium, where processive motor enzymes might operate in crowded conditions. In this work, we therefore extend contemporary statistical kinetic analysis to study collective transport phenomena of motors in terms of lattice gas models belonging to the exclusion process class. Via numerical simulations, we show how to interpret and use the randomness calculated from single particle trajectories in crowded conditions. Importantly, we also show that time fluctuations and non-Poissonian behavior are intrinsically related to spatial correlations and the emergence of large, but finite, clusters of comoving motors. The properties unveiled by our analysis have important biological implications on the collective transport characteristics of processive motor enzymes in crowded conditions.     

Polarization-dependent selective transport to the apical membrane by KIF5B in MDCK cells

Microtubule-based vesicular transport is well documented in epithelial cells, but the specific motors involved and their regulation during polarization are largely unknown. We demonstrate that KIF5B mediates post-Golgi transport of an apical protein in epithelial cells, but only after polarity has developed. Time-lapse imaging of EB1-GFP in polarized MDCK cells showed microtubule plus ends growing toward the apical membrane, implying that plus end-directed N-kinesins might be used to transport apical proteins. Indeed, time-lapse microscopy revealed that expression of a KIF5B dominant negative or microinjection of function-blocking KIF5 antibodies inhibited selectively post-Golgi transport of the apical marker, p75-GFP, after polarization of MDCK cells. Expression of other KIF dominant negatives did not alter p75-GFP trafficking. Immunoprecipitation experiments demonstrated an interaction between KIF5B and p75-GFP in polarized, but not in subconfluent, MDCK cells. Our results demonstrate that apical protein transport depends on selective microtubule motors and that epithelial cells switch kinesins for post-Golgi transport during acquisition of polarity.     

Stepping and Crowding of Molecular Motors: Statistical Kinetics from an Exclusion Process Perspective

Motor enzymes are remarkable molecular machines that use the energy derived from the hydrolysis of a nucleoside triphosphate to generate mechanical movement, achieved through different steps that constitute their kinetic cycle. These macromolecules, nowadays investigated with advanced experimental techniques to unveil their molecular mechanisms and the properties of their kinetic cycles, are implicated in many biological processes, ranging from biopolymerization (e.g., RNA polymerases and ribosomes) to intracellular transport (motor proteins such as kinesins or dyneins). Although the kinetics of individual motors is well studied on both theoretical and experimental grounds, the repercussions of their stepping cycle on the collective dynamics still remains unclear. Advances in this direction will improve our comprehension of transport process in the natural intracellular medium, where processive motor enzymes might operate in crowded conditions. In this work, we therefore extend contemporary statistical kinetic analysis to study collective transport phenomena of motors in terms of lattice gas models belonging to the exclusion process class. Via numerical simulations, we show how to interpret and use the randomness calculated from single particle trajectories in crowded conditions. Importantly, we also show that time fluctuations and non-Poissonian behavior are intrinsically related to spatial correlations and the emergence of large, but finite, clusters of comoving motors. The properties unveiled by our analysis have important biological implications on the collective transport characteristics of processive motor enzymes in crowded conditions.     

Interactions of mitochondria with the actin cytoskeleton

Interactions between mitochondria and the cytoskeleton are essential for normal mitochondrial morphology, motility and distribution. While microtubules and their motors have been established as important factors for mitochondrial transport, emerging evidence indicates that mitochondria interact with the actin cytoskeleton in many cell types. In certain fungi, such as the budding yeast and Aspergillus, or in plant cells mitochondrial motility is largely actin-based. Even in systems such as neurons, where microtubules are the primary means of long-distance mitochondrial transport, the actin cytoskeleton is required for short-distance mitochondrial movements and for immobilization of the organelle at the cell cortex. The actin cytoskeleton is also involved in the immobilization of mitochondria at the cortex in cultured tobacco cells and in budding yeast. While the exact nature of these immobilizations is not known, they may be important for retaining mitochondria at sites of high ATP utilization or at other cellular locations where they are needed. Recent findings also indicate that mutations in actin or actin-binding proteins can influence mitochondrial pathways leading to cell death. Thus, mitochondria-actin interactions contribute to apoptosis.     

Mitochondrial morphology is dynamic and varied

The morphology of mitochondria is dynamic, often changing within a cell and from one cell type to the next. In the past few years, significant advances have been made in the study of mechanisms that help determine the morphologies of mitochondria and their intracellular distributions. It has become apparent that the distribution of mitochondria is determined by movement along the cytoskeleton, driven by molecular motors, and attachment to the cytoskeleton, using specific connector proteins. However, not all cells use the same cytoskeletal elements and motor proteins for mitochondrial movement and attachment. The shapes of mitochondria are also influenced by the extent of mitochondrial division and fusion. A number of proteins that affect mitochondrial division and fusion were recently discovered. Here, we review the proteins involved in the distribution and morphology of mitochondria and discuss how they may be physiologically regulated.     

Lactate shuttles in nature

Once thought to be the consequence of oxygen lack in contracting skeletal muscle, the glycolytic product lactate is formed and utilized continuously under fully aerobic conditions. "Cell-cell" and "intracellular lactate shuttle" concepts describe the roles of lactate in the delivery of oxidative and gluconeogenic substrates, as well as in cell signalling. Examples of cell-cell shuttles include lactate exchanges between white-glycolytic and red-oxidative fibres within a working muscle bed, between working skeletal muscle and heart, and between tissues of net lactate release and gluconeogenesis. Lactate exchange between astrocytes and neurons that is linked to glutamatergic signalling in the brain is an example of a lactate shuttle supporting cell-cell signalling. Lactate uptake by mitochondria and pyruvate-lactate exchange in peroxisomes are examples of intracellular lactate shuttles. Lactate exchange between sites of production and removal is facilitated by monocarboxylate transport proteins, of which there are several isoforms, and, probably, also by scaffolding proteins. The mitochondrial lactate-pyruvate transporter appears to work in conjunction with mitochondrial lactate dehydrogenase, which permits lactate to be oxidized within actively respiring cells. Hence mitochondria function to establish the concentration and proton gradients necessary for cells with high mitochondrial densities (e.g. cardiocytes) to take up and oxidize lactate. Arteriovenous difference measurements on working cardiac and skeletal muscle beds as well as NMR spectral analyses of these tissues show that lactate is formed and oxidized within the cells of formation in vivo. Glycolysis and lactate oxidation within cells permits high flux rates and the maintenance of redox balance in the cytosol and mitochondria. Other examples of intracellular lactate shuttles include lactate uptake and oxidation in sperm mitochondria and the facilitation of beta-oxidation in peroxisomes by pyruvate-lactate exchange. An ancient origin to the utility of lactate shuttling is implied by the observation that mitochondria of Saccharomyces cerevisiae contain flavocytochrome b(2), a lactate-cytochrome c oxidoreductase that couples lactate dehydrogenation to the reduction of cytochrome c. The presence of cell-cell and intracellular lactate shuttles gives rise to the notion that glycolytic and oxidative pathways can be viewed as linked, as opposed to alternative, processes, because lactate, the product of one pathway, is the substrate for the other.     

Motor Reattachment Kinetics Play a Dominant Role in Multimotor-Driven Cargo Transport

Kinesin-based cargo transport in cells frequently involves the coordinated activity of multiple motors, including kinesins from different families that move at different speeds. However, compared to the progress at the single-molecule level, mechanisms by which multiple kinesins coordinate their activity during cargo transport are poorly understood. To understand these multimotor coordination mechanisms, defined pairs of kinesin-1 and kinesin-2 motors were assembled on DNA scaffolds and their motility examined in vitro. Although less processive than kinesin-1 at the single-molecule level, addition of kinesin-2 motors more effectively amplified cargo run lengths. By applying the law of total expectation to cargo binding durations in ADP, the kinesin-2 microtubule reattachment rate was shown to be fourfold faster than that of kinesin-1. This difference in microtubule binding rates was also observed in solution by stopped-flow. High-resolution tracking of a gold-nanoparticle-labeled motor with 1 ms and 2 nm precision revealed that kinesin-2 motors detach and rebind to the microtubule much more frequently than does kinesin-1. Finally, compared to cargo transported by two kinesin-1, cargo transported by two kinesin-2 motors more effectively navigated roadblocks on the microtubule track. These results highlight the importance of motor reattachment kinetics during multimotor transport and suggest a coordinated transport model in which kinesin-1 motors step effectively against loads whereas kinesin-2 motors rapidly unbind and rebind to the microtubule. This dynamic tethering by kinesin-2 maintains the cargo near the microtubule and enables effective navigation along crowded microtubules.