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.     

How molecular motors are arranged on a cargo is important for vesicular transport

The spatial organization of the cell depends upon intracellular trafficking of cargos hauled along microtubules and actin filaments by the molecular motor proteins kinesin, dynein, and myosin. Although much is known about how single motors function, there is significant evidence that cargos in vivo are carried by multiple motors. While some aspects of multiple motor function have received attention, how the cargo itself--and motor organization on the cargo--affects transport has not been considered. To address this, we have developed a three-dimensional Monte Carlo simulation of motors transporting a spherical cargo, subject to thermal fluctuations that produce both rotational and translational diffusion. We found that these fluctuations could exert a load on the motor(s), significantly decreasing the mean travel distance and velocity of large cargos, especially at large viscosities. In addition, the presence of the cargo could dramatically help the motor to bind productively to the microtubule: the relatively slow translational and rotational diffusion of moderately sized cargos gave the motors ample opportunity to bind to a microtubule before the motor/cargo ensemble diffuses out of range of that microtubule. For rapidly diffusing cargos, the probability of their binding to a microtubule was high if there were nearby microtubules that they could easily reach by translational diffusion. Our simulations found that one reason why motors may be approximately 100 nm long is to improve their 'on' rates when attached to comparably sized cargos. Finally, our results suggested that to efficiently regulate the number of active motors, motors should be clustered together rather than spread randomly over the surface of the cargo. While our simulation uses the specific parameters for kinesin, these effects result from generic properties of the motors, cargos, and filaments, so they should apply to other motors as well.     

Importance of anisotropy in detachment rates for force production and cargo transport by a team of motor proteins

Many cellular processes are driven by collective forces generated by a team consisting of multiple molecular motor proteins. One aspect that has received less attention is the detachment rate of molecular motors under mechanical force/load. While detachment rate of kinesin motors measured under backward force increases rapidly for forces beyond stall‐force; this scenario is just reversed for non‐yeast dynein motors where detachment rate from microtubule decreases, exhibiting a catch‐bond type behavior. It has been shown recently that yeast dynein responds anisotropically to applied load, i.e. detachment rates are different under forward and backward pulling. Here, we use computational modeling to show that these anisotropic detachment rates might help yeast dynein motors to improve their collective force generation in the absence of catch‐bond behavior. We further show that the travel distance of cargos would be longer if detachment rates are anisotropic. Our results suggest that anisotropic detachment rates could be an alternative strategy for motors to improve the transport properties and force production by the team.     

Effect of fuel concentration and force on collective transport by a team of dynein motors

Motor proteins are essential components of intracellular transport inside eukaryotic cells. These protein molecules use chemical energy obtained from hydrolysis of ATP to produce mechanical forces required for transporting cargos inside cells, from one location to another, in a directed manner. Of these motors, cytoplasmic dynein is structurally more complex than other motor proteins involved in intracellular transport, as it shows force and fuel (ATP) concentration dependent step‐size. Cytoplasmic dynein motors are known to work in a team during cargo transport and force generation. Here, we use a complete Monte‐Carlo model of single dynein constrained by in vitro experiments, which includes the effect of both force and ATP on stepping as well as detachment of motors under force. We then use our complete Monte‐Carlo model of single dynein motor to understand collective cargo transport by a team of dynein motors, such as dependence of cargo travel distance and velocity on applied force and fuel concentration. In our model, cargos pulled by a team of dynein motors do not detach rapidly under higher forces, confirming the experimental observation of longer persistence time of dynein team on microtubule under higher forces.     

Opposite-polarity motors activate one another to trigger cargo transport in live cells

Intracellular transport is typically bidirectional, consisting of a series of back and forth movements. Kinesin-1 and cytoplasmic dynein require each other for bidirectional transport of intracellular cargo along microtubules; i.e., inhibition or depletion of kinesin-1 abolishes dynein-driven cargo transport and vice versa. Using Drosophilamelanogaster S2 cells, we demonstrate that replacement of endogenous kinesin-1 or dynein with an unrelated, peroxisome-targeted motor of the same directionality activates peroxisome transport in the opposite direction. However, motility-deficient versions of motors, which retain the ability to bind microtubules and hydrolyze adenosine triphosphate, do not activate peroxisome motility. Thus, any pair of opposite-polarity motors, provided they move along microtubules, can activate one another. These results demonstrate that mechanical interactions between opposite-polarity motors are necessary and sufficient for bidirectional organelle transport in live cells.     

Vacuolar transport in tobacco leaf epidermis cells involves a single route for soluble cargo and multiple routes for membrane cargo

We tested if different classes of vacuolar cargo reach the vacuole via distinct mechanisms by interference at multiple steps along the transport route. We show that nucleotide-free mutants of low molecular weight GTPases, including Rab11, the Rab5 members Rha1 and Ara6, and the tonoplast-resident Rab7, caused induced secretion of both lytic and storage vacuolar cargo. In situ analysis in leaf epidermis cells indicates a sequential action of Rab11, Rab5, and Rab7 GTPases. Compared with Rab5 members, mutant Rab11 mediates an early transport defect interfering with the arrival of cargo at prevacuoles, while mutant Rab7 inhibits the final delivery to the vacuole and increases cargo levels in prevacuoles. In contrast with soluble cargo, membrane cargo may follow different routes. Tonoplast targeting of an α-TIP chimera was impaired by nucleotide-free Rha1, Ara6, and Rab7 similar to soluble cargo. By contrast, the tail-anchored tonoplast SNARE Vam3 shares only the Rab7-mediated vacuolar deposition step. The most marked difference was observed for the calcineurin binding protein CBL6, which was insensitive to all Rab mutants tested. Unlike soluble cargo, α-TIP and Vam3, CBL6 transport to the vacuole was COPII independent. The results indicate that soluble vacuolar proteins follow a single route to vacuoles, while membrane spanning proteins may use at least three different transport mechanisms.     

Regulated motion of glycoproteins revealed by direct visualization of a single cargo in the endoplasmic reticulum

The quality of cargo proteins in the endoplasmic reticulum (ER) is affected by their motion during folding. To understand how the diffusion of secretory cargo proteins is regulated in the ER, we directly analyze the motion of a single cargo molecule using fluorescence imaging/fluctuation analyses. We find that the addition of two N-glycans onto the cargo dramatically alters their diffusion by transient binding to membrane components that are confined by hyperosmolarity. Via simultaneous observation of a single cargo and ER exit sites (ERESs), we could exclude ERESs as the binding sites. Remarkably, actin cytoskeleton was required for the transient binding. These results provide a molecular basis for hypertonicity-induced immobilization of cargo, which is dependent on glycosylation at multiple sites but not the completion of proper folding. We propose that diffusion of secretory glycoproteins in the ER lumen is controlled from the cytoplasm to reduce the chances of aggregation.     

The lectin ERGIC-53 is a cargo transport receptor for glycoproteins

Soluble secretory proteins are transported from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC) in vesicles coated with COP-II coat proteins. The sorting of secretory cargo into these vesicles is thought to involve transmembrane cargo-receptor proteins. Here we show that a cathepsin-Z-related glycoprotein binds to the recycling, mannose-specific membrane lectin ERGIC-53. Binding occurs in the ER, is carbohydrate- and calcium-ion-dependent and is affected by untrimmed glucose residues. Binding does not, however, require oligomerization of ERGIC-53, although oligomerization is required for exit of ERGIC-53 from the ER. Dissociation of ERGIC-53 occurs in the ERGIC and is delayed if ERGIC-53 is mislocalized to the ER. These results strongly indicate that ERGIC-53 may function as a receptor facilitating ER-to-ERGIC transport of soluble glycoprotein cargo.     

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.     

Fluctuation driven transport and models of molecular motors and pumps

Non-equilibrium fluctuations can drive vectorial transport along an anisotropic structure in an isothermal medium by biasing the effect of thermal noise (k _B T). Mechanisms based on this principle are often called Brownian ratchets and have been invoked as a possible explanation for the operation of biomolecular motors and pumps. We discuss the thermodynamics and kinetics for the operation of microscopic ratchet motors under conditions relevant to biology, showing how energy provided by external fluctuations or a non-equilibrium chemical reaction can cause unidirectional motion or uphill pumping of a substance. Our analysis suggests that molecular pumps such as Na,K-ATPase and molecular motors such as kinesin and myosin may share a common underlying mechanism. Received: 18 February 1998 / Revised version: 5 May 1998 / Accepted: 14 May 1998     

To team up or not: single versus team driving in European road freight transport

Flexible Services and Manufacturing Journal - The last decades have seen a tremendous amount of research being devoted to effectively managing vehicle fleets and minimizing empty mileage. However,...     

Theoretical formalism for bead movement powered by single two-headed motors in a motility assay

Kinesins and dyneins are protein motors that can use the free energy of ATP hydrolysis to carry a cargo and move uni-directionally along a microtubule filament. The purpose of this paper is to derive the formalism connecting the ATP-driven translocation reactions of these motors on microtubule filaments and the movement of the bead carried by the motor in a motility assay in which the bead is clamped at an arbitrary constant force. The formalism is thus useful in elucidating the load-dependent kinetic mechanism of the free-energy transduction of the motor using the mechanical data obtained from the motility assay. The formalism is also useful in assessing the effect on the measured motility data of various physical and hydrodynamic parameters of the assay, such as the size of the bead, the viscosity of the medium, the stiffness of the elastic element connecting the motor and the bead, etc. In a previous paper [Biophys. J. 67 (2000) 313] (hereafter referred to as paper I), we have derived the formalism for the case that the motor in the assay has only one head. In this paper we extend the derivation to the case that the motor is two-headed. The formalism is derived based on a simple two-state hand-over-hand model for the movement of the motor on microtubule, but can be easily extended to more complicated kinetic models. Effects of various hydrodynamic parameters on the velocity of the bead are studied with numerical calculations of the model. The difference between the formalism presented in this paper and the widely used "chemical" formalism, in which the movement of the kinesin and the bead is described by pure chemical reactions, is discussed.     

A single amino acid mutation in the mouse MEIG1 protein disrupts a cargo transport system necessary for sperm formation

Mammalian spermatogenesis is a highly coordinated process that requires cooperation between specific proteins to coordinate diverse biological functions. For example, mouse Parkin coregulated gene (PACRG) recruits meiosis-expressed gene 1 (MEIG1) to the manchette during normal spermiogenesis. Here we mutated Y68 of MEIG1 using the CRISPR/cas9 system and examined the biological and physiological consequences in mice. All homozygous mutant males examined were completely infertile, and sperm count was dramatically reduced. The few developed sperm were immotile and displayed multiple abnormalities. Histological staining showed impaired spermiogenesis in these mutant mice. Immunofluorescent staining further revealed that this mutant MEIG1 was still present in the cell body of spermatocytes, but also that more MEIG1 accumulated in the acrosome region of round spermatids. The mutant MEIG1 and a cargo protein of the MEIG1/PACRG complex, sperm-associated antigen 16L (SPAG16L), were no longer found to be present in the manchette; however, localization of the PACRG component was not changed in the mutants. These findings demonstrate that Y68 of MEIG1 is a key amino acid required for PACRG to recruit MEIG1 to the manchette to transport cargo proteins during sperm flagella formation. Given that MEIG1 and PACRG are conserved in humans, small molecules that block MEIG1/PACRG interaction are likely ideal targets for the development of male contraconception drugs.     

Moving on to the cargo problem of microtubule-dependent motors in neurons

Vigorous investigation has finally begun to shed light on the cargo problem of the microtubule-dependent motors, kinesin and dynein superfamily proteins. Biochemical observations have suggested that the potential cargoes of certain populations of motor proteins seem to be in vesicle-form, each vesicle possessing specific functional marker molecules. In addition to the close relationship between microtubule-dependent motors and cargoes in vesicle-form, kinesin has also been highlighted as an apparent driving force for another cargo in non-vesicle-form, cytoplasmic protein. On the basis of new biophysical and cell-biological evidence, the controversy over the movement of cytoplasmic cargoes has entered a new phase.     

The cargo in vacuolar storage protein transport vesicles is stratified

Developing pea seeds contain two functionally distinct vacuoles--lytic vacuoles and protein storage vacuoles (PSV). The Golgi apparatus of these cells has to discriminate between proteins destined for these vacuolar compartments. Whereas it is known that sorting into the lytic vacuole is performed via the conserved clathrin-coated vesicle pathway, sorting of proteins into the protein storage vacuole remains enigmatic. In developing pea cotyledons, the major storage proteins are sorted via 'dense vesicles'. In this report we examined the sorting of a minor protein of the protein storage vacuole, the sucrose-binding-protein homolog (SBP), along the secretory pathway employing immunoelectron microscopy on cryosectioned pea cotyledons. SBP follows the same vesicular route into the PSV as the main storage proteins legumin and vicilin, via the dense-vesicles. Furthermore, legumin and SBP are sorted together into the same dense vesicle population at the stack. Although soluble cargo proteins of the dense vesicles, they show a stratified distribution in the lumen of the dense vesicles. Whereas the legumin label is equally distributed across the lumen, the SBP label is concentrated at the membrane of the vesicle. This observation is discussed with respect to a putative receptor-mediated sorting of the proteins into the dense vesicles.     

The direct transport of oxygen in insects by large tracheae

Vast numbers of large and small tracheae in the abdomen of adult Calliphora are so modified that they can transfer oxygen directly by diffusion to the developing ovaries. Tracheoles are scarce but. instead, a system of distended tubules, evaginated into the cytoplasmic surface of the trachea, cover the entire tracheal surface with permeable tubes diffusing oxygen to the developing tissues. A similar development occurs also in the male fly.     

Efficient nuclear transport of structurally disturbed cargo: mutations in a cargo protein switch its cognate karyopherin

The Karyopherin (Kap) family of nuclear transport receptors enables trafficking of proteins to and from the nucleus in a precise, regulated manner. Individual members function in overlapping pathways, while simultaneously being very specific for their main cargoes. The details of this apparent contradiction and rules governing pathway preference remain to be further elucidated. S. cerevisiae Lhp1 is an abundant protein that functions as an RNA chaperone in a variety of biologically important processes. It localizes almost exclusively to the nucleus and is imported by Kap108. We show that mutation of 3 of the 275 residues in Lhp1 alters its import pathway to a Kap121-dependent process. This mutant does not retain wild-type function and is bound by several chaperones. We propose that Kap121 also acts as a chaperone, one that can act as a genetic buffer by transporting mutated proteins to the nucleus.     

The influence of small direct electric currents on the transport of auxin in intact plants

When a d.c. potential of 9.0 V was applied to the stem of intact pea seedlings (Pisum sativum L. cv. Meteor and cv. Alderman) via 10 mM KCl-soaked filter paper electrodes placed ca. 50 mm apart the stem passed a steady current of 15–20 A (resistance ca. 100 k cm^-1). The basipetal transport of [1-¹⁴C]IAA applied to the apical bud was completely inhibited over the portion of the stem through which current flowed and ¹⁴C-labelled compounds accumulated in the vicinity of the upper electrode. The inhibition of transport was independent of the polarity of the applied potential. The basipetal transport of IAA in the stem above the electrode was not affected.Labelled auxin accumulated at the upper electrode both as unchanged IAA and as a compound tentatively identified as indol-3yl-acetyl aspartic acid (IAAsp). These compounds were only slowly remobilised when the current was interrupted. However, the ability of the transport system to move freshly-applied IAA was rapidly and fully restored when the potential was removed. No injury to the plant was detected after maintaining a current flow for up to 72 h. No leakage of ¹⁴C-labelled compounds into the KCl solution bathing the electrodes was detected.Abbreviations IAA indol-3yl-acetic acid - IAAsp indol-3yl-acetyl aspartic acid