Modeling bidirectional transport of quantum dot nanoparticles in membrane nanotubes

This paper develops a model of transport of quantum dot (QD) nanoparticles in membrane nanotubes (MNTs). It is assumed that QDs are transported inside intracellular organelles (called here nanoparticle-loaded vesicles, NLVs) that are propelled by either kinesin or dynein molecular motors while moving on microtubules (MTs). A vesicle may have both types of motors attached to it, but the motors are assumed to work in a cooperative fashion, meaning that at a given time the vesicle is moved by either kinesin or dynein motors. The motors are assumed not to work against each other, when one type of motors is pulling the vesicle, the other type is inactive. From time to time the motors may switch their roles: passive motors can become active motors and vice versa, resulting in the change of the vesicle’s direction of motion. It is further assumed that QDs can escape NLVs and become free QDs, which are then transported by diffusion. Free QDs can be internalized by NLVs. The effects of two possible types of MT orientation in MNTs are investigated: when all MTs have a uniform polarity orientation, with their plus-ends directed toward one of the cells connected by an MNT, and when MTs have a mixed polarity orientation, with half of MTs having their plus-ends directed toward one of the cells and the other half having their plus-ends directed toward the other cell. Computational results are presented for three cases. The first case is when organelles are as likely to be transported by kinesin motors as by dynein motors. The second case is when organelles are more likely to be transported by kinesin motors than by dynein motors, and the third case is when NLVs do not associate with dynein motors at all.     

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.     

Autoinhibition of the kinesin-2 motor KIF17 via dual intramolecular mechanisms

Long-distance transport in cells is driven by kinesin and dynein motors that move along microtubule tracks. These motors must be tightly regulated to ensure the spatial and temporal fidelity of their transport events. Transport motors of the kinesin-1 and kinesin-3 families are regulated by autoinhibition, but little is known about the mechanisms that regulate kinesin-2 motors. We show that the homodimeric kinesin-2 motor KIF17 is kept in an inactive state in the absence of cargo. Autoinhibition is caused by a folded conformation that enables nonmotor regions to directly contact and inhibit the enzymatic activity of the motor domain. We define two molecular mechanisms that contribute to autoinhibition of KIF17. First, the C-terminal tail interferes with microtubule binding; and second, a coiled-coil segment blocks processive motility. The latter is a new mechanism for regulation of kinesin motors. This work supports the model that autoinhibition is a general mechanism for regulation of kinesin motors involved in intracellular trafficking events.     

Molecular motors: strategies to get along

The majority of active transport in the cell is driven by three classes of molecular motors: the kinesin and dynein families that move toward the plus-end and minus-end of microtubules, respectively, and the unconventional myosin motors that move along actin filaments. Each class of motor has different properties, but in the cell they often function together. In this review we summarize what is known about their single-molecule properties and the possibilities for regulation of such properties. In view of new results on cytoplasmic dynein, we attempt to rationalize how these different classes of motors might work together as part of the intracellular transport machinery. We propose that kinesin and myosin are robust and highly efficient transporters, but with somewhat limited room for regulation of function. Because cytoplasmic dynein is less efficient and robust, to achieve function comparable to the other motors it requires a number of accessory proteins as well as multiple dyneins functioning together. This necessity for additional factors, as well as dynein's inherent complexity, in principle allows for greatly increased control of function by taking the factors away either singly or in combination. Thus, dynein's contribution relative to the other motors can be dynamically tuned, allowing the motors to function together differently in a variety of situations.     

The dynein microtubule motor

Dyneins are large multi-component microtubule-based molecular motors involved in many fundamental cellular processes including vesicular transport, mitosis and ciliary/flagellar beating. In order to achieve useful work, these enzymes must contain motor, cargo-binding and regulatory components. The ATPase and microtubule motor domains are located within the very large dynein heavy chains that form the globular heads and stems of the complex. Cargo-binding activity involves the intermediate chains and several classes of light chain that associate in a subcomplex at the base of the soluble dynein particle. Regulatory control of dynein motor function is thought to involve the phosphorylation of various components as well as a series of light chain proteins that are directly associated with the heavy chains. These latter polypeptides have a variety of intriguing attributes, including redox-sensitive vicinal dithiols and Ca(2+)-binding, suggesting that the activity of individual dyneins may be subject to multiple regulatory inputs. Recent molecular, genetic and structural studies have revealed insight into the roles played by these various components and the mechanisms of dynein-based motility.     

Coupling viruses to dynein and kinesin-1

It is now clear that transport on microtubules by dynein and kinesin family motors has an important if not critical role in the replication and spread of many different viruses. Understanding how viruses hijack dynein and kinesin motors using a limited repertoire of proteins offers a great opportunity to determine the molecular basis of motor recruitment. In this review, we discuss the interactions of dynein and kinesin-1 with adenovirus, the α herpes viruses: herpes simplex virus (HSV1) and pseudorabies virus (PrV), human immunodeficiency virus type 1 (HIV-1) and vaccinia virus. We highlight where the molecular links to these opposite polarity motors have been defined and discuss the difficulties associated with identifying viral binding partners where the basis of motor recruitment remains to be established. Ultimately, studying microtubule-based motility of viruses promises to answer fundamental questions as to how the activity and recruitment of the dynein and kinesin-1 motors are coordinated and regulated during bi-directional transport.     

Dynein, dynactin, and kinesin II's interaction with microtubules is regulated during bidirectional organelle transport

The microtubule motors, cytoplasmic dynein and kinesin II, drive pigmented organelles in opposite directions in Xenopus melanophores, but the mechanism by which these or other motors are regulated to control the direction of organelle transport has not been previously elucidated. We find that cytoplasmic dynein, dynactin, and kinesin II remain on pigment granules during aggregation and dispersion in melanophores, indicating that control of direction is not mediated by a cyclic association of motors with these organelles. However, the ability of dynein, dynactin, and kinesin II to bind to microtubules varies as a function of the state of aggregation or dispersion of the pigment in the cells from which these molecules are isolated. Dynein and dynactin bind to microtubules when obtained from cells with aggregated pigment, whereas kinesin II binds to microtubules when obtained from cells with dispersed pigment. Moreover, the microtubule binding activity of these motors/dynactin can be reversed in vitro by the kinases and phosphatase that regulate the direction of pigment granule transport in vivo. These findings suggest that phosphorylation controls the direction of pigment granule transport by altering the ability of dynein, dynactin, and kinesin II to interact with microtubules.     

A direct interaction between cytoplasmic dynein and kinesin I may coordinate motor activity

Cytoplasmic dynein and kinesin I are both unidirectional intracellular motors. Dynein moves cargo toward the cell center, and kinesin moves cargo toward the cell periphery. There is growing evidence that bi-directional motility is regulated in the cell, potentially through direct interactions between oppositely oriented motors. We have identified a direct interaction between cytoplasmic dynein and kinesin I. Using the yeast two-hybrid assay and affinity chromatography, we demonstrate that the intermediate chain of dynein binds to kinesin light chains 1 and 2. The interaction is both direct and specific. Co-immunoprecipitation experiments demonstrate an interaction between endogenous proteins in rat brain cytosol. Double-label immunocytochemistry reveals a partial co-localization of vesicle-associated motor proteins. Together these observations suggest that soluble motors can interact, potentially allowing kinesin I to actively localize dynein to cellular sites of function. There is also a vesicle population with both dynein and kinesin I bound that may be capable of bi-directional motility along cellular microtubules.     

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.     

Cargo transport: molecular motors navigate a complex cytoskeleton

Intracellular cargo transport requires microtubule-based motors, kinesin and cytoplasmic dynein, and the actin-based myosin motors to maneuver through the challenges presented by the filamentous meshwork that comprises the cytoskeleton. Recent in vitro single molecule biophysical studies have begun to explore this process by characterizing what occurs as these tiny molecular motors happen upon an intersection between two cytoskeletal filaments. These studies, in combination with in vivo work, define the mechanism by which molecular motors exchange cargo while traveling between filamentous tracks and deliver it to its destination when going from the cell center to the periphery and back again.     

Quantifying Protein Copy Number in Super Resolution Using an Imaging-Invariant Calibration

The use of super-resolution microscopy in recent years has revealed that proteins often form small assemblies inside cells and are organized in nanoclusters. However, determining the copy number of proteins within these nanoclusters constitutes a major challenge because of unknown labeling stoichiometries and complex fluorophore photophysics. We previously developed a DNA-origami-based calibration approach to extract protein copy number from super-resolution images. However, the applicability of this approach is limited by the fact that the calibration is dependent on the specific labeling and imaging conditions used in each experiment. Hence, the calibration must be repeated for each experimental condition, which is a formidable task. Here, using cells stably expressing dynein intermediate chain fused to green fluorescent protein (HeLa IC74 cells) as a reference sample, we demonstrate that the DNA-origami-based calibration data we previously generated can be extended to super-resolution images taken under different experimental conditions, enabling the quantification of any green-fluorescent-protein-fused protein of interest. To do so, we first quantified the copy number of dynein motors within nanoclusters in the cytosol and along the microtubules. Interestingly, this quantification showed that dynein motors form assemblies consisting of more than one motor, especially along microtubules. This quantification enabled us to use the HeLa IC74 cells as a reference sample to calibrate and quantify protein copy number independently of labeling and imaging conditions, dramatically improving the versatility and applicability of our approach.     

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.     

A requirement for cytoplasmic dynein and dynactin in intermediate filament network assembly and organization

We present evidence that vimentin intermediate filament (IF) motility in vivo is associated with cytoplasmic dynein. Immunofluorescence reveals that subunits of dynein and dynactin are associated with all structural forms of vimentin in baby hamster kidney-21 cells. This relationship is also supported by the presence of numerous components of dynein and dynactin in IF-enriched cytoskeletal preparations. Overexpression of dynamitin biases IF motility toward the cell surface, leading to a perinuclear clearance of IFs and their redistribution to the cell surface. IF-enriched cytoskeletal preparations from dynamitin-overexpressing cells contain decreased amounts of dynein, actin-related protein-1, and p150Glued relative to controls. In contrast, the amount of dynamitin is unaltered in these preparations, indicating that it is involved in linking vimentin cargo to dynactin. The results demonstrate that dynein and dynactin are required for the normal organization of vimentin IF networks in vivo. These results together with those of previous studies also suggest that a balance among the microtubule (MT) minus and plus end-directed motors, cytoplasmic dynein, and kinesin are required for the assembly and maintenance of type III IF networks in interphase cells. Furthermore, these motors are to a large extent responsible for the long recognized relationships between vimentin IFs and MTs.     

Dynactin: coordinating motors with opposite inclinations

In eukaryotic cells, many organelles are transported bidirectionally along microtubules by kinesin and dynein. These opposite-polarity motors appear to be coordinated to avoid interfering with each other's function. New work has provided the first molecular insight into how such coordination might occur.     

Regulation of kinesin-directed movements

Bidirectional organelle transport along microtubules is most likely mediated by the opposing forces generated by two microtubule-based motors: kinesin and cytoplasmic dynein. Because the direction and timing of organelle movements are controlled by the cell, the activity of one or both of these motor molecules must be regulated. Recent studies demonstrate that kinesin, kinesin-like proteins and kinesin-associated proteins can be phosphorylated, and suggest that changes in their phosphorylation state may modulate kinesin's ability to interact with either microtubules or organelles. Thus, it is possible that phosphorylation regulates kinesin-driven movements.     

Motor Regulation Results in Distal Forces that Bend Partially Disintegrated Chlamydomonas Axonemes into Circular Arcs

The bending of cilia and flagella is driven by forces generated by dynein motor proteins. These forces slide adjacent microtubule doublets within the axoneme, the motile cytoskeletal structure. To create regular, oscillatory beating patterns, the activities of the axonemal dyneins must be coordinated both spatially and temporally. It is thought that coordination is mediated by stresses or strains, which build up within the moving axoneme, and somehow regulate dynein activity. During experimentation with axonemes subjected to mild proteolysis, we observed pairs of doublets associating with each other and forming bends with almost constant curvature. By modeling the statics of a pair of filaments, we show that the activity of the motors concentrates at the distal tips of the doublets. Furthermore, we show that this distribution of motor activity accords with models in which curvature, or curvature-induced normal forces, regulates the activity of the motors. These observations, together with our theoretical analysis, provide evidence that dynein activity can be regulated by curvature or normal forces, which may, therefore, play a role in coordinating the beating of cilia and flagella.     

The role of motor proteins in endosomal sorting

Microtubule motor proteins play key roles in the spatial organization of intracellular organelles as well as the transfer of material between them. This is well illustrated both by the vectorial transfer of biosynthetic cargo from the endoplasmic reticulum to the Golgi apparatus as well as the sorting of secretory and endocytic cargo in the endosomal system. Roles have been described for dynein and kinesin motors in each of these steps. Cytoplasmic dynein is a highly complex motor comprising multiple subunits that provide functional specialization. The family of human kinesins includes over 40 members. This complexity provides immense functional diversity, yet little is known of the specific requirements and functions of individual motors during discrete membrane trafficking steps. In the present paper, we describe some of the latest findings in this area that seek to define the mechanisms of recruitment and control of activity of microtubule motors in spatial organization and cargo trafficking through the endosomal network.     

Motor Regulation Results in Distal Forces that Bend Partially Disintegrated Chlamydomonas Axonemes into Circular Arcs

The bending of cilia and flagella is driven by forces generated by dynein motor proteins. These forces slide adjacent microtubule doublets within the axoneme, the motile cytoskeletal structure. To create regular, oscillatory beating patterns, the activities of the axonemal dyneins must be coordinated both spatially and temporally. It is thought that coordination is mediated by stresses or strains, which build up within the moving axoneme, and somehow regulate dynein activity. During experimentation with axonemes subjected to mild proteolysis, we observed pairs of doublets associating with each other and forming bends with almost constant curvature. By modeling the statics of a pair of filaments, we show that the activity of the motors concentrates at the distal tips of the doublets. Furthermore, we show that this distribution of motor activity accords with models in which curvature, or curvature-induced normal forces, regulates the activity of the motors. These observations, together with our theoretical analysis, provide evidence that dynein activity can be regulated by curvature or normal forces, which may, therefore, play a role in coordinating the beating of cilia and flagella.     

Novel roles for saccharomyces cerevisiae mitotic spindle motors.

The single cytoplasmic dynein and five of the six kinesin-related proteins encoded by Saccharomyces cerevisiae participate in mitotic spindle function. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle. Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell. This study reveals that kinesin-related Kar3p and Kip3p are unique in that they perform roles both inside and outside the nucleus. Kar3p, like Kip3p, was found to be required for spindle positioning in the absence of dynein. The spindle positioning role of Kar3p is performed in concert with the Cik1p accessory factor, but not the homologous Vik1p. Kar3p and Kip3p were also found to overlap for a function essential for the structural integrity of the bipolar spindle. The cytoplasmic and nuclear roles of both these motors could be partially substituted for by the microtubule-destabilizing agent benomyl, suggesting that these motors perform an essential microtubule-destabilizing function. In addition, we found that yeast cell viability could be supported by as few as two microtubule-based motors: the BimC-type kinesin Cin8p, required for spindle structure, paired with either Kar3p or Kip3p, required for both spindle structure and positioning.     

The dynein heavy chain: structure, mechanics and evolution

The translocation of dynein along microtubules is the basis for a variety of essential cellular movements. Despite a general domain organization that is found in all the cytoskeletal motors, there are structural features of dynein that set it apart from the other motors. These include a track-binding site that is located at the tip of a long projection, and six nucleotide-binding modules that together form the globular head of dynein. These unique features suggest that dynein produces movement by a mechanism that is different from that used by the other motors.