Kinesin motors and disease

Kinesins are motor proteins that move cargoes such as vesicles, organelles and chromosomes along microtubules. They are best known for their role in axonal transport and in mitosis. There is a diverse family of kinesins, members of which differ in composition and functions. Roles of kinesins in diseases typically involve defective transport of cell components, transport of pathogens, or cell division.     

Kinesin motors and primary cilia

Cilia and flagella play important roles in human health by contributing to cellular motility as well as sensing and responding to environmental cues. Defects in ciliary assembly and/or function can lead to a range of human diseases, collectively known as the ciliopathies, including polycystic kidney, liver and pancreatic diseases, sterility, obesity, situs inversus, hydrocephalus and retinal degeneration. A basic understanding of how cilia form and function is essential for deciphering ciliopathies and generating therapeutic treatments. The cilium is a unique compartment that contains a distinct complement of protein and lipid. However, the molecular mechanisms by which soluble and membrane protein components are targeted to and trafficked into the cilium are not well understood. Cilia are generated and maintained by IFT (intraflagellar transport) in which IFT cargoes are transported along axonemal microtubules by kinesin and dynein motors. A variety of genetic, biochemical and cell biological approaches has established the heterotrimeric kinesin-2 motor as the 'core' IFT motor, whereas other members of the kinesin-2, kinesin-3 and kinesin-4 families function as 'accessory' motors for the transport of specific cargoes in diverse cell types. Motors of the kinesin-9 and kinesin-13 families play a non-IFT role in regulating ciliary beating or axonemal length, respectively. Entry of kinesin motors and their cargoes into the ciliary compartment requires components of the nuclear import machinery, specifically importin-β2 (transportin-1) and Ran-GTP (Ran bound to GTP), suggesting that similar mechanisms may regulate entry into the nuclear and ciliary compartments.     

Coordination of Kinesin motors pulling on fluid membranes

Intracellular transport relies on the action of motor proteins, which work collectively to either carry small vesicles or pull membranes tubes along cytoskeletal filaments. Although the individual properties of kinesin-1 motors have been extensively studied, little is known on how several motors coordinate their action and spatially organize on the microtubule when pulling on fluid membranes. Here we address these questions by studying, both experimentally and numerically, the growth of membrane tubes pulled by molecular motors. Our in vitro setup allows us to simultaneously control the parameters monitoring tube growth and measure its characteristics. We perform numerical simulations of membrane tube growth, using the experimentally measured values of all parameters, and analyze the growth properties of the tube considering various motor cooperation schemes. The comparison of the numerical results and the experimental data shows that motors use simultaneously several protofilaments of a microtubule to pull a single tube, as motors moving along a single protofilament cannot generate the forces required for tube extraction. In our experimental conditions, we estimate the average number of motors pulling the tube to be approximately nine, distributed over three contiguous protofilaments. Our results also indicate that the motors pulling the tube do not step synchronously.     

Kinesin motors: no strain, no gain

The processive movement of the dimeric motor protein kinesin 1 along microtubules requires communication between the two motor domains. Yildiz et al. (2008) now show that tension between the motor domains not only is necessary for normal processivity but also may be sufficient for motor motility under some conditions.     

Nucleosome-remodelling machines and other molecular motors observed at the single-molecule level

Through its capability to transiently pack and unpack our genome, chromatin is a key player in the regulation of gene expression. Single-molecule approaches have recently complemented conventional biochemical and biophysical techniques to decipher the complex mechanisms ruling chromatin dynamics. Micromanipulations with tweezers (magnetic or optical) and imaging with molecular microscopy (electron or atomic force) have indeed provided opportunities to handle and visualize single molecules, and to measure the forces and torques produced by molecular motors, along with their effects on DNA or nucleosomal templates. By giving access to dynamic events that tend to be blurred in traditional biochemical bulk experiments, these techniques provide critical information regarding the mechanisms underlying the regulation of gene activation and deactivation by nucleosome and chromatin structural changes. This minireview describes some single-molecule approaches to the study of ATP-consuming molecular motors acting on DNA, with applications to the case of nucleosome-remodelling machines.     

Kinesin proteins: A phylum of motors for microtubule-based motility

The cellular processes of transport, division and, possibly, early development all involve microtubule-based motors. Recent work shows that, unexpectedly, many of these cellular functions are carried out by different types of kinesin and kinesin-related motor proteins. The kinesin proteins are a large and rapidly growing family of microtubule-motor proteins that share a 340-amino-acid motor domain. Phylogenetic analysis of the conserved motor domains groups the kinesin proteins into a number of subfamilies, the members of which exhibit a common molecular organization and related functions. The kinesin proteins that belong to different subfamilies differ in their rates and polarity of movement along microtubules, and probably in the particles/organelles that they transport. The kinesins arose early in eukaryotic evolution and gene duplication has allowed functional specialization to occur, resulting in a surprisingly large number of different classes of these proteins adapted for intracellular transport of vesicles and organelles, and for assembly and force generation in the meiotic and mitotic spindles.     

Mitochondrial preprotein translocases as dynamic molecular machines

Proteomic studies have demonstrated that yeast mitochondria contain roughly 1000 different proteins. Only eight of these proteins are encoded by the mitochondrial genome and are synthesized on mitochondrial ribosomes. The remaining 99% of mitochondrial precursors are encoded within the nuclear genome and after their synthesis on cytosolic ribosomes must be imported into the organelle. Targeting of these proteins to mitochondria and their import into one of the four mitochondrial subcompartments--outer membrane, intermembrane space (IMS), inner membrane and matrix--requires various membrane-embedded protein translocases, as well as numerous chaperones and cochaperones in the aqueous compartments. During the last years, several novel protein components involved in the import and assembly of mitochondrial proteins have been identified. The picture that emerges from these exciting new findings is that of highly dynamic import machineries, rather than of regulated, but static protein complexes. In this review, we will give an overview on the recent progress in our understanding of mitochondrial protein import. We will focus on the presequence translocase of the inner mitochondrial membrane, the TIM23 complex and the presequence translocase-associated motor, the PAM complex. These two molecular machineries mediate the multistep import of preproteins with cleavable N-terminal signal sequences into the matrix or inner membrane of mitochondria.     

Kinesin walks the line: single motors observed by atomic force microscopy

Motor proteins of the kinesin family move actively along microtubules to transport cargo within cells. How exactly a single motor proceeds on the 13 narrow lanes or protofilaments of a microtubule has not been visualized directly, and there persists controversy on the relative position of the two kinesin heads in different nucleotide states. We have succeeded in imaging Kinesin-1 dimers immobilized on microtubules with single-head resolution by atomic force microscopy. Moreover, we could catch glimpses of single Kinesin-1 dimers in their motion along microtubules with nanometer resolution. We find in our experiments that frequently both heads of one dimer are microtubule-bound at submicromolar ATP concentrations. Furthermore, we could unambiguously resolve that both heads bind to the same protofilament, instead of straddling two, and remain on this track during processive movement.     

Kinesin velocity increases with the number of motors pulling against viscoelastic drag

Although the properties of single kinesin molecular motors are well understood, it is not clear whether multiple motors pulling a single vesicle in a cell cooperate or interfere with one another. To learn how small numbers of motors interact, microtubule gliding assays were carried out with full-length Drosophila kinesin in a novel motility medium containing xanthan, a stiff, water-soluble polysaccharide. At 2 mg/ml xanthan, the zero-shear viscosity of this medium is 1,000 times the viscosity of water, similar to cellular viscosity. To mimic the rheological drag force on the motors when attached to a vesicle in a cell, we attached a 2 μm bead to one end of the microtubule (MT). During gliding assays in our novel medium, the moving bead exerted a drag force of 4–15 pN on the kinesins pulling the MT. The velocity of MTs with an attached bead increased with MT length and with kinesin concentration. The increase with MT length arose because the number of motors is directly proportional to MT length. Our results show that small numbers of kinesins cooperate constructively when pulling against a viscoelastic drag. In the absence of a bead but still in the viscous medium, MT velocity was independent of MT length and kinesin concentration because the thin MT, like a snake moving through grass, was able to move between xanthan molecules with little resistance. A minimal shared-load model in which the number of motors is proportional to MT length fits the observed dependence of gliding velocity on MT length and kinesin concentration.     

Fluctuation as a tool of biological molecular machines

The mechanism for biological molecular machines is different from that of man-made ones. Recently single molecule measurements and other experiments have revealed unique operations where biological molecular machines exploit thermal fluctuation in response to small inputs of energy or signals to achieve their function. Understanding and applying this mechanism to engineering offers new artificial machine designs.     

Kinesin as an Electrostatic Machine

Kinesin and related motor proteins utilize ATP fuel to propel themselves along the external surface of microtubules in a processive and directional fashion. We show that the observed step-like motion is possible through time-varying charge distributions furnished by the ATP hydrolysis cycle while the static charge configuration on the microtubule provides the guide for motion. Thus, while the chemical hydrolysis energy induces appropriate local conformational changes, the motor translational energy is fundamentally electrostatic. Numerical simulations of the mechanical equations of motion show that processivity and directionality are direct consequences of the ATP-dependent electrostatic interaction between the different charge distributions of kinesin and the microtubule.     

Multiple conformations of the nucleotide site of Kinesin family motors in the triphosphate state

Identifying conformational changes in kinesin family motors associated with nucleotide and microtubule (MT) binding is essential to determining an atomic-level model for force production and motion by the motors. Using the mobility of nucleotide analog spin probes bound at the active sites of kinesin family motors to monitor conformational changes, we previously demonstrated that, in the ADP state, the open nucleotide site closes upon MT binding [Naber, N., Minehardt, T. J., Rice, S., Chen, X., Grammer, J., Matuska, M., et al. (2003). Closing of the nucleotide pocket of kinesin family motors upon binding to microtubules. Science, 300, 798-801]. We now extend these studies to kinesin-1 (K) and ncd (nonclaret disjunctional protein) motors in ATP and ATP-analog states. Our results reveal structural differences between several triphosphate and transition-state analogs bound to both kinesin and ncd in solution. The spectra of kinesin/ncd in the presence of SLADP•AlFx/BeFx and kinesin, with the mutation E236A (K-E236A; does not hydrolyze ATP) bound to ATP, show an open conformation of the nucleotide pocket similar to that seen in the kinesin/ncd•ADP states. In contrast, the triphosphate analogs K•SLAMPPNP and K-E236A•SLAMPPNP induce a more immobilized component of the electron paramagnetic resonance spectrum, implying closing of the nucleotide site. The MT-bound states of all of the triphosphate analogs reveal two novel spectral components. The equilibrium between these two components is only weakly dependent on temperature. Both components have more restricted mobility than observed in MT-bound diphosphate states. Thus, the closing of the nucleotide pocket when the diphosphate state binds to MTs is amplified in the triphosphate state, perhaps promoting accelerated ATP hydrolysis. Consistent with this idea, molecular dynamics simulations show a good correlation between our spectroscopic data, X-ray crystallography, and the electron microscopy of MT-bound triphosphate-analog states.     

Millennial musings on molecular motors

In a universe that is dominated by increasing entropy, living organisms are a curious anomaly. The organization that distinguishes living organisms from their inanimate surroundings relies upon their ability to execute vectorial processes, such as directed movements and the assembly of macromolecules and organelle systems. Many of these phenomena are executed by molecular motors that harness chemical potential energy to perform mechanical work and unidirectional motion. This article explores how these remarkable protein machines might have evolved and what roles they could play in biological and medical research in the coming decades.     

Millennial musings on molecular motors

In a universe that is dominated by increasing entropy, living organisms are a curious anomaly. The organization that distinguishes living organisms from their inanimate surroundings relies upon their ability to execute vectorial processes, such as directed movements and the assembly of macromolecules and organelle systems. Many of these phenomena are executed by molecular motors that harness chemical potential energy to perform mechanical work and unidirectional motion. This article explores how these remarkable protein machines might have evolved and what roles they could play in biological and medical research in the coming decades.     

Interaction of the molecular motors

A review addresses the up-to-date evidence on the regulation of the organelle transport along microtubules in a very specific aspect of the interaction of the molecular motors of the opposite directions.     

Transport characteristics of molecular motors

Properties of transport of molecular motors are investigated. A simplified model based on the concept of Brownian ratchets is applied. We analyze a stochastic equation of motion by means of numerical methods. The transport is systematically studied with respect to its energetic efficiency and quality expressed by an effective diffusion coefficient. We demonstrate the role of friction and non-equilibrium driving on the transport quantifiers and identify regions of a parameter space where motors are optimally transported.     

Millennial musings on molecular motors

In a universe that is dominated by increasing entropy, living organisms are a curious anomaly. The organization that distinguishes living organisms from their inanimate surroundings relies upon their ability to execute vectorial processes, such as directed movements and the assembly of macromolecules and organelle systems. Many of these phenomena are executed by molecular motors that harness chemical potential energy to perform mechanical work and unidirectional motion. This article explores how these remarkable protein machines might have evolved and what roles they could play in biological and medical research in the coming decades.