Kinesin Molecular Motors Research Articles

Structural Basis for Nucleotide Exchange and Power Stroke Generation by the Kinesin Molecular Motor

Kinesin molecular motors use energy derived from ATP to step along microtubules, driving many essential processes in eukaryotic cells, including mitosis, vesicle transport and cytoskeletal remodeling. Several conformational states of kinesin have been identified by X-ray crystallography, but the structural transitions used by kinesin to generate force and movement along intact microtubules have remained unclear. We used recent improvements in cryo-EM methodology and instrumentation to capture the conformation of microtubule-attached kinesin at the beginning (no-nucleotide) and end (ATP analog-bound) of the force generation process at 5-6A resolution. We derived all-atom models for these two maps from a crystal structure of the tubulin-kinesin complex, using explicitly solvated molecular dynamics simulations combined with restraints derived from the maps. This analysis revealed that, contrary to existing models, kinesin's central beta sheet serves as the primary transducer in the motor's force-generation mechanism, twisting to drive ADP release and subsequently untwisting upon ATP binding to trigger a power stroke. We identified conserved residues on the motor domain, supported by additional structural and biochemical analysis of site-directed mutations, which serve as allosteric latches during the motor's microtubule-attached phase. These latches regulate the sheet-twisting motion and couple key properties of motor function to each other, including nucleotide binding, hydrolysis, and generation of a power stroke. These findings reveal how interactions with the microtubule can fundamentally alter kinesin's energetic landscape in order to initiate productive motility.

Covalent immobilization of microtubules on glass surfaces for molecular motor force measurements and other single-molecule assays.

Rigid attachment of microtubules (MTs) to glass cover slip surfaces is a prerequisite for a variety of microscopy experiments in which MTs are used as substrates for MT-associated proteins, such as the molecular motors kinesin and cytoplasmic dynein. We present an MT-surface coupling protocol in which aminosilanized glass is formylated using the cross-linker glutaraldehyde, fluorescence-labeled MTs are covalently attached, and the surface is passivated with highly pure beta-casein. The technique presented here yields rigid MT immobilization while simultaneously blocking the remaining glass surface against nonspecific binding by polystyrene optical trapping microspheres. This surface chemistry is straightforward and relatively cheap and uses a minimum of specialized equipment or hazardous reagents. These methods provide a foundation for a variety of optical tweezers experiments with MT-associated molecular motors and may also be useful in other assays requiring surface-immobilized proteins.

A molecular motor finds its track

The detailed mechanism by which the molecular motors kinesin and myosin travel along their respective protein tracks as they generate force during motile processes is still poorly understood. In a recent breakthrough, a crystal structure of kinesin in complex with tubulin illuminates the atomic-level details of a motor-track interaction, answering many questions yet leaving a number of mysteries unresolved.

A strategy to capture and characterize the synaptic transcriptome

Here we describe a strategy designed to identify RNAs that are actively transported to synapses during learning. Our approach is based on the characterization of RNA transport complexes carried by molecular motor kinesin. Using this strategy in Aplysia, we have identified 5,657 unique sequences consisting of both coding and noncoding RNAs from the CNS. Several of these RNAs have key roles in the maintenance of synaptic function and growth. One of these RNAs, myosin heavy chain, is critical in presynaptic sensory neurons for the establishment of long-term facilitation, but not for its persistence.

ZKSCAN3 Is a Master Transcriptional Repressor of Autophagy

Autophagy constitutes a major cell-protective mechanism that eliminates damaged components and maintains energy homeostasis via recycling nutrients under normal/stressed conditions. Although the core components of autophagy have been well studied, regulation of autophagy at the transcriptional level is poorly understood. Herein, we establish ZKSCAN3, a zinc finger family DNA-binding protein, as a transcriptional repressor of autophagy. Silencing of ZKSCAN3 induced autophagy and increased lysosome biogenesis. Importantly, we show that ZKSCAN3 represses transcription of a large gene set (>60) integral to, or regulatory for, autophagy and lysosome biogenesis/function and that a subset of these genes, including Map1lC3b and Wipi2, represent direct targets. Interestingly, ZKSCAN3 and TFEB are oppositely regulated by starvation and in turn oppositely regulate lysosomal biogenesis and autophagy, suggesting that they act in conjunction. Altogether, our study uncovers an autophagy master switch regulating the expression of a transcriptional network of genes integral to autophagy and lysosome biogenesis/function.

Single-Axonal Organelle Analysis Method Reveals New Protein–Motor Associations

Axonal transport of synaptic vesicle proteins is required to maintain neurons' ability to communicate via synaptic transmission. Neurotransmitter-containing synaptic vesicles are assembled at synaptic terminals via highly regulated endocytosis of membrane proteins. These synaptic vesicle membrane proteins are synthesized in the cell body and transported to synapses in carrier vesicles that make their way down axons via microtubule-based transport utilizing kinesin molecular motors. Identifying the cargos that each kinesin motor protein carries from the cell bodies to the synapse is key to understanding both diseases caused by motor protein dysfunction and how synaptic vesicles are assembled. However, obtaining a bulk sample of axonal transport complexes from central nervous system (CNS) neurons to use for identification of their contents has posed a challenge to researchers. To obtain axonal carrier vesicles from primary cultured neurons, we fabricated a microfluidic chip designed to physically isolate axons from dendrites and cell bodies and developed a method to remove bulk axonal samples and label their contents. Synaptic vesicle protein carrier vesicles in these samples were labeled with antibodies to the synaptic vesicle proteins p38, SV2A, and VAMP2, and the anterograde axonal transport motor KIF1A, after which antibody overlap was evaluated using single-organelle TIRF microscopy. This work confirms a previously discovered association between KIF1A and p38 and shows that KIF1A also transports SV2A- and VAMP2-containing carrier vesicles.

Effects of Confinement on Molecular Motor-Driven Self-Assembly of Ring Structures

Active transport by kinesin molecular motors has been used to assemble ring nanocomposites comprised of biotinylated microtubules and streptavidin-coated quantum dots. Here we studied the effects of two-dimensional surface confinement on ring self-assembly using substrates of microfluidic channels or periodic post arrays. Microfabricated devices were composed of gold–silicon oxide surfaces where the gold surfaces were functionalized with thiol-based self-assembled monolayers, which enabled selective adsorption of kinesin to silicon surfaces. Confinement of ring self-assembly within microfluidic channels was observed as a change in the distribution of ring diameters, specifically by placing an upper limit on the diameter capable of forming in the channels. Confining assembly using periodic post arrays where the edge-to-edge spacing was 2 μm resulted in a significantly smaller average diameter when compared against those formed in arrays with 5 and 10 μm spacing. Differences in diameters formed in 5 and 10 μm arrays were not observed. Observations of ring composite assembly along channel edges on the top surface, as well as around posts in the arrays confirm the fundamental role of active transport-induced mechanical strain in initiating the self-assembly process.

Intrinsic disorder in the kinesin superfamily

Kinesin molecular motors perform a myriad of intracellular transport functions. While their mechanochemical mechanisms are well understood and well-conserved throughout the superfamily, the cargo-binding and regulatory mechanisms governing the activity of kinesins are highly diverse and in general, are incompletely characterized. Here we present evidence from bioinformatic predictions indicating that most kinesin superfamily members contain significant regions of intrinsically disordered (ID) residues. ID regions can bind to multiple partners with high specificity, and are highly labile to post-translational modification and degradation signals. In kinesins, the predicted ID regions are primarily found in areas outside the motor domains, where primary sequences diverge by family, suggesting that ID may be a critical structural element for determining the functional specificity of individual kinesins. To support this idea, we present a systematic analysis of the kinesin superfamily, family by family, for predicted regions of ID. We combine this analysis with a comprehensive review of kinesin binding partners and post-translational modifications. We find two key trends across the entire kinesin superfamily. First, ID residues tend to be in the tail regions of kinesins, opposite the superfamily-conserved motor domains. Second, predicted ID regions correlate to regions that are known to bind to cargoes and/or undergo post-translational modifications. We therefore propose that ID is a structural element utilized by the kinesin superfamily in order to impart functional specificity to individual kinesins.

Engineering of a novel Ca2+-regulated kinesin molecular motor using a calmodulin dimer linker

The kinesin–microtubule system holds great promise as a molecular shuttle device within biochips. However, one current barrier is that such shuttles do not have “on–off” control of their movement. Here we report the development of a novel molecular motor powered by an accelerator and brake system, using a kinesin monomer and a calmodulin (CaM) dimer. The kinesin monomer, K355, was fused with a CaM target peptide (M13 peptide) at the C-terminal part of the neck region (K355–M13). We also prepared CaM dimers using CaM mutants (Q3C), (R86C), or (A147C) and crosslinkers that react with cysteine residues. Following induction of K355–M13 dimerization with CaM dimers, we measured K355–M13 motility and found that it can be reversibly regulated in a Ca2+-dependent manner. We also found that velocities of K355–M13 varied depending on the type and crosslink position of the CaM dimer used; crosslink length also had a moderate effect on motility. These results suggest Ca2+-dependent dimerization of K355–M13 could be used as a novel molecular shuttle, equipped with an accelerator and brake system, for biochip applications.

Reconstructing the free-energy landscape associated to molecular motors processivity

We propose a biochemical model providing the kinetic and energetic descriptions of the processivity dynamics of kinesin and dinein molecular motors. Our approach is a modified version of a well known model describing kinesin dynamics and considers the presence of a competitive inhibition reaction by ADP. We first reconstruct a continuous free-energy landscape of the cycle catalyst process that allows us to calculate the number of steps given by a single molecular motor. Then, we calculate an analytical expression associated to the translational velocity and the stopping time of the molecular motor in terms of time and ATP concentration. An energetic interpretation of motor processivity is discussed in quantitative form by using experimental data. We also predict a time duration of collective processes that agrees with experimental reports.

Nesca, a novel neuronal adapter protein, links the molecular motor kinesin with the pre‐synaptic membrane protein, syntaxin‐1, in hippocampal neurons

Vesicular transport in neurons plays a vital role in neuronal function and survival. Nesca is a novel protein that we previously identified and herein describe its pattern of expression, subcellular localization and protein-protein interactions both in vitro and in vivo. Specifically, a large proportion of Nesca is in tight association with both actin and microtubule cytoskeletal proteins. Nesca binds to F-actin, microtubules, βIII and acetylated α-tubulin, but not neurofilaments or the actin-binding protein drebrin, in in vitro-binding assays. Nesca co-immunoprecipitates with kinesin heavy chain (KIF5B) and kinesin light-chain motors as well as with the synaptic membrane precursor protein, syntaxin-1, and is a constituent of the post-synaptic density. Moreover, in vitro-binding assays indicate that Nesca directly binds KIF5B, kinesin light-chain and syntaxin-1. In contrast, Nesca does not co-immunoprecipitate with the kinesin motors KIF1B, KIF3A nor does it bind syntaxin-4 or the synaptosome-associated protein 25 kDa (SNAP-25) in vitro. Nesca expression in neurons is highly punctuate, co-stains with syntaxin-1, and is found in fractions containing markers of early endosomes and Golgi suggesting that it is involved in vesicular transport. Collectively, these data suggest that Nesca functions as an adapter involved in neuronal vesicular transport including vesicles containing soluble N-ethylmaleimide sensitive factor attachment protein receptors that are essential to exocytosis.

Classically Activated Macrophages Use Stable Microtubules for Matrix Metalloproteinase-9 (MMP-9) Secretion

As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-γ causes enhanced microtubule (MT) stabilization and secretion of MMPs. Macrophages up-regulate MMP-9 expression and secretion upon immunological challenge and require its activity for migration during the inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution as well as the mechanisms responsible for its trafficking are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contained calreticulin and protein-disulfide isomerase in activated RAW 264.7 macrophages. We demonstrated vesicular organelles of MMP-9 aligned along stable subsets of MTs and showed that selective modulation of MT dynamics contributes to the enhanced trafficking of MMP-9 extracellularly. We found a Rab3D-dependent association of MMP-9 vesicles with the molecular motor kinesin, whose association with the MT network was greatly enhanced after macrophage activation. Finally, we implicated kinesin 5B and 3B isoforms in the effective trafficking of MMP-9 extracellularly.

Amyotrophic lateral sclerosis-associated mutant VAPBP56S perturbs calcium homeostasis to disrupt axonal transport of mitochondria

A proline-to-serine substitution at position 56 in the gene encoding vesicle-associated membrane protein-associated protein B (VAPB; VAPBP56S) causes some dominantly inherited familial forms of motor neuron disease, including amyotrophic lateral sclerosis (ALS) type-8. Here, we show that expression of ALS mutant VAPBP56S but not wild-type VAPB in neurons selectively disrupts anterograde axonal transport of mitochondria. VAPBP56S-induced disruption of mitochondrial transport involved reductions in the frequency, velocity and persistence of anterograde mitochondrial movement. Anterograde axonal transport of mitochondria is mediated by the microtubule-based molecular motor kinesin-1. Attachment of kinesin-1 to mitochondria involves the outer mitochondrial membrane protein mitochondrial Rho GTPase-1 (Miro1) which acts as a sensor for cytosolic calcium levels ([Ca2+]c); elevated [Ca2+]c disrupts mitochondrial transport via an effect on Miro1. To gain insight into the mechanisms underlying the VAPBP56S effect on mitochondrial transport, we monitored [Ca2+]c levels in VAPBP56S-expressing neurons. Expression of VAPBP56S but not VAPB increased resting [Ca2+]c and this was associated with a reduction in the amounts of tubulin but not kinesin-1 that were associated with Miro1. Moreover, expression of a Ca2+ insensitive mutant of Miro1 rescued defective mitochondrial axonal transport and restored the amounts of tubulin associated with the Miro1/kinesin-1 complex to normal in VAPBP56S-expressing cells. Our results suggest that ALS mutant VAPBP56S perturbs anterograde mitochondrial axonal transport by disrupting Ca2+ homeostasis and effecting the Miro1/kinesin-1 interaction with tubulin.

RNA Granules and Stress Granules in Virus Systems

Viruses initiate a number of cellular stress responses and modulate gene regulation and compartmentalization of RNA upon infection to be successful parasites. Virus infections may induce or impair stress granule (SG) formation to maximize replication efficiency. SGs and processing bodies (PBs) are the RNA granules, which contain translationally inactive pool of transcripts as the mRNA silencing foci. PBs and SGs, the highly conserved macromolecular aggregates, can release mRNAs to allow their translations. Unlike constitutively existing PBs that can respond to stimuli and affect mRNA translation and decay, SGs are specifically induced upon cellular stress and can triggers a global translational silencing by several pathways, including phosphorylation of the key translation initiation factor eIF2alpha, tRNA cleavage, and sequestration of cellular components and so on. The dynamics of PBs and SGs are regulated by several signaling pathways, including histone deacetylase 6, and depend on microfilaments and microtubules, and the cognate molecular motors myosin, dynein, and kinesin. SGs share features with aggresomes and related aggregates of unfolded proteins and may play a role in the pathology. The recent advances in understanding the relationship between viruses and mRNA stress granules are summarized.

Incorporation of Photochromic Molecule into the Functional Loop L5 of Kinesin for the Purpose of Photo-Regulation

ATP driven molecular motor kinesin has several unique loops, which may determine the characteristic properties of kinesin. L5 is one of the unique loops locates in the vicinity of ATP binding site. We have previously demonstrated that the point mutations in the loop drastically affect ATPase activity. Moreover, it seems that the plus-end directed kinesins have longer L5 loop than that of minus-end directed kinesins. Therefore, the loop may determine the directionality of kinesin. Rice plant kinesin K16 has much shorter L5 than that of conventional kinesin. We have prepared the K16 mutant Q101C and H102C that have a single reactive cysteine reside in the L5. SH group reactive photochromic molecule composed of azobenzene derivative, N- (4-phenylazophenyl) maleimide (PAM) was incorporated into the cysteine residue in L5 to induce conformational change of L5 by ultraviolet (UV) and visible (VIS) light irradiation. The kinesin modified by PAM slightly altered the ATPase activity by UV and VIS light irradiation reversibly. We have also focused on the mitotic kinesin Eg5 that has an unusually elongated L5. It is known that the L5 of Eg5 has an important role to stabilize sterically its specific inhibitors (monastrol, STLC) that bind to the pocket near the ATPase site. We designed Eg5 mutants, E116C, E118C, D130C, A133C and Y125C to introduce photochromic molecule into the L5 of Eg5. The Effect of photoisomerization of azobenzene derivatives incorporated into L5 upon UV-VIS light irradiation for the inhibition of ATPase activity by monastrol or STLC was studied.

Molecular Motor Proteins and Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting motor neurons in the brain, brainstem and spinal cord, which is characterized by motor dysfunction, muscle dystrophy and progressive paralysis. Both inherited and sporadic forms of ALS share common pathological features, however, the initial trigger of neurodegeneration remains unknown. Motor neurons are uniquely targeted by ubiquitously expressed proteins in ALS but the reason for this selectively vulnerability is unclear. However motor neurons have unique characteristics such as very long axons, large cell bodies and high energetic metabolism, therefore placing high demands on cellular transport processes. Defects in cellular trafficking are now widely reported in ALS, including dysfunction to the molecular motors dynein and kinesin. Abnormalities to dynein in particular are linked to ALS, and defects in dynein-mediated axonal transport processes have been reported as one of the earliest pathologies in transgenic SOD1 mice. Furthermore, dynein is very highly expressed in neurons and neurons are particularly sensitive to dynein dysfunction. Hence, unravelling cellular transport processes mediated by molecular motor proteins may help shed light on motor neuron loss in ALS.

Association of the testis-specific TRIM/RBCC protein RNF33/TRIM60 with the cytoplasmic motor proteins KIF3A and KIF3B

The Rnf33/Trim60 gene is temporally transcribed in the preimplantation embryo before being silenced at the blastocyst stage but Rnf33 expression is detected in adult testis of the mouse. The putative RNF33 protein is a tripartite motif (TRIM)/RBCC protein composed of a typical RING zinc finger, a B-box 2, two α-helical coiled-coil segments, and a B30.2 domain. As a first step towards the elucidation of the biologic function of RNF33, we aimed in this study to elucidate proteins that associate with RNF33. RNF33-interacting proteins were first derived by the yeast two-hybrid system followed by co-immunoprecipitation assays. Interacting domains were determined by deletion mapping in genetic and biochemical analyzes. RNF33 was shown to interact with the kinesin-2 family members 3A (KIF3A) and 3B (KIF3B) motor proteins in the heterodimeric form known to transport cargos along the microtubule. Domain mapping showed that the RB and B30.2 domains of RNF33 interacted with the respective carboxyl non-motor domains of KIF3A and KIF3B. Since RNF33 interacted with the carboxyl-terminal tail of the KIF3A-KIF3B heterodimer, the motor head section of KIF3A-KIF3B was free and available for association with designated cargo(s) and movement along the microtubule. Data also suggest that RNF33 most likely interacted with KIF3A-KIF3B independent of the adaptor kinesin-associated protein KAP3. This study is a first demonstration of a TRIM protein, namely RNF33, that interacts with the kinesin molecular motors possibly contributing to kinesin-dependent mobilization of specific cargo(s) along the microtubule in the testis of the mouse.

Bidirectional transport and pulsing states in a multi-lane ASEP model

In this paper, we introduce an ASEP-like transport model for bidirectional motion ofparticles on a multi-lane lattice. The model is motivated by in vivo experiments onorganelle motility along a microtubule (MT), consisting of thirteen protofilaments, whereparticles are propelled by molecular motors (dynein and kinesin). In the model, organelles(particles) can switch directions of motion due to ‘tug-of-war’ events between counteractingmotors. Collisions of particles on the same lane can be cleared by switching to adjacentprotofilaments (lane changes).We analyse transport properties of the model with no-flux boundary conditions at one endof a MT (‘plus end’ or tip). We show that the ability to make lane changes can affect thetransport efficiency and the particle-direction change rate obtained from experiments isclose to optimal for achieving efficient motor and organelle transport in a living cell. Inparticular, we find a nonlinear scaling of the mean tip size (the number of particlesaccumulated at the tip) with injection rate and an associated phase transitionleading to pulsing states characterized by periodic filling and emptying of thesystem.

Kinesin molecular motor Eg5 functions during polypeptide synthesis

The kinesin-related molecular motor Eg5 plays roles in cell division, promoting spindle assembly. We show that during interphase Eg5 is associated with ribosomes and is required for optimal nascent polypeptide synthesis. When Eg5 was inhibited, ribosomes no longer bound to microtubules in vitro, ribosome transit rates slowed, and polysomes accumulated in intact cells, suggesting defects in elongation or termination during polypeptide synthesis. These results demonstrate that the molecular motor Eg5 associates with ribosomes and enhances the efficiency of translation.

The Aspergillus nidulans CENP-E kinesin KipA is able to dimerize and to move processively along microtubules

Kinesin molecular motors serve a variety of cellular functions usually in dynamic processes. One characteristic feature of many kinesins is their ATP-dependent processive movement along polymerized microtubules. However, many kinesins work as stationary polymerases or depolymerases. Therefore, it needs to be determined for each motor, whether it moves processively along microtubules or not. The Schizosaccharomyces pombe kinesin-7, Tea2, has been shown to be involved in cell end marker transportation towards the cortex to organize the actin cytoskeleton. In human, kinesin 7 promotes microtubule polymerization. In Aspergillus nidulans, the machinery for determining growth directionality is conserved, but there is no evidence yet that kinesin 7, KipA is potentially involved in the transportation of the cell end marker proteins, TeaA or TeaR or newly identified proteins such as KatA. We expressed KipA in Escherichia coli and determined the catalytic properties of this kinesin. Here we show that KipA is an active ATPase, which is able to dimerize and move processively along microtubules with a speed of 9.48μm/min.