Kinesin Function Research Articles

Microtubule Defects Influence Kinesin-Based Transport In Vitro

Microtubules are protein polymers that form “molecular highways” for long-range transport within living cells. Molecular motors actively step along microtubules to shuttle cellular materials between the nucleus and the cell periphery; this transport is critical for the survival and health of all eukaryotic cells. Structural defects in microtubules exist, but whether these defects impact molecular motor-based transport remains unknown. Here, we report a new, to our knowledge, approach that allowed us to directly investigate the impact of such defects. Using a modified optical-trapping method, we examined the group function of a major molecular motor, conventional kinesin, when transporting cargos along individual microtubules. We found that microtubule defects influence kinesin-based transport in vitro. The effects depend on motor number: cargos driven by a few motors tended to unbind prematurely from the microtubule, whereas cargos driven by more motors tended to pause. To our knowledge, our study provides the first direct link between microtubule defects and kinesin function. The effects uncovered in our study may have physiological relevance in vivo.

Engineered kinesin motor proteins amenable to small-molecule inhibition.

The human genome encodes 45 kinesin motor proteins that drive cell division, cell motility, intracellular trafficking and ciliary function. Determining the cellular function of each kinesin would benefit from specific small-molecule inhibitors. However, screens have yielded only a few specific inhibitors. Here we present a novel chemical-genetic approach to engineer kinesin motors that can carry out the function of the wild-type motor yet can also be efficiently inhibited by small, cell-permeable molecules. Using kinesin-1 as a prototype, we develop two independent strategies to generate inhibitable motors, and characterize the resulting inhibition in single-molecule assays and in cells. We further apply these two strategies to create analogously inhibitable kinesin-3 motors. These inhibitable motors will be of great utility to study the functions of specific kinesins in a dynamic manner in cells and animals. Furthermore, these strategies can be used to generate inhibitable versions of any motor protein of interest.

Why motor proteins team up - Intraflagellar transport inC. eleganscilia

ABSTRACTInside the cell, vital processes such as cell division and intracellular transport are driven by the concerted action of different molecular motor proteins. In C. elegans chemosensory cilia, 2 kinesin-2 family motor proteins, kinesin-II and OSM-3, team up to drive intraflagellar transport (IFT) in the anterograde direction, from base to tip, whereas IFT dynein hitchhikes toward the tip and subsequently drives IFT in the opposite, retrograde direction, thereby recycling both kinesins. While it is evident that at least a retrograde and an anterograde motor are necessary to drive IFT, it has remained puzzling why 2 same-polarity kinesins are employed. Recently, we addressed this question by combining advanced genome-engineering tools with ultrasensitive, quantitative fluorescence microscopy to study IFT with single-molecule sensitivity.1,2 Using this combination of approaches, we uncovered a differentiation in kinesin-2 function, in which the slower kinesin-II operates as an ‘importer’, loading IFT trains into the cilium before gradually handing them over to the faster OSM-3. OSM-3 subsequently acts as a long-range ‘transporter’, driving the IFT trains toward the tip. The two kinesin-2 motors combine their unique motility properties to achieve something neither motor can achieve on its own; that is to optimize the amount of cargo inside the cilium. In this commentary, we provide detailed insight into the rationale behind our research approach and comment on our recent findings. Moreover, we discuss the role of IFT dynein and provide an outlook on future studies.

A Screen for Modifiers of Cilia Phenotypes Reveals Novel MKS Alleles and Uncovers a Specific Genetic Interaction between osm-3 and nphp-4.

Nephronophthisis (NPHP) is a ciliopathy in which genetic modifiers may underlie the variable penetrance of clinical features. To identify modifiers, a screen was conducted on C. elegans nphp-4(tm925) mutants. Mutations in ten loci exacerbating nphp-4(tm925) ciliary defects were obtained. Four loci have been identified, three of which are established ciliopathy genes mks-1, mks-2, and mks-5. The fourth allele (yhw66) is a missense mutation (S316F) in OSM-3, a kinesin required for cilia distal segment assembly. While osm-3(yhw66) mutants alone have no overt cilia phenotype, nphp-4(tm925);osm-3(yhw66) double mutants lack distal segments and are dye-filling (Dyf) and osmotic avoidance (Osm) defective, similar to osm-3(mn357) null mutants. In osm-3(yhw66) mutants anterograde intraflagellar transport (IFT) velocity is reduced. Furthermore, expression of OSM-3(S316F)::GFP reduced IFT velocities in nphp-4(tm925) mutants, but not in wild type animals. In silico analysis indicates the S316F mutation may affect a phosphorylation site. Putative phospho-null OSM-3(S316F) and phospho-mimetic OSM-3(S316D) proteins accumulate at the cilia base and tip respectively. FRAP analysis indicates that the cilia entry rate of OSM-3(S316F) is slower than OSM-3 and that in the presence of OSM-3(S316F), OSM-3 and OSM-3(S316D) rates decrease. In the presence OSM-3::GFP or OSM-3(S316D)::GFP, OSM-3(S316F)::tdTomato redistributes along the cilium and accumulates in the cilia tip. OSM-3(S316F) and OSM-3(S316D) are functional as they restore cilia distal segment formation in osm-3(mn357) null mutants; however, only OSM-3(S316F) rescues the osm-3(mn357) null Dyf phenotype. Despite rescue of cilia length in osm-3(mn357) null mutants, neither OSM-3(S316F) nor OSM-3(S316D) restores ciliary defects in nphp-4(tm925);osm-3(yhw66) double mutants. Thus, these OSM-3 mutations cause NPHP-4 dependent and independent phenotypes. These data indicate that in addition to regulating cilia protein entry or exit, NPHP-4 influences localization and function of a distal ciliary kinesin. Moreover, data suggest human OSM-3 homolog (Kif17) could act as a modifying locus affecting disease penetrance or expressivity in NPHP patients.

The natural diterpene tonantzitlolone A and its synthetic enantiomer inhibit cell proliferation and kinesin-5 function

Tonantzitlolone A, a diterpene isolated from the Mexican plant Stillingia sanguinolenta, shows cytostatic activity. Both the natural product tonantzitlolone A and its synthetic enantiomer induce monoastral spindle formation in cell experiments which indicates inhibitory activity on kinesin-5 mitotic motor molecules. These inhibitory effects on kinesin-5 could be verified in in vitro single-molecule motility assays, where both tonantzitlolones interfered with kinesin-5 binding to its cellular interaction partner microtubules in a concentration-dependent manner, yet with a larger effect of the synthetic enantiomer. In contrast to kinesin-5 inhibition, both tonantzitlolone A enantiomers did not affect conventional kinesin-1 function; hence tonantzitlolones are not unspecific kinesin inhibitors. The observed stronger inhibitory effect of the synthetic enantiomer demonstrates the possibility to enhance the overall moderate anti-proliferative effect of the lead compound tonantzitlolon A by chemical modification.

Impacts of Microtubule Structural Defects on Kinesin-Based Transport

Microtubules are protein polymers that form “molecular highways” for long-range transport within living cells. Molecular motors actively step along microtubules to shuttle cellular materials between the nucleus and the cell periphery; this transport is critical for the survival and health of all eukaryotic cells. Structural defects in microtubules exist, but whether these structural defects impact molecular-motor based transport remains unknown. Here we report a new approach that allowed us to directly investigate the impact of such structural defects. Using a modified optical-trapping method, we examined the group function of a major molecular motor, conventional kinesin, when transporting cargoes along individual microtubules in vitro. We found that structural defects in microtubules are a determining factor in kinesin-based transport. This impact depends on motor number: cargoes driven by two motors tended to dissociate prematurely from the microtubule, whereas cargoes driven by more motors tended to pause. Our study provides the first direct link between structural defects in microtubules and kinesin function, and suggests how alterations in cargo-motor number nontrivially tune this effect. Our study highlights the potential of kinesins as non-invasive biomarkers of structural defects in microtubules, and raises the possibility of microtubule defects as a previously unexplored mechanism for regulating kinesin-based transport in cells.

Synthesis of Fluorescent-NTA and its Application to the Labeling of Photo-Controlled Kinesin Eg5

All members of the kinesin superfamily contain a structurally conserved loop L5 near the ATP-binding site. The length and the amino acids composition of L5 vary among the kinesin superfamily members. It is believed that L5 may be related to the enzymatic and motor function of kinesin. Interestingly, L5 of Eg5 is uniquely longer than that of other kinesins. Moreover, it was demonstrated that this loop undergoes conformational changes that are related to the nucleotide binding and neck linker docking. Previously we attempted to control Eg5 function photo-reversibly by incorporating photochromic molecules into the L5. We prepared Eg5 mutants, E116C, E118C, T125C, W127C, D130C, which have a single cysteine residue in L5 in order to incorporate photochromic molecules specifically. The Eg5 mutants modified with azobenzene and spiropyran derivatives showed photo-reversible alteration of microtubules dependent ATPase activities.In this study, we synthesized a novel thiol reactive photochromic molecule, monoiodoacetyl-flugide (IAFG). Fulgimide performs photo-reversible isomerization between non-polar opened-ring form and polar closed-ring form upon visible light and ultraviolet light, respectively. IAFG was incorporated into Eg5 mutant W127C stoichiometrically. Although the modified Eg5 mutant W127C-IAFG showed slightly decreased ATPase activity, the ATPase activity showed photo-reversible alteration upon UV and visible light irradiations. We utilized fluorescent probe bound NTA-Ni to label the His-tagged Eg5 modified with photochromic molecules. According to the methods of Soh et al., Dansyl-NTA and other fluorescent-NTA were synthesized and conjugated with Ni2+. The Dansyl-NTA-Ni2+ bound to His tagged Eg5. Using the fluorescent NTA-Ni2+, interaction of photo-regulated Eg5 with microtubules was monitored.

Identification of tail binding effect of kinesin-1 using an elastic network model.

Kinesin is a motor protein that delivers cargo inside a cell. Kinesin has many different families, but they perform basically same function and have same motions. The walking motion of kinesin enables the cargo delivery inside the cell. Autoinhibition of kinesin is important because it explains how function of kinesin inside a cell is stopped. Former researches showed that tail binding is related to autoinhibition of kinesin. In this work, we performed normal mode analysis with elastic network model using different conformation of kinesin to determine the effect of tail binding by considering four models such as functional form, autoinhibited form, autoinhibited form without tail, and autoinhibited form with carbon structure. Our calculation of the thermal fluctuation and cross-correlation shows the change of tail-binding region in structural motion. Also strain energy of kinesin showed that elimination of tail binding effect leads the structure to have energetically similar behavior with the functional form.

Kinesins in cell migration.

Human cells express 45 kinesins, microtubule motors that transport a variety of molecules and organelles within the cell. Many kinesins also modulate the tracks they move on by either bundling or sliding or regulating the dynamic assembly and disassembly of the microtubule polymer. In migrating cells, microtubules control the asymmetry between the front and rear of the cell by differentially regulating force generation processes and substrate adhesion. Many of these functions are mediated by kinesins, transporters as well as track modulators. In this review, we summarize the current knowledge on kinesin functions in cell migration.

Microtubule-depolymerizing kinesins in the regulation of assembly, disassembly, and length of cilia and flagella.

Defects in ciliary assembly, maintenance, and signaling are associated with various human diseases and developmental disorders, termed ciliopathies. Eukaryotic flagella and cilia (interchangeable terms) are microtubule-based organelles. Thus, microtubule dynamics and microtubule-dependent transport are predicted to affect the structural integrity and functionality of cilia profoundly. Kinesin-2 is well known for its role in intraflagellar transport to transport ciliary precursors and signaling molecules. Recently, microtubule-depolymerizing kinesins found in kinesin-8, -13, and -14A families have emerged as regulators of cilia. We first discuss ciliary kinesins identified in the flagellar or ciliary proteome, and then focus on the function and regulation of microtubule-depolymerizing kinesins. Lastly, we review the recent advances of microtubule-depolymerizing kinesins in controlling ciliary assembly, disassembly, and length.

Development of Photo-Controlled Molecular Shuttle Utilizing ATP Driven Motor Kinesin

The ATP driven motor kinesin has many possibilities for the application to the molecular machines. Previously, we introduced photochromic molecules into the functional key region of the kinesin as a photo-switch and tried to control the function of kinesin by ultraviolet (UV) and visible (VIS) light irradiations. The kinesin mutant S275C modified with thiol reactive azobenzene derivative exhibited photo-controlled ATPase activity correlating to photo isomerization of photochromic molecules.In this study, we tried to prepare the photo-controlled molecular shuttles using kinesin and liposome modified with photochromic molecule.We prepared the kinesin motor domain (K355) mutant that has a single cysteine at neck linker region and His-tag at C-terminal. Subsequently the kinesin mutant was dimerized with photochromic bifunctional cross-linker, azobenzene dimaleimide (ABDM). And the photo-reversible regulation of the ATPase and motor activities of the kinesin dimer cross-linked with ABDM was studied.We also tried to develop photo-responsive vesicle composed of photochromic molecules as a cargo for the photo-controlled kinesin. Diacyl glycerol was coupled with carboxypropyl-spiropyran to be phospholipid analogue using carbonyldiimidazole condensation reagent. The spiropyran moiety performs photo-reversible isomerization between hydrophobic spiro form and merocyanine zwitterion form upon visible light and ultraviolet light, respectively. Therefore, it is expected that the merocyanine form of the mimic phospholipid results in formation liposome like vesicle. The photo-reversible formation of the vesicle was studied using water-soluble fluorescent probe.

Synthesis and biological evaluation of optimized inhibitors of the mitotic kinesin Kif18A.

The mitotic spindle, a highly dynamic structure composed of microtubules, mediates the segregation of the previously duplicated genome into the two nascent daughter cells. Errors in this process contribute to pathology including tumor formation. Key for the shape and function of the mitotic spindle are kinesins, molecular motor proteins that convert chemical energy into mechanical work. Due to their fast mode of action, small molecules are valuable tools to dissect the dynamic functions of kinesins during mitosis. In this study, we report the identification of optimized small molecule inhibitors of the mitotic kinesin Kif18A. Using BTB-1, the first identified Kif18A inhibitor, as a lead compound, we synthesized a collection of derivatives. We demonstrate that some of the synthesized derivatives potently inhibited the ATPase activity of Kif18A with a half maximal inhibitory concentration (IC50) value in the low micromolar range. In vitro analysis of a panel of Kif18A-related kinesins revealed that the two most potent compounds show improved selectivity compared to BTB-1. Structure-activity relationship studies identified substituents mediating undesired inhibitory effects on microtubule polymerization. In summary, our study provides key insights into the mechanism of action of BTB-1 and its analogs, which will have a great impact on the further development of highly selective and bioactive Kif18A inhibitors. Since Kif18A is frequently overexpressed in solid tumors, such compounds are not only of great interest for basic research but also have the potential to open up new strategies for the treatment of human diseases.

High-throughput screening for small molecule modulators of motor protein Kinesin.

The kinesin superfamily of motor proteins are involved in the active transport of a large number of cargos such as organelles, proteins, and RNAs from the neuronal cell body to distal neuronal processes. Previously, we have shown that kinesin-mediated axonal transport of proteins and RNAs are important for long-term memory storage. Identification of small molecules that can activate or inhibit kinesins is of specific interest due to the significance of kinesin-mediated functions in neuronal health and plasticity. Here, we describe a high-throughput screening assay designed to specifically identify compounds that inhibit or activate adenosine triphosphatase activity of the kinesin 5B of humans. The luminescence-based assay that we developed is highly reproducible and robust. Using this approach, we screened a pharmacologically characterized compound collection and have identified small molecules with either activator or inhibitor-like activity. To further characterize screening hits, we also developed an orthogonal assay based on absorbance and a counter screen assay based on luminescence. Development of such assays is important to help identify small molecules that can be used in potential drug development efforts targeted at modulating the function of kinesin.

Tug-of-war of microtubule filaments at the boundary of a kinesin- and dynein-patterned surface.

Intracellular cargo is transported by multiple motor proteins. Because of the force balance of motors with mixed polarities, cargo moves bidirectionally to achieve biological functions. Here, we propose a microtubule gliding assay for a tug-of-war study of kinesin and dynein. A boundary of the two motor groups is created by photolithographically patterning gold to selectively attach kinesin to the glass and dynein to the gold surface using a self-assembled monolayer. The relationship between the ratio of two antagonistic motor numbers and the velocity is derived from a force-velocity relationship for each motor to calculate the detachment force and motor backward velocity. Although the tug-of-war involves >100 motors, values are calculated for a single molecule and reflect the collective dynein and non-collective kinesin functions when they work as a team. This assay would be useful for detailed in vitro analysis of intracellular motility, e.g., mitosis, where a large number of motors with mixed polarities are involved.

Transport of c-MYC by Kinesin-1 for proteasomal degradation in the cytoplasm

c-MYC is an oncogenic transcription factor that is degraded by the proteasome pathway. However, the mechanism that regulates delivery of c-MYC to the proteasome for degradation is not well characterized. Here, the results show that the motor protein complex Kinesin-1 transports c-MYC to the cytoplasm for proteasomal degradation. Inhibition of Kinesin-1 function enhanced ubiquitination of c-MYC and induced aggregation of c-MYC in the cytoplasm. Transport studies showed that the c-MYC aggregates moved from the nucleus to the cytoplasm and KIF5B is responsible for the transport in the cytoplasm. Furthermore, inhibition of the proteasomal degradation process also resulted in an accumulation of c-MYC aggregates in the cytoplasm. Moreover, Kinesin-1 was shown to interact with c-MYC and the proteasome subunit S6a. Inhibition of Kinesin-1 function also reduced c-MYC-dependent transformation activities. Taken together, the results strongly suggest that Kinesin-1 transports c-MYC for proteasomal degradation in the cytoplasm and the proper degradation of c-MYC mediated by Kinesin-1 transport is important for transformation activities of c-MYC. In addition, the results indicate that Kinesin-1 transport mechanism is important for degradation of a number of other proteins as well.

Endogenous localizome identifies 43 mitotic kinesins in a plant cell

Kinesins are microtubule (MT)-based motor proteins that have been identified in every eukaryotic species. Intriguingly, land plants have more than 60 kinesins in their genomes, many more than that in yeasts or animals. However, many of these have not yet been characterized, and their cellular functions are unknown. Here, by using endogenous tagging, we comprehensively determined the localization of 72 kinesins during mitosis in the moss Physcomitrella patens. We found that 43 kinesins are localized to mitotic structures such as kinetochores, spindle MTs, or phragmoplasts, which are MT-based structures formed during cytokinesis. Surprisingly, only one of them showed an identical localization pattern to the animal homolog, and many were enriched at unexpected sites. RNA interference and live-cell microscopy revealed postanaphase roles for kinesin-5 in spindle/phragmoplast organization, chromosome segregation, and cytokinesis, which have not been observed in animals. Our study thus provides a list of MT-based motor proteins associated with the cell division machinery in plants. Furthermore, our data challenge the current generalization of determining mitotic kinesin function based solely on studies using yeast and animal cells.

Development of Molecular Shuttle Regulated by External Stimulation Utilizing Kinesin ATP Driven Motor

Kinesin is known as a dimeric motor protein, which carries cellular cargoes along microtubules by hydrolyzing ATP. Its structure and the molecular mechanism of energy transduction to move along microtubule are well studied. The kinesin has many possibility of application to the molecular machines. Previously, we introduced photochromic molecules into the functional key region of the kinesin as a photoswitch and tried to control the function of kinesin by ultraviolet (UV) and visible (VIS) light irradiations. The kinesin mutant S275C modified with thiol reactive azobenzene derivative exhibited photocontrolled ATPase activity correlating to photo isomerization. We have also demonstrated that kinesin fused with CaM at the C-terminal binds reversibly to M13-Qdots in a calcium dependent manner. As mentioned above, kinesin works as a nanodevice to be a component of bionanomachines. In this study, we tried to prepare the molecular shuttles regulated by external stimulation utilizing kinesin and other functional proteins. We designed the two chimeric kinesins. One is the kinesin motor domain (K355) fused with calmodulin (CaM) and the other is K355 fused with calmodulin target peptide M13. The chimeras were expressed by E. coli. and purified by Co-Chelate column. These kinesin chimeras showed normal ATPase activities. K355-CaM bound to K355-M13 in a calcium dependent manner and formed dimer. The dimer composed of the two chimeras induced exhibited motor activity to induce microtubule gliding. Moreover, we also tried to prepare the chimeric kinesins, K355-CaM-split GFP C-terminal domain and K355-split GFP N-terminal domain to be dimerized and fluorescently labelled spontaneously.

Microtubule-regulating kinesins

Microtubule-regulating kinesins

P31comet inactivates the chemically induced Mad2-dependent spindle assembly checkpoint and leads to resistance to anti-mitotic drugs

Mad2 is a key component of the spindle assembly checkpoint (SAC) that delays the onset of anaphase until all kinetochores are attached to the spindle. It binds to Cdc20 and prevents it from promoting destruction of an anaphase inhibitor, Securin. Previously, we showed that a Mad2-binding protein, p31comet, formed a complex with Mad2 upon the completion of spindle attachment. Here, we showed that the overexpression of p31comet can abolish the Mad2-dependent SAC that is induced by anti-mitotic drugs, including nocodazole, taxol, and monastrol; these drugs, except monastrol, cause aneuploidy in HeLa cells. In the absence of Eg5, which is a target of monastrol, overexpression of p31comet caused premature destruction of Securin and premature sister chromatid separation, but it did not cause aneuploidy. These results indicated that Eg5 kinesin function might be required for checkpoint exit and mitotic progression. Moreover, overexpression of p31comet led to resistance against apoptosis that was induced by nocodazole and taxol in human cells, and taxol resistance was dependent on the p31comet/Mad2 protein expression level ratio of in cancer cell lines. These results indicated that p31comet is an indicator of resistance to anti-mitotic drugs in cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-562) contains supplementary material, which is available to authorized users.

Structure of a kinesin–tubulin complex and implications for kinesin motility

The typical function of kinesins is to transport cargo along microtubules. Binding of ATP to microtubule-attached motile kinesins leads to cargo displacement. To better understand the nature of the conformational changes that lead to the power stroke that moves a kinesin's load along a microtubule, we determined the X-ray structure of human kinesin-1 bound to αβ-tubulin. The structure defines the mechanism of microtubule-stimulated ATP hydrolysis, which releases the kinesin motor domain from microtubules. It also reveals the structural linkages that connect the ATP nucleotide to the kinesin neck linker, a 15-amino acid segment C terminal to the catalytic core of the motor domain, to result in the power stroke. ATP binding to the microtubule-bound kinesin favors neck-linker docking. This biases the attachment of kinesin's second head in the direction of the movement, thus initiating each of the steps taken.