RESUMEN
Nanocapsules that collapse in response to guanosine triphosphate (GTP) have the potential as drug carriers for efficiently curing diseases caused by cancer and RNA viruses because GTP is present at high levels in such diseased cells and tissues. However, known GTP-responsive carriers also respond to adenosine triphosphate (ATP), which is abundant in normal cells as well. Here, we report the elaborate reconstitution of microtubule into a nanocapsule that selectively responds to GTP. When the tubulin monomer from microtubule is incubated at 37 °C with a mixture of GTP (17 mol%) and nonhydrolysable GTP* (83 mol%), a tubulin nanosheet forms. Upon addition of photoreactive molecular glue to the resulting dispersion, the nanosheet is transformed into a nanocapsule. Cell death results when a doxorubicin-containing nanocapsule, after photochemically crosslinked for properly stabilizing its shell, is taken up into cancer cells that overexpress GTP.
Asunto(s)
Nanocápsulas , Tubulina (Proteína) , Adenosina Trifosfato/metabolismo , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Guanosina Trifosfato/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
The movement of single kinesin molecules was observed while applying noisy external forces that mimic intracellular active fluctuations. We found kinesin accelerates under noise, especially when a large hindering load is added. The behavior quantitatively conformed to a theoretical model that describes the kinesin movement with simple two-state reactions. The universality of the kinetic theory suggests that intracellular enzymes share a similar noise-induced acceleration mechanism, i.e., active fluctuations in cells are not just noise but are utilized to promote various physiological processes.
Asunto(s)
Aceleración , Células/metabolismo , Cinesinas/metabolismo , Células/enzimología , Cinética , Modelos BiológicosRESUMEN
Molecular motors are nonequilibrium open systems that convert chemical energy to mechanical work. Their energetics are essential for various dynamic processes in cells, but largely remain unknown because fluctuations typically arising in small systems prevent investigation of the nonequilibrium behavior of the motors in terms of thermodynamics. Recently, Harada and Sasa proposed a novel equality to measure the dissipation of nonequilibrium small systems. By utilizing this equality, we have investigated the nonequilibrium energetics of the single-molecule walking motor kinesin-1. The dissipation from kinesin movement was measured through the motion of an attached probe particle and its response to external forces, indicating that large hidden dissipation exists. In this short review, aiming to readers who are not familiar with nonequilibrium physics, we briefly introduce the theoretical basis of the dissipation measurement as well as our recent experimental results and mathematical model analysis and discuss the physiological implications of the hidden dissipation in kinesin. In addition, further perspectives on the efficiency of motors are added by considering their actual working environment: living cells.
RESUMEN
Nonequilibrium energetics of single molecule translational motor kinesin was investigated by measuring heat dissipation from the violation of the fluctuation-response relation of a probe attached to the motor using optical tweezers. The sum of the dissipation and work did not amount to the input free energy change, indicating large hidden dissipation exists. Possible sources of the hidden dissipation were explored by analyzing the Langevin dynamics of the probe, which incorporates the two-state Markov stepper as a kinesin model. We conclude that internal dissipation is dominant.
RESUMEN
Single-molecule fluorescence observation of adenosine triphosphate (ATP) is a powerful tool to elucidate the chemomechanical coupling of ATP with a motor protein. However, in total internal reflection fluorescence microscopy (TIRFM), available ATP concentration is much lower than that in the in vivo environment. To achieve single-molecule observation with a high signal-to-noise ratio, zero-mode waveguides (ZMWs) are utilized even at high fluorescent molecule concentrations in the micromolar range. Despite the advantages of ZMWs, the use of cytoskeletal filaments for single-molecule observation has not been reported because of difficulties in immobilization of cytoskeletal filaments in the cylindrical aperture of ZMWs. Here, we propose linear ZMWs (LZMWs) to visualize enzymatic reactions on cytoskeletal filaments, specifically kinesin-driven microtubule motility accompanied by ATP binding/unbinding. Finite element method simulation revealed excitation light confinement in a 100 nm wide slit of LZMWs. Single-molecule observation was then demonstrated with up to 1 µM labeled ATP, which was 10-fold higher than that available in TIRFM. Direct observation of binding/unbinding of ATP to kinesins that propel microtubules enabled us to find that a significant fraction of ATP molecules bound to kinesins were dissociated without hydrolysis. This highlights the advantages of LZMWs for single-molecule observation of proteins that interact with cytoskeletal filaments such as microtubules, actin filaments, or intermediate filaments.
Asunto(s)
Adenosina Trifosfato/química , Citoesqueleto/química , Colorantes Fluorescentes/química , Cinesinas/química , Microtúbulos/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Citoesqueleto/metabolismo , Cinesinas/metabolismo , Microscopía Fluorescente , Microtúbulos/metabolismo , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
The dimeric motor protein kinesin-1 walks along microtubules by alternatingly hydrolyzing ATP and moving two motor domains ('heads'). Nanometer-precision single-molecule studies demonstrated that kinesin takes regular 8-nm steps upon hydrolysis of each ATP; however, the intermediate states between steps have not been directly visualized. Here, we employed high-temporal resolution dark-field microscopy to directly visualize the binding and unbinding of kinesin heads to or from microtubules during processive movement. Our observations revealed that upon unbinding from microtubules, the labeled heads were displaced rightward and underwent tethered diffusive movement. Structural and kinetic analyses of wild-type and mutant kinesins with altered neck linker lengths provided evidence that rebinding of the unbound head to the rear-binding site is prohibited by a tension increase in the neck linker and that ATP hydrolysis by the leading head is suppressed when both heads are bound to the microtubule, thereby explaining how the two heads coordinate to move in a hand-over-hand manner.
Asunto(s)
Adenosina Trifosfato/metabolismo , Cinesinas/química , Cinesinas/metabolismo , Microtúbulos/metabolismo , Biotinilación , Escherichia coli/genética , Oro/química , Cinesinas/genética , Cinética , Microscopía Fluorescente , Modelos Biológicos , Movimiento , Mutación , Pinzas Ópticas , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Transporte de ProteínasRESUMEN
We developed two types of high-speed angle-resolved imaging methods for single gold nanorods (SAuNRs) using objective-type vertical illumination dark-field microscopy and a high-speed CMOS camera to achieve microsecond temporal and one-degree angle resolution. These methods are based on: (i) an intensity analysis of focused images of SAuNR split into two orthogonally polarized components and (ii) the analysis of defocused SAuNR images. We determined the angle precision (statistical error) and accuracy (systematic error) of the resultant SAuNR (80 nm × 40 nm) images projected onto a substrate surface (azimuthal angle) in both methods. Although both methods showed a similar precision of â¼1° for the azimuthal angle at a 10 µs temporal resolution, the defocused image analysis showed a superior angle accuracy of â¼5°. In addition, the polar angle was also determined from the defocused SAuNR images with a precision of â¼1°, by fitting with simulated images. By taking advantage of the defocused image method's full revolution measurement range in the azimuthal angle, the rotation of the rotary molecular motor, F1-ATPase, was measured with 3.3 µs temporal resolution. The time constants of the pauses waiting for the elementary steps of the ATP hydrolysis reaction and the torque generated in the mechanical steps have been successfully estimated. The high-speed angle-resolved SAuNR imaging methods will be applicable to the monitoring of the fast conformational changes of many biological molecular machines.
Asunto(s)
Bacillus/enzimología , Oro/química , Microscopía/instrumentación , Nanotubos/química , ATPasas de Translocación de Protón/análisis , Adenosina Trifosfato/metabolismo , Diseño de Equipo , Hidrólisis , Procesamiento de Imagen Asistido por Computador/instrumentación , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía/métodos , Modelos Moleculares , Nanotubos/ultraestructura , ATPasas de Translocación de Protón/metabolismoRESUMEN
Kinesin is the canonical plus-end microtubule motor and has been the focus of intense study since its discovery in 1985. We previously demonstrated a time-dependent inactivation of kinesin in vitro that was fully reversible by the addition of purified casein kinase 2 (CK2) and showed that this inactivation/reactivation pathway was relevant in cells. Here we show that kinesin inactivation results from a conformational change that causes the neck linker to be positioned closer to the motor domain. Furthermore, we show that treatment of kinesin with CK2 prevents and reverses this repositioning. Finally, we demonstrate that CK2 treatment facilitates ADP dissociation from the motor, resulting in a nucleotide-free state that promotes microtubule binding. Thus, we propose that kinesin inactivation results from neck-linker repositioning and that CK2-mediated reactivation results from CK2's dual ability to reverse this repositioning and to promote ADP release.
Asunto(s)
Quinasa de la Caseína II/química , Quinasa de la Caseína II/metabolismo , Cinesinas/química , Cinesinas/metabolismo , Transducción de Señal/fisiología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Activación Enzimática/fisiología , Humanos , Microtúbulos/fisiología , Modelos Moleculares , Estructura Terciaria de ProteínaRESUMEN
A water-soluble dendron with a fluorescein isothiocyanate (FITC) fluorescent label and bearing nine pendant guanidinium ion (Gu(+))/benzophenone (BP) pairs at its periphery (Glue(BP)-FITC) serves as a "photoclickable molecular glue". By multivalent salt-bridge formation between Gu(+) ions and oxyanions, Glue(BP)-FITC temporarily adheres to a kinesin/microtubule hybrid. Upon subsequent exposure to UV light, this noncovalent binding is made permanent via a cross-linking reaction mediated by carbon radicals derived from the photoexcited BP units. This temporal-to-permanent transformation by light occurs quickly and efficiently in this preorganized state, allowing the movements of microtubules on a kinesin-coated glass plate to be photochemically controlled. A fundamental difference between such temporal and permanent bindings was visualized by the use of "optical tweezers".
Asunto(s)
Benzofenonas/química , Fluoresceína-5-Isotiocianato/química , Colorantes Fluorescentes/química , Guanidina/química , Cinesinas/química , Microtúbulos/química , Animales , Cationes Monovalentes/química , Bovinos , Cinesinas/ultraestructura , Microtúbulos/ultraestructura , Modelos Moleculares , Procesos Fotoquímicos , Albúmina Sérica Bovina/química , Rayos UltravioletaRESUMEN
Kinesin-1 (conventional kinesin) is a molecular motor that transports various cargo such as endoplasmic reticulum and mitochondria in cells. Its two head domains walk along microtubule by hydrolyzing ATP, while the tail domains at the end of the long stalk bind to the cargo. When a kinesin is not carrying cargo, its motility and ATPase activity is inhibited by direct interactions between the tail and head. However, the mechanism of this tail regulation is not well understood. Here, we apply single molecule fluorescence resonance energy transfer (smFRET) to observe this interaction in stalk-truncated kinesin. We found that kinesin with two tails forms a folding conformation and dissociates from microtubules, whereas kinesin with one tail remains bound to the micro-tubule and is immobile even in the presence of ATP. We further investigated the head-tail interaction as well as head-head coordination on the microtubule at various nucleotide conditions. From these results, we propose a two-step inhibition model for kinesin motility.
RESUMEN
Kinesin advances 8 nm along a microtubule per ATP hydrolyzed, but the mechanism responsible for coordinating the enzymatic cycles of kinesin's two identical motor domains remains unresolved. Here, we have tested whether such coordination is mediated by intramolecular tension generated by the "neck linkers," mechanical elements that span between the motor domains. When tension is reduced by extending the neck linkers with artificial peptides, the coupling between ATP hydrolysis and forward stepping is impaired and motor's velocity decreases as a consequence. However, speed recovers to nearly normal levels when external tension is applied by an optical trap. Remarkably, external load also induces bidirectional stepping of an immotile kinesin that lacks its mechanical element (neck linker) and fuel (ATP). Our results indicate that the kinesin motor domain senses and responds to strain in a manner that facilitates its plus-end-directed stepping and communication between its two motor domains.
Asunto(s)
Cinesinas/química , Cinesinas/metabolismo , Microtúbulos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Axonema/metabolismo , Movimiento , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Erizos de MarRESUMEN
Kinesin-5 family members cross-link and slide parallel microtubules of opposite polarity, an activity that is essential for the formation of a bipolar spindle during mitosis. In this issue, Kapitein et al. (Kapitein, L.C., B.H. Kwok, J.S. Weinger, C.F. Schmidt, T.M. Kapoor, and E.J.G. Peterman. 2008. J. Cell Biol. 182:421-428) demonstrate that microtubule cross-linking triggers the conversion of kinesin-5 motility from a diffusive mode to a directional mode, initiating antiparallel microtubule sliding.
Asunto(s)
Cinesinas/metabolismo , Microtúbulos/metabolismo , Mitosis , Animales , Difusión , Dimerización , Cinesinas/química , Transporte de Proteínas , Porcinos , Xenopus , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismoRESUMEN
Kinesin-1 (conventional kinesin) is a dimeric motor protein that carries cellular cargoes along microtubules by hydrolysing ATP and moving processively in 8-nm steps. The mechanism of processive motility involves the hand-over-hand motion of the two motor domains ('heads'), a process driven by a conformational change in the neck-linker domain of kinesin. However, the 'waiting conformation' of kinesin between steps remains controversial-some models propose that kinesin adopts a one-head-bound intermediate, whereas others suggest that both the kinesin heads are bound to adjacent tubulin subunits. Addressing this question has proved challenging, in part because of a lack of tools to measure structural states of the kinesin dimer as it moves along a microtubule. Here we develop two different single-molecule fluorescence resonance energy transfer (smFRET) sensors to detect whether kinesin is bound to its microtubule track by one or two heads. Our FRET results indicate that, while moving in the presence of saturating ATP, kinesin spends most of its time bound to the microtubule with both heads. However, when nucleotide binding becomes rate-limiting at low ATP concentrations, kinesin waits for ATP in a one-head-bound state and makes brief transitions to a two-head-bound intermediate as it walks along the microtubule. On the basis of these results, we suggest a model for how transitions in the ATPase cycle position the two kinesin heads and drive their hand-over-hand motion.
Asunto(s)
Cinesinas/química , Cinesinas/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Hidrólisis , Isomerismo , Cinesinas/genética , Cinética , Microtúbulos/metabolismo , Modelos Moleculares , Movimiento , Conformación ProteicaRESUMEN
Kinesin-1 is a dimeric motor protein that moves cargo processively along microtubules. Kinesin motility has been proposed to be driven by the coordinated forward extension of the neck linker (a approximately 12-residue peptide) in one motor domain and the rearward positioning of the neck linker in the partner motor domain. To test this model, we have introduced fluorescent dyes selectively into one subunit of the kinesin dimer and performed 'half-molecule' fluorescence resonance energy transfer to measure conformational changes of the neck linker. We show that when kinesin binds with both heads to the microtubule, the neck linkers in the rear and forward heads extend forward and backward, respectively. During ATP-driven motility, the neck linkers switch between these conformational states. These results support the notion that neck linker movements accompany the 'hand-over-hand' motion of the two motor domains.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Cinesinas/química , Proteínas Motoras Moleculares/química , Sitios de Unión , Dimerización , Cinesinas/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Coloración y Etiquetado , Relación Estructura-ActividadRESUMEN
Kinesin is a processive motor that takes 8.3-nm center-of-mass steps along microtubules for each adenosine triphosphate hydrolyzed. Whether kinesin moves by a "hand-over-hand" or an "inchworm" model has been controversial. We have labeled a single head of the kinesin dimer with a Cy3 fluorophore and localized the position of the dye to within 2 nm before and after a step. We observed that single kinesin heads take steps of 17.3 +/- 3.3 nm. A kinetic analysis of the dwell times between steps shows that the 17-nm steps alternate with 0-nm steps. These results strongly support a hand-over-hand mechanism, and not an inchworm mechanism. In addition, our results suggest that kinesin is bound by both heads to the microtubule while it waits for adenosine triphosphate in between steps.
Asunto(s)
Cinesinas/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Proteínas Motoras Moleculares/metabolismo , Adenosina Trifosfato , Carbocianinas , Dimerización , Fluorescencia , Colorantes Fluorescentes , Humanos , Cinesinas/química , Cinesinas/genética , Cinética , Modelos Moleculares , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/genética , Mutación , Conformación ProteicaRESUMEN
Unc104/KIF1A belongs to a class of monomeric kinesin motors that have been thought to possess an unusual motility mechanism. Unlike the unidirectional motion driven by the coordinated actions of the two heads in conventional kinesins, single-headed KIF1A was reported to undergo biased diffusional motion along microtubules. Here, we show that Unc104/KIF1A can dimerize and move unidirectionally and processively with rapid velocities characteristic of transport in living cells. These results suggest that Unc104/KIF1A operates in vivo by a mechanism similar to conventional kinesin and that regulation of motor dimerization may be used to control transport by this class of kinesins.
Asunto(s)
Cinesinas/química , Microtúbulos/fisiología , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/fisiología , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/fisiología , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/fisiología , Difusión , Dimerización , Humanos , Cinesinas/fisiología , Liposomas , Datos de Secuencia Molecular , Movimiento , Mutación , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/químicaRESUMEN
Unc104 (KIF1A) kinesin transports membrane vesicles along microtubules in lower and higher eukaryotes. Using an in vitro motility assay, we show that Unc104 uses a lipid binding pleckstrin homology (PH) domain to dock onto membrane cargo. Through its PH domain, Unc104 can transport phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2)-containing liposomes with similar properties to native vesicles. Interestingly, liposome movement by monomeric Unc104 motors shows a very steep dependence on PtdIns(4,5)P2 concentration (Hill coefficient of approximately 20), even though liposome binding is noncooperative. This switch-like transition for movement can be shifted to lower PtdIns(4,5)P2 concentrations by the addition of cholesterol/sphingomyelin or GM1 ganglioside/cholera toxin, conditions that produce raft-like behavior of Unc104 bound to lipid bilayers. These studies suggest that clustering of Unc104 in PtdIns(4,5)P2-containing rafts provides a trigger for membrane transport.