RESUMO
Eg5, a mitotic kinesin, has been a target for anticancer drug development. Clinical trials of small-molecule inhibitors of Eg5 have been stymied by the development of resistance, attributable to mitotic rescue by a different endogenous kinesin, KIF15. Compared with Eg5, relatively little is known about the properties of the KIF15 motor. Here, we employed single-molecule optical-trapping techniques to define the KIF15 mechanochemical cycle. We also studied the inhibitory effects of KIF15-IN-1, an uncharacterized, commercially available, small-molecule inhibitor, on KIF15 motility. To explore the complementary behaviors of KIF15 and Eg5, we also scored the effects of small-molecule inhibitors on admixtures of both motors, using both a microtubule (MT)-gliding assay and an assay for cancer cell viability. We found that (i) KIF15 motility differs significantly from Eg5; (ii) KIF15-IN-1 is a potent inhibitor of KIF15 motility; (iii) MT gliding powered by KIF15 and Eg5 only ceases when both motors are inhibited; and (iv) pairing KIF15-IN-1 with Eg5 inhibitors synergistically reduces cancer cell growth. Taken together, our results lend support to the notion that a combination drug therapy employing both inhibitors may be a viable strategy for overcoming chemotherapeutic resistance.
Assuntos
Antineoplásicos/farmacologia , Cinesinas/antagonistas & inibidores , Microtúbulos/patologia , Neoplasias/patologia , Bibliotecas de Moléculas Pequenas/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células HeLa , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Fuso Acromático/efeitos dos fármacosRESUMO
During eukaryotic translation initiation, the small ribosomal subunit, assisted by initiation factors, locates the messenger RNA start codon by scanning from the 5' cap. This process is powered by the eukaryotic initiation factor 4A (eIF4A), a DEAD-box helicase. eIF4A has been thought to unwind structures formed in the untranslated 5' region via a nonprocessive mechanism. Using a single-molecule assay, we found that eIF4A functions instead as an adenosine triphosphate-dependent processive helicase when complexed with two accessory proteins, eIF4G and eIF4B. Translocation occurred in discrete steps of 11 ± 2 base pairs, irrespective of the accessory factor combination. Our findings support a memory-less stepwise mechanism for translation initiation and suggest that similar factor-dependent processivity may be shared by other members of the DEAD-box helicase family.
Assuntos
Trifosfato de Adenosina/química , Fator de Iniciação 4A em Eucariotos/química , Fator de Iniciação Eucariótico 4G/química , Fatores de Iniciação em Eucariotos/química , Biossíntese de Proteínas , DNA/química , Humanos , Conformação de Ácido Nucleico , RNA/químicaRESUMO
Kinesin-1 is a dimeric motor that transports cargo along microtubules, taking 8.2-nm steps in a hand-over-hand fashion. The ATP hydrolysis cycles of its two heads are maintained out of phase by a series of gating mechanisms, which lead to processive runs averaging ~1 µm. A key structural element for inter-head coordination is the neck linker (NL), which connects the heads to the stalk. To examine the role of the NL in regulating stepping, we investigated NL mutants of various lengths using single-molecule optical trapping and bulk fluorescence approaches in the context of a general framework for gating. Our results show that, although inter-head tension enhances motor velocity, it is crucial neither for inter-head coordination nor for rapid rear-head release. Furthermore, cysteine-light mutants do not produce wild-type motility under load. We conclude that kinesin-1 is primarily front-head gated, and that NL length is tuned to enhance unidirectional processivity and velocity.
Assuntos
Cinesinas/metabolismo , Proteínas dos Microtúbulos/metabolismo , Modelos Biológicos , Proteínas Motores Moleculares/metabolismo , Transporte Proteico/fisiologia , Fluorescência , Humanos , Pinças ÓpticasRESUMO
Kinesin-1 is a dimeric motor protein, central to intracellular transport, that steps hand-over-hand toward the microtubule (MT) plus-end, hydrolyzing one ATP molecule per step. Its remarkable processivity is critical for ferrying cargo within the cell: over 100 successive steps are taken, on average, before dissociation from the MT. Despite considerable work, it is not understood which features coordinate, or "gate," the mechanochemical cycles of the two motor heads. Here, we show that kinesin dissociation occurs subsequent to, or concomitant with, phosphate (P(i)) release following ATP hydrolysis. In optical trapping experiments, we found that increasing the steady-state population of the posthydrolysis ADP · P(i) state (by adding free P(i)) nearly doubled the kinesin run length, whereas reducing either the ATP binding rate or hydrolysis rate had no effect. The data suggest that, during processive movement, tethered-head binding occurs subsequent to hydrolysis, rather than immediately after ATP binding, as commonly suggested. The structural change driving motility, thought to be neck linker docking, is therefore completed only upon hydrolysis, and not ATP binding. Our results offer additional insights into gating mechanisms and suggest revisions to prevailing models of the kinesin reaction cycle.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Cinesinas/química , Cinesinas/metabolismo , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Fenômenos Biofísicos , Proteínas de Drosophila/genética , Hidrólise , Cinesinas/genética , Cinética , Modelos Biológicos , Proteínas Motores Moleculares/genética , Pinças Ópticas , Fosfatos/metabolismo , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Kinesin-1 is an ATP-driven, processive motor that transports cargo along microtubules in a tightly regulated stepping cycle. Efficient gating mechanisms ensure that the sequence of kinetic events proceeds in the proper order, generating a large number of successive reaction cycles. To study gating, we created two mutant constructs with extended neck-linkers and measured their properties using single-molecule optical trapping and ensemble fluorescence techniques. Owing to a reduction in the inter-head tension, the constructs access an otherwise rarely populated conformational state in which both motor heads remain bound to the microtubule. ATP-dependent, processive backstepping and futile hydrolysis were observed under moderate hindering loads. On the basis of measurements, we formulated a comprehensive model for kinesin motion that incorporates reaction pathways for both forward and backward stepping. In addition to inter-head tension, we found that neck-linker orientation is also responsible for ensuring gating in kinesin.
Assuntos
Cinesinas/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Fenômenos Biomecânicos , Humanos , Hidrólise , Cinesinas/metabolismo , Cinesinas/fisiologia , Cinética , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Moleculares , Pinças ÓpticasRESUMO
Kinesin is a homodimeric motor with two catalytic heads joined to a stalk via short neck linkers (NLs). We measured the torsional properties of single recombinant molecules by tracking the thermal angular motions of fluorescently labeled beads bound to the C terminus of the stalk. When kinesin heads were immobilized on microtubules (MTs) under varied nucleotide conditions, we observed bounded or unbounded angular diffusion, depending on whether one or both heads were attached to the MT. Free rotation implies that NLs act as swivels. From data on constrained diffusion, we conclude that the coiled-coil stalk domains are approximately 30-fold stiffer than its flexible "hinge" regions. Surprisingly, while tracking processive kinesin motion at low ATP concentrations, we observed occasional abrupt reversals in the directional orientations of the stalk. Our results impose constraints on kinesin walking models and suggest a role for rotational freedom in cargo transport.
Assuntos
Proteínas de Drosophila/química , Cinesinas/química , Microtúbulos/química , Estrutura Terciária de Proteína , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Cinesinas/metabolismo , Cinética , Microtúbulos/metabolismo , Modelos Moleculares , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Ligação Proteica , Multimerização Proteica , RotaçãoRESUMO
Using a high-resolution optical trapping instrument, we directly observed the processive motions of individual Eg5 dimers over a range of external loads and ATP, ADP, and phosphate concentrations. To constrain possible models for dissociation from the microtubule, we measured Eg5 run lengths and also compared the duration of the last step of a processive run to all previous step durations. We found that the application of large longitudinal forces in either hindering or assisting directions could induce Eg5-microtubule dissociation. At a constant moderate force, maintained with a force clamp, the premature binding of ADP strongly promoted microtubule release by Eg5, whereas the addition of ATP or phosphate had little effect on dissociation. These results imply that run length is determined not only by the load, but also by the concentration and type of nucleotides present, and therefore that the biochemical cycles of the two motor domains of the Eg5 dimer are coordinated to promote processive stepping.
Assuntos
Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Cinesinas/química , Cinesinas/metabolismo , Movimento/efeitos dos fármacos , Multimerização Proteica , Trifosfato de Adenosina/farmacologia , Animais , Bovinos , Humanos , Cinesinas/antagonistas & inibidores , Cinética , Microtúbulos/metabolismo , Fosfatos/farmacologia , Probabilidade , Ligação Proteica , Estrutura Quaternária de ProteínaRESUMO
The dimeric motor protein kinesin-1 converts chemical energy from ATP hydrolysis into mechanical work used to transport cargo along microtubules. Cargo attached to the kinesin stalk moves processively in 8-nm increments as its twin motor domains (heads) carry out an asymmetric, 'hand-over-hand' walk. The extent of individual head interactions with the microtubule during stepping, however, remains controversial. A major experimental limitation has been the lack of a means to monitor the attachment of an individual head to the microtubule during movement, necessitating indirect approaches. Here we report the development of a single-molecule assay that can directly report head binding in a walking kinesin molecule, and show that only a single head is bound to the microtubule between steps at low ATP concentrations. A bead was linked to one of the two kinesin heads by means of a short DNA tether and used to apply rapidly alternating hindering and assisting loads with an optical trap. The time-dependent difference between forwards and backwards displacements of the bead alternated between two discrete values during stepping, corresponding to those intervals when the linked head adopted a bound or an unbound state. The linked head could only rebind the microtubule once ATP had become bound to its partner head.
Assuntos
Cinesinas/química , Cinesinas/metabolismo , Microtúbulos/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , DNA/química , DNA/metabolismo , Drosophila melanogaster , Microesferas , Movimento/efeitos dos fármacos , Pinças Ópticas , Ligação Proteica/efeitos dos fármacos , Fatores de TempoRESUMO
We present a method for sequencing DNA that relies on the motion of single RNA polymerase molecules. When a given nucleotide species limits the rate of transcription, polymerase molecules pause at positions corresponding to the rare base. An ultrastable optical trapping apparatus capable of base pair resolution was used to monitor transcription under limiting amounts of each of the four nucleotide species. From the aligned patterns of pauses recorded from as few as four molecules, we determined the DNA sequence. This proof of principle demonstrates that the motion of a processive nucleic acid enzyme may be used to extract sequence information directly from DNA.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Análise de Sequência de DNA/métodos , Trifosfato de Adenosina/metabolismo , Pareamento de Bases , Citidina Trifosfato/metabolismo , Escherichia coli/enzimologia , Guanosina Trifosfato/metabolismo , Movimento (Física) , Moldes Genéticos , Uridina Trifosfato/metabolismoRESUMO
The two-headed kinesin motor harnesses the energy of ATP hydrolysis to take 8-nm steps, walking processively along a microtubule, alternately stepping with each of its catalytic heads in a hand-over-hand fashion. Two persistent challenges for models of kinesin motility are to explain how the two heads are coordinated ("gated") and when the translocation step occurs relative to other events in the mechanochemical reaction cycle. To investigate these questions, we used a precision optical trap to measure the single-molecule kinetics of kinesin in the presence of substrate analogs beryllium fluoride or adenylyl-imidodiphosphate. We found that normal stepping patterns were interspersed with long pauses induced by analog binding, and that these pauses were interrupted by short-lived backsteps. After a pause, processive stepping could only resume once the kinesin molecule took an obligatory, terminal backstep, exchanging the positions of its front and rear heads, presumably to allow release of the bound analog from the new front head. Preferential release from the front head implies that the kinetics of the two heads are differentially affected when both are bound to the microtubule, presumably by internal strain that is responsible for the gating. Furthermore, we found that ATP binding was required to reinitiate processive stepping after the terminal backstep. Together, our results support stepping models in which ATP binding triggers the mechanical step and the front head is gated by strain.
Assuntos
Biofísica/métodos , Cinesinas/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Animais , Dineínas/química , Hidrólise , Cinética , Loligo , Proteínas Motores Moleculares , Poliestirenos/química , Conformação Proteica , Fatores de TempoRESUMO
Eg5, a member of the kinesin superfamily of microtubule-based motors, is essential for bipolar spindle assembly and maintenance during mitosis, yet little is known about the mechanisms by which it accomplishes these tasks. Here, we used an automated optical trapping apparatus in conjunction with a novel motility assay that employed chemically modified surfaces to probe the mechanochemistry of Eg5. Individual dimers, formed by a recombinant human construct Eg5-513-5His, stepped processively along microtubules in 8-nm increments, with short run lengths averaging approximately eight steps. By varying the applied load (with a force clamp) and the ATP concentration, we found that the velocity of Eg5 was slower and less sensitive to external load than that of conventional kinesin, possibly reflecting the distinct demands of spindle assembly as compared with vesicle transport. The Eg5-513-5His velocity data were described by a minimal, three-state model where a force-dependent transition follows nucleotide binding.
Assuntos
Cinesinas/química , Cinesinas/metabolismo , Mitose , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Fenômenos Biomecânicos , Bovinos , Dimerização , Histidina , Humanos , Cinética , Oligopeptídeos , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Kinesin is a double-headed motor protein that moves along microtubules in 8-nanometer steps. Two broad classes of model have been invoked to explain kinesin movement: hand-over-hand and inchworm. In hand-over-hand models, the heads exchange leading and trailing roles with every step, whereas no such exchange is postulated for inchworm models, where one head always leads. By measuring the stepwise motion of individual enzymes, we find that some kinesin molecules exhibit a marked alternation in the dwell times between sequential steps, causing these motors to "limp" along the microtubule. Limping implies that kinesin molecules strictly alternate between two different conformations as they step, indicative of an asymmetric, hand-over-hand mechanism.
Assuntos
Drosophila melanogaster/enzimologia , Cinesinas/química , Cinesinas/fisiologia , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Simulação por Computador , Decapodiformes/enzimologia , Dimerização , Proteínas de Drosophila/química , Proteínas de Drosophila/fisiologia , Humanos , Cinética , Microesferas , Microtúbulos/metabolismo , Modelos Moleculares , Movimento , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , RotaçãoRESUMO
The ability of kinesin to travel long distances on its microtubule track without dissociating has led to a variety of models to explain how this remarkable degree of processivity is maintained. All of these require that the two motor domains remain enzymatically "out of phase," a behavior that would ensure that, at any given time, one motor is strongly attached to the microtubule. The maintenance of this coordination over many mechanochemical cycles has never been explained, because key steps in the cycle could not be directly observed. We have addressed this issue by applying several novel spectroscopic approaches to monitor motor dissociation, phosphate release, and nucleotide binding during processive movement by a dimeric kinesin construct. Our data argue that the major effect of the internal strain generated when both motor domains of kinesin bind the microtubule is to block ATP from binding to the leading motor. This effect guarantees the two motor domains remain out of phase for many mechanochemical cycles and provides an efficient and adaptable mechanism for the maintenance of processive movement.
Assuntos
Cinesinas/química , Cinesinas/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Dimerização , Humanos , Cinética , Microtúbulos/química , Movimento , Fosfatos/química , Plasmídeos/metabolismo , Cloreto de Potássio/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Espectrometria de FluorescênciaRESUMO
With every step it takes, the kinesin motor undergoes a mechanochemical reaction cycle that includes the hydrolysis of one ATP molecule, ADPP(i) release, plus an unknown number of additional transitions. Kinesin velocity depends on both the magnitude and the direction of the applied load. Using specialized apparatus, we subjected single kinesin molecules to forces in differing directions. Sideways and forward loads up to 8 pN exert only a weak effect, whereas comparable forces applied in the backward direction lead to stall. This strong directional bias suggests that the primary working stroke is closely aligned with the microtubule axis. Sideways loads slow the motor asymmetrically, but only at higher ATP levels, revealing the presence of additional, load-dependent transitions late in the cycle. Fluctuation analysis shows that the cycle contains at least four transitions, and confirms that hydrolysis remains tightly coupled to stepping. Together, our findings pose challenges for models of kinesin motion.
Assuntos
Biofísica/métodos , Cinesinas/química , Cinesinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Encéfalo/metabolismo , Calibragem , Bovinos , Vidro , Hidrólise , Cinética , Modelos Químicos , Moluscos , Ligação ProteicaRESUMO
The microtubule-severing enzyme katanin uses ATP hydrolysis to disrupt noncovalent bonds between tubulin dimers within the microtubule lattice. Although its microtubule severing activity is likely important for fundamental processes including mitosis and axonal outgrowth, its mechanism of action is poorly understood. To better understand this activity, an in vitro assay was developed to enable the real-time observation of katanin-mediated severing of individual, mechanically unconstrained microtubules. To interpret the experimental observations, a number of theoretical models were developed and compared quantitatively to the experimental data via Monte Carlo simulation. Models that assumed that katanin acts on a uniform microtubule lattice were incompatible with the in vitro data, whereas a model that assumed that katanin acts preferentially on spatially infrequent microtubule lattice defects was found to correctly predict the experimentally observed breaking rates, number and spatial frequency of severing events, final levels of severing, and sensitivity to katanin concentration over the range 6-300 nM. As a result of our analysis, we propose that defects in the microtubule lattice, which are known to exist but previously not known to have any biological function, serve as sites for katanin activity.
Assuntos
Adenosina Trifosfatases/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Fenômenos Biofísicos , Biofísica , Dimerização , Hidrólise , Técnicas In Vitro , Katanina , Cinética , Microscopia de Vídeo , Microtúbulos/ultraestrutura , Modelos Biológicos , Modelos Moleculares , Método de Monte Carlo , Ouriços-do-Mar , Tubulina (Proteína)/ultraestruturaRESUMO
Many cargoes move bidirectionally, frequently reversing course between plus- and minus-end microtubule travel. For such cargoes, the extent and importance of interactions between the opposite-polarity motors is unknown. In this paper we test whether opposite-polarity motors on lipid droplets in Drosophila embryos are coordinated and avoid interfering with each other's activity, or whether they engage in a tug of war. To this end we impaired the minus-end transport machinery using dynein and dynactin mutations, and then investigated whether plus-end motion was improved or disrupted. We observe a surprisingly severe impairment of plus-end motion due to these alterations of minus-end motor activity. These observations are consistent with a coordination hypothesis, but cannot be easily explained with a tug of war model. Our measurements indicate that dynactin plays a crucial role in the coordination of plus- and minus-end-directed motors. Specifically, we propose that dynactin enables dynein to participate efficiently in bidirectional transport, increasing its ability to stay "on" during minus-end motion and keeping it "off" during plus-end motion.