Processive movement of single kinesins on crowded microtubules visualized using quantum dots

Seitz, Arne; Surrey, Thomas

doi:10.1038/sj.emboj.7600937

Cited by 141 publications

(179 citation statements)

References 52 publications

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“…For this purpose, we used a CENP-E dimer-to-quantum-dot ratio of 1:4 in our assays (21,22). At this ratio, we observed that quantum dots traveled long distances [supporting information (SI) Movie S1], implying that individual CENP-E dimers can move processively along microtubules.…”

Section: Resultsmentioning

confidence: 99%

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The mitotic kinesin CENP-E is a processive transport motor

Yardimci¹,

Duffelen²,

Mao

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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In vivo studies suggest that centromeric protein E (CENP-E), a kinesin-7 family member, plays a key role in the movement of chromosomes toward the metaphase plate during mitosis. How CENP-E accomplishes this crucial task, however, is not clear. Here we present single-molecule measurements of CENP-E that demonstrate that this motor moves processively toward the plus end of microtubules, with an average run length of 2.6 ؎ 0.2 m, in a hand-over-hand fashion, taking 8-nm steps with a stall force of 6 ؎ 0.1 pN. The ATP dependence of motor velocity obeys MichaelisMenten kinetics with K M,ATP ‫؍‬ 35 ؎ 5 M. All of these features are remarkably similar to those for kinesin-1-a highly processive transport motor. We, therefore, propose that CENP-E transports chromosomes in a manner analogous to how kinesin-1 transports cytoplasmic vesicles.mitotic motor ͉ single molecule C ell division requires proper attachment of chromosomes to spindle microtubules, which occurs by means of a multiprotein complex called the kinetochore. Centromeric protein E (CENP-E), a kinetochore-associated member of the kinesin superfamily, plays an essential role in capturing and positioning chromosomes to the mitotic spindle during metaphase (1). CENP-E localizes to kinetochores throughout chromosome congression and remains there until anaphase, at which point it relocates to the spindle midzone and is subsequently degraded (2).Interfering with CENP-E function significantly affects chromosome movement. Injection of an anti-CENP-E antibody leads to mitotic arrest, with either mono-oriented chromosomes positioned close to spindle poles or bi-oriented chromosomes that cannot align on the metaphase plate (1). Depletion of CENP-E from Xenopus egg extracts disturbs metaphase chromosome alignment (3), and gene silencing of CENP-E by RNA interference in HeLa cells produces unaligned chromosomes (4). A recent study by Kapoor et al. (5) suggests that CENP-E can transport mono-oriented chromosomes to the metaphase plate along the spindle fibers that are attached to already bi-oriented chromosomes. It has further been proposed (6) that CENP-E is responsible for silencing the mitotic checkpoint signaling, through its capture of spindle microtubules at the kinetochore.These roles for CENP-E represent a diverse set of functions and thus do not provide us with a unifying mechanism to explain how this kinetochore protein functions in mitosis. One approach to addressing this question is to compare the structure of CENP-E with that of other kinesins of known function. However, the crystallographic model of CENP-E resembles that of kinesin-1 (a transport motor) in some respects, and Eg5 (a mitotic motor designed to generate sustained force) in others (7). Previous in vitro functional studies of the CENP-E motor also have not been helpful in defining how this kinesin functions physiologically. A study of CENP-E purified from HeLa cells (8) demonstrated that it can bind to microtubules but does not generate microtubule-gliding activity. Another study (9) suggested that...

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Section: Resultsmentioning

confidence: 99%

“…We labeled our dimeric CENP-E construct with quantum dots (20,21) to visualize individual motor molecules through their movement on microtubules. Streptavidin-conjugated quantum dots were initially functionalized with biotinylated anti-histidine antibody and were then coupled to CENP-E dimers through the hexahistidine tag on their tail region.…”

Section: Resultsmentioning

confidence: 99%

The mitotic kinesin CENP-E is a processive transport motor

Yardimci¹,

Duffelen²,

Mao

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…In some experiments, it has been shown that kinesins stay tightly bound to the MT and wait when being blocked by an obstacle [337]. Although the general direction of kinesin motion is along individual protofilaments [301,41], recent experiments seem to show that occasional side steps onto neighboring protofilaments of the same MT are possible.…”

Section: Experimental Observationsmentioning

confidence: 99%

“…The length of the displacement performed along the MT by a molecular motor between two phases of diffusion in the cytosol is called its run length. Typical run lengths for kinesin are of the order of one hundred steps [337] which in combination with the step size of 8 nm gives runs of about 1 µm in length. Kinesin is thus a processive motor.…”

Section: Kinesinmentioning

confidence: 99%

Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

2015

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Cells are the elementary units of living organisms, which are able to carry out many vital functions. These functions rely on active processes on a microscopic scale. Therefore, they are strongly out-of-equilibrium systems, which are driven by continuous energy supply. The tasks that have to be performed in order to maintain the cell alive require transportation of various ingredients, some being small, others being large. Intracellular transport processes are able to induce concentration gradients and to carry objects to specific targets. These processes cannot be carried out only by diffusion, as cells may be crowded, and quite elongated on molecular scales. Therefore active transport has to be organized.The cytoskeleton, which is composed of three types of filaments (microtubules, actin and intermediate filaments), determines the shape of the cell, and plays a role in cell motion. It also serves as a road network for a special kind of vehicles, namely the cytoskeletal motors. These molecules can attach to a cytoskeletal filament, perform directed motion, possibly carrying along some cargo, and then detach.It is a central issue to understand how intracellular transport driven by molecular motors is regulated. The interest for this type of question was enhanced when it was discovered that intracellular transport breakdown is one of the signatures of some neuronal diseases like the Alzheimer.We give a survey of the current knowledge on microtubule based intracellular transport. Our review includes on the one hand an overview of biological facts, obtained from experiments, and on the other hand a presentation of some modeling attempts based on cellular automata. We present some background knowledge on the original and variants of the TASEP (Totally Asymmetric Simple Exclusion Process), before turning to more application oriented models. After addressing microtubule based transport in general, with a focus on in vitro experiments, and on cooperative effects in the transportation of large cargos by multiple motors, we concentrate on axonal transport, because of its relevance for neuronal diseases. Some important characteristics of axonal transport is that it takes place in a confined environment; Besides several types of motors are involved, that move in opposite directions. It is a challenge to understand how this bidirectional transport is organized. We review several features that could contribute to the efficiency of bidirectional transport in the axon, including in particular the role of motor-motor interactions and of the dynamics of the underlying microtubule network. Finally, we also discuss some open questions that may be relevant for future research in this field.

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“…First, blockages or alteration of the microtubule surface (via MAPs) or post-translational modifications could alter the motor velocity. Indeed, when blockages are present on a MT, it can alter how fast other motors move, and MT acetylation can alter velocity as well [27,28]. Second, there could be alteration of a motor's enzymatic cycle via phosphorylation, or accessory proteins.…”

Section: The Significance Of Changes In In Vivo Cargo Velocitymentioning

confidence: 99%

On the use of in vivo cargo velocity as a biophysical marker

Martínez

Vershinin

Shubeita

et al. 2007

Biochemical and Biophysical Research Communications

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Molecular motors move many intracellular cargos along microtubules. Recently it has been hypothesized that in vivo cargo velocity can be used to determine the number of engaged motors. We use theoretical and experimental approaches to investigate these assertions, and find that this hypothesis is inconsistent with previously described motor behavior, surveyed and re-analyzed in this paper. Studying lipid droplet motion in Drosophila embryos, we compare transport in a mutant, Δ(halo), with that in wild-type embryos. The minus-end moving cargos in the mutant appear to be driven by more motors (based on in vivo stall force observations). Periods of minus-end motion are indeed longer than in wild-type embryos but the corresponding velocities are not higher. We conclude that velocity is not a definitive read-out of the number of motors propelling a cargo.

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Processive movement of single kinesins on crowded microtubules visualized using quantum dots

Cited by 141 publications

References 52 publications

The mitotic kinesin CENP-E is a processive transport motor

The mitotic kinesin CENP-E is a processive transport motor

Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

On the use of in vivo cargo velocity as a biophysical marker

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