Tracking Individual Kinesin Motors in Living Cells Using Single Quantum-Dot Imaging

Courty, Sébastien; Luccardini, Camilla; Bellaı̈che, Yohanns; Cappello, Giovanni; Dahan, Maxime

doi:10.1021/nl060921t

Cited by 311 publications

(288 citation statements)

References 33 publications

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“…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 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%

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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“…Studies have revealed various characteristics of these motor proteins in vitro and in vivo with reference to endocytotic organelles being transported. Friedman et al measured the in vivo mobility of kinesin unattached to a surface using single molecule assays to be 600 to 800 nm/s [46][47][48]. Further studies revealed that average velocities strongly depend on the size of the attached cargo that is transported by these motors: a larger size appears to correlate with slower motions [49].…”

Section: Introductionmentioning

confidence: 99%

Gold nanoparticles explore cells: Cellular uptake and their use as intracellular probes

Huefner

Septiadi

Wilts

et al. 2014

Methods

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Understanding uptake of nanomaterials by cells and their use for intracellular sensing is important for studying their interaction and toxicology as well as for obtaining new biological insight. Here, we investigate cellular uptake and intracellular dynamics of gold nanoparticles and demonstrate their use in reporting chemical information from the endocytotic pathway and cytoplasm. The intracellular gold nanoparticles serve as probes for surface-enhanced Raman spectroscopy (SERS) allowing for biochemical characterisation of their local environment. In particular, in this work we compare intracellular SERS using non-functionalised and functionalised nanoparticles in their ability to segregate different but closely related cell phenotypes. The results indicate that functionalised gold nanoparticles are more efficient in distinguishing between different types of cells. Our studies 2 pave the way for understanding the uptake of gold nanoparticles and their utilisation for SERS to give rise to a greater biochemical understanding in cell-based therapies.

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“…These properties enable a broad range of applications such as multiplexed imaging of fixed cells, tissues and even clinical tissue samples, enhancing the predictability of outcome of cancer progression at more early stages [5,6]. Owing to their high photostability and brightness, QDots are also extensively used for kinetic single molecule studies on cellular proteins such as kinesin or individual glycine receptors, providing detailed information on their intracellular mobility and processing that was not previously possible using organic fluorophores [7,8].…”

Section: Introductionmentioning

confidence: 99%

The performance of gradient alloy quantum dots in cell labeling

Soenen¹,

Manshian²,

Himmelreich³

et al. 2014

Biomaterials

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The interest in using quantum dots (QDots) as highly fluorescent and photostable nanoparticles in biomedicine is vastly increasing. One major hurdle that slows down the (pre)clinical translation of QDots is their potential toxicity. Several strategies have been employed to optimize common coreshell QDots, such as the use of gradient alloy (GA)-QDots. These particles no longer have a sizedependent emission wavelength, but the emission rather depends on the chemical composition of the gradient layer. Therefore, particles of identical sizes but with emission maxima spanning the entire visible spectrum can be generated. In the present study, two types of GA-QDots are studied with respect to their cytotoxicity and cellular uptake. A multiparametric cytotoxicity approach reveals concentration-dependent effects on cell viability, oxidative stress, cell morphology and cell functionality (stem cell differentiation and neurite outgrowth), where the particles are very robust against environmentally-induced breakdown. Non-toxic concentrations are defined and compared to common core-shell QDots analyzed under identical conditions. Additionally, this value is translated into a functional value by analyzing the potential of the particles for cell visualization. Interestingly, these particles result in clear endosomal localization, where different particles result in identical intracellular distributions. This is in contrast with CdTe QDots with the same surface coating, which resulted in clearly distinct intracellular distributions as a result of differences in nanoparticle diameter.The GA-QDots are therefore ideal platforms for cell labeling studies given their high brightness, low cytotoxicity and identical sizes, resulting in highly similar intracellular particle distributions which offer a lot of potential for optimizing drug delivery strategies.

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Tracking Individual Kinesin Motors in Living Cells Using Single Quantum-Dot Imaging

Cited by 311 publications

References 33 publications

The mitotic kinesin CENP-E is a processive transport motor

The mitotic kinesin CENP-E is a processive transport motor

Gold nanoparticles explore cells: Cellular uptake and their use as intracellular probes

The performance of gradient alloy quantum dots in cell labeling

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