Kinesin motion in the absence of external forces characterized by interference total internal reflection microscopy

Physico Chimie Curie, UMR CNRS/IC 168, 26 rue d'Ulm, 75248 Paris Cedex 05, France.
Physical Review E (Impact Factor: 2.29). 09/2003; 68(2 Pt 1):021907. DOI: 10.1103/PhysRevE.68.021907
Source: PubMed


We study the motion of the kinesin molecular motor along microtubules using interference total internal reflection microscopy. This technique achieves nanometer scale resolution together with a fast time response. We describe the first in vitro observation of kinesin stepping at high ATP concentration in the absence of an external load, where the 8-nm step can be clearly distinguished. The short-time resolution allows us to measure the time constant related to the relative motion of the bead-motor connection; we deduce the associated bead-motor elastic modulus.

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    • "8 Author to whom any correspondence should beaddressed. molecule motor experiments, employing techniques such as optical tweezers [1] [2], single fluorophore imaging/tracking [3] [4] and bead motility assays [5] [6] have provided insight into the mechano-chemical coupling that ultimately drives the individual motor stepping. On the other hand, approaches involving multi-motor systems have been performed in the socalled gliding assay geometry. "
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    Physical Biology 02/2008; 5(4):046004. DOI:10.1088/1478-3975/5/4/046004 · 2.54 Impact Factor
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    ABSTRACT: In eukaryotic cells, nanotubes represent a substantial fraction of transport intermediates between organelles. They are extracted from membranes by molecular motors walking along microtubules. We previously showed that kinesins fixed on giant unilamellar vesicles in contact with microtubules are sufficient to form nanotubes in vitro. Motors were attached to the membrane through beads, thus facilitating cooperative effects. Koster et al. proposed that motors could dynamically cluster at the tip of tubes when they are individually attached to the membrane. We demonstrate, in a recently designed experimental system, the existence of an accumulation of motors allowing tube extraction. We determine the motor density along a tube by using fluorescence intensity measurements. We also perform a theoretical analysis describing the dynamics of motors and tube growth. The only adjustable parameter is the motor binding rate onto microtubules, which we measure to be 4.7 +/- 2.4 s(-1). In addition, we quantitatively determine, for a given membrane tension, the existence of a threshold in motor density on the vesicle above which nanotubes can be formed. We find that the number of motors pulling a tube can range from four at threshold to a few tens away from it. The threshold in motor density (or in membrane tension at constant motor density) could be important for the understanding of membrane traffic regulation in cells.
    Proceedings of the National Academy of Sciences 01/2005; 101(49):17096-101. DOI:10.1073/pnas.0406598101 · 9.67 Impact Factor
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    ABSTRACT: Les tubes de membrane sont omnipr´esents dans les cellules vivantes eucaryotes. Ce sont des structures tr`es dynamiques qui permettent en particulier la communication entre les diff´erents compartiments de la cellule. Pour comprendre les m´ecanismes impliqu´es dans le trafic intracellulaire, il paraˆıt essentiel d'isoler le rˆole des diff´erents constituants impliqu´es. Dans ce but, un syst`eme minimal qui permet de mimer in vi- tro les diff´erentes ´etapes d'extraction, de croissance et d'arrˆet des tubes de membrane avec des ´el´ements purifi´es ou artificiels (kin´esines, microtubules, v´esicules g´eantes unilamellaires) a ´et´e utilis´e. La comparaison des r´esultats exp´erimentaux avec ceux obtenus par une analyse th´eorique du syst`eme a ainsi permis de caract´eriser de fa¸con compl`ete ces diff´erentes ´etapes. Nous avons notamment montr´e l'existence d'un seuil de formation de tubes qui d´epend essentiellement de deux param`etres non locaux supramol´eculaires : la tension de membrane et la quantit´e de kin´esines `a la surface des v´esicules. Lorsque le tube est form´e, nous avons ´evalu´e le nombre de moteurs qui le tirent et montr´e qu'ils s'accumulent de fa¸con dynamique au bout du tube. De la mesure de la longueur caract´eristique d'accumulation, nous avons d´eduit un param`etre mol´eculaire : le taux d'attachement des kin´esines sur un microtubule dans une g´eom´etrie proche de celle observ´ee in vivo. Enfin, nous avons mis en ´evidence un ph´enom`ene d'oscillations li´ees au comportement collectif de moteurs processifs pour des tubes tr`es longs. Ce syst`eme, bien que simplifi´e, permet d'apporter une nouvelle approche du trafic intracellulaire, en proposant des m´ecanismes physiques qui sont souvent masqu´es, dans les cellules, par des m´ecanismes mol´eculaires.
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