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Kinesin KIFC1 actively transports bare double-stranded DNA.

Farina F, Pierobon P, Delevoye C, Monnet J, Dingli F, Loew D, Quanz M, Dutreix M, Cappello G - Nucleic Acids Res. (2013)

Bottom Line: We used an in vitro motility assay, in which the motion of single-DNA molecules along cytoskeleton filaments in cell extracts is monitored; we demonstrate that microtubule-associated motors are involved in this transport.Precipitation of DNA-bound proteins and mass spectrometry analyses reveal the preferential binding of the kinesin KIFC1 on DNA.Cell extract depletion of kinesin KIFC1 significantly decreases DNA motion, confirming the active implication of this molecular motor in the intracellular DNA transport.

View Article: PubMed Central - PubMed

Affiliation: Physico-Chimie-Curie/UMR168 Institut Curie, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, 75231 Paris, France.

ABSTRACT
During the past years, exogenous DNA molecules have been used in gene and molecular therapy. At present, it is not known how these DNA molecules reach the cell nucleus. We used an in cell single-molecule approach to observe the motion of exogenous short DNA molecules in the cytoplasm of eukaryotic cells. Our observations suggest an active transport of the DNA along the cytoskeleton filaments. We used an in vitro motility assay, in which the motion of single-DNA molecules along cytoskeleton filaments in cell extracts is monitored; we demonstrate that microtubule-associated motors are involved in this transport. Precipitation of DNA-bound proteins and mass spectrometry analyses reveal the preferential binding of the kinesin KIFC1 on DNA. Cell extract depletion of kinesin KIFC1 significantly decreases DNA motion, confirming the active implication of this molecular motor in the intracellular DNA transport.

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In cell single-particle experiments with DNA:QD complexes. (A) Bright field image of a HeLa cell plated onto a glass coverslip: we have superposed the trajectories of the DNA:QD complexes observed in fluorescence (in red). The blue boxes correspond to the particles in the sequences shown in (B) and (C). Stars indicate the beginning of the tracks. The scale is 10 micron. (B) and (C) Two examples of directed trajectories. Left: frame sequence of two DNA:QD complexes extracted from a movie acquired at 100 frames per second. The scale is 2 micron. Right: MSD for both trajectories. The MSD is fitted to the power law MSD(t) = a·tb. The coefficients are b = 1.8 and b = 2 in (B) and (C), respectively, indicating a directed motion.
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gkt204-F1: In cell single-particle experiments with DNA:QD complexes. (A) Bright field image of a HeLa cell plated onto a glass coverslip: we have superposed the trajectories of the DNA:QD complexes observed in fluorescence (in red). The blue boxes correspond to the particles in the sequences shown in (B) and (C). Stars indicate the beginning of the tracks. The scale is 10 micron. (B) and (C) Two examples of directed trajectories. Left: frame sequence of two DNA:QD complexes extracted from a movie acquired at 100 frames per second. The scale is 2 micron. Right: MSD for both trajectories. The MSD is fitted to the power law MSD(t) = a·tb. The coefficients are b = 1.8 and b = 2 in (B) and (C), respectively, indicating a directed motion.

Mentions: The DNA:QD complexes were internalized into the HeLa cells (see ‘Materials and Methods’ section), and their motion was followed by real-time epifluorescence. Each HeLa cell was observed both by phase contrast microscopy (Figure 1A) and fluorescence. The fluorescent spots correspond to DNA:QD complexes or small aggregates. We noticed that most of the DNA:QD complexes were localized in the cytoplasm, whereas few remained trapped at cell membrane. None seemed to be distributed within the nucleus. From real-time observations, two populations of cytoplasmic DNA:QD complexes were observed: most of them exhibited a diffusive motion (∼90%), whereas a small fraction had a directional motion with constant speed (∼10%). Examples of such directional motions are given, respectively, in Figure 1B and C showing two stacks of images acquired with a time resolution of 10 ms. These directed movements are incompatible with pure diffusion and more likely because of an active transport. Interestingly, only half of the observed movements were directed towards the nucleus. This does not suggest any preferential direction of this active transport.Figure 1.


Kinesin KIFC1 actively transports bare double-stranded DNA.

Farina F, Pierobon P, Delevoye C, Monnet J, Dingli F, Loew D, Quanz M, Dutreix M, Cappello G - Nucleic Acids Res. (2013)

In cell single-particle experiments with DNA:QD complexes. (A) Bright field image of a HeLa cell plated onto a glass coverslip: we have superposed the trajectories of the DNA:QD complexes observed in fluorescence (in red). The blue boxes correspond to the particles in the sequences shown in (B) and (C). Stars indicate the beginning of the tracks. The scale is 10 micron. (B) and (C) Two examples of directed trajectories. Left: frame sequence of two DNA:QD complexes extracted from a movie acquired at 100 frames per second. The scale is 2 micron. Right: MSD for both trajectories. The MSD is fitted to the power law MSD(t) = a·tb. The coefficients are b = 1.8 and b = 2 in (B) and (C), respectively, indicating a directed motion.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643607&req=5

gkt204-F1: In cell single-particle experiments with DNA:QD complexes. (A) Bright field image of a HeLa cell plated onto a glass coverslip: we have superposed the trajectories of the DNA:QD complexes observed in fluorescence (in red). The blue boxes correspond to the particles in the sequences shown in (B) and (C). Stars indicate the beginning of the tracks. The scale is 10 micron. (B) and (C) Two examples of directed trajectories. Left: frame sequence of two DNA:QD complexes extracted from a movie acquired at 100 frames per second. The scale is 2 micron. Right: MSD for both trajectories. The MSD is fitted to the power law MSD(t) = a·tb. The coefficients are b = 1.8 and b = 2 in (B) and (C), respectively, indicating a directed motion.
Mentions: The DNA:QD complexes were internalized into the HeLa cells (see ‘Materials and Methods’ section), and their motion was followed by real-time epifluorescence. Each HeLa cell was observed both by phase contrast microscopy (Figure 1A) and fluorescence. The fluorescent spots correspond to DNA:QD complexes or small aggregates. We noticed that most of the DNA:QD complexes were localized in the cytoplasm, whereas few remained trapped at cell membrane. None seemed to be distributed within the nucleus. From real-time observations, two populations of cytoplasmic DNA:QD complexes were observed: most of them exhibited a diffusive motion (∼90%), whereas a small fraction had a directional motion with constant speed (∼10%). Examples of such directional motions are given, respectively, in Figure 1B and C showing two stacks of images acquired with a time resolution of 10 ms. These directed movements are incompatible with pure diffusion and more likely because of an active transport. Interestingly, only half of the observed movements were directed towards the nucleus. This does not suggest any preferential direction of this active transport.Figure 1.

Bottom Line: We used an in vitro motility assay, in which the motion of single-DNA molecules along cytoskeleton filaments in cell extracts is monitored; we demonstrate that microtubule-associated motors are involved in this transport.Precipitation of DNA-bound proteins and mass spectrometry analyses reveal the preferential binding of the kinesin KIFC1 on DNA.Cell extract depletion of kinesin KIFC1 significantly decreases DNA motion, confirming the active implication of this molecular motor in the intracellular DNA transport.

View Article: PubMed Central - PubMed

Affiliation: Physico-Chimie-Curie/UMR168 Institut Curie, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, 75231 Paris, France.

ABSTRACT
During the past years, exogenous DNA molecules have been used in gene and molecular therapy. At present, it is not known how these DNA molecules reach the cell nucleus. We used an in cell single-molecule approach to observe the motion of exogenous short DNA molecules in the cytoplasm of eukaryotic cells. Our observations suggest an active transport of the DNA along the cytoskeleton filaments. We used an in vitro motility assay, in which the motion of single-DNA molecules along cytoskeleton filaments in cell extracts is monitored; we demonstrate that microtubule-associated motors are involved in this transport. Precipitation of DNA-bound proteins and mass spectrometry analyses reveal the preferential binding of the kinesin KIFC1 on DNA. Cell extract depletion of kinesin KIFC1 significantly decreases DNA motion, confirming the active implication of this molecular motor in the intracellular DNA transport.

Show MeSH
Related in: MedlinePlus