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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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(A–D) Immunoprecipitation of HeLa cell lysates using rabbit anti-human IgG as a control (CTRL) or antibodies against the four kinesins. Input and immunoprecipitated materials are revealed by western blot using specific antibodies for the four kinesins. (E) Directed movements observed in the control test and in depleted tests reported to the control test.
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gkt204-F4: (A–D) Immunoprecipitation of HeLa cell lysates using rabbit anti-human IgG as a control (CTRL) or antibodies against the four kinesins. Input and immunoprecipitated materials are revealed by western blot using specific antibodies for the four kinesins. (E) Directed movements observed in the control test and in depleted tests reported to the control test.

Mentions: To evaluate the implication in the intracellular DNA transport of the kinesins identified in co-purification experiments, we performed in vitro experiments on microtubules in presence and in absence of each kinesin. The DNA molecules were incubated with depleted cell lysates prepared as described in ‘Materials and Methods’ section and then mixed with the streptavidin QDs. Immunoprecipitation efficiency was controlled by western blot using specific antibodies for every kinesin (Figure 4A–D). The control test (CTRL) was performed using the cell extracts incubated with irrelevant rabbit anti-human IgG. The complexes obtained in control and depleted conditions were diluted in the motility buffer and injected in the flow chamber pre-coated with microtubules (see ‘Material and Methods’ section). The movements of the complexes were observed by TIRFM to discard the signal coming from the QDs in solution. The motions were tracked, and the trajectories were analyzed as described in the in vitro experiments (see ‘Materials and Methods’ section). We focused only on the directed motions selected by imposing the MSD exponent b > 1.5 and the trajectory length > 0.5 μm. Figure 4E shows the percentages of directed motions reported to the control test for every depleted condition. We observe that KIFC1 significantly influences the motion of DNA fragments along the microtubules, with a reduction of 74 ± 26% of the DNA motion in the absence of KIFC1. The immunodepletion of the three other kinesins (KIF1C, KIF4A and KIF14) also induces a reduction of the DNA motility in the order of 30%. Considering the large dispersion between different runs (also in the order of ±30%), this reduction cannot be considered as statistically relevant. Nevertheless, a possible role of these kinesins in the transport of cytoplasmic DNA cannot be ruled out a priori.Figure 4.


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)

(A–D) Immunoprecipitation of HeLa cell lysates using rabbit anti-human IgG as a control (CTRL) or antibodies against the four kinesins. Input and immunoprecipitated materials are revealed by western blot using specific antibodies for the four kinesins. (E) Directed movements observed in the control test and in depleted tests reported to the control test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt204-F4: (A–D) Immunoprecipitation of HeLa cell lysates using rabbit anti-human IgG as a control (CTRL) or antibodies against the four kinesins. Input and immunoprecipitated materials are revealed by western blot using specific antibodies for the four kinesins. (E) Directed movements observed in the control test and in depleted tests reported to the control test.
Mentions: To evaluate the implication in the intracellular DNA transport of the kinesins identified in co-purification experiments, we performed in vitro experiments on microtubules in presence and in absence of each kinesin. The DNA molecules were incubated with depleted cell lysates prepared as described in ‘Materials and Methods’ section and then mixed with the streptavidin QDs. Immunoprecipitation efficiency was controlled by western blot using specific antibodies for every kinesin (Figure 4A–D). The control test (CTRL) was performed using the cell extracts incubated with irrelevant rabbit anti-human IgG. The complexes obtained in control and depleted conditions were diluted in the motility buffer and injected in the flow chamber pre-coated with microtubules (see ‘Material and Methods’ section). The movements of the complexes were observed by TIRFM to discard the signal coming from the QDs in solution. The motions were tracked, and the trajectories were analyzed as described in the in vitro experiments (see ‘Materials and Methods’ section). We focused only on the directed motions selected by imposing the MSD exponent b > 1.5 and the trajectory length > 0.5 μm. Figure 4E shows the percentages of directed motions reported to the control test for every depleted condition. We observe that KIFC1 significantly influences the motion of DNA fragments along the microtubules, with a reduction of 74 ± 26% of the DNA motion in the absence of KIFC1. The immunodepletion of the three other kinesins (KIF1C, KIF4A and KIF14) also induces a reduction of the DNA motility in the order of 30%. Considering the large dispersion between different runs (also in the order of ±30%), this reduction cannot be considered as statistically relevant. Nevertheless, a possible role of these kinesins in the transport of cytoplasmic DNA cannot be ruled out a priori.Figure 4.

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