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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) and (B) Co-purification experiment. (A) Gel example. Proteins are loaded and separated by SDS–PAGE. Alkaline phosphatase (ALP), used as control of streptavidin-biotin bound, control using streptavidin-magnetic beads (CTRL) and proteins bound to the DNA. (B) Molecular motors identified by LC-MS/MS using comparative proteomics. Hits are represented according to the number of peptides identified, the Molecular Weight (MW) and the protein coverage and the number of times that they were found in all three.
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gkt204-F3: (A) and (B) Co-purification experiment. (A) Gel example. Proteins are loaded and separated by SDS–PAGE. Alkaline phosphatase (ALP), used as control of streptavidin-biotin bound, control using streptavidin-magnetic beads (CTRL) and proteins bound to the DNA. (B) Molecular motors identified by LC-MS/MS using comparative proteomics. Hits are represented according to the number of peptides identified, the Molecular Weight (MW) and the protein coverage and the number of times that they were found in all three.

Mentions: To identify molecular motors interacting with the DNA fragments, we performed the co-purification experiments of biotinylated-DNA incubated with CE. Pre-cleared cytoplasmic lysates were mixed to the DNA molecules overnight, and then streptavidin-magnetic beads were added to the mix (see ‘Materials and Methods’ section). The proteins bound to the DNA fragments were separated according to their electrophoretic mobility by using the SDS–PAGE. Biotinylated-alkaline phosphatase and pre-cleared cytoplasmic lysates without DNA were used as control. Figure 3A shows an example of a gel. From the left: the alkaline phosphatase, the proteins in the control condition (CTRL) and in the co-purification with the DNA molecules (DNA).Figure 3.


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) and (B) Co-purification experiment. (A) Gel example. Proteins are loaded and separated by SDS–PAGE. Alkaline phosphatase (ALP), used as control of streptavidin-biotin bound, control using streptavidin-magnetic beads (CTRL) and proteins bound to the DNA. (B) Molecular motors identified by LC-MS/MS using comparative proteomics. Hits are represented according to the number of peptides identified, the Molecular Weight (MW) and the protein coverage and the number of times that they were found in all three.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt204-F3: (A) and (B) Co-purification experiment. (A) Gel example. Proteins are loaded and separated by SDS–PAGE. Alkaline phosphatase (ALP), used as control of streptavidin-biotin bound, control using streptavidin-magnetic beads (CTRL) and proteins bound to the DNA. (B) Molecular motors identified by LC-MS/MS using comparative proteomics. Hits are represented according to the number of peptides identified, the Molecular Weight (MW) and the protein coverage and the number of times that they were found in all three.
Mentions: To identify molecular motors interacting with the DNA fragments, we performed the co-purification experiments of biotinylated-DNA incubated with CE. Pre-cleared cytoplasmic lysates were mixed to the DNA molecules overnight, and then streptavidin-magnetic beads were added to the mix (see ‘Materials and Methods’ section). The proteins bound to the DNA fragments were separated according to their electrophoretic mobility by using the SDS–PAGE. Biotinylated-alkaline phosphatase and pre-cleared cytoplasmic lysates without DNA were used as control. Figure 3A shows an example of a gel. From the left: the alkaline phosphatase, the proteins in the control condition (CTRL) and in the co-purification with the DNA molecules (DNA).Figure 3.

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