Limits...
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.

Show MeSH

Related in: MedlinePlus

Immunofluorescence co-localization experiment. Top: control experiment is performed in absence of DNA molecules. Kinesin KIFC1 bound to the microtubules is visualized in the FITC channel (image on the left). On the right is the FRET channel to confirm that there is no fluorescence passage from KIFC1. Bottom: DNA experiment is performed by using a DNA marked with Cy3. On the left is the KIFC1 in the FITC channel. On the right we observe the DNA molecules excited by the secondary antibody bound to KIFC1. Scale bar is 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3643607&req=5

gkt204-F5: Immunofluorescence co-localization experiment. Top: control experiment is performed in absence of DNA molecules. Kinesin KIFC1 bound to the microtubules is visualized in the FITC channel (image on the left). On the right is the FRET channel to confirm that there is no fluorescence passage from KIFC1. Bottom: DNA experiment is performed by using a DNA marked with Cy3. On the left is the KIFC1 in the FITC channel. On the right we observe the DNA molecules excited by the secondary antibody bound to KIFC1. Scale bar is 10 μm.

Mentions: Using FRET microscopy, we investigated the co-localization between the kinesin KIFC1 and DNA molecules in vitro. Complexes DNA/cytoplasmic proteins were prepared by mixing fluorescently labeled DNA-Cy3 molecules with CE (see ‘Materials and Methods’ section). These complexes were incubated with microtubules in a flow chamber, in presence of 1 mM of AMP–PNP. Afterwards, the KIFC1 proteins were labeled with anti-KIFC1 primary antibody and a fluorescent secondary antibody (Alexa488). To verify that we do not observe any signal coming from the secondary antibody in the FRET channel, we performed a control experiment without DNA: KIFC1 was revealed by immunofluorescence as described in ‘Materials and Methods’ section. Figure 5 shows an example of FRET image. On the left is the fluorescein isothiocyanate (FITC) channel to visualize the secondary antibody binding KIFC1; on the right is the FRET channel. We do not observe any signal in the FRET channel in absence of DNA (Figure 5, top). On the contrary, in presence of DNA (Figure 5, bottom), we observe a FRET signal of DNA-Cy3 molecules excited from the secondary antibody, supporting the hypothesis of a direct interaction between KIFC1 and DNA molecules.Figure 5.


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)

Immunofluorescence co-localization experiment. Top: control experiment is performed in absence of DNA molecules. Kinesin KIFC1 bound to the microtubules is visualized in the FITC channel (image on the left). On the right is the FRET channel to confirm that there is no fluorescence passage from KIFC1. Bottom: DNA experiment is performed by using a DNA marked with Cy3. On the left is the KIFC1 in the FITC channel. On the right we observe the DNA molecules excited by the secondary antibody bound to KIFC1. Scale bar is 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt204-F5: Immunofluorescence co-localization experiment. Top: control experiment is performed in absence of DNA molecules. Kinesin KIFC1 bound to the microtubules is visualized in the FITC channel (image on the left). On the right is the FRET channel to confirm that there is no fluorescence passage from KIFC1. Bottom: DNA experiment is performed by using a DNA marked with Cy3. On the left is the KIFC1 in the FITC channel. On the right we observe the DNA molecules excited by the secondary antibody bound to KIFC1. Scale bar is 10 μm.
Mentions: Using FRET microscopy, we investigated the co-localization between the kinesin KIFC1 and DNA molecules in vitro. Complexes DNA/cytoplasmic proteins were prepared by mixing fluorescently labeled DNA-Cy3 molecules with CE (see ‘Materials and Methods’ section). These complexes were incubated with microtubules in a flow chamber, in presence of 1 mM of AMP–PNP. Afterwards, the KIFC1 proteins were labeled with anti-KIFC1 primary antibody and a fluorescent secondary antibody (Alexa488). To verify that we do not observe any signal coming from the secondary antibody in the FRET channel, we performed a control experiment without DNA: KIFC1 was revealed by immunofluorescence as described in ‘Materials and Methods’ section. Figure 5 shows an example of FRET image. On the left is the fluorescein isothiocyanate (FITC) channel to visualize the secondary antibody binding KIFC1; on the right is the FRET channel. We do not observe any signal in the FRET channel in absence of DNA (Figure 5, top). On the contrary, in presence of DNA (Figure 5, bottom), we observe a FRET signal of DNA-Cy3 molecules excited from the secondary antibody, supporting the hypothesis of a direct interaction between KIFC1 and DNA molecules.Figure 5.

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