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Comment
. 2011 Jan;13(1):3-5; author reply 5-7.
doi: 10.1038/ncb0111-3.

Reducing background fluorescence reveals adhesions in 3D matrices

Kristopher E Kubow, Alan Rick Horwitz
Comment

Reducing background fluorescence reveals adhesions in 3D matrices

Kristopher E Kubow et al. Nat Cell Biol. 2011 Jan.

Erratum in

  • Nat Cell Biol. 2012 Dec;14(12):1344
No abstract available

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Figures

Figure 1
Figure 1
Cell-matrix adhesions are detected in 3D collagen gels. (a) Z-projection of a multi-polar U2OS cell expressing EGFP–paxillin under the control of a truncated promoter, with adhesions on the distal sections of the protrusions (arrows). See Supplementary Information, Fig. S1a–f for more images. (b) Z-projection images of protrusions from cells expressing the indicated construct. The cell expressing a low level of normal EGFP–paxillin (upper left) has a diffuse cytoplasmic fluorescence, similar in appearance to the cell expressing the fluorescent protein TagRFP-T alone (upper-right). Adhesions (arrows) are detectable in U2OS cells (bottom left) and HT-1080 cells (bottom right) transfected with a plasmid encoding promoter-truncated EGFP–paxillin, which expresses at a lower level than EGFP–paxillin and decreases background cytoplasmic fluorescence. (c) Z-projection of a U2OS cell expressing promoter-truncated EGFP–vinculin. Scale bars, 5 µm.
Figure 2
Figure 2
Dynamics of cell-matrix adhesions in 3D culture. Frames are taken from Supplementary Information, Video S1. The top row shows Z-projections of a protrusion end from a U2OS cell that was transfected with a plasmid encoding promoter-truncated EGFP–paxillin. The bottom row shows the image from the top row (green) overlayed with a reflectance image of the collagen fibres (magenta). Time indicates the min:s since the beginning of the movie. At 0:20, a small adhesion (arrowhead) moves rearward, pulling a collagen fibre. At 2:30, a new protrusion (boundary shown by dotted line) pauses, two new adhesions form at the leading edge (arrows), and the earlier adhesion (arrowhead) has elongated while travelling rearward. At 3:10, the two new adhesions (arrows) continue to grow and translocate with the attached collagen fibres. By 4:50, the new adhesions have continued to grow and move rearward while the early adhesion (arrowhead in previous panels) is no longer visible. Scale bar,2 µm
See this image and copyright information in PMC

Comment on

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