9.11.2008

Robots and wound healing

OK, so the title is a bit of a stretch. But the paper is kind of about wound healing. And they use a robot. And given the current political climate of massive misrepresentation, it's not all that bad. At least I didn't call it Lipstick on a Pig.

Identification of genes that regulate epithelial cell migration using an siRNA approach
Simpson et al., Nature Cell Biology, September 2008.

As part of the efforts of the multi-laboratory Cell Migration Consortium, the Brugge lab published the results of an siRNA screen to identify genes involved in epithelial cell migration. Although not a genome-wide screen, they used libraries of all known human kinases (576) and phosphatases (192), as well as a custom library targeting 313 genes with known or predicted roles in migration or adhesion.

The technique they used is elegantly simple, based on the "scratch-wound" assay performed in numerous laboratories. Basically, cells are grown to confluency, and then a region of cells in the middle is "scratched" away. Cells will then migrate into the scratched area in an attempt to close the "wound". The Brugge laboratory grew human MCF-10A (breast epithelial) cells to confluency, incubated cells with the siRNA, then used a robotic pinning device to create predictable scratch in the monolayer. Cells were allowed to migrate into the wound for 12 hours and the size of the resulting wound was compared to control. siRNA that caused increased rates of migration had smaller wounds, while siRNA that caused lower rates of migration had larger wounds.


Unlike many other screening papers, the final product of this paper was not simply a list of "hits" - positive results from the screen - with a bit of follow-up on their favorite hit. Instead, the group repeated the experiments of all hits and performed time-lapse, video microscopy of the 12 hour migration period for each. This allowed them to begin to parse the mechanistic basis for the change in migration rate for each. For example, knocking down p120-catenin and MLCK caused similar increases in migration rate (as assayed by the extent to which the wound closed). However, time-lapse microscopy showed that the increased rates were for completely different reasons; p120-catenin knockdown decreased cell-cell adhesion allowing cells to migrate more freely into the wound, while knockdown of MLCK had normal adhesion but an increase in wound-directed protrusion.

All hits from the inital screen were measured on the basis of four parameters:
1) extent and nature of adhesion impairment
2) directionality of movement
3) alterations in cell polarity
4) leading edge morphology and dynamics

As an added bonus, all of the videos from their analysis, as well as their annotation of each parameter can be found at www.cellmigration.org/pubs/wound_rnai.htm.

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