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.

Seeing three colors in one channel

Sometimes you would like to be able to image two or more different proteins in the same channel, and be able to separate them? For instance, wouldn't be great to see GFP-actin and GFP-mitochondria in separate channels? That is now possible with the publication of two new reversible switchable fluorescent proteins (RSFPs).

Nature Biotechnology 26, 1035 - 1040 (2008)| doi:10.1038/nbt.1493
Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopyMartin Andresen1, Andre C Stiel1, Jonas Fölling1, Dirk Wenzel2, Andreas Schönle1, Alexander Egner1, Christian Eggeling1, Stefan W Hell1 & Stefan Jakobs1

What is a reversible switchable fluorescent protein (RSFP)? These fluorescent proteins have excitation and emission properties similar to GFP, but they can be turned ON and OFF reversibly using a lower wavelength light source. Up to now all the RSFP identified, such as Drompa and rsFastLime (Drompa-Val157Gly), are turned OFF with irradiation of blue light (around 500nm) and turned ON with UV light (405 nm).
In this paper the authors describe a new variant of rsFastLime that behaves inversily regarding turning ON and OFF. rsFastLime is turned OFF with UV light and turned ON with blue light. they called it "Padron".

Using this two fluorofores (rsFastLime and Padron) to label mitochondria and actin cable in yeast, they were able to image the dynamics of both these structures over time, using a single "GFP fluorescent channel". They achieved this by alternatively turning ON and OFF rsFastLime and Padron (see image).

The other advantage of distinguishing two proteins in the same imaging channel is to avoid chromatic aberrations present in the different microscope components. This is particulary important when imaging at nanoscopic scale, as shown in this paper.

In addition, the authors identified another mutant with similar ON-OFF properties of rsFastLime, but with a broader absorption peak towards the UV. This allow them to excite and detect this new fluorofore (called bsDronpa) with UV light even in the presence of active rsFastLime. Furthermore, bsDronpa has better photobleaching properties than EBFP2, making it a good alternative for a UV-exciting genetically encoded fluorofore.

Overall, these new fluorofores are very usefull addition to the growing list of genetically encoded fluorofores.