Faculty of 1000

Post-publication peer review

Come Home

Posted by rpg on 25 January, 2010

Back when I was an acolyte in the service of science, I worked on an interesting little big protein by the name of talin. This 270 kDa sucker is involved in focal adhesions: the ‘ankle’ of the cell, joining the actin cytoskeleton to the outside world. Focal adhesions are fascinating and complex, and if I had access to my thesis right now I’d draw you a picture. You’ll just have to take my word for it.

Talin is a molecule that back then was probably too big a problem for a grad student to tackle more or less single-handedly; the post-doc was was concentrating on some genetic analysis and my supervisor was taken up with the department’s computing services, which left very little lab time. I did manage to show that talin didn’t, as had been proposed, cap or nucleate actin polymerization (a negative result that was essentially unpublishable), and I also developed a long-term love of immunofluorescence microscopy. It appears we were ahead of our time: talin is closely involved with integrins (which see) and seems to be enjoying a (re-)surgence of interest lately.

Back to focal adhesions. Formation of these structures, completely essential to cell adhesion and migration, has been pretty thoroughly prodded. What’s interesting is how focal adhesions disassemble, so that the moving cell doesn’t get stuck. Again, this is something I had a professional interest in: one of my projects in Cambridge involved  the determination of how moving cells generate the required motile force, or how they put their ‘feet’ forward’. We used a model system and discovered that essentially it’s gel effects. You take a load of little rods (= actin filaments), grow them, and the space they fill is disproportionately large, driving protrusion. What we didn’t do was look at the trailing edge of the cell, how the ‘foot’ comes up again. That was something I would have dearly loved to work on, and if I’d stayed in Cambridge, or even in science, I might have worked on it.

Clathrin

Good job I didn’t, because we’ve just published an evaluation of a paper showing that focal adhesion disassembly is just as complex as assembly. It turns out that our old friend clathrin, along with two of its adaptors, gets directed to focal adhesions by microtubules, and, as you might expect seeing as clathrin is involved, the integrins are reincorporated into the cell by endocytosis, and recycled (rather than being left behind as the cell walks away. I don’t think any of us thought much of that hypothesis anyway, but I mention it for completeness.)

The researchers used total internal reflection fluorescence and watched individual molecules on the underside of migrating cells scooting around. The clathrin sidled up to focal adhesions, hooked up with integrin; and the two left the party together.

time series of leaving the partyGet your coat, you’ve pulled

Just as we don’t think that our spaghetti/copper wire/gel effects produce all the force required for forward motion, neither is it certain that all focal adhesion disassembly is driven this way (the paper says that depleting clathrin reduces disassembly by 60-80%) . Talin itself has a head domain and an extended domain, and there is a calpain protease recognition site at the join (this was an immense pain when purifying the native protein; I had to make sure everything was swimming in protease inhibitors). Similarly, it’s possible that calpain actively degrades one or more focal adhesion components to make sure the whole thing gets packed away nicely, even if that does seem expensive in energy terms.

It doesn’t stop there, of course. Somehow these integrins have to get recycled to the front of the cell. It would makes sense for the little blighters to make their way through known endocytotic pathways and be ready for reassembly into focal adhesions at the business end of the cell, this hasn’t yet been demonstrated directly. It’s probably a mass effect. He said, airily.

Ezratty, E., Bertaux, C., Marcantonio, E., & Gundersen, G. (2009). Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells The Journal of Cell Biology, 187 (5), 733-747 DOI: 10.1083/jcb.200904054

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