Putting this all together, our data suggests that zyxin is performing the same roles in strain sites as in FAs - it's helping to build a stress fiber while it's under tension by recognizing strained actin filaments

To test this we looked at how VASP responds to our laser ablation in WT and zyxin KO MEFs. We only saw a change in VASP intensity at the FA when zyxin was present! We also measured retrograde flow of actin at FAs and found that it was slower in the zyxin KO cells!

If we looked at cell-cell adhesions we saw the exact same thing! Both Trip6 and LIMD1, tension sensitive LIM domain proteins, showed sudden large drops in intensity in response to changes in tension on the adhesion.

So we next looked at other FA proteins, including paxillin, kindlin2, and PINCH. Only paxillin, another tension sensitive LIM domain protein showed changes in response to drops in tension!

Vinculin clearly stretches in response to forces though (PMID: 20613844), so we looked at molecular tension using the VinTS tension sensor. Shockingly it also was unperturbed by the change in tension on the adhesion, but it did change in response to Y-27 (ROCK inh.)

We took the same optogenetic approach and saw the same results. Vinculin intensity in FAs was completely unresponsive to changes in traction stresses!

We next tried the same experiments with vinculin, a known mechanosensitive FA protein. What we found was a total surprise - while zyxin was responsive to changes in tension, vinculin accumulation was completely unchanged!

We then used an optogenetic RhoA probe to modulate tension in the cell. We identified FAs where forces increased, remained stable, or decreased and looked at what happened to zyxin intensity. The responses were perfectly coordinated!

We used a laser to damage a stress fiber (SF) upstream of an adhesion and see what happened to zyxin at the adhesion. We saw both zyxin intensity and traction stresses drop immediately in response to the laser ablation.

This machine learning model also directly predicted a relationship between zyxin intensity and traction stress on the focal adhesion (FA). Stefano set out to explicitly test this prediction.

It'll be a slightly smaller crew than previous years but we'll still be at #cellbio24 #ASCB! If you like LIM proteins or septins come check out our posters:

There are some good local RhoA activation movies in one of our old papers for this pubmed.ncbi.nlm.nih.gov/28604737/

this is myosin being recruited

From the archive - integrins and actin. I never get tired of looking at the cytoskeleton!

We have a bunch of posters at the ASCB meeting in Boston for those that are around. Come check out everything we’ve been working on!