Focal Adhesions and Cell Motility

This is for cell motility aficionados.

How do cells crawl? Well most in the field would say that actin polymerization generated by the Arp2/3 complex at the leading edge acts to generate an actin meshwork (see pic). The addition of actin monomers right under the membrane (arrows) act as a Brownian ratchet to push membrane forward. But for anyone who has actually monitored cell motility in the microscope, you must think that this idea is ludicrous. First of all, motility is more that pushing membrane, it's about transporting all your organelles and cytoplasmic components forward while retracting the cell's rear. Second, this Arp2/3 complexes generates membrane ruffling, but many migratory cells don't ruffle their membrane.

So what is the solution? Yesterday I saw a great talk by Claire Waterman-Storer who gave some insights into the crucial cellular behavior.

Before Claire, large scale latices within cells where visualized by incorporating monomers conjugated with fluorophores. Thus people were able to visualize the microtubule and actin cytoskeletons in realtime. This gave you general information of who these polymers behaved in cells, but you couldn't distinguish between whether a lattice was moving or whether it was assembling at one end and disassembling at another end. So you got some info, but there was some uncertainty as to what was really going on.

When Claire was a postdoc at Edward Salmon's lab at UNC, she developed speckle microscopy. The basic idea is to visualize how a lattice moves as oppose to how a lattice is assembled or disassembled, you incorporate small amounts of fluorescent monomers into the lattice. Heterogeneous incorporation of the fluorophores within the lattice could be visualized as speckles and thus the motion of the lattice could now be seen. So was the lattice moving and what extra info did we get from this technique?

A lot. Claire who has had her own lab for quite a while now has been exploiting this technology to get at all sorts of issues dealing with cell morphology and cell migration.

I won't talk about her work on microtubules here, but focus on recent actin data. It turns out that a cell is like an ocean full of actin currents. And just like the gulf stream explains quite a bit about the world we live in, actin currents dictate quite a bit of cell physiology. So what's going on. Well at the very front of a cell there is a tremendous amount of actin being polymerized which is immediately swept inside just a few micrometers (or microns) only to be rapidly depolymerized. This forms the actin meshwork shown in the micrograph above and is responsible for membrane ruffles. Further in, there is a slow rearward movement of actin driven by the motor myosin. This second rearward movement occurs in a part of the cell called the lamella. At the rear of the lamella, near the cell-center, the rear-flowing actin is bundled into large cables by another type of myosin. At the back of the cell, actin flows frontwards. But the real action turns out to be in the lamella. Click here for a time lapse of speckle microscopy of actin at the front and in the lamella of a migrating cell.

So what's up with the slow rearward movement of actin in the lamella?

It turns out that this "centripetal actin flow" is like the engine of the cell. It is the most significant intracellular mechanical force that sets up polarity and that rearranges the organelles. This rearward actin flow is orchestrated by microtubules which turn on and off actin motors and modulators of actin polarity. The rearward actin flow is also determined by membrane cues and cell/cell contact cues. (Note to all cell "signalling people" - get this idea into your head - I read far too many papers where the authors have not thought about the spatial-temporal distribution of enzymatic activities in cells. And it's not just that certain proteins are at the front, you must consider how he proteins are dynamically organized.)

But how does this all relate to cell motility? Back when imaging proteins in live cells was in its infancy, the Gundersen lab was imaging how focal adhesions (the part of the cells contacting the extra-cellular matrix) were organized. These large macromolecular complexes have over 100 different types of proteins in them including matrix receptors that spanthe membrane, signalling molecules and actin binding proteins. Focal adhersions formed right near the ruffle/lamella interface and mature into big cellular-matrix contacts within the bulk ofthe lamella. Focal adhesions turned out to be very dynamic structures. In stationary cells these complexes moved rearwards while in migrating cells they tended to be anchored to the crawling surface. It was proposed that cells moved forward due to an increase in traction. So that if intracellular actin was constantly flowing backwards and now this actin was latched onto a stable substrate across the membrane, the cell could now be moved forwards by relative motion. Thus what regulates cell motility was the engagement of actin to the extracellular matrix. This idea - the "clutch hypothesis" was then placed aside while actin people worked out the Arp2/3 actin polymerization machine that drives membrane pushing, but now Claire has new data that support the clutch hypothesis. By measuring force generation between the cell and the substrate at the front of cells at focal adhesions and measuring cell migration, Claire makes a convincing case for the clutch. She is now performing speckle microscopy on focal adhesion molecules to figure out the nature of the clutch, how the clutch is engaged and how the cell turns on the clutch. I won't tell you anymore, just watch for her next couple of papers and I'm sure that some mysteries of cell motility will be revealed.

Ref:
Ponti A., M. Machacek, S. L. Gupton, C. M. Waterman-Storer, and G. Danuser
Two distinct actin networks drive the protrusion of migrating cells.
Science (2004) 305:1782-1786.