Our exploration into the world of biochemistry and molecular cell biology has provided us with an impressive view of how cells, tissues, organs and body systems can be controlled on a day-to-day basis. A myriad of vital regulators have been identified and integrated into linear pathways of increasing complexity, which elegantly talk to each other to keep our cells in order.

As we delve deeper into this world, what is increasingly apparent is that these once linear pathways have another tier to their complexity. If we are to understand how a handful of instructions generate diverse cell behaviours we must now address how they behave in both space and time.

So what do I mean by space and time? Well for me, this means what happens when cells move? What sets of instructions will be activated and will they change their location within the cell? As I’ve mentioned before, when cells are told to move, they become “polarised” and with this we see key proteins moving to new locations to carry out their function. The difficulty in studying these changes comes when we try to dissect out these localised effects from all the other signals whizzing around the cell.

A common way to study the different locations within a cell is to separate the cell into its constituent parts. This is a process called “fractionation” and is reliant on the varying densities of different parts of the cell, which can be exploited along with their solubility to separate these parts by centrifugation. However, the complement of proteins important for cell adhesion and movement are notoriously difficult to isolate, especially if you wish to maintain an “activated” state to provide a snapshot of “what” is active “where” when a cell is moving.

At the start of my PhD I pondered in great depth how to gain access to this complement of proteins and regulators. I tried techniques from the 80’s where cells are forced to swell with water and then sprayed with liquid. The aim (of what sounds like a crude technique) is to shear away the majority of the cell and leave behind the adhesion/migration associated membranes and proteins that remain attached to the surface of your dish (see below). 

This technique worked with varying degrees of success, but I could never get it consistent enough, on a large enough scale, to do the experiments I wanted. So, I was very excited when reading some recent publications where variations of this technique had been skilfully applied to a number of adhesion based questions (1-3).

Variations on the core idea of this technique are seen in these papers. Humphries et al and Schiller et al use a kind of glue, which is added to the cells for just a few minutes. This really is the key, as this glue will first contact the adhesion proteins, their receptors and binding partners. This more strongly links these factors to the world around the cell and enables you to be a bit rougher when shearing away the rest of the cell. In another variation, Jean-Cheng Kuo et al showed how depletion of the annoying “hanger-on” proteins that make your sample messy gives cleaner results.

Once established (the vital figure 1 in these papers!), these techniques could be applied to different conditions. Cells were given varying substrates to stick to, engaged different adhesion receptors and were exposed to treatments that affect the cells skeleton. Ultimately, the protein changes could then be assessed on a large scale using protein fingerprinting and the connections and conclusions made using computer based analyses.

Although the techniques differ slightly and the conditions the cells are presented with vary, these papers demonstrate how much we can understand by successfully accessing this exclusive piece of sub-cellular real estate. We can only hope that this technique is scrutinised and improved so more and more questions can be asked and we can all have a look through the adhesion keyhole. 

References:

1.     Humphries et al (2009) Proteomic Analysis of Integrin-Associated Complexes Identifies RCC2 as a Dual Regulator of Rac1 and Arf6. Science Signaling. 2. ra51.

2.     Schiller et al (2011) Quantitative proteomics of the Integrin adhesome show a myosin II-dependent recruitment of LIM domain proteins. EMBO reports. 12. pp259-266. 

3.     Jean-Cheng Kuo et al (2011) Analysis of the Myosin-II-responsive focal adhesion proteome reveals a role for β-Pix in negative regulation of focal adhesion maturation. Nature Cell Biology. 13. pp383-393.