Again some interesting thoughts about problem solving in biological systems ... ripped off of a seminar intro from this weekend's retreat. Here goes ...
Scientists are tinkerers. We need tools to get inside a system and manipulated it to understand what is going on and to ultimately test our theories. So what are our tools? And what are the major parameters that we must consider when using our tools.
This weekend I heard a great talk by Kevan Shokat of UCSF. In his intro Dr Shokat made a great point in detailing the two most important features in manipulating biological systems: specificity and speed.
Specificity is crucial. When manipulating the constituents of a living organism we can revert to several methods. Of course most of the time we are manipulating the products of divergent evolution, mostly transcriptional products (i.e. proteins). We can use drugs, however drugs tend to inhibit many related enzymes and thus have low specificity. Genetic manipulations are much better as we can isolate a single genetic product and eliminate it. To ensure that the effects are specific we can reintroduce the gene of interest back into the organism.
Another important concern is speed. Living organisms are very dynamic organisms and many processes that we study are temporally regulated and extremely dynamic. To study the role of gene X in brain function it would be best to remove molecule X from a fully functional brain. If we remove it from birth, many (what we jokingly call) "Biological Heisenberg Effects" kick in. The animal must develop a brain in the absence of this molecule. This will not only affect the how the brain and other organs are constructed, but will commonly activate compensatory mechanisms that will mask the role that the molecule plays in a normal situation. In other words, the measured object has been altered. In contrast, drugs tend to take affect immediately and can be used to ascertain the role that a molecule plays within the unaltered biological system.
So there is a balance between specificity and temporal resolution best summed up in this graph shown by Shokat:
Now this intro leads into the main goal of the Shokat lab. To combine the speed of pharmaceutical manipulations with the specificity of genetic manipulations. I won't get into the nitty gritty of what the Shokat lab has done, since most of it is published, but in summary he has been altering gene products to increase the specificity of their pharmaceutical inhibitors. For example he will manipulate a protein that adds phosphates (i.e. a kinase) by enlarging the catalytic/inhibitor binding site. He can then inhibit this kinase, but not other highly related kinases by using a bulkier inhibitor that only fits into the enlarged binding site. Of course you can test whether this altered chemical has any unspecific effects by treating the genetically unaltered system. Thus the speed of drug delivery has been combined with the specificity inferred by genetics.
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I don't know if he got into this, but in addition to molecular specificity, cellular specificity is also very important. This is particularly true when studying the nervous system, where there are probably millions of functionally distinct neuronal cell types (in mammals).
One of the great advantages of using Drosophila as a model system is that we have access to a wonderful technique that allows us to genetically target molecular manipulations solely to a highly restricted group of about twenty functionally related neurons in the background of an otherwise completely normal nervous system. This level of cellular specificity is not currently achievable in mammals.
So, when trying to understand integrative physiology in vivo, the keys are temporal specificity, molecular specificity, *and* cellular specificity.
Good point PhysioProf. I should also add that with Shokat's technique you can express the modified kinase in your 20 neurons and then throw on inhibitor. The beauty of his system is that you can combine every manipulation accessible to genetics with the temporal resolution of drug treatments.