James Moody and Ben Werner with Dr. Marc Hansen, Physiology and Developmental Biology
Background
The purpose of this project is to identify the organization and mechanism of the proteins in the Zyxin-VASP actin regulatory system using biochemical analysis and in vitro reconstitution.
Cross-bead precipitation
A cross-bead precipitation assay is used to determine which proteins will bind to one another under physiological conditions. Using cross-bead precipitation assays we learned that VASP binds the zyxin LIMs region in a 1:1 ratio and that individual VASP monomers also bind the zyxin Act-A region in an apparent 1:1 ratio. This leads us to propose a structure for a functional actin-zyxin-VASP complex. Four VASP proteins form a tetramer by interlocking their tails. Each of those VASP proteins binds an individual actin filament and accelerates the rate of filament growth. Each VASP protein also binds transiently an individual zyxin protein. The zyxin can either clamp down on its associated VASP with its LIMs, inactivating it, or release VASP to allow it to function. Zyxin may be bound by regulatory protein Rac1, and we believe that the Rac1 toggles zyxin between open and closed forms.
Actin Sedimentation
Actin sedimentation assays are used to determine which proteins will bind to actin filaments and under what conditions. Using actin sedimentation assays, we determined that not only does VASP bind actin, but LIMs do not prevent the interaction. Instead, LIMs will also bind to the VASP-actin complex. We also learned that the other portion of zyxin, Act A, prevents some, but not all of VASP binding to actin, not what we had expected. A recent paper indicates that VASP binding to Act A is transient1. From this information, we redrew our model, hypothesizing that it is the Act A that prevents VASP binding to actin, but only if VASP stays bound to it for longer than a transient period of time. We believe that LIMs acts like a clamp to force VASP to stay attached to Act A long enough for Act A to prevent VASP binding to actin.
Critical Concentration
Critical concentration assays are used to determine if a protein of interest affects the amount of actin monomers required in solution to allow formation of stable filaments. Proteins may affect this amount by stabilizing existing filaments, by blocking addition or attrition of monomers from existing filaments, or by sequestering un-polymerized monomers. Using this type of assay, we determined that VASP-LIMs complexes do not stabilize actin filaments, block addition or attrition of monomers from existing filaments, or sequester un-polymerized monomers.
Pyrene actin
Pyrene actin assays measure the rate and amount of growth of actin filaments while under the influence of other proteins of interest. Using pyrene actin assays, we determined that VASP appears to double the rate of actin filament growth and that LIMs do not interfere with this function.
Problems
Protein biochemistry is tedious work, and we ran into many problems that had to be overcome. First, purified proteins are extremely fragile and short-lived. VASP, for example, must be kept at 4ºC at all times and has a fridge life of no more than two weeks before it loses its precise three-dimensional structure and becomes non-functional. We had a crew of several students which continuously grew and purified our needed proteins from bacteria, thereby supplying us with an endless supply of fresh VASP, LIMs, and zyxin∆LIM.
Due to their delicacy, proteins will only function under specific conditions, such as specific salt concentrations and pH ranges. We had to carefully adjust our experimental conditions to ensure that VASP and the other proteins functioned as they would in living cells.
Some proteins, such as the LIMs construct, cannot be purified from bacteria without the use of a non-ionic detergent, such as Triton-X, which releases LIMs from the bacterial cells and allows the protein to be soluble so that it can be used in experiments. Unfortunately, the presence of Triton-X interferes with the spectrographic monitoring of certain experiments. As Triton-X is indispensable for working with LIMs, we have found alternative methods to monitor our experiments.
Publications
Some of the above data was presented as a poster at the American Society of Cell Biologists held last December in San Diego. Another poster on this work is being presented at the same meeting this December. A research article containing further results is nearing completion and will soon be submitted to the appropriate scientific journals.
Future Direction
We still need to see whether the Act A-LIMs complex prevents VASP interaction with actin. We need to determine whether Rac1 does indeed bind zyxin and what effect this binding has on zyxin and hence VASP function. The lab is currently in the process of producing a source of full-length zyxin protein so they can test how Act A and LIMs behave when they are parts of a single molecule, as this is how they exist in the cell. Further pyrene actin assays will also be conducted to determine how the various combinations of VASP, LIMs, Act A, zyxin, and Rac1 affect the growth and stability of actin filaments.
References
- Applewhite DA, Barzik M, Kojima S, Svitkina TM, Gertler FB, Borisy GG. (2007) Ena/VASP Proteins Have an Anti-Capping Independent Function in Filopodia Formation. Molecular Biology of the Cell 18, 2579-91.