The History of Tubulin Detyrosination

(OK folks I'll be back this afternoon, this post is the last in serries of entries from my old blogs ... and this one is a hard core science entry from about 1 year ago ... enjoy)

Late last week I posted an entry on tubulin modification ... an area of research that one well respected cytoskeletal researcher described as "a cottage industry based on antibodies" ... Due to increased interest in the field, I'll recount here the tale of how tubulin modifications were first discovered.

In 1973 group of researchers (a team from Argentina!) decided to test whether proteins were repaired in neurons, since in these cells, the nerve terminal is located far away from the cell nuclei (where the instruction material to make new proteins is found). To test this hypothesis, they gave mice drugs to stop protein synthesis from the canonical protein synthesis pathway (e.i. by the ribosome - see the entry on the Central Dogma of Biology) and then feed these poor mice radio-labeled amino acids. If the small criters "repaired" proteins using the fed amino acids, then the radio-label should incorporate into "fixed" proteins - since protein synthesis was inhibited, there should be no other radioacive incorporation. Sure enough one, and only one amino acid was being incorporated into neuronal protein ... tyrosine. Eventually the Argentines looked to see what was being modified, and it was a single protein, alpha-tubulin. One amino-acid, one protein.

Turns out that tyrosine is reversibly ligated to the end (or Carboxyl-terminal) of tubulin and this occurs in almost every cell in the body. The two modifying enzymes (the one that adds tyrosine to tubulin - the tubulin tyrosine ligase, and the one that takes tyrosine off - the tubulin carboxypeptidase) had been elusive for decades, until the tubulin tyrosine ligase was identified. As I mentioned in a previous blog entry, this enzyme is special in that it is one of a few enzymes (outside of the ribosome) that can catalyze the formation of a peptide bond.

Antibodies raised against tyrosinated and detyrosinated tubulin are very specific(they don't cross react) and can bind to their respective targets at incredible dilutions. These antibody studies suggested that the tubulin tail (where the reversible tyrosine is situated) is highly antigenic (i.e. is reactive against antibodies and thus very "bindable") and a change of a single amino acid can drastically change it's "affinity profile" (i.e. it's ability to bind stuff). The first surprise was when antibodies against the tyrosinated and detyrosinated forms were used to stain fixed cells. All the modifications (in this case detyrosinated tubulin) were segregated to a subset of microtubules that were oriented along the cell's "axis of polarity", such as towards the front in a migrating cell. Thus microtubule differentiation was discovered. Then came the famous discovery that microtubules, which are distributed in an astral patern in cells, grow and shrink from their plus ends (which are located on the periphery) and are inert at their minus ends (which are located in the cell center at the MTOC). Tim Mitchison and Marc Kirschner speculated about what this all means and came up with their famous review Beyond self-assembly: from microtubules to morphogenesis. The idea is simple - microtubule plus ends grow and shrink, probing the cellular space to find specialized targets (such as chromosomes, waiting to be pulled apart by microtubule plus ends). When they do find these targets, the microtubule plus ends are "captured" and now serve as a track conecting the cell center (where the microtubule minus ends are) and the specialized site (where the captured microtubule plus ends are). Elements like chromosomes can then be pulled to the new cell center. In the case of migrating cells, the specialized site (the front of the cell) captures and stabilize microtubules. It was then shown that the stable microtubules accumulate modifications (like detyrosination) and can be used by the cell to allow communication between the front of the cell and the cell center (see image above, modified microtubules in green/yellow are all poited towards the wound while non-modified microtubules in red are everywhere).

Well is tubulin detyrosination important after all? I won't go into details about how detyrosinated microtubules bind to stuff differently from tyrosinated "tubes" ... all I'll mention is that Jurgen Wehland and Didier Job's groups finally knocked out the tubulin tyrosine ligase (TTL) gene ... and the knock-out mice died due to a lack of neuronal organization in the cerebral cortex ... basically no modification = the brain is a mess. Why neurons - well as the Argentine scientists will tell you, those are long cells - and they have a lot of modified tubulin!

Ref:

1- The crazy observation that started it all off -
Barra HS, Rodriguez JA, Arce CA, Caputto R.J, A soluble preparation from rat brain that incorporates into its own proteins ( 14 C)arginine by a ribonuclease-sensitive system and ( 14 C)tyrosine by a ribonuclease-insensitive system. Neurochem. (1973) 20:97-108.

2- The first look at modified microtubules in cells -
Gundersen GG, Kalnoski MH, Bulinski JC. Distinct populations of microtubules: tyrosinated and nontyrosinated alpha tubulin are distributed differently in vivo. Cell. (1984) 38:779-89.

3- The great hypothesis -
Kirschner M, Mitchison T., Beyond self-assembly: from microtubules to morphogenesis. Cell. (1986) 45:329-42.

4- The TTL knockout -
Erck et al. A vital role of tubulin-tyrosine-ligase for neuronal organization. PNAS (2005) 102:7853-7858.