Antonina Roll-Mecak: Decoding the secrets of tubulin complexity

Deceptively uniform ultrastructurally, microtubules are composed of multiple tubulin isoforms that bear a bewildering range of posttranslational modifications, including acetylation, detyrosination, phosphorylation, glutamylation, and glycylation. Tubulin α/β heterodimers consist of a compact folded body and intrinsically disordered C-terminal tails. These tails form a dense lawn on the microtubule surface and serve as binding sites for molecular motors and microtubule-associated proteins. The majority of sequence variation between tubulin isoforms as well as posttranslational modifications concentrates on these intrinsically disordered tails. Some of the modifications we study, such as glutamylation and glycylation, can add amino acid chains that are significantly longer than the tubulin tails themselves. I want to understand how these modifications affect the intrinsic biophysical properties of the microtubule (dynamics and mechanical properties) and how it interacts with cellular effectors. What are the signaling events that lead to these modifications?

We want to crack the tubulin code. To crack this code we first need to understand how it is written: the mechanism and regulation of the enzymes that introduce tubulin posttranslational modifications. Second, we need to elucidate how posttranslational modifications and isoform variability affect the basic properties of the microtubule polymer itself. Third, we need to know how spatial and temporal patterns of microtubule modifications are established and propagated. Ultimately, we need to understand how tubulin isoform composition and modifications are interpreted by cellular effectors, regulating their recruitment or activity. My laboratory has developed a biochemical platform to obtain recombinant, isotypically pure human tubulin (1) as well as quantitatively defined, differentially modified tubulins (2) that allow a mechanistic look at the tubulin code. We will continue to try to uncover the basic principles that give rise to the differential specificities of tubulin modification enzymes (3, 4) and how they cooperate and compete to modify the microtubule substrate; to discover how the tubulin code modulates the structure, dynamics, and mechanical properties of microtubules; and to understand how motors and microtubule-associated proteins are influenced by the tubulin code (2) and how their action in turn modulates the behavior of this dynamic polymer. Moreover, we and others have discovered that some of the modifications that were thought to be characteristic of tubulin in fact regulate other molecular players such as histone chaperones and DNA sensors involved in the innate immune response. We have recently shown that glutamylation, the addition of glutamates to tubulin tails, acts as molecular rheostat to control the activity of the microtubule-severing enzyme spastin, suggesting a possible mechanism by which stereotyped subcellular patterns of microtubule glutamylation can regulate local microtubule dynamics through severing. The majority of tubulin in the adult mammalian brain is glutamylated and the number of glutamates on tubulin tails in neurons is distributed in stereotyped patterns: axonal microtubules have long glutamate chains, while the soma and growth cones mostly lack glutamylated microtubules. We anticipate that other microtubule regulators display such graded, spatially controlled responses to glutamylation. In addition, a recent collaboration with Tiansen Li’s laboratory at the Eye Institute has allowed us to show that glutamylation by tubulin tyrosine ligase-like glutamylase TTLL5 is required for the function of a protein essential for normal vision, RPGR, which localizes to the transition zone in photoreceptor cilia (5). It is mutated in retinitis pigmentosa, a degenerative eye disease. Thus, we have become interested in how glutamylation is used by cells to modulate other important pathways.

We use a highly mechanistic and interdisciplinary approach. Like a complex music score that can be broken down into its constituent phrases, so can complex biological phenomena be distilled down and reconstituted in vitro to understand their governing principles that can then serve as the foundations to reconstitute the complex score. We use a variety of techniques in our work from structural biology and classic enzymology to single molecule biophysics, live cell imaging, and modeling.

I had a long dry spell during my PhD and only experienced success at the end of my fourth year. There were small signs along the way that things might work out, but there was also a long history of failure from many other groups on the project. In structural biology, you have nothing until you all of a sudden have everything: a picture of how a protein is put together that nobody else has seen and that you can now use to extract its secrets. While it definitely had its dark moments, my PhD experience gave me the confidence that I can crack problems that remained unsolved for some time. My postdoc with Ron Vale was highly formative. Ron taught me to trust my scientific nose and use any technique at my disposal, either directly or through collaborations to solve a problem. I also learned how to maintain a competitive environment that was collaborative and friendly.

The paperwork! But I have learned to navigate it efficiently now.

My biggest accomplishment has been to set up a vibrant laboratory where people challenge themselves and work collaboratively in opening up an exciting realm in microtubule biology where there are so many unsolved basic questions.

Deciding when to give up on projects or on people.

My parents taught me that even the greatest effort seems easy if it is done for something that you love, while my piano teacher taught me focus and discipline. I was a bit of a street urchin in my childhood and my parents were quite concerned about my academic performance until my piano teacher Eniko Orth took me under her wing when I was nine. In addition to the guidance offered by my doctoral and postdoctoral advisors, I benefitted from mentorship by Rod MacKinnon, who introduced me to the experimental craft of protein biochemistry while working side by side with me at the bench when I started graduate school. I was also influenced by Günter Blobel, who impressed on me the importance of in vitro reconstitution in understanding cell biology.

Follow your nose and ignore the prevailing fashions.

Running a laboratory and having a toddler does not leave much time for hobbies. I enjoy classical music. When I was a graduate student I was able to take in two to three concerts or opera performances a week in between running chromatography columns or sucrose gradients. Going out for performances is a lot harder now with a toddler at home.

I might have been a pianist, but I really have a hard time picturing myself doing anything else other than science. I really love showing up to work in my laboratory.

Go after a problem you are interested in and don’t try to hedge your bets too much. Go all in!