Microtubules are polymers essential for cell morphogenesis, cell division and intracellular transport. Microtubules execute their diverse cellular roles by forming suprastructures with highly distinctive geometries: the radial cytoplasmic array, the short, highly parallel axonemal array, the spindle array or the tiled long axonal array. The microtubule cytoskeleton is a complex function of many unit operations, the individual actions of cytoskeletal regulators: nucleation, growth and shrinkage, severing and motor movement. Moreover, the microtubule itself is more than just a naive roadway for cellular components to transit along. Alpha and beta tubulins have multiple isoforms and are subject to highly diverse, abundant and evolutionarily conserved post-translational modifications that mark subpopulations of microtubules (Yu et al, 2015). Given the central role microtubules play in basic cellular processes, it is not surprising that microtubule regulators have been implicated in many human diseases, including cancers, cardiovascular disease, fungal, bacterial and viral infections, as well as neurodegenerative disorders such as Parkinson's, Alzheimer's and Amyotrophic lateral sclerosis. Our efforts concentrate on two families of microtubule regulators: microtubule severing enzymes and enzymes that post-translationally modify tubulin. Our research plan is highly interdisciplinary, integrating techniques and concepts from biophysics, structural, molecular and cell biology to answer two closely interdigitated questions: how is the structure of the microtubule locally perturbed when it is engaged by these regulators and how do these regulators affect microtubule architecture and dynamics at the cellular level? Perturbation of microtubule dynamics has emerged as a common theme in a variety of neurodegenerative diseases and our work has implications for the etiologies of all these disorders. In the last year we initiated several studies aimed at understanding the mechanistic underpinnings of the functions of microtubule post-translational modifications as well as continued our work on the mechanism of microtubule severing by spastin. (1) My group has been responsible in the last year for the development of novel methods for the production of recombinant human tubulin. While studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure and dynamics has been held back due to a lack of dynamics measurements using homogenous, engineered tubulin. My laboratory has recently reported the first 4.2 cryo-EM structure (collaboration with Carolyn Moores, Birkbeck College, University of London) and in vitro dynamics parameters of recombinant, isotypically pure human neuronal alpha1A/betaIII tubulin, an essential and important initial step in quantitatively establishing the correlates between sequence and dynamics for mammalian microtubules (Vemu et al., 2015 - listed in report bibliography). (2) Using our platform for generating quantitatively defined modified microtubules, we investigated the effects of the tubulin code on the activity of an important neuronal cytoskeletal effector, the hereditary spastic paraplegia protein spastin. Glutamylation is the most abundant tubulin modification in the adult mammalian brain. It has the potential to be informationally highly complex as it involves the addition of variable numbers of glutamates to tubulin tails. 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 have no glutamylated microtubules and dendrites contain microtubules with shorter glutamate chains. We discovered that the microtubule-severing enzyme spastin, an AAA ATPase, is under rheostatic control by the number of glutamates attached to the tubulin tails of its substrate, suggesting a possible mechanism by which stereotyped subcellular patterns of microtubule glutamylation can regulate local microtubule dynamics through severing (Valenstein and Roll-Mecak, 2016 - listed in report bibliography). Our results provided the first quantitative evidence for a graded, spatially controlled response to a tubulin posttranslational modification. Moreover, we demonstrated that the graded response to glutamylation enables regulation at a distance of microtubule severing within a microtubule network. Our work is an initial important and essential step towards understanding how the cell interprets the tubulin code and addresses the fundamental problem of how functional specificity is imparted to microtubules in cells. The mechanism of spastin regulation by glutamylation on the tubulin tails echoes the rheostatic regulation by additive phosphorylation of intrinsically disordered protein regions and has wider implications for signaling via glutamylation as a large array of molecular players, most notable histone chaperones, are glutamylated in cells. (3) We have also extended our studies of glutamylation to non-tubulin substrates as proteomic studies revealed that many proteins in addition to tubulin are glutamylated. As part of a new collaboration with Tiansen Lis laboratory at the National Eye Institute we discovered that the retinitis pigmentosa GTPase regulator (RPGR), the gene mutated in the majority of patients with inherited retinal degeneration, is glutamylated by TTLL5, and that loss of glutamylation compromises its function in photoreceptor cilia, leading to opsin mislocalization (Sun et al., 2016 - listed in report bibliography) . Interestingly, TTLL5 glutamylates a Glu-Gly rich repetitive region with motifs homologous to the -tubulin C-terminal tail. This region is present only in a photoreceptor specific ORF15 variant of RPGR (RPGRORF15). TTLL5 mutations were reported in patients with retinal degeneration for the first time last year. Thus, our study resolves at the molecular level the pathophysiology of TTLL5 gene mutations that cause retinal dystrophy and connects the RPGR and TTLL5 disease mutations into a common pathway. Lastly, we are actively working on purifying to homogeneity and in biophysical quantities several tubulin modification enzymes that show different substrate specificities and modes of regulation. We will use these enzyme preparations to modify microtubules in vitro in order to investigate the effects of the introduced tubulin modification on microtubule dynamics and the recruitment and activity of motors and microtubule associated proteins.

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7
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2016
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Sun, Xun; Park, James H; Gumerson, Jessica et al. (2016) Loss of RPGR glutamylation underlies the pathogenic mechanism of retinal dystrophy caused by TTLL5 mutations. Proc Natl Acad Sci U S A 113:E2925-34
Meyer, Peter A; Socias, Stephanie; Key, Jason et al. (2016) Data publication with the structural biology data grid supports live analysis. Nat Commun 7:10882
Valenstein, Max L; Roll-Mecak, Antonina (2016) Graded Control of Microtubule Severing by Tubulin Glutamylation. Cell 164:911-21
Vemu, Annapurna; Atherton, Joseph; Spector, Jeffrey O et al. (2016) Structure and Dynamics of Single-isoform Recombinant Neuronal Human Tubulin. J Biol Chem 291:12907-15
Roll-Mecak, Antonina (2015) Intrinsically disordered tubulin tails: complex tuners of microtubule functions? Semin Cell Dev Biol 37:11-9
Yu, Ian; Garnham, Christopher P; Roll-Mecak, Antonina (2015) Writing and Reading the Tubulin Code. J Biol Chem 290:17163-72
Garnham, Christopher P; Vemu, Annapurna; Wilson-Kubalek, Elizabeth M et al. (2015) Multivalent Microtubule Recognition by Tubulin Tyrosine Ligase-like Family Glutamylases. Cell 161:1112-23
Vemu, Annapurna; Garnham, Christopher P; Lee, Duck-Yeon et al. (2014) Generation of differentially modified microtubules using in vitro enzymatic approaches. Methods Enzymol 540:149-66
Szyk, Agnieszka; Deaconescu, Alexandra M; Spector, Jeffrey et al. (2014) Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferase. Cell 157:1405-15
Ziolkowska, Natasza E; Roll-Mecak, Antonina (2013) In vitro microtubule severing assays. Methods Mol Biol 1046:323-34

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