Readout of the tubulin code by cellular effectors

National Institute Of Neurological Disorders And Stroke

Investigators

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Abstract

Microtubules are polymers essential for cell morphogenesis, cell division and intracellular transport. They are subject to highly diverse, abundant and evolutionarily conserved post-translational modifications. Disruption of tubulin modification levels and patterns leads to cancers, neuropathologies and defective axonal regeneration. Our long-term goal is to understand how cells use tubulin isoform diversity and posttranslational modifications to regulate the structure and dynamics of microtubules as well as their interactions with molecular motors and microtubule associated proteins (MAPs). Although discovered over thirty years ago, an understanding of the roles of the chemical and genetic complexity of tubulin has remained elusive. My group integrates techniques and concepts from biophysics, proteomics, structural and cell biology to address this fundamental problem in microtubule cell biology. My laboratory has made significant progress towards these goals. These include: (1) development of novel methods for generating homogenous engineered single isoform recombinant unmodified human tubulin (Vemu et al., J. Biol. Chem., 2016); (2) determination of the first structure and dynamic instability parameters of recombinant isotopically pure recombinant neuronal tubulin (Vemu et al., J. Biol. Chem., 2016; Vemu et al. 2020); (3) demonstration that microtubules with different isoform compositions exhibit different dynamic properties and that these properties can be proportionally tuned by varying tubulin isoform composition (Vemu et al., Mol. Biol. Cell, 2017).(4) development of a biochemical platform for obtaining tubulin with quantitatively defined levels of posttranslational modifications (Valenstein and Roll-Mecak, Cell 2016) and use of this platform to (5) showing the graded response of an important microtubule regulator, the hereditary spastic paraplegia protein spastin, to tubulin glutamylation (Valenstein and Roll-Mecak, Cell 2016) thus furnishing strong support for the tubulin code hypothesis. Using our platform for generating quantitatively defined modified microtubules as well recombinant engineered human microtubules, we are currently investigating how the tubulin code, both through genetic variation and posttranslational modifications, regulates the basic biophysical properties of microtubules as well as molecular motors and neuronal MAPs with strong involvement in neurodegenerative disorders. Specifically, this year we elucidated how glutamylation, a modification long associated with microtubule stability in cells affects microtubule dynamics. Cell biologists from diverse fields use antibodies against this modification as a proxy for stable microtubules in cells. Using our recent advances in obtaining differentially glutamylated tubulin coupled with microtubule dynamics reconstitution we showed that, surprisingly, glutamylation acts as a negative regulator of microtubule growth (Chen and Roll-Mecak, 2023)and does not stabilize microtubules. Thus, the higher stability of glutamylated microtubules in cells must be due to trans effects. Our future efforts will focus on identifying the proteome that is recruited to glutamylated microtubules. Taken together, our most recent work on glutamylation, as well as our previous work on tyrosination, detyrosination and Delta2 tubulin established that none of the posttranslational modifications long associated with increased microtubule stability in cells, confer increased stability directly. Thus, these modifications function as signals to recruit effectors in a temporally and spatially controlled manner to the microtubule.