Structural basis for α-tubulin-specific and modification state-dependent glutamylation.

Microtubules have spatiotemporally complex posttranslational modification patterns. Tubulin tyrosine ligase-like (TTLL) enzymes introduce the most prevalent modifications on α-tubulin and ß-tubulin. How TTLLs specialize for specific substrate recognition and ultimately modification-pattern generation is largely unknown. TTLL6, a glutamylase implicated in ciliopathies, preferentially modifies tubulin α-tails in microtubules. Cryo-electron microscopy, kinetic analysis and single-molecule biochemistry reveal an unprecedented quadrivalent recognition that ensures simultaneous readout of microtubule geometry and posttranslational modification status. By binding to a ß-tubulin subunit, TTLL6 modifies the α-tail of the longitudinally adjacent tubulin dimer. Spanning two tubulin dimers along and across protofilaments (PFs) ensures fidelity of recognition of both the α-tail and the microtubule. Moreover, TTLL6 reads out and is stimulated by glutamylation of the ß-tail of the laterally adjacent tubulin dimer, mediating crosstalk between α-tail and ß-tail. This positive feedback loop can generate localized microtubule glutamylation patterns. Our work uncovers general principles that generate tubulin chemical and topographic complexity.

Combinatorial and antagonistic effects of tubulin glutamylation and glycylation on katanin microtubule severing.

Microtubules have spatiotemporally complex posttranslational modification patterns. How cells interpret this tubulin modification code is largely unknown. We show that C. elegans katanin, a microtubule severing AAA ATPase mutated in microcephaly and critical for cell division, axonal elongation, and cilia biogenesis, responds precisely, differentially, and combinatorially to three chemically distinct tubulin modifications-glycylation, glutamylation, and tyrosination-but is insensitive to acetylation. Glutamylation and glycylation are antagonistic rheostats with glycylation protecting microtubules from severing. Katanin exhibits graded and divergent responses to glutamylation on the α- and ß-tubulin tails, and these act combinatorially. The katanin hexamer central pore constrains the polyglutamate chain patterns on ß-tails recognized productively. Elements distal to the katanin AAA core sense α-tubulin tyrosination, and detyrosination downregulates severing. The multivalent microtubule recognition that enables katanin to read multiple tubulin modification inputs explains in vivo observations and illustrates how effectors can integrate tubulin code signals to produce diverse functional outcomes.

ER proteins decipher the tubulin code to regulate organelle distribution.

Organelles move along differentially modified microtubules to establish and maintain their proper distributions and functions1,2. However, how cells interpret these post-translational microtubule modification codes to selectively regulate organelle positioning remains largely unknown. The endoplasmic reticulum (ER) is an interconnected network of diverse morphologies that extends promiscuously throughout the cytoplasm3, forming abundant contacts with other organelles4. Dysregulation of endoplasmic reticulum morphology is tightly linked to neurologic disorders and cancer5,6. Here we demonstrate that three membrane-bound endoplasmic reticulum proteins preferentially interact with different microtubule populations, with CLIMP63 binding centrosome microtubules, kinectin (KTN1) binding perinuclear polyglutamylated microtubules, and p180 binding glutamylated microtubules. Knockout of these proteins or manipulation of microtubule populations and glutamylation status results in marked changes in endoplasmic reticulum positioning, leading to similar redistributions of other organelles. During nutrient starvation, cells modulate CLIMP63 protein levels and p180-microtubule binding to bidirectionally move endoplasmic reticulum and lysosomes for proper autophagic responses.

Author Correction: Structural basis for polyglutamate chain initiation and elongation by TTLL family enzymes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structural basis for polyglutamate chain initiation and elongation by TTLL family enzymes.

Glutamylation, introduced by tubulin tyrosine ligase-like (TTLL) enzymes, is the most abundant modification of brain tubulin. Essential effector proteins read the tubulin glutamylation pattern, and its misregulation causes neurodegeneration. TTLL glutamylases post-translationally add glutamates to internal glutamates in tubulin carboxy-terminal tails (branch initiation, through an isopeptide bond), and additional glutamates can extend these (elongation). TTLLs are thought to specialize in initiation or elongation, but the mechanistic basis for regioselectivity is unknown. We present cocrystal structures of murine TTLL6 bound to tetrahedral intermediate analogs that delineate key active-site residues that make this enzyme an elongase. We show that TTLL4 is exclusively an initiase and, through combined structural and phylogenetic analyses, engineer TTLL6 into a branch-initiating enzyme. TTLL glycylases add glycines post-translationally to internal glutamates, and we find that the same active-site residues discriminate between initiase and elongase glycylases. These active-site specializations of TTLL glutamylases and glycylases ultimately yield the chemical complexity of cellular microtubules.

Differences in Intrinsic Tubulin Dynamic Properties Contribute to Spindle Length Control in Xenopus Species.

The function of cellular organelles relates not only to their molecular composition but also to their size. However, how the size of dynamic mesoscale structures is established and maintained remains poorly understood [1-3]. Mitotic spindle length, for example, varies several-fold among cell types and among different organisms [4]. Although most studies on spindle size control focus on changes in proteins that regulate microtubule dynamics [5-8], the contribution of the spindle's main building block, the αß-tubulin heterodimer, has yet to be studied. Apart from microtubule-associated proteins and motors, two factors have been shown to contribute to the heterogeneity of microtubule dynamics: tubulin isoform composition [9, 10] and post-translational modifications [11]. In the past, studying the contribution of tubulin and microtubules to spindle assembly has been limited by the fact that physiologically relevant tubulins were not available. Here, we show that tubulins purified from two closely related frogs, Xenopus laevis and Xenopus tropicalis, have surprisingly different microtubule dynamics in vitro. X. laevis microtubules combine very fast growth and infrequent catastrophes. In contrast, X. tropicalis microtubules grow slower and catastrophe more frequently. We show that spindle length and microtubule mass can be controlled by titrating the ratios of the tubulins from the two frog species. Furthermore, we combine our in vitro reconstitution assay and egg extract experiments with computational modeling to show that differences in intrinsic properties of different tubulins contribute to the control of microtubule mass and therefore set steady-state spindle length.