CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC.

γ-Tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here we reconstituted this process in vitro using purified components. We found that all CAMSAPs could bind to the minus ends of γ-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends but not the minus-end tracking protein CAMSAP1, induced microtubule release from γ-TuRC. CDK5RAP2, a γ-TuRC-interactor, and CLASP2, a regulator of microtubule growth, strongly stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 suppressed CAMSAP binding to γ-TuRC-anchored minus ends and their release. CDK5RAP2 also improved selectivity of γ-tubulin-containing complexes for 13- rather than 14-protofilament microtubules in microtubule-capping assays. Knockout and overexpression experiments in cells showed that CDK5RAP2 inhibits the formation of CAMSAP2-bound microtubules detached from the microtubule-organizing centre. We conclude that CAMSAPs can release newly nucleated microtubules from γ-TuRC, whereas nucleation-promoting factors can differentially regulate this process.

Structural insights into the mechanism of GTP initiation of microtubule assembly.

In eukaryotes, the dynamic assembly of microtubules (MT) plays an important role in numerous cellular processes. The underlying mechanism of GTP triggering MT assembly is still unknown. Here, we present cryo-EM structures of tubulin heterodimer at their GTP- and GDP-bound states, intermediate assembly states of GTP-tubulin, and final assembly stages of MT. Both GTP- and GDP-tubulin heterodimers adopt similar curved conformations with subtle flexibility differences. In head-to-tail oligomers of tubulin heterodimers, the inter-dimer interface of GDP-tubulin exhibits greater flexibility, particularly in tangential bending. Cryo-EM of the intermediate assembly states reveals two types of tubulin lateral contacts, "Tube-bond" and "MT-bond". Further, molecular dynamics (MD) simulations show that GTP triggers lateral contact formation in MT assembly in multiple sequential steps, gradually straightening the curved tubulin heterodimers. Therefore, we propose a flexible model of GTP-initiated MT assembly, including the formation of longitudinal and lateral contacts, to explain the nucleation and assembly of MT.

DCX-EMAP is a core organizer for the ultrastructure of Drosophila mechanosensory organelles.

Mechanoreceptor cells develop specialized mechanosensory organelles (MOs), where force-sensitive channels and supporting structures are organized in an orderly manner to detect forces. It is intriguing how MOs are formed. Here, we address this issue by studying the MOs of fly ciliated mechanoreceptors. We show that the main structure of the MOs is a compound cytoskeleton formed of short microtubules and electron-dense materials (EDMs). In a knock-out mutant of DCX-EMAP, this cytoskeleton is nearly absent, suggesting that DCX-EMAP is required for the formation of the MOs and in turn fly mechanotransduction. Further analysis reveals that DCX-EMAP expresses in fly ciliated mechanoreceptors and localizes to the MOs. Moreover, it plays dual roles by promoting the assembly/stabilization of the microtubules and the accumulation of the EDMs in the MOs. Therefore, DCX-EMAP serves as a core ultrastructural organizer of the MOs, and this finding provides novel molecular insights as to how fly MOs are formed.

TUBright: A Peptide Probe for Imaging Microtubules.

In vitro assays using reconstituted microtubules have provided molecular insights into the principles of microtubule dynamics and the roles of microtubule-associated proteins. Emerging questions that further uncover the complexity in microtubule dynamics, especially those on tubulin isotypes and post-translational modifications, raise new technical challenges on how to visualize microtubules composed of tubulin purified from limited sources, primarily due to the low efficiency of the conventional tubulin labeling protocol. Here, we develop a peptide probe, termed TUBright, that labels in vitro reconstituted microtubules. TUBright, when coupled with different fluorescent dyes, provides flexible labeling of microtubules with a high signal-to-noise ratio. TUBright does not interfere with the dynamic behaviors of microtubules and microtubule-associated proteins. Therefore, TUBright is a useful tool for imaging microtubules, making it feasible to use tubulin from limited sources for answering many open questions on microtubule dynamics.

α1A and α1C form microtubules to display distinct properties mainly mediated by their C-terminal tails.

Microtubules consisting of α/ß-tubulin dimers play critical roles in cells. More than seven genes encode α-tubulin in vertebrates. However, the property of microtubules composed of different α-tubulin isotypes is largely unknown. Here, we purified recombinant tubulin heterodimers of mouse α-tubulin isotypes including α1A and α1C with ß-tubulin isotype ß2A. In vitro microtubule reconstitution assay detected that α1C/ß2A microtubules grew faster and underwent catastrophe less frequently than α1A/ß2A microtubules. Generation of chimeric tail-swapped and point-mutation tubulins revealed that the carboxyl-terminal (C-terminal) tails of α-tubulin isotypes largely accounted for the differences in polymerization dynamics of α1A/ß2A and α1C/ß2A microtubules. Kinetics analysis showed that in comparison to α1A/ß2A microtubules, α1C/ß2A microtubules displayed higher on-rate, lower off-rate, and similar GTP hydrolysis rate at the plus-end, suggesting a contribution of higher plus-end affinity to faster growth and less frequent catastrophe of α1C/ß2A microtubules. Furthermore, EB1 had a higher binding ability to α1C/ß2A microtubules than to α1A/ß2A ones, which could also be attributed to the difference in the C-terminal tails of these two α-tubulin isotypes. Thus, α-tubulin isotypes diversify microtubule properties, which, to a great extent, could be accounted by their C-terminal tails.

The microtubule end-binding affinity of EB1 is enhanced by a dimeric organization that is susceptible to phosphorylation.

In cells, microtubule dynamics are regulated by plus-end tracking proteins (+TIPs). End-binding protein 1 (EB1, also known as MAPRE1) acts as a master regulator of +TIP networks by targeting the growing ends of microtubules and recruiting other factors. However, the molecular mechanism underlying high-affinity binding of EB1 to microtubule ends remains an open area of research. Using single-molecule imaging, we show that the end-binding kinetics of EB1 change when the polymerization and hydrolysis rates of tubulin dimers are altered, confirming that EB1 binds to GTP-tubulin and/or GDP-Pi-tubulin at microtubule growing ends. The affinity of wild-type EB1 to these sites is higher than that of monomeric EB1 mutants, suggesting that both calponin homology domains present in the EB1 dimer contribute to end binding. Introduction of phosphomimetic mutations into the EB1 linker domain weakens the end-binding affinity and confers a more curved conformation on the EB1 dimer without compromising dimerization, suggesting that the overall architecture of EB1 is important for its end-binding affinity. Taken together, our results provide insights into how the high-affinity end-binding of EB1 is achieved and how this activity may be regulated in cells.

Spectraplakin Shot Maintains Perinuclear Microtubule Organization in Drosophila Polyploid Cells.

Polyploid cells endoreplicate their DNA through a modified cell cycle that skips mitosis as part of their differentiation programs. Upon cell-cycle exit and differentiation, non-centrosomal sites govern microtubule distribution in most cells. Little is known on how polyploid cells, differentiated but cycling, organize their microtubules. We show that microtubules in Drosophila adipocytes and other polyploid tissues form a dense perinuclear cortex responsible for nuclear size and position. Confirming a relation between this perinuclear cortex and the polyploid endocycle, polyploidization of normally diploid cells was sufficient for cortex formation. A critical component of the perinuclear microtubule organizer (pnMTOC) is Shot, absence of which caused collapse of the perinuclear network into a condensed organizer through kinesin-dependent microtubule sliding. Furthermore, this ectopic organizer was capable of directing partial assembly of a deeply disruptive cytokinesis furrow. In all, our study revealed the importance of perinuclear microtubule organization for stability of endocycling Drosophila cells.

Microtubule-binding protein FOR20 promotes microtubule depolymerization and cell migration.

Microtubules are highly dynamic filaments assembled from αß-tubulin heterodimers and play important roles in many cellular processes, including cell division and migration. Microtubule dynamics is tightly regulated by microtubule-associated proteins (MAPs) that function by binding to microtubules or free tubulin dimers. Here, we report that FOR20 (FOP-related protein of 20 kDa), a conserved protein critical for ciliogenesis and cell cycle progression, is a previously uncharacterized MAP that facilitates microtubule depolymerization and promotes cell migration. FOR20 not only directly binds to microtubules but also regulates microtubule dynamics in vitro by decreasing the microtubule growth rate and increasing the depolymerization rate and catastrophe frequency. In the in vitro microtubule dynamics assays, FOR20 appears to preferentially interact with free tubulin dimers over microtubules. Depletion of FOR20 inhibits microtubule depolymerization and promotes microtubule regrowth after the nocodazole treatment in HeLa cells. In addition, FOR20 knockdown significantly inhibits both individual and collective migration of mammalian cells. Taken together, these data suggest that FOR20 functions as a MAP to promote microtubule depolymerization and cell migration.