SummaryThe dynamic instability of microtubules plays a key role in controlling their organization and function, but the cellular mechanisms regulating this process are poorly understood. Here, we show that cytoplasmic linker-associated proteins (CLASPs) suppress transitions from microtubule growth to shortening, termed catastrophes, including those induced by microtubule-destabilizing agents and physical barriers. Mammalian CLASPs encompass three TOG-like domains, TOG1, TOG2, and TOG3, none of which bind to free tubulin. TOG2 is essential for catastrophe suppression, whereas TOG3 mildly enhances rescues but cannot suppress catastrophes. These functions are inhibited by the C-terminal domain of CLASP2, while the TOG1 domain can release this auto-inhibition. TOG2 fused to a positively charged microtubule-binding peptide autonomously accumulates at growing but not shrinking ends, suppresses catastrophes, and stimulates rescues. CLASPs suppress catastrophes by stabilizing growing microtubule ends, including incomplete ones, preventing their depolymerization and promoting their recovery into complete tubes. TOG2 domain is the key determinant of these activities.

SummaryCentrioles are fundamental and evolutionarily conserved microtubule-based organelles whose assembly is characterized by microtubule growth rates that are orders of magnitude slower than those of cytoplasmic microtubules. Several centriolar proteins can interact with tubulin or microtubules, but how they ensure the exceptionally slow growth of centriolar microtubules has remained mysterious. Here, we bring together crystallographic, biophysical, and reconstitution assays to demonstrate that the human centriolar protein CPAP (SAS-4 in worms and flies) binds and “caps” microtubule plus ends by associating with a site of β-tubulin engaged in longitudinal tubulin-tubulin interactions. Strikingly, we uncover that CPAP activity dampens microtubule growth and stabilizes microtubules by inhibiting catastrophes and promoting rescues. We further establish that the capping function of CPAP is important to limit growth of centriolar microtubules in cells. Our results suggest that CPAP acts as a molecular lid that ensures slow assembly of centriolar microtubules and, thereby, contributes to organelle length control.

BKM120 (Buparlisib) is one of the most advanced phosphoinositide 3-kinase (PI3K) inhibitors for the treatment of cancer, but it interferes as an off-target effect with microtubule polymerization. Here, we developed two chemical derivatives that differ from BKM120 by only one atom. We show that these minute changes separate the dual activity of BKM120 into discrete PI3K and tubulin inhibitors. Analysis of the compounds cellular growth arrest phenotypes and microtubule dynamics suggest that the antiproliferative activity of BKM120 is mainly due to microtubule-dependent cytotoxicity rather than through inhibition of PI3K. Crystal structures of BKM120 and derivatives in complex with tubulin and PI3K provide insights into the selective mode of action of this class of drugs. Our results raise concerns over BKM120's generally accepted mode of action, and provide a unique mechanistic basis for next-generation PI3K inhibitors with improved safety profiles and flexibility for use in combination therapies.

Feedback-Driven Assembly of the Axon Initial Segment Highlights d Ankyrin-G in complex with EBs recruits microtubule bundles to the plasma membrane d TRIM46 is a rescue factor that forms stable parallel microtubule bundles d TRIM46-bound microtubules direct Neurofascin-186 trafficking to the proximal axon d Ankyrin-G controls Neurofascin-186 retention in the axon initial segment

Highlights d CLASP stabilizes damaged microtubule lattices d CLASP converts partial protofilament assemblies into complete tubes d CLASP promotes complete repair of microtubule lattice defects d CLASP inhibits softening of microtubules bent by hydrodynamic flow