CLASP Suppresses Microtubule Catastrophes through a Single TOG Domain

Abstract

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.

Cytolinker Gas2L1 regulates axon morphology through microtubule‐modulated actin stabilization

“…All Gas2L1 expression constructs were N-terminal fusions generated from PCR-based strategies using GFP-Gas2L1 or GFP-Gas2L1-SxAA as templates, which were based on human Gas2L1 (GenBank Accession No NM_152236.1) [20]. The following backbones were used to create Gas2L1 plasmids: GW1-GFP/HA for neuronal expression, bioGFP and GW1-HA for pull-down experiments and StrepII-EGFP for protein purification [46]. To create GW1-GFP/HA-ABD and GW1-GFP/HA-ABD-Tail, a-actinin CH domains were amplified from Gallus gallus cDNA (amino acids For shRNA-mediated depletion, shRNA sequences were inserted into HindIII and BglII sites of the pSuper backbone [47].…”

CLASP mediates microtubule repair by promoting tubulin incorporation into damaged lattices

“…However, in cells, it is likely to be regulated by specific factors, the nature of which is currently unknown. CLASP is an interesting candidate for microtubule repair, because it induces microtubule nucleation, stimulates rescue and suppresses catastrophes by stabilizing incomplete growing plus ends with lagging protofilaments and promoting their conversion into complete ones [9][10][11][12][13][14][15][16]. Here, we used in vitro reconstitution assays combined with laser microsurgery and microfluidics to show that CLASP2α indeed stimulates microtubule lattice repair.…”

“…Microtubules were grown from GMPCPP-stabilized seeds, visualized by adding fluorescently labeled tubulin and observed by Total Internal Reflection Fluorescence (TIRF) microscopy [11,17]. In this assay, GFP-CLASP2α ( Figure 1A) shows some binding to microtubule lattices and a weak enrichment at growing microtubule tips [11]. To explore the capacity of CLASP to repair microtubule lattices, we performed laser-mediated microsurgery experiments on dynamic microtubules.…”

“…Mammalian CLASPs contain three TOG-like domains, TOG1, TOG2 and TOG3, connected by flexible positively charged linkers, and a C-terminal domain (CLIP-ID) that binds to different partners and targets CLASPs to various subcellular locations [16,18] (Figure 1A). Our previous work has shown that an isolated TOG2 domain has a very low affinity for microtubules and does not bind to free tubulin [11]. However, when TOG2 was fused to the adjacent intrinsically disordered positively charged protein region (a fusion protein termed TOG2-S, Figure 1A), TOG2 could bind to microtubule lattice, show some autonomous enrichment at growing microtubule ends and suppress catastrophes even in the absence of End Binding (EB) proteins, which normally target CLASPs to growing microtubule plus ends [11].…”

“…Our previous work has shown that an isolated TOG2 domain has a very low affinity for microtubules and does not bind to free tubulin [11]. However, when TOG2 was fused to the adjacent intrinsically disordered positively charged protein region (a fusion protein termed TOG2-S, Figure 1A), TOG2 could bind to microtubule lattice, show some autonomous enrichment at growing microtubule ends and suppress catastrophes even in the absence of End Binding (EB) proteins, which normally target CLASPs to growing microtubule plus ends [11]. By performing laser damage experiments in the presence of TOG2-S, we found that it could also autonomously enrich at the damaged lattice site 4 and promote microtubule repair (Figures 1E,F and S1A,B, Video S1).…”