CLASP mediates microtubule repair by promoting tubulin incorporation into damaged lattices

Abstract

Microtubule network plays a key role in cell division, motility and intracellular trafficking.Microtubule lattices are generally regarded as stable structures that undergo turnover through dynamic instability of their ends [1]. However, recent evidence suggests that microtubules also exchange tubulin dimers at the sites of lattice defects, which can either be induced by mechanical stress or occur spontaneously during polymerization [2][3][4]. Tubulin incorporation can restore microtubule integrity; moreover, "islands" of freshly incorporated GTP-tubulin can inhibit microtubule disassembly and promote rescues [3][4][5][6][7]. Microtubule repair occurs in vitro in the presence of tubulin alone [2][3][4]8]. 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. CLASP2α promoted tubulin incorporation into damaged lattice sites thereby restoring microtubule integrity. Furthermore, it induced the formation of complete tubes from partial protofilament assemblies and inhibited microtubule softening caused by hydrodynamic flowinduced bending. A single CLASP2α domain, TOG2, which suppresses catastrophes when tethered to microtubules, was sufficient to stimulate microtubule repair, indicating that catastrophe suppression and lattice repair are mechanistically similar. Our results suggest that the cellular machinery controlling microtubule nucleation and growth can also help to maintain microtubule integrity.3 CLASP stalls depolymerization and promotes repair of microtubule lattices damaged by photoablationTo investigate whether CLASPs can promote microtubule repair, we modified previously described in vitro reconstitution assays with full length GFP-tagged CLASP2α purified from HEK293T cells ( Figure S1A) [11]. 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. Lattice damage induced by laser irradiation next to a microtubule often led to progressive bending and eventual breakage of the microtubule at the damage site (Figures 1B,C and Video S1). This was most likely due to dissociation of tubulin dimers from the damaged microtubule lattice and subsequent loss of mechanical integrity. In the presence of ...

CLASPs at a glance

“…Thus, CLASP may compete with katanin for binding to sites of microtubule defects, as well as target intermediate lattice structures that are generated during severing (Vemu et al, 2018) (see poster). Although enhanced accumulation of CLASP at microtubule crossovers has not been observed (Lindeboom et al, 2019), the localized action of even a small number of CLASP molecules may be sufficient to stabilize the growing microtubule end (Aher et al, 2018(Aher et al, , 2019. Therefore, CLASP at severing sites may allow immediate continuous growth of nascent microtubule plus ends, while the minus ends are simultaneously protected by SPIRAL2 (Nakamura et al, 2018).…”

The lifecycle of the neuronal microtubule transport machinery

“…Although these experiments were carried out with in vitro reconstituted systems, mid lattice repair has been observed in cells [58]. Rather than relying solely on free tubulin for repair, cells are likely to make use of proteins currently best known for suppressing catastrophe, such as CLASP [59]. However, this observation highlights the importance of maintaining free tubulin pools in distal axons in order to support repair and transport.…”