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.
Highlights
Microtubules (MTs) are dynamic cytoskeletal polymers composed of tubulin dimers, which attach to each other in a head-to-tail fashion to form protofilaments that interact laterally to form a hollow tube (Desai and Mitchison, 1997)
We demonstrate that a single TOG-like domain of CLASP2, TOG2, which does not bind to free tubulin, is sufficient to induce rescues and, when targeted to MT plus ends, suppress catastrophes
A Complex of CLASP2a and EB3 Suppresses Catastrophes and Promotes Templated MT Nucleation To investigate the impact of CLASP2a on MT dynamics, we purified it from HEK293T cells (Figure S1A) and analyzed its activity using an in vitro reconstitution assay (Bieling et al, 2007; Doodhi et al, 2016), in which MT growth from GMPCPP-stabilized seeds is observed by total internal reflection fluorescence microscopy (TIRFM)
Summary
Microtubules (MTs) are dynamic cytoskeletal polymers composed of tubulin dimers, which attach to each other in a head-to-tail fashion to form protofilaments that interact laterally to form a hollow tube (Desai and Mitchison, 1997). Switching to MT depolymerization, termed catastrophe, can be induced by intrinsic MT properties, such as fluctuations in the size of the protective guanosine triphosphate (GTP) cap (Brouhard, 2015; Howard and Hyman, 2009). MT catastrophes can be induced by MT depolymerases that can cause protofilament peeling, by an encounter with obstacles that block protofilament elongation or by MT-destabilizing agents that can induce structural defects at MT tips (Akhmanova and Steinmetz, 2015; Gardner et al, 2013). Blocking just one MT protofilament at the growing MT end can disrupt growth and induce a catastrophe (Doodhi et al, 2016), but it is unclear how severe the accompanying aberrations in MT structure can be, and whether and how they can be repaired
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