Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus ends

Abstract

Growing microtubule ends provide platforms for the accumulation of plus-end tracking proteins that organize into comets of mixed protein composition. Using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions appear to facilitate the non-stoichiometric accumulation of cargo Tip1. We dissected the contribution of two disordered protein regions in Mal3 and found that both are required for the ability to form droplets and Tip1 accumulation, while autonomous Mal3 comet formation only requires one of them. Using theoretical modeling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends.

Phase separation of +TIP-networks regulates microtubule dynamics

“…Is phase separation a common feature of +TIPs? Studies performed in parallel to our work show that this phenomenon is conserved across evolution: +TIPs in budding yeast, fission yeast, and higher eukaryotes have recently been demonstrated to undergo phase separation (Maan et al, 2021;Meier et al, 2021;Song et al, 2021, Jijumon et al, 2022. Intriguingly, in line with our results, the yeast studies confirmed that the CLIP-170 homolog played a key role in the phase separation process, whereas LLPS potency of EB homologs varied between organisms.…”

“…The role of different mammalian EB family members in regulating LLPS will be an interesting direction for future studies. The ability of these +TIP-networks to phase separate depends on intrinsically disordered regions (Maan et al, 2021;Song et al, 2021) and multivalent interaction modules (Figure 2; Meier et al, 2021), consistent with the observation that these features are highly evolutionarily conserved across +TIPs (Wu et al, 2021). Further studies will be necessary to investigate whether additional +TIPs contribute to the formation and regulation of +TIP-droplets.…”

Phase separation of +TIP-networks regulates microtubule dynamics

“…Further studies will be necessary to investigate whether additional +TIPs contribute to the formation and regulation of +TIP-droplets. Our work here and recent studies demonstrate that +TIP networks can behave like liquid condensates (Wu et al, 2021;Maan et al, 2021;Meier et al, 2021;Song et al, 2021). This work adds to the growing list of microtubule-related processes that are driven by LLPS and provides an exciting new paradigm for how cells can spatiotemporally control microtubule dynamics through local tubulin concentration (Zhang et al, 2015;Woodruff et al, 2017;Hernández-Vega et al, 2017;King and Petry, 2020;Jiang et al, 2021;Maan et al, 2021;Meier et al, 2021;Song et al, 2021).…”

“…Our work here and recent studies demonstrate that +TIP networks can behave like liquid condensates (Wu et al, 2021;Maan et al, 2021;Meier et al, 2021;Song et al, 2021). This work adds to the growing list of microtubule-related processes that are driven by LLPS and provides an exciting new paradigm for how cells can spatiotemporally control microtubule dynamics through local tubulin concentration (Zhang et al, 2015;Woodruff et al, 2017;Hernández-Vega et al, 2017;King and Petry, 2020;Jiang et al, 2021;Maan et al, 2021;Meier et al, 2021;Song et al, 2021). Interrogating the mechanical properties and composition of +TIP-droplets, as well as studying their regulation throughout the cell cycle, will be exciting avenues for future research.…”

“…The role of different mammalian EB family members in regulating LLPS will be an interesting direction for future studies. The ability of +TIP-networks to phase separate depends on intrinsically disordered regions (Maan et al, 2021;Song et al, 2021) and multivalent interaction modules (Meier et al, 2021), consistent with the observation that these features are highly evolutionarily conserved across +TIPs (Wu et al, 2021). Further studies will be necessary to investigate whether additional +TIPs contribute to the formation and regulation of +TIP-droplets.…”