Masters of the tips: End Binding proteins orchestrate microtubule plus- and minus-end dynamics

Chao Yang

Research output: ThesisDoctoral thesis 1 (Research UU / Graduation UU)

Abstract

Microtubules are dynamic polymers built of dimers of α- and β-tubulin, which attach to each other in a head-to-tail fashion. Microtubules can rapidly switch between phases of growth and shortening, a behavior known as “dynamic instability”. In cells, microtubule dynamics are also tightly controlled by numerous factors including microtubule plus end tracking proteins (+TIPs). End Binding proteins (EBs) are highly conserved +TIPs. In mammalian cells, the EB family includes three members, EB1, EB2 and EB3. EBs form the core of the +TIP network by recruiting to microtubule ends many structurally and functionally diverse +TIPs. EBs can also alter the properties of microtubule plus ends by promoting GTP hydrolysis by β-tubulin and possibly by modifying microtubule end structure. In Chapter 2, we applied CRISPR/Cas9 technology to generate a series of EB1, EB2 and EB3 mutant cell lines. Surprisingly, we found that EBs strongly affect microtubule minus-end organization: microtubule minus ends stabilized by CAMSAP2 were detached from Golgi membranes and the Golgi apparatus became more compact. Further studies showed that co-organization of microtubules and Golgi membranes depends on the EB1/EB3-myomegalin-AKAP450 complex, which tethers microtubules to the Golgi and counteract compaction of Golgi stacks. In Chapter 3, we investigated how EBs and their partners affect the properties of growing microtubule plus ends. We generated new EB1, EB2 and EB3 triple knockout HeLa cell lines. We compare the length of EB comets in vivo and in vitro. We found a stable, depolymerization-resistant microtubule zone in the tip-proximal region. We also found that EBs inhibit mobility of microtubule tips and make their growth more persistent. Experiments with EB deletion mutants showed that these properties depend on the recruitment of EB binding partners. In Chapter 4, we explored the possibility that liquid-liquid phase separation (LLPS) plays a role in microtubule tip regulation. We found that SLAIN2 can form droplet in cells, particularly in EB1, EB2 and EB3 triple knockout cells. We also found that EBs and chTOG were recruited to SLAIN2 droplets, whereas CLASP and CLIP170 were not. We also found that SLAIN2 and chTOG, but not EB3, could efficiently form droplets in vitro, and the droplets containing chTOG could nucleate microtubule asters. In Chapter 5, we investigated how different microtubule binding domains could affect microtubule dynamics in vitro when targeted to microtubule tips by the N-terminal part of EB3. We found that fusions of SLAIN2 and spectraplakin caused no obvious changes, fusion of the TOG domains of CLASP2 suppressed catastrophes or induced rescues. Fusion of different TOG domain of chTOG increased catastrophe frequency. Fusion of the GAR domain of spectraplakin and the CAP-Gly domains of CLIP170 induced rescues, fusion of the microtubule binding domain of MAP7 reduced microtubule depolymerization rate. The same fusion proteins did not affect microtubule dynamics in EB1, EB2 and EB3 triple knockout cells as they did in vitro. In conclusion, we have obtained new insights into how EBs control microtubule networks and generated new tools that will help in future to unravel cellular mechanisms controlling microtubule polymerization.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Utrecht University
Supervisors/Advisors
  • Akhmanova, Anna, Primary supervisor
Award date20 Apr 2020
Publisher
Print ISBNs978-94-6375-797-3
Publication statusPublished - 20 Apr 2020

Keywords

  • End binding proteins
  • microtubule dynamics
  • EB1,EB2,EB3 triple knockout
  • microtubule minus end organization
  • microtubule tip properties
  • SLAIN2
  • phase separation
  • microtubule binding domains.

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