EB1 interacts with outwardly curved and straight regions of the microtubule lattice

Abstract

EB1 is a microtubule plus-end tracking protein that recognizes GTP-tubulin dimers in microtubules and thus represents a unique probe to investigate the architecture of the GTP cap of growing microtubule ends. Here, we conjugated EB1 to gold nanoparticles (EB1-gold) and imaged by cryo-electron tomography its interaction with dynamic microtubules assembled in vitro from purified tubulin. EB1-gold forms comets at the ends of microtubules assembled in the presence of GTP, and interacts with the outer surface of curved and straight tubulin sheets as well as closed regions of the microtubule lattice. Microtubules assembled in the presence of GTP, different GTP analogues or cell extracts display similarly curved sheets at their growing ends, which gradually straighten as their protofilament number increases until they close into a tube. Together, our data provide unique structural information on the interaction of EB1 with growing microtubule ends. They further offer insights into the conformational changes that tubulin dimers undergo during microtubule assembly and the architecture of the GTP-cap region.

EB1 contributes to microtubule bundling and organization, along with root growth, in Arabidopsis thaliana

“…In both animals and yeasts, the end-binding-1 protein (EB1) accumulates at growing MT plus-ends where it regulates MT dynamics and polarization (Akhmanova and Steinmetz, 2008, 2010; Coquelle et al, 2009; Galjart, 2010; Guesdon et al, 2016; Morrison, 2007), thus contributing to the shaping of MT arrays in both interphase and mitosis (Lee et al, 2000; Rogers et al, 2002; Tirnauer et al, 2002; Wen et al, 2004). Three EB1 orthologs have been identified in Arabidopsis thaliana and other related plants (Bisgrove et al, 2008; Chan et al, 2003; Gardiner and Marc, 2003; Komaki et al, 2010; Mathur et al, 2003; Meagher and Fechheimer, 2003).…”

Steady-state EB cap size fluctuations are determined by stochastic microtubule growth and maturation

“…These studies revealed that the cap consists of hundreds of tubulins in the stabilizing conformation and that they have a roughly monoexponential distribution starting from the growing microtubule end, giving rise to the comet-shaped appearance of the EB binding region (13,15,17,18). The majority of this EB cap is lost during a period of several seconds before catastrophe occurs (16,19), indicating that the EB binding region is critical for stability.…”

Multivalent Chelators for In Vivo Protein Labeling

“…Thec ontrolled protein immobilization further allowed the investigation of the prokaryotic neurotransmitter:sodium symporter LeuT by smFRET. [39].C opyright Nature Publishing Group, 2016. analysis (Figure 2b). Combining electron microscopy and FRET,s ubunits of RNAP were mapped with nanometer precision.…”