EB1 Accelerates Two Conformational Transitions Important for Microtubule Maturation and Dynamics

Abstract

SummaryBackgroundThe dynamic properties of microtubules depend on complex nanoscale structural rearrangements in their end regions. Members of the EB1 and XMAP215 protein families interact autonomously with microtubule ends. EB1 recruits several other proteins to growing microtubule ends and has seemingly antagonistic effects on microtubule dynamics: it induces catastrophes, and it increases growth velocity, as does the polymerase XMAP215.ResultsUsing a combination of in vitro reconstitution, time-lapse fluorescence microscopy, and subpixel-precision image analysis and convolved model fitting, we have studied the effects of EB1 on conformational transitions in growing microtubule ends and on the time course of catastrophes. EB1 density distributions at growing microtubule ends reveal two consecutive conformational transitions in the microtubule end region, which have growth-velocity-independent kinetics. EB1 binds to the microtubule after the first and before the second conformational transition has occurred, positioning it several tens of nanometers behind XMAP215, which binds to the extreme microtubule end. EB1 binding accelerates conformational maturation in the microtubule, most likely by promoting lateral protofilament interactions and by accelerating reactions of the guanosine triphosphate (GTP) hydrolysis cycle. The microtubule maturation time is directly linked to the duration of a growth pause just before microtubule depolymerization, indicating an important role of the maturation time for the control of dynamic instability.ConclusionsThese activities establish EB1 as a microtubule maturation factor and provide a mechanistic explanation for its effects on microtubule growth and catastrophe frequency, which cause microtubules to be more dynamic.

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

“…Image acquisition and channel alignment were carried out as explained previously (Maurer et al , 2014). All time‐lapse movies were recorded at 1 fps for 500 s. The exposure time was always 200 ms for all the channels.…”

Biased Brownian motion as a mechanism to facilitate nanometer-scale exploration of the microtubule plus end by a kinesin-8

“…This distance corresponds to roughly 24 tubulin subunits on a 13-protofilament MT. Kif18B joins a growing list of proteins that localize to the MT plus-end terminus, the best characterized of which is ch-TOG (32,33). To determine whether ch-TOG and Kif18B occupy similar or distinct regions of the plus end, we compared the plusend distributions of the two proteins in fixed mitotic cells.…”

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

“…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. In agreement with this notion, faster-growing microtubules that have larger caps were found to be more stable after sudden tubulin removal (14).…”

“…In the simplest kinetic model cap sites are generated by tubulin incorporation into the microtubule lattice, followed by a growth-speed-independent maturation process that forms the mature lattice (13,19,20). Maturation corresponds to a conformational change, most likely associated with GTP hydrolysis or phosphate release (15,16,19,21).…”