The +TIP coordinating protein EB1 is highly dynamic and diffusive on microtubules, sensitive to GTP analog, ionic strength, and EB1 concentration

Abstract

Using single-molecule fluorescence microscopy, we investigated the dynamics of dye-labeled EB1, a +TIP microtubule binding protein. To promote EB1 binding along the entire microtubule length, we formed microtubules using the nonhydrolyzable GTP analogs GMPCPP and GTPγS. Through precise tracking of the motions of individual dye-labeled proteins, we found EB1 to be highly dynamic and continuously diffusive while bound to a microtubule, with a diffusion coefficient and characteristic binding lifetime that were sensitive to both the choice of GTP analog and the buffer ionic strength. Using fluorescence-based equilibrium binding measurements, we found EB1 binding to be cooperative and also sensitive to GTP analog and ionic strength. By tracking the motion of a small number of individually-labeled EB1 proteins within a bath of unlabeled EB1 proteins, we determined the effects of increasing the total EB1 concentration on binding and dynamics. We found that the diffusion coefficient decreased with increasing EB1 concentration, which may be due at least in part, to the cooperativity of EB1 binding. Our results may have important consequences for the assembly and organization of the growing microtubule plus-end.

Structural state recognition facilitates tip tracking of EB1 at growing microtubule ends

“…Despite the very similar dwell times for all six pools, the differences in on-rate leads to K d values that decrease from GDP to GMPCPP to GTPγS microtubules, and with each disrupted pool having a lower K d than in the control pool. ( F ) Left: Predicted binding curves based on the calculated K d values in panel E. As previously described, the data was fit to a Hill Equation for cooperative binding of EB1 onto microtubules (Lopez and Valentine, 2016; Zhu et al, 2009). The experimental EB1-GFP concentrations used in Figure 2C–E are shown as a dotted line on each graph.…”

α-Tubulin Tyrosination and CLIP-170 Phosphorylation Regulate the Initiation of Dynein-Driven Transport in Neurons

“…Vesicles co-purify with an endogenous complement of dynein, dynactin, and kinesins (Figure S2), are positive for markers of late-endosomes/ lysosomes (Rab7/ LAMP-1) but not early endosomes (Rab5), and exhibit dynein-dependent motility in vitro (Hendricks et al 2010). To mimic recruitment of organelles to the GTP-like microtubule lattice at the plus end, we used Guanosine-5′-[(α,β)-methyleno]triphosphate (GMPCPP) stabilized microtubules (Alushin et al 2014) that specifically recruit +TIPs including EB1 and CLIP-170 (Dixit et al 2009; Zanic et al 2009; Lopez & Valentine 2015), and provide a stable landing platform more tractable for automated analysis of landing rates than dynamic microtubules. To this, we added purified recombinant unlabeled EB1 and high speed supernatant (HSS) from COS-7 cells transfected with either HaloTag (HT) CLIP-A or CLIP-E, or HSS from mock-transfected cells (Mock-HSS)(Figure 2A).…”

Microtubule end conversion mediated by motors and diffusing proteins with no intrinsic microtubule end-binding activity

“…Our model employs Brownian dynamics simulations of ensemble of MAPs and motors interacting with MT. To describe these stochastic molecular interactions, we used force-dependent characteristics for motors and MAPs stepping and unbinding from the MT, which were determined in vitro or based on prior publications 54–63 . Stochastic simulations 64 were carried out using Mathematica (Wolfram Research).…”