The Microtubule Plus-End Proteins EB1 and Dynactin Have Differential Effects on Microtubule Polymerization

Abstract

Several microtubule-binding proteins including EB1, dynactin, APC, and CLIP-170 localize to the plus-ends of growing microtubules. Although these proteins can bind to microtubules independently, evidence for interactions among them has led to the hypothesis of a plus-end complex. Here we clarify the interaction between EB1 and dynactin and show that EB1 binds directly to the N-terminus of the p150Glued subunit. One function of a plus-end complex may be to regulate microtubule dynamics. Overexpression of either EB1 or p150Glued in cultured cells bundles microtubules, suggesting that each may enhance microtubule stability. The morphology of these bundles, however, differs dramatically, indicating that EB1 and dynactin may act in different ways. Disruption of the dynactin complex augments the bundling effect of EB1, suggesting that dynactin may regulate the effect of EB1 on microtubules. In vitro assays were performed to elucidate the effects of EB1 and p150Glued on microtubule polymerization, and they show that p150Glued has a potent microtubule nucleation effect, whereas EB1 has a potent elongation effect. Overall microtubule dynamics may result from a balance between the individual effects of plus-end proteins. Differences in the expression and regulation of plus-end proteins in different cell types may underlie previously noted differences in microtubule dynamics

Microtubule binding proteins CLIP‐170, EB1, and p150Glued form distinct plus‐end complexes

“…microtubule plus-ends As CLIP-170 and EB1 co-localize to dynamic microtubule plus ends in the cell, we tested whether these two proteins may interact directly, similar to our previous observation that EB1 binds to the related N-terminal CAP-Gly domain of p150 Glued [17]. We found that recombinant EB1 was specifically retained on a CLIP-170-H2 column but not on a BSA control column (Fig.…”

“…Recombinant constructs of CLIP-170 (an in-frame T7 tag was fused to the N-terminus of the previously characterized H2 construct [16] generously provided by Holly Goodson, Notre Dame University) and p150 Glued (residues 1-333) [17], which span the microtubule binding domains of these proteins, as well as full length EB1 [17], were expressed as His-tagged fusion proteins in E. coli and purified by Ni 2+ -chromatography.…”

“…For assembly assays these microtubules were sheared to generate seeds [17]. For polarity marked microtubules, rhodamine-conjugated tubulin (Cytoskeleton) was polymerized at a 1:20 ratio to unlabeled tubulin to form brightly labeled seeds, followed by polymerization in 15 lM tubulin at a ratio of 1:50 rhodamine-labeled to unlabeled tubulin dimers to generate more dimly labeled polymer.…”

“…Microtubules were fixed with gluteraldehyde and pelleted onto coverslips as previously described [17], then labeled with primary antibodies to tubulin (Sigma, St. Louis, MO; Cytoskeleton), EB1 (BD Transduction Laboratories, San Jose, CA), T7 (Novagen, Madison, WI; Covance, Berkeley, CA), p150 Glued (UP235 generated in our lab), and Alexa-conjugated secondary antibodies (Molecular Probes). Micrographs were acquired with consistent exposure times between comparable images; only linear image enhancement algorithms were applied.…”

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

“…EBs recruit a multitude of other plus‐end tracking proteins (Akhmanova & Steinmetz, 2015), including dynactin, which is additionally required for plus‐end tracking of dynein in cells (Vaughan et al , 1999; Ligon et al , 2003; Dixit et al , 2008). The critical dynactin subunit for plus‐end tracking is p150 Glued (called p150 here).…”