Single-Molecule Analysis of the Microtubule Cross-Linking Protein MAP65-1 Reveals a Molecular Mechanism for Contact-Angle-Dependent Microtubule Bundling

Abstract

Bundling of microtubules (MTs) is critical for the formation of complex MT arrays. In land plants, the interphase cortical MTs form bundles specifically following shallow-angle encounters between them. To investigate how cells select particular MT contact angles for bundling, we used an in vitro reconstitution approach consisting of dynamic MTs and the MT-cross-linking protein MAP65-1. We found that MAP65-1 binds to MTs as monomers and inherently targets antiparallel MTs for bundling. Dwell-time analysis showed that the affinity of MAP65-1 for antiparallel overlapping MTs is about three times higher than its affinity for single MTs and parallel overlapping MTs. We also found that purified MAP65-1 exclusively selects shallow-angle MT encounters for bundling, indicating that this activity is an intrinsic property of MAP65-1. Reconstitution experiments with mutant MAP65-1 proteins with different numbers of spectrin repeats within the N-terminal rod domain showed that the length of the rod domain is a major determinant of the range of MT bundling angles. The length of the rod domain also determined the distance between MTs within a bundle. Together, our data show that the rod domain of MAP65-1 acts both as a spacer and as a structural element that specifies the MT encounter angles that are conducive for bundling.

MAP65 Coordinate Microtubule Growth during Bundle Formation

“…Nevertheless this observation suggest that MAP65-1 cross-links between parallel MTs are labile. These observations are in agreement with a recent study showing that the dwell-time of MAP65-1 on parallel MTs is larger than its dwell-time on anti-parallel MTs [27]. Altogether these data demonstrate that in bundles, MAP65-1 binds to both parallel and anti-parallel MTs.…”

“…Furthermore, our model predicts that bonds between parallel MTs control the amplitude of bundle elongation, despite the fact that they are very sensitive to MT depolymerization. In agreement with these data, a recent study showed that the dwell time of MAP65-1 is three times longer on anti-parallel MTs than on parallel MTs and on single MTs [27].…”

“…In the assays, knowing that apparent Kd of MAP65-1 for MTs is 0.57 µM ([26]; 1.03 µM as determined in [27]), we used MAP65-1 concentrations ranging from 0.1 µM to 1.5 µM. In the presence of MAP65-1, we observed that MT bundle length increased linearly with time and generated a dense array of MT bundles (Figure 1B, Movies S1 and S2).…”

MAP65/Ase1 promote microtubule flexibility

“…The MT-binding domain is responsible for the preferential antiparallel orientation of the bundled MTs, whereas the projection domain length determines MT interspacing (Gaillard et al. , 2008; Tulin et al. , 2012).…”

Microtubules, MAPs, and motor patterns

“…The protocol we present was adapted from a protocol he provided (Tulin, McClerklin, Huang, & Dixit, 2012). …”