title: 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.
authors.affiliation: Biology Department, Washington University in St. Louis, St. Louis, Missouri, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: Tulin
authors.forename: Sheri
authors.initials: S
authors.lastname: McClerklin
authors.forename: Yue
authors.initials: Y
authors.lastname: Huang
authors.forename: Ram
authors.initials: R
authors.lastname: Dixit
mesh_terms: D017360:Arabidopsis / Q000166:cytology / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D017434:Protein Structure, Tertiary
chemical_list: D029681:Arabidopsis Proteins; C492901:MAP65-1 protein, Arabidopsis; D008869:Microtubule-Associated Proteins