FIGURE 3:

Topology of p-MTs and pa-MTs in wild-type and γ-tubS360A spindles. (A) Schematic representation of microtubule pitch–length pairing analysis. Pitch is defined by the angle of projection of the microtubule from its origin relative to the imaginary plane defined by the two spindle poles (spindle axis). The contour length (nanometers) of individual spindle microtubules and their intersection and spacing (gap) are extracted from the tomographic models using IMOD. Pairing events are represented by two types of antiparallel interactions: p-MTs (blue dots) and pa-MTs (yellow dots). p-MTs are defined as two microtubules (usually both >500 nm) projecting from opposite poles that interact over a contour length of >300 nm with gap of 45 nm. pa-MT pairs consist of two microtubules projecting from opposite poles that intersect over 10–290 nm of contour length with gap of <45 nm. (B) Distribution of p-MT pairing angles in wild-type spindles as a function of microtubule length (nanometers). The pitch of wild-type p-MTs is 2.7 ± 1.68° (n = 39). Three pa-MTs were identified in one wild-type spindle and are indicated with lines and shown in the inset. (C) Distribution of pa-MT and p-MT pairing angles in γ-tubS360A spindles as a function of microtubule length (nanometers). The pitch of γ-tubS360A p-MTs is 4.1 ± 2.58° (n = 35). pa-MT pairs are frequently composed of one long (>600 nm) microtubule projecting at <10° paired to at least one short (<500 nm) microtubule projecting at a pitch of >10°. High angle (>5°) antiparallel pairing is correlated with pa-MT bending. Bending is frequently observed in γ-tubS360A spindles (inset, C) and in one medial wild-type spindle that contained three pa-MTs (inset, B). p-MTs in γ-tubS360A spindles project from the poles at an increased pitch.