FIGURE 1:

Reconstitution of SPB–microtubule attachments. (A) Arrangement of components within the SPB. Top, microtubule minus ends attach γ-tubulin small complexes (γ-TuSCs) on the nuclear and cytoplasmic faces of the SPB. Spc110 molecules tether γ-TuSCs on the nuclear side to the inner core of the SPB. Spc72 molecules tether γ-TuSCs on the cytoplasmic side. Bottom, in cells, ∼20 microtubules emanate from the nuclear side to form the intranuclear mitotic spindle. Only 2 or 3 emanate from the cytoplasmic side. Dashed box indicates the region depicted in the top diagram. (B–D) Assay for observing individual SPB–microtubule attachments. Purified SPBs adhere tightly to glass coverslips (B). After incubation with free αβ-tubulin to promote nucleation and growth, the SPBs are associated with microtubule minus ends (C). Beads decorated with kinesins form strong linkages to the microtubules (D). (E) Time-lapse VE-DIC images showing pivoting of a microtubule attached to a coverslip-anchored SPB. (F) Fluctuations in the orientation of an SPB-attached microtubule over time (left) and distribution of angles (right, gray histogram) fit with a normal distribution (right, black curve; SD, 9.4°). Additional examples of microtubule pivoting and statistical analyses are shown in Supplemental Figure S2. (G) An individual fluorescent SPB (Spc42-mCherry) bound to a microtubule (MT) and viewed by TIRF microscopy. Pivoting of the filament around its SPB-attached end is evident from blurring of its distal end. (H) In the presence of ATP, a kinesin-decorated bead walks toward the plus end of an SPB-nucleated microtubule. See also Supplemental Movie S1.