Figure 3.
Together the Ndc80 and Dam1 complexes maintain persistent, load-bearing attachments to growing and shortening microtubule tips. (A) Schematic of force clamp assay. A bead coated with Ndc80 complex and in the presence of free Dam1 complex is initially placed onto the side (lattice) of a microtubule. Laser scissors are then used to sever the microtubule tip (top panel), which induces tip shortening (middle panel). Eventually, the shortening microtubule tip reaches the bead, whereupon a constant force is applied to the bead as it tracks with the dynamic tip (bottom panel). (B) A coupler can detach from a microtubule tip either during growth (assembly) or during shortening (disassembly), with distinct rates for each type of detachment event. The tip can interconvert between growth and shortening via transitions called catastrophe and rescue. (C) Examples of tip-coupled movement recorded while constant tensile force was applied using a feedback-controlled laser trap. The upper five traces, plotted in the unshaded (white) region, were recorded using beads decorated with either wild-type Ndc80 complex (red), or with mutant Ndc80 complexes carrying insertions that disrupted regions ANdc80p (purple), BNdc80p (green), or CNdc80p (turquoise), all in the presence of free wild-type Dam1 complex. The lower four traces, plotted in the yellow shaded region, were recorded using beads decorated with wild-type Ndc80 complex in the presence of free phosphodeficient 6A Dam1 complex (red), as a control, or in the presence of Dam1 complex phosphorylated at region ADam1p (purple), BAsk1p (green), or CSpc34p (turquoise). Traces above the dashed line were recorded with 1.5 pN of continuously applied tension, and those below the dashed line were recorded with 3.5 pN. Arrowheads mark catastrophe (▼) and rescue (▲) events. Open circles indicate detachments.