Fig. 4

She1 binds directly to dynein, and recognizes a specific nucleotide-bound state. a Schematic of experimental setup. Note that for these experiments, a GFP-tagged monomeric dynein₃₃₁ (GFP–dynein₃₃₁) fragment was used. b–d Representative fluorescence images (b) and quantitation (c) of She1-TMR recruitment (fixed at 40 nM) to control (“+E-Hook”) or subtilisin-digested (“−E-hook”) microtubules by increasing concentrations of GFP–dynein₃₃₁ (scale bars, 2 µm; error bars, standard deviation; n ≥ 19 microtubules, and ≥75 µm of MT length for each condition). d Schematic of experimental setup. Note that the absence of nucleotide elicits a conformational state that is distinct from that of dynein in the presence of ATP and vanadate (see text). e Relative recruitment of She1-TMR by GFP–dynein₃₃₁ in the presence of either no nucleotide (apo) or 3 mM ATP and vanadate (ADP–vanadate). Different points reflect the mean fluorescence intensity values (along with standard deviations) of She1-TMR (fixed at 40 nM) vs. increasing concentrations of GFP–dynein₃₃₁. Given the different microtubule-binding affinity of GFP–dynein₃₃₁ in each nucleotide state (Supplementary Fig. 3a), the extent of She1-TMR microtubule recruitment was directly compared to the relative microtubule binding by GFP–dynein₃₃₁ (n ≥ 10 microtubules, and ≥36 µm of MT length for each condition). f Cartoon (left) and three example kymographs (right) depicting that on subtilisin-digested microtubules, She1 remains bound to GST–dynein₃₃₁ as it walks, and thus transitions through many iterations of its mechanochemical cycle (horizontal scale bar, 1 µm; vertical scale bar, 30 s; Supplementary Fig. 3)