A Tubulin Binding Switch Underlies Kip3/Kinesin-8 Depolymerase Activity

Abstract

Kinesin-8 motors regulate the size of microtubule structures, using length-dependent accumulation at the plus end to preferentially disassemble long microtubules. Despite extensive study, the kinesin-8 depolymerase mechanism remains under debate. Here, we provide evidence for an alternative, tubulin curvature-sensing model of microtubule depolymerization by the budding yeast kinesin-8, Kip3. Kinesin-8/Kip3 uses ATP hydrolysis, like other kinesins, for stepping on the microtubule lattice, but at the plus end Kip3 undergoes a switch: its ATPase activity is suppressed when it binds tightly to the curved conformation of tubulin. This prolongs plus-end binding, stabilizes protofilament curvature, and ultimately promotes microtubule disassembly. The tubulin curvature-sensing model is supported by our identification of Kip3 structural elements necessary and sufficient for plus-end binding and depolymerase activity, as well as by the identification of an α-tubulin residue specifically required for the Kip3-curved tubulin interaction. Together, these findings elucidate a major regulatory mechanism controlling the size of cellular microtubule structures.

Keywords: depolymerization; kinesins; microtubule associated proteins; microtubule dynamics; spindle scaling.

Figure 1. Motility is not required for Kip3 depolymerase activity

A. Diagrams of the Kip3_FL, Kip3₄₃₈ and KHC₃₂₅ constructs. Subscripts denote last residue of the protein construct. FL=Full length. B. Kip3₄₃₈ is monomeric and non-motile. Representative S200 gel filtration elution profile for Kip3₄₃₈. The graph shows the intensity of the coomassie-stained elution fractions (normalized to the maximum, n= 3 runs). In blue are molecular weights of protein standards. The molecular weight of Kip3₄₃₈, calculated from its sequence, is 74.6 KDa. Right: Representative kymograph of fluorescently labelled Kip3₄₃₈ on taxol-stabilized microtubules showing that Kip3₄₃₈ lacks motility (n=17 experiments). Scale bar: 1um (horizontal), 20s (vertical). C. Concentration-dependent depolymerization of GMPCPP-stabilized microtubules by monomeric Kip3₄₃₈. Top: kymographs showing GMPCPP-stabilized microtubules over time in the absence (control) or in the presence of 1.5 uM Kip3₄₃₈. Scale bar: 1um (vertical), 1 min (horizontal). Bottom: Quantification of GMPCPP-stabilized microtubule depolymerization rates (mean +/− SEM (standard error of the mean); n_MT=151–278) of Kip3₄₃₈. The depolymerization rate of 1uM monomeric kinesin-1 (KHC₃₂₅) and 24 nM dimeric Kip3_FL are shown as controls (mean +/−SEM; n_MT=87, 241). D. Non-motile monomeric Kip3 can reduce the end-residence time of plus-end-bound dimeric Kip3_FL. Top panel: Cartoons illustrating the experimental design. Middle: (left) Control kymograph of fluorescently labelled reporter Kip3_FL (<0.05 nM Kip3_FL-TMR) on taxol-stabilized microtubules. Unlabeled Kip3_FL (middle) or Kip3₄₃₈ (right) both decrease the plus-end dwell time of the labeled Kip3_FL reporter. The microtubule plus-end is marked as (+). Scale bar: 1um (vertical), 1 min (horizontal) left kymograph (0 nM unlabeled Kip3); 30 s (horizontal) for both unlabeled Kip3_FL and Kip3₄₃₈ kymographs. Bottom: Left: The plus-end dwell time (mean +/− SEM; n=50–175 events) of fluorescently labelled Kip3_FL is shown in the presence of spiked-in unlabeled Kip3_FL (<0.05 nM Kip3_FL -TMR, 0 nM to 4 nM unlabeled Kip3_FL dimer). Right: The plus-end dwell time (mean +/− SEM; n=149–242 events) of fluorescently labelled Kip3_FL is shown in the presence of varying concentrations of Kip3₄₃₈ (<0.05 nM Kip3_FL-TMR, 0 to 100 nM unlabeled Kip3₄₃₈). The data points were weighted by the SEM and were fit to a single exponential (dotted blue line). E. Kymographs of dynamic microtubules illustrating Kip3₄₃₈-induced catastrophes. Microtubules grown in 14 uM tubulin rarely catastrophe (top) whereas 100 nM Kip3₄₃₈ increases the frequency of catastrophes (bottom. Scale bar: 2 um (vertical), 30s (horizontal). F. Microtubule catastrophe induced by varying concentrations of Kip3₄₃₈. Top: The catastrophe frequency of dynamic microtubules grown (14uM tubulin) at the indicated concentrations of Kip3₄₃₈ (mean +/− SE (standard error); n_MT=35–273). Middle: Kip3₄₃₈ subtly increases, rather than decreases, microtubule growth rates. Microtubule growth velocity as a function of Kip3₄₃₈ concentration (mean +/− SE; n_MT=35–273). G. No preferential binding of Kip3 to GMPCPP (GTP-like) microtubules; Kip3 instead exhibits a slight preference for the GDP lattice. Ratio of the fluorescence intensity of Kip3₄₃₈ bound to taxol-stabilized microtubules (GDP-like) relative to GMPCPP-stabilized microtubules (GTP-like) at [Kip3₄₃₈]: 12 nM Kip3 (N=12 flow-cells) or 50 nM Kip3 (N=6 flow-cells). For dynamic microtubules (GDP lattice), the fluorescence intensity of 100 nM Kip3₄₃₈ was measured. The ratio of the intensity of Kip3₄₃₈ on the microtubule lattice (GDP-like), to the GMPCPP-seed (GTP-like) is quantified in the right column (N=12 flow-cells). (Mean+/−SE). In all assays, the ends of microtubules (600 nm from end) were excluded from quantification. H. Kymographs illustrating the preferential binding of Kip3 to the GDP microtubule lattice. Dynamic microtubules were grown at 14 uM tubulin (green, left) from GMPCPP seeds (blue, middle). The fluorescence intensity of Kip3₄₃₈ on the GDP-lattice and the GMPCPP seed (red, right) was imaged under TIRF microscopy and quantified as show in G.

Figure 2. Structural elements required for Kip3 microtubule depolymerase activity

A. Homology model of Kip3 bound to tubulin based on the human Kif18A structure (3LRE). β-tubulin is shown in dark green (marked +) and α-tubulin in pink (4LNU). Loop2 is in red, Loop11 in blue. Right: diagrams of the Kip3_FL, Kip3_FL-L2^KHC and Kip3_FL-L11^KHC constructs. Superscripts denote the origin of the swapped region. B. L2 is required for Kip3 processivity. Quantification of the motility (run-length, velocity and plus-end dwell time) of Kip3_FL and Kip3_FL-L2^KHC on taxol-stabilized microtubules imaged by TIRF microscopy (mean +/− SEM; n=75–870 events). The kymograph illustrates the reduced plus-end dwell time and run length of the Kip3_FL-L2^KHC construct. Scale bar: 1 min (horizontal), 2um (vertical). C. Kip3 Loop2 is dispensable for microtubule depolymerization. Kip3_FL or Kip3_FL-L2^KHC have comparable depolymerization rates of GMPCPP-stabilized microtubules (mean +/−SEM; n_MT=96–407). Depolymerization rates are shown at concentrations of the input motors has been corrected to achieve comparable flux to the plus-end (~170 motors/min; see methods, Fig S4B, input: 12nM and 40nM respectively). Kymograph showing depolymerization of a GMPCPP-stabilized microtubule over time by Kip3_FL-L2^KHC as quantified above. Scale bar: 2 min (horizontal), 2um (vertical). D. The motility of Kip3_FL-L11^KHC is comparable to Kip3_FL. Quantification of the run length (left) and velocity (right) of Kip3_FL (grey) and Kip3_FL -L11^KHC (blue) on taxol-stabilized microtubules (mean +/− SEM; n=155–1022 events). E. Specific requirement of Kip3 Loop11 for prolonged plus-end binding. Plus-end residence time of fluorescently labelled Kip3_FL and Kip3_FL-L11^KHC (mean +/− SEM; n=149–493 events) on taxol-stabilized microtubules. Right: Representative kymographs from single molecule assays quantified in D, E showing the motility and plus-end residence of Kip3_FL and Kip3_FL-L11^KHC. Scale bar: 1 um (vertical), 1 min (horizontal). F. Kip3 Loop11 is required for depolymerase activity. Depolymerization rate of GMPCPP-stabilized microtubules by the indicated concentrations of Kip3_FL (grey) or Kip3_FL-L11^KHC (dark blue, mean +/− SEM; n_MT=69–439 and n_MT=121–265 respectively). Bottom: Kymographs illustrating the depolymerization of GMPCPP-stabilized microtubules by Kip3_FL or Kip3_FL-L11^KHC at 40 nM input. Scale bar: 2 um (vertical), 2 min (horizontal). G. The Loop11 sequence is required for KIP3 function. KIP3 strains are sensitive to benomyl whereas the kip3-L11^KHC strain exhibits resistance comparable to the null allele (kip3Δ). kip3-L2^KHC strains display intermediate benomyl resistance. L2^KHC and L11^KHC experiments were performed in a kip3Δ strain background. The indicated strains were spotted at 10-fold dilutions. Partial complementation by L2^KHC is likely explained, at least in part, by its low steady state expression (see Fig S4A).

Figure 3. Kip3’s L11 is sufficient to convey plus-end binding to KHC and Kip3’s L2 is sufficient to increase the processivity of KHC

A. Domain swaps of Kip3 segments into dimeric KHC. Diagrams of the chimeric constructs used. Superscripts denote the origin of the inserted segment. B. Domain swap showing that Kip3 Loop11 is a transportable segment sufficient for prolonged plus-end binding. Kip3 and a KHC chimera containing Kip3’s L2, L11 and the family-specific neck segment (KHC₅₆₀-K8^Kip3) have comparable plus-end residence times. Shown is the end-residence time (mean +/− SEM; n=149–394 events) of the indicated KHC variant constructs compared to unmodified KHC₅₆₀ and Kip3_FL. The superscript denotes the origin of the indicated loop that was grafted into KHC₅₆₀. C. Either Kip3 L2 or L11 reduce the velocity of KHC₅₆₀. The velocity (mean +/− SEM; n=155–795 events) of KHC₅₆₀, KHC chimeras and Kip3 on taxol-stabilized microtubules. D. Kip3 and a KHC chimera containing Kip3’s L2, L11 and family-specific neck segment (KHC₅₆₀-K8^Kip3) have comparable run lengths (mean +/− SEM; n=155–795 events.) E. Representative kymographs from single molecule assays illustrating the motility and plus-end residence of KHC₅₆₀ and KHC₅₆₀ variant motor constructs. Scale bar: 1 min (horizontal), 1 um (vertical). F. KHC-K8^Kip3 is a synthetic microtubule depolymerase. Depolymerization rate of GMPCPP-stabilized microtubules by the indicated kinesin-1 chimeras (120nM, mean +/− SEM; n_MT=72–278). The KHC₅₆₀-Neck^Kip3 construct has a velocity of 7.4 +/− 0.1 um/min and a run length of 1.2 +/− 0.1 (n=547), similar to KHC. Bottom: GMPCPP-stabilized microtubules shown at 29s intervals after the addition of 120 nM KHC₅₆₀-K8^Kip3 (n_MT=84). Scale bars: 2 um (vertical), 1 min (horizontal).

Figure 4. Strong tubulin dimer binding by depolymerase proficient Kip3 variants

A. Diagram of monomeric and dimeric Kip3 variant constructs. Superscripts denote the origin of the grafted segment. The neck in Kip3_FL has been replaced with a leucine zipper (LZ, purple) in Kip3_FL-CC. B. Loop11 is required for strong tubulin binding. Affinity measurements of the binding of tubulin to immobilized Kip3₄₃₈. Biolayer interferometry example traces of tubulin binding to Kip3₄₃₈ at 0.5 uM tubulin (light grey), 1uM tubulin (dark grey) versus Kip3₄₃₈-L11^KHC at 1 uM tubulin (light blue) or at 2 uM tubulin (dark blue). Dotted orange line indicates the time point of tubulin washout, allowing measurement of k_off values. K_D values are listed in the inset (mean +/− SE, n_{Kip3 438}=30, n_{Kip3 438-L11KHC}=28 concentrations). C. Depolymerization proficient constructs have a higher relative affinity for tubulin relative to microtubules. Left: Michaelis-Menten constants for microtubules (blue) or tubulin (red) for Kip3₄₃₈, Kip3₄₃₈-L2^KHC, Kip3₄₃₈-L11^KHC and Kip3-CC (K_M, uM, mean +/− SEM, n=3–4 curves). K_M values are from steady-state ATPase assays shown in D. ATPase curves are shown in Fig. S5. A line at 100 nM is drawn for visualization of the results. Right: Relative affinity of the constructs for tubulin over the microtubule shown as (K_M^Tubulin)⁻¹ / (K_M^Microtubule)⁻¹. D. Values from steady state ATPase assays of the indicated Kip3 constructs with microtubules or free tubulin as substrate (0–10 uM). Shown are mean +/− SEM; n=3−4 curves. Raw data and fits are presented in Fig. S5. E. Preferential binding of Kip3₄₃₈ to regions of higher curvature on bent microtubules. Left: Representative images of fluorescently labelled Kip3₄₃₈ bound to taxol-stabilized microtubules bent under flow. Scale bar: 2 um. Integrated brightness for Kip3₄₃₈ (middle, black, n_MT=138) or Kip3₄₃₈-L11^KHC (right, blue, n_MT=528) plotted against the local curvature (κ) of the microtubule (mean +/− SE). Solid lines are linear fits to the data with slope mKip3₄₃₈=68.4 AU/um⁻¹ and mKip3_{438_L11KHC}= 27.9 AU/um⁻¹. Dashed lines represent a line with a slope of 0.

Figure 5. Kip3 Loop11 mediates tubulin-specific suppression of ATP turnover

A. Diagrams of monomeric constructs: Kip3₄₄₈ and Kip3₄₄₈-L11^KHC. B. & C. The Kip3 Loop11 segment specifically suppresses tubulin-stimulated ATPase activity but not its microtubule stimulated ATPase activity. Steady state ATPase assays comparing Kip3₄₄₈ and Kip3₄₄₈-L11^KHC stimulated by: taxol-stabilized microtubules (B) or tubulin (C) (mean +/− SEM; n=3–4 curves). The data was fitted to a Michaelis-Menten curve, except for the tubulin-activated ATPase curve of Kip3₄₄₈ which showed concentration dependent inhibition of activity. D. Values from steady state ATPase assays with Kip3₄₄₈ or Kip3₄₄₈-L11^KHC shown in B–C, using microtubules or free tubulin as substrate (0–10 uM). Shown are mean +/− SEM; n=3–4 curves. E. Slow tubulin-stimulated ADP release by Kip3₄₄₈. mantADP-incubated Kip3₄₄₈ (middle) or Kip3₄₄₈-L11^KHC (right) was rapidly mixed with varying concentrations of microtubules or tubulin in a stopped-flow apparatus. Left, top: scheme of the experiment. Left, bottom: Representative trace of fluorescent signal decay as mantADP unbinds the motor. Observed microtubule- and tubulin-dependent mantADP release rates are plotted for Kip3₄₄₈ (middle) and Kip3₄₄₈-L11^KHC (right). mantADP release rates (k_off) for Kip3₄₄₈ and Kip3₄₄₈-L11^KHC in saturating microtubule (MT) or tubulin (Tub) are shown in the figure in blue and red, respectively. N=5–6 per point. The x-axis scale ([Tubulin] (uM)) for each construct was chosen as the concentration where the motor reaches a constant maximal mantADP release rate. Each construct shows different kinetics and therefore plateaus at different substrate concentrations. The reported mantADP release rate constants are derived from a fit to the curves shown here as described in the methods section. F. Tubulin binding to Kip3₄₄₈ does not inhibit ATP binding. Left, top: scheme of the experiment. Left, bottom: representative trace of the fluorescent signal detected upon mixing of motor-tubulin complex with mantATP. A bi-exponential signal arises with two distinct phases. The initial fast phase corresponds to mantATP exchange to tubulin-bound (APO-state) motor. The slow phase (shown in Fig. S4G) combines ADP dissociation from tubulin-motors with mantATP exchange in the following cycle. The data were fitted to the equation: k_obs=k_on[mATP]+k_off (see methods). The mantATP on-rate (k_on) and off-rate (k_off) of each construct are shown in the figure. Right, top: Kip3₄₄₈, in the presence of 2.5 uM tubulin. The slow nucleotide off-rate is consistent with the slow hydrolysis turnover in ATPase assay in (C) and the slow mantADP off-rates in (E). Right, bottom: Kip3₄₄₈-L11^KHC, in the presence of 2.5 uM tubulin. N=5–6 per point. G. ATP hydrolysis is not required for Kip3 depolymerase activity. Shown are the depolymerization rates (mean +/− SEM; n_MT=102–229) for Kip3₄₄₈ or ATPase deficient Kip3₄₄₈^E345A. Bottom left: Control validating that Kip3₄₄₈^E345A lacks microtubule (1 uM)-stimulated ATPase activity (phosphate produced over time) Kip3₄₄₈^E345A (mean+/− SD; n=3 events per condition comparing Kip3₄₄₈^E345A with Kip3₄₄₈); input 100 nM motor. Bottom right: Kymographs of GMPCPP-stabilized microtubules showing depolymerization by Kip3₄₄₈^E345A. Scale bar: 2 um (vertical), 2 min (horizontal).

Figure 6. An α-tubulin residue specifically required for Kip3 curved tubulin-binding and depolymerase activity

A. Structure of KHC bound to tubulin (4LNU, light grey) with Loop11 highlighted in blue. Structures of straight αβ-tubulin (3J6G, dark grey) and curved αβ-tubulin (4FFB, α in pink, β in green) are aligned using the β subunit. Selected residues on α-tubulin, D118 and E156/E157 are shown in space filling mode. These residues were chosen from a larger set of α-tubulin residues that are likely to be more proximal to Loop11 in curved tubulin relative to straight tubulin. B. D118A yeast microtubules are insensitive to depolymerization by Kip3_FL. Quantification of the depolymerization rate of the Kip3_FL dimer on GTPγS-stabilized yeast microtubules (black), Tub1^{E156A E157A} microtubules (green) and Tub1^D118A microtubules (red) (mean +/− SEM; n=10–30). C. Tub1^D118A microtubules are depolymerized by MCAK or TOG domains at the same rate as control microtubules. Depolymerization rate of Tub1 (black) or Tub1^D118A (red) GTPyS-stabilized yeast microtubules at various concentrations of Stu2 TOG domains (Left) or MCAK (right). Mean +/− SEM (n_MT=20). D. Tub1^D118A does not affect Kip3_FL microtubule binding. Integrated fluorescence intensity of Kip3_FL binding to either wild-type or Tub1^D118A GTPyS-stabilized yeast microtubules; imaged under TIRF microscopy. Shown are mean +/− SEM; N=5 flow cells per condition. Bottom: representative images of Kip3_FL bound to Tub1 or Tub1^D118A microtubules. E. Soluble tubulin can compete for Kip3_FL binding to the microtubule lattice. Left: Tubulin competition assay schematic. Middle: Representative images of 10nM Kip3 bound to microtubules at 0 nM tubulin, 2 uM tubulin and 0.5 uM, 2 uM D118A tubulin. Scale bar= 2 um. Right: Integrated fluorescence intensity of Kip3_FL and Kip3⁴⁴⁸ on the microtubule lattice with or without competition from soluble tubulin. Data was normalized to fluorescence intensity at 0 nM tubulin and fit to a competition model. Shown are the mean +/−SEM, N=5 flow cells per condition. F. Kip3_FL has comparable motility on control and Tub1^D118A microtubules. Shown are velocity and run length measurements (mean +/− SEM; n=100 events). G. The Tub1^D118A mutation abolishes Kip3_FL‘s ability to dwell at the microtubule plus-end. Plus-end residence time (mean +/−SEM; n=100 events per condition) of Kip3_FL on Tub1 (grey) and Tub1^D118A microtubules (red). Scale bar: 5 um (vertical), 1 min (horizontal).

Figure 7. Two-state binding switch model for Kip3 length dependent microtubule regulation

Schematic of the two modes for Kip3 binding to tubulin: a motile kinesin and a depolymerase, binding straight (left) and curved (right) tubulin respectively. On the microtubule lattice, Kip3 uses ATP-dependent processive stepping to walk to the plus-end. Similar to other kinesins, straight tubulin in the microtubule lattice stimulates ATP hydrolysis. Binding of Kip3 to curved tubulin found at the microtubule plus-end and in solution suppresses ATPase activity, induces high affinity binding, and promotes microtubule catastrophe or the disassembly of stabilized microtubules. In the cartoon, the proximity of Kip3 L11 to the D118 residue on α-tubulin, both of which are required for the curved tubulin-selective interaction, is highlighted.