Journal: bioRxiv

Article Title: The minus end depolymerase KIF2A drives flux-like treadmilling of γTuRC-uncapped microtubules

doi: 10.1101/2023.04.06.535808

Figure Lengend Snippet: A Schematic of fluorescently-tagged KIF2A and MCAK constructs used in this study, indicating the fluorescent tag fused via a flexible linker to the N terminus of each protein, the conserved internal motor domain, and the class-specific “neck” and predicted dimerization domain N and C terminal to the motor domain. Below, schematic of TIRF microscopy experiment set-up. B TIRF microscopy image of 20 nM mScarlet-KIF2A (cyan) binding to GMPCPP-microtubule seeds (AlexaFluor647, 5%; magenta) bound to the glass surface. A kymograph of a depolymerizing microtubule seed indicated by the yellow arrow is on the right. C Kymographs of GMPCPP-microtubule seeds with a dimly labeled plus end and brightly labeled minus end (AlexaFluor647; magenta) being depolymerized by 20 nM mScarlet-KIF2A (left; cyan) or 20 nM mCherry-MCAK (right; gray). D Depolymerization rates of each end of polarity-marked GMPCPP seeds by 20 nM mScarlet-KIF2A ( n = 29 microtubules) and 20 nM mCherry-MCAK ( n = 33 microtubules). Error bars are SD. E Depolymerization rates of GMPCPP-microtubule seeds at varying concentrations of mScarlet-KIF2A. Polarity of the seed was assigned assuming the minus end depolymerizes at the faster rate. Number of microtubule seeds measured per condition: mScarlet-KIF2A: 20 nM, n = 46; 40 nM, n = 61; 100 nM, n = 78; 250 nM, n = 64. Error bars are SD. F Localization of mScarlet-KIF2A (20 nM) on polarity-marked microtubule seeds in AMPPNP and ADP. Images are averages of 10 frames. Top row: merged channels (AlexaFluor647 tubulin, magenta; mScarlet-KIF2A, cyan). Bottom row, mScarlet-KIF2A channel alone. Bottom inset, cropped and enlarged images of mScarlet-KIF2A binding to microtubule ends, showing its localization to both ends in AMPPNP, and its localization to the minus end, and not the plus end, in ADP. Inset scale bars: 1 µm. G Ratio of intensity of mScarlet-KIF2A or mCherry-MCAK at microtubule tips to their intensity on the microtubule lattice (see Methods). Dashed line at a ratio of 1 indicates equal binding to the tip and the lattice. Number of microtubule seeds measured per condition: mScarlet-KIF2A: ATP, n = 26; AMPPNP, n = 29; ADP, n = 36: mCherry-MCAK: ATP, n = 47; AMPPNP, n = 40; ADP, n = 32. Error bars are SEM. H “Raw” mScarlet-KIF2A intensities at minus ends and on the lattice in ATP and ADP (see Methods). Error bars are SEM.

Article Snippet: Before use in experiments, frozen aliquots were thawed and dialyzed against KIF2A gel-filtration buffer (Slide-A-Lyzer MINI, Thermo Fisher).

Techniques: Construct, Microscopy, Binding Assay, Labeling

Journal: bioRxiv

Article Title: The minus end depolymerase KIF2A drives flux-like treadmilling of γTuRC-uncapped microtubules

doi: 10.1101/2023.04.06.535808

Figure Lengend Snippet: A Snapshot of dynamic microtubules polymerizing from brightly labeled GMPCPP seeds (Alexa647 tubulin, magenta) in the presence of mScarlet-KIF2A (cyan). Below, mScarlet-KIF2A channel alone. B Sequence of kymographs for representative microtubules polymerizing from bright seeds (Alexa647 tubulin, magenta) in the presence of increasing concentrations of mScarlet-KIF2A (cyan). mScarlet-KIF2A channels were additionally background subtracted and contrast was independently adjusted for each condition to show minus-end binding at a similar brightness. C Dynamics of microtubule plus-ends growing in the presence of increasing concentrations of mScarlet-KIF2A (left) or mCherry-MCAK (right), with a tubulin concentration of 12.5 µM. Both proteins show no major impact on growth speed (top) and a similar increase in catastrophe rate (bottom) with increasing concentrations. Number of growth speeds measured per condition: mScarlet-KIF2A: 0 nM, n = 502; 0.5 nM, n = 379; 2 nM, n = 508; 5 nM, n = 405; 20 nM, n = 572; 100 nM, n = 259: mCherry-MCAK: 0 nM, n = 275; 5 nM, n = 275; 10 nM, n = 406; 20 nM, n = 317; 40 nM, n = 164. Number of catastrophe rates measured per condition: mScarlet-KIF2A: 0 nM, n = 127; 0.5 nM, n = 90; 2 nM, n = 104; 5 nM, n = 71; 20 nM, n = 64; 100 nM, n = 26: mCherry-MCAK: 0 nM, n = 57; 5 nM, n = 60; 10 nM, n = 61; 20 nM, n = 41; 40 nM, n = 14. Error bars are SD. D Fraction of time in growth phase for minus and plus ends of dynamic microtubules in the presence of increasing concentrations of mScarlet-KIF2A or mCherry-MCAK and 12.5 µM tubulin. Lines are drawn as a guide to the eye. Number of minus end traces measured per condition: mScarlet-KIF2A: 0 nM, n = 127; 0.5 nM, n = 99; 2 nM, n = 60; 5 nM, n = 63: mCherry-MCAK: 0 nM, n = 59; 5 nM, n = 21; 10 nM, n = 33; 20 nM, n = 35. Number of plus end traces measured per condition: mScarlet-KIF2A: 0 nM, n = 131; 0.5 nM, n = 93; 2 nM, n = 95; 5 nM, n = 70; 20 nM, n = 67; 100 nM, n = 32: mCherry-MCAK: 0 nM, n = 58; 5 nM, n = 65; 10 nM, n = 67; 20 nM, n = 49; 40 nM, n = 34. Measurements of 0% were inferred by manual inspection of conditions in which seed ends only depolymerized. Error bars are SD. E Snapshot of microtubules (magenta) growing from brightly-labeled seeds in the presence of mScarlet-KIF2A (top, cyan) or mCherry-MCAK (bottom, gray) in the presence of ADP and absence of ATP. Yellow arrowheads indicate mScarlet-KIF2A accumulated at the slower growing minus ends; mCherry-MCAK did not significantly decorate either end of the growing microtubule in the presence of ADP. Images are averages of two frames taken 5 seconds apart. F Representative kymographs showing minus end tracking of ADP-bound mScarlet-KIF2A (cyan) and partially suppressed minus end growth of microtubules growing from brightly-labeled seeds (magenta). G Microtubule minus-end growth speeds in the presence of increasing concentrations of mScarlet-KIF2A (left) or 100 nM mCherry-MCAK (right) in ADP. Number of growth speeds measured per condition: mScarlet-KIF2A: 0, n = 404; 5, n = 303; 10, n = 330; 20, n = 336; 40, n = 397: mCherry-MCAK: 100, n = 323.

Article Snippet: Before use in experiments, frozen aliquots were thawed and dialyzed against KIF2A gel-filtration buffer (Slide-A-Lyzer MINI, Thermo Fisher).

Techniques: Labeling, Sequencing, Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: The minus end depolymerase KIF2A drives flux-like treadmilling of γTuRC-uncapped microtubules

doi: 10.1101/2023.04.06.535808

Figure Lengend Snippet: A Time sequence snapshots of microtubules growing from brightly-labeled seeds (magenta, Atto647) in the presence of 20 nM untagged KIF2A. Circles track the depolymerizing minus ends of selected microtubules that undergo treadmilling. A solid circle indicates the microtubule is still attached to the brightly labeled GMPCPP seed, whereas a dashed circle indicates the seed has completely depolymerized and the microtubule (including the minus end) is composed of only GTP/GDP tubulin. Arrows indicate the direction of “travel” across the glass surface. The asterisk indicates the microtubule was lost from view after this time-point following a plus-end catastrophe. B Representative kymographs of microtubules (magenta) that are released from the stabilized GMPCPP seed after depolymerization by mScarlet-KIF2A (cyan), indicated by the black arrowhead. Minus-end depolymerization was heterogeneous: asterisks indicate instances of increased depolymerization speed. Insets: enlarged regions of kymographs showing mScarlet-KIF2A localization at minus ends undergoing periods of “slow” (10 nM KIF2A inset) and “fast” (20 nM KIF2A inset) depolymerization. Inset scale bars: horizontal, 1 µm; vertical, 30 sec. C Average depolymerization rates of minus ends of treadmilling microtubules, measured from microtubules that are no longer attached to seeds, in the presence of various concentrations of mScarlet-KIF2A. Each measurement is plotted as a semi-transparent dot (with random x-jitter to improve visualization) and the mean is overlaid as a black bar. Inset: the same data with a 0 nM KIF2A timepoint included (where each measurement is made from each instance of depolymerization after catastrophe) and an extended y axis. Number of depolymerization speeds measured per condition: mScarlet-KIF2A: 0 nM, n = 264; 10 nM, n = 74; 20 nM, n = 76; 40 nM, n = 62; 60 nM, n = 68.

Article Snippet: Before use in experiments, frozen aliquots were thawed and dialyzed against KIF2A gel-filtration buffer (Slide-A-Lyzer MINI, Thermo Fisher).

Techniques: Sequencing, Labeling

Journal: bioRxiv

Article Title: The minus end depolymerase KIF2A drives flux-like treadmilling of γTuRC-uncapped microtubules

doi: 10.1101/2023.04.06.535808

Figure Lengend Snippet: (A) Schematic of the TIRF microscopy-based γTuRC-nucleation assay. Biotinylated and mBFP-tagged γTuRC is immobilized on a biotin-PEG-functionalized glass surface via NeutrAvidin followed by the addition of tubulin and KIF2A. ( B) TIRF microscopy images of γTuRC-nucleated microtubules in the absence (left) and presence of mScarlet-KIF2A (cyan) at the indicated concentrations and 12.5 µM of tubulin (AlexaFluor647, 5,4%; magenta), 20 minutes after the start of imaging. Surface-immobilized γTuRC is not shown. (C) Representative kymographs of microtubules nucleating from γTuRC (yellow) for the conditions shown in B. (D) Numbers of microtubules nucleated over time, (E) microtubule nucleation rates for mScarlet-KIF2A and mCherry-MCAK, (F) catastrophe frequencies and (G) microtubule plus-end growth speeds for the conditions in B. Circles represent mean values and error bars are SEM (D, E) and SD (F, G). For symbols without visible error bars, error bars are smaller than the symbol size. The lines in (D) represent linear regressions. The dashed line in (G) represents the mean microtubule plus-end grow speeds in the absence of mScarlet-KIF2A. Nucleation rates (E) were calculated from the slope of the linear regression (mScarlet-KIF2A: D; mCherry-MCAK: not shown). Number of microtubules analyzed for the catastrophe frequency (F) per mScarlet-KIF2A concentration: 0 nM, n = 191; 20 nM, n = 186; 40 nM, n = 122; 100 nM, n = 65; Number of microtubule growth speeds (G) measured per mScarlet-KIF2A concentration: 0 nM, n = 187; 20 nM, n = 169; 40 nM, n = 79; 100 nM, n = 66. Data for plots was pooled from three independent experiments. (H) Percentage of microtubules that either remain protected by γTuRC or are released after nucleation, for the indicated concentrations of mScarlet-KIF2A and mCherry-MCAK. Number of microtubules analyzed per condition: mScarlet-KIF2A - 0 nM, n = 256; 20 nM, n = 249; 40 nM, n = 190; 100 nM, n = 91; mCherry-MCAK - 0 nM, n = 332; 5 nM, n = 281; 10 nM, n = 182; 20 nM, n = 156. Data was pooled from three independent experiments. Error bars shown for mScarlet-KIF2A concentrations are SEM. (I) Representative kymographs of occasionally observed spontaneously nucleated microtubules, recognized by growth at both ends after nucleation, in a γTuRC nucleation assay, in the absence (top image) or presence (bottom image) of 20 nM of mScarlet-KIF2A (cyan). (J) Representative kymographs of γTuRC-nucleated microtubules that are released after nucleation in the presence of 20 nM of mScarlet-KIF2A (cyan). Yellow arrowheads indicate the moment of γTuRC-uncapping and the beginning of accumulation of mScarlet-KIF2A at the released and slowly depolymerizing microtubule minus end. 2 nM γTuRC was used for initial surface immobilization in all experiments.

Article Snippet: Before use in experiments, frozen aliquots were thawed and dialyzed against KIF2A gel-filtration buffer (Slide-A-Lyzer MINI, Thermo Fisher).

Techniques: Microscopy, Imaging, Concentration Assay

Journal: bioRxiv

Article Title: The minus end depolymerase KIF2A drives flux-like treadmilling of γTuRC-uncapped microtubules

doi: 10.1101/2023.04.06.535808

Figure Lengend Snippet: (A) Time sequence of TIRF microscopy images and kymographs of microtubules (magenta) nucleated by surface-immobilized γTuRC in the presence of 11 µM Cy5-tubulin (5.4% labelling percentage), which remain unaffected by 100 nM of spastin. Arrowheads point to γTuRC-capped microtubule minus ends. (B) Time sequence of TIRF microscopy images and kymograph of γTuRC-nucleated microtubules (magenta) that were severed in the presence of 11 µM Cy5-tubulin, 20 nM mScarlet-KIF2A (cyan) and 100 nM of spastin. Yellow arrowheads and asteriks indicate γTuRC-capped microtubule minus-ends and severing events, respectively. (C) Time sequence of TIRF microscopy images and kymographs of γTuRC-nucleated microtubules (magenta) that were released from γTuRC by the combined action of mScarlet-KIF2A (cyan) and spastin. Conditions as in (B). Yellow arrowheads point to the γTuRC-nucleation sites of microtubules and the yellow asterisks indicate the release of the microtubules from γTuRC, leaving the minus end uncapped for depolymerization by mScarlet-KIF2A (cyan). (D) Percentage of microtubules that either remain protected by γTuRC or are released after nucleation, for the indicated concentrations of mScarlet-KIF2A and spastin. Number of microtubules analyzed per condition: mScarlet-KIF2A and spastin - 0 nM, n = 228; mScarlet-KIF2A 0 nM and Spastin 100 nM, n = 152; mScarlet-KIF2A 20 nM and spastin 0 nM, n = 160; mScarlet-KIF2A 20 nM and spastin 100 nM, n = 222. Data for plots was pooled from at least two independent experiments. Error bars are SEM. (E) Percentage of released and severed γTuRC-nucleated microtubules for the indicated concentration of spastin and mScarlet-KIF2A. Number of microtubules analyzed per condition: mScarlet-KIF2A 20 nM and spastin 100 nM, n = 222. In all experiments, 2.5 nM γTuRC was used for surface-immobilization. The time stamps indicate min:s.

Article Snippet: Before use in experiments, frozen aliquots were thawed and dialyzed against KIF2A gel-filtration buffer (Slide-A-Lyzer MINI, Thermo Fisher).

Techniques: Sequencing, Microscopy, Concentration Assay