Figure 1.
The depolymerase KIF2A preferentially accumulates and depolymerizes the minus ends of microtubules. (A) Schematic of fluorescently tagged KIF2A and MCAK constructs used in this study, indicating the fluorescent protein (FP) fused via a flexible linker to the N-terminus of each protein, the conserved internal motor domain, and the class-specific neck and predicted coiled-coil dimerization domain (CC) 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 SEM. (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 SEM. (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) The ratio of intensity of mScarlet-KIF2A or mCherry-MCAK (both 20 nM) at microtubule ends to their intensity on the microtubule lattice (see Materials and methods). A dashed line at a ratio of 1 indicates equal binding to the end 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 Materials and methods). Error bars are SEM. (I) Kymographs of polarity-marked GMPCPP seeds (Atto647; magenta) in the presence of 20 nM mScarlet-KIF2A (cyan) and either 2 µM iE5 (middle) or 15 µM (D1)2 (right). (J) Depolymerization speeds of plus and minus ends of polarity-marked GMPCPP seeds in the presence of 20 nM mScarlet-KIF2A (n = 173 microtubules) and either iE5 (2 µM, n = 149 microtubules; 10 µM, n = 125) or (D1)2 (3 µM, n = 149 microtubules; 15 µM, n = 141 microtubules). Error bars are SEM. (K) Ratio of microtubule end and lattice intensities of mScarlet-KIF2A (n = 44 microtubules) in the absence or presence of iE5 (2 µM, n = 44 microtubules; 10 µM, n = 55) or (D1)2 (3 µM, n = 52 microtubules; 15 µM, n = 37 microtubules) at the indicated concentrations. Dashed line indicates equal binding to the end and the lattice (ratio = 1). Error bars are SEM.

The depolymerase KIF2A preferentially accumulates and depolymerizes the minus ends of microtubules. (A) Schematic of fluorescently tagged KIF2A and MCAK constructs used in this study, indicating the fluorescent protein (FP) fused via a flexible linker to the N-terminus of each protein, the conserved internal motor domain, and the class-specific neck and predicted coiled-coil dimerization domain (CC) 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 SEM. (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 SEM. (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) The ratio of intensity of mScarlet-KIF2A or mCherry-MCAK (both 20 nM) at microtubule ends to their intensity on the microtubule lattice (see Materials and methods). A dashed line at a ratio of 1 indicates equal binding to the end 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 Materials and methods). Error bars are SEM. (I) Kymographs of polarity-marked GMPCPP seeds (Atto647; magenta) in the presence of 20 nM mScarlet-KIF2A (cyan) and either 2 µM iE5 (middle) or 15 µM (D1)2 (right). (J) Depolymerization speeds of plus and minus ends of polarity-marked GMPCPP seeds in the presence of 20 nM mScarlet-KIF2A (n = 173 microtubules) and either iE5 (2 µM, n = 149 microtubules; 10 µM, n = 125) or (D1)2 (3 µM, n = 149 microtubules; 15 µM, n = 141 microtubules). Error bars are SEM. (K) Ratio of microtubule end and lattice intensities of mScarlet-KIF2A (n = 44 microtubules) in the absence or presence of iE5 (2 µM, n = 44 microtubules; 10 µM, n = 55) or (D1)2 (3 µM, n = 52 microtubules; 15 µM, n = 37 microtubules) at the indicated concentrations. Dashed line indicates equal binding to the end and the lattice (ratio = 1). Error bars are SEM.

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