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. 2019 Nov 15;30(24):2953–2968. doi: 10.1091/mbc.E19-05-0247

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© 2019 Drechsler et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 2: — starPEG-(KA7)₄ diffuses along the microtubule lattice, switches tracks at microtubule intersections, and tracks the depolymerizing plus end of dynamic microtubules. (A) Representative kymographs showing the diffusion of 1 nM starPEG-(KA7)₄-TAMRA along the lattice of taxol-stabilized microtubules at 25°C in BRB80. (B) Mean square displacement analysis (top) and covariance-based estimator of the diffusion coefficient D (bottom) of starPEG-(KA7)₄-TAMRA at 25°C in BRB80. Error bars in MSD plot (top) are the SE. Data are fitted with a linear regression. The dashed line in the CVE plot (bottom) indicates the 95% confidence interval and the calculated mean D value is weighted for molecule trajectory length/data points. (C) Track-switching of single starPEG-(KA7)₄-TAMRA at microtubule intersections. Top pictures show the geometry of intersecting taxol-stabilized Atto647N-labeled microtubules superimposed by the trajectories (blue) of single starPEG-(KA7)₄-TAMRA molecules (i.e., M1 and M2) tracked by FIESTA. Bottom kymographs derived by line-scans along the respective substrate microtubules (MT1-4) show the diffusional movement of single starPEG-(KA7)₄-TAMRA molecules (i.e., molecule 1 and molecule 2). Switch events are indicated by orange bars above and below the respective kymographs. (D) starPEG-(KA7)₄-TAMRA (top row and cyan, bottom row) tracks the depolymerizing plus end of dynamic Alexa 647–labeled microtubules (red, bottom row) grown from GMP-CPP-stabilized seeds at 35°C. White arrowheads indicate end-tracking starPEG-(KA7)₄-TAMRA molecules and blue arrowheads their maximal compression at the end of depolymerization events. (E) Tracking efficiency of starPEG-(KA7)₄-TAMRA. Left: schematic definition of the parameters and time points used for the graphs on the right. Magenta data points show the fraction of total microtubule-associated starPEG-(KA7)₄-TAMRA intensity that remains bound to the microtubules during individual tracking events (n = 19). Cyan data points show the relative microtubule length remaining at the end of individual tracking events. Blue data points are the normalized starPEG-(KA7)₄-TAMRA intensity values divided by the normalized microtubule length values; values above 1 indicate accumulation of starPEG-(KA7)₄-TAMRA in terms of intensity per length.