Extended Data Fig. 6: CLASP and EB1 differentially localize on microtubules in response to axial confinement.

(a) Representative timelapse images of a 1205Lu cell stably overexpressing EB1-meGFP pre and post-axial confinement (unconfined and confined, respectively, 3 microns). (b) Temporal projection of EB1 comet tracks (42 seconds duration), pre and post-axial confinement. (c) Kymograph of regions marked in b. (white dashed line). (d) Quantitation of EB1 track displacement and (e) speed relative to confinement condition and time. EB1 comet speeds slow in response to confinement however EB1 remains associated with growing microtubule plus-ends. Measurement of EB1 track displacement and speed is from n = 60 individual tracks taken from an individual cell. Data shown represents one experiment. Experiments were performed two times with similar results. Data displayed as violin plot indicating minimum, first quartile, median, third quartile, and maximum, with width indicating frequency of values. d, and e, statistical analysis by Kruskal-Wallis one-way ANOVA test with Dunn’s multiple comparison. (f) Representative timelapse images of a 1205Lu cell stably overexpressing meGFP-tagged N-terminal fragment of CLASP1 (meGFP-CLASP1 N-term; TOG1 and TOG 2) pre- and post-axial confinement (3 microns). In the absence of confinement, meGFP-CLASP1 N-term localizes to the cytoplasm. Induction of confinement results in an association with microtubules (g), progressively increasing over time (24 min). (h) 3D kymographs of the cell in f. (i). 1205Lu cells stably overexpressing HaloTag-tagged C-terminal fragment of CLASP1 (HaloTag-CLASP1-C-term; SxIP-TOG3-CLIP-ID), labelled with Halo-JF549. HaloTag-CLASP1-C-term tracks microtubule plus-ends irrespective of axial confinement (pre-confinement 0’, and post-confinement 4’30”), similar to EB1 (j) 3D kymographs of cell in i. Images were gamma adjusted (0.3) to increase contrast of microtubule structures. Source numerical data are available in Source data.

Source data