Amol Aher, Dipti Rai, Laura Schaedel, Jeremie Gaillard, Karin John, Qingyang Liu, Maarten Altelaar, Laurent Blanchoin, Manuel Thery, Anna Akhmanova
(A) A scheme of full-length CLASP2α and its TOG2-S fragment. Vertical lines labeled SxIP (Ser-any amino acid-Ile-Pro) represent EB-binding motifs located in the unstructured positively charged region adjacent to the TOG2 domain.
(B) Schematic for an experiment to monitor the possible outcomes of a 532 nm pulsed laser induced damage at a site along the dynamic lattice. (I) Microtubule bending at the site of damage, which can lead to either straightening of the lattice or microtubule breakage. (II) Reduction of the tubulin intensity. (III) Microtubule severing resulting in direct appearance of two microtubule ends.
(C) Stills from a time-lapse video showing photodamage of a dynamic microtubule grown in the presence of Rhodamine-tubulin alone followed by bending and subsequent breakage (outcome I). Scale bar, 2 μm.
(D) Percentage of total events for outcome I resulting in either microtubule breakage or straightening at the point of photodamage in the presence of tubulin alone (n = 22 microtubules analyzed from 4 experiments) or together with either 30 nM GFP-CLASP2α (n = 53 microtubule analyzed from 6 experiments) or 30 nM GFP-TOG2-S (n = 54 from 8 experiments). Error bars denote SD.
(E and F) Normalized mean intensity at the site of photodamage in case of outcome I for the GFP channel for CLASP2α and TOG2-S (E) and Rhodamine-tubulin channel in the presence of tubulin alone or together with either CLASP2α or TOG2-S (F); before damage (black), immediately after damage (orange) and after microtubule straightening (blue). Tubulin alone: n = 4 microtubules, 4 experiments; CLASP2α: n = 21 microtubules, 4 experiments; TOG2-S: n = 20 microtubules, 6 experiments. Error bars denote SD.
(G) Stills from a time-lapse video showing a dynamic microtubule grown in the presence of Rhodamine-tubulin together with 30 nM GFP-CLASP2α for outcome I. Normalized intensity profiles along the microtubule for the CLASP (green) and tubulin channel (magenta) at different time points are shown in the bottom panels, with the arrow pointing to the site of photodamage. The purple circle on the plot indicates the end of the microtubule. Scale bars, 2 μm.
(H) Normalized mean tubulin fluorescence intensity over time at the site of local photodamage (outcome II); microtubules were grown in the presence of Rhodamine tubulin alone (gray) (n = 35 microtubules, 2 experiments) or together with 30 nM GFP-CLASP2α (blue) (n = 44 microtubules, 2 experiments). Straight lines were fitted to the initial increase in tubulin intensity until saturation for the respective mean values yielding slopes as indicated.
(I and J) Stills and the corresponding kymograph of a microtubule grown in the presence of Rhodamine-tubulin alone (I) or together with GFP-CLASP2α (30 nM) (J) severed with a 532 nm laser as indicated (outcome III). Scale bars: still image, 2 μm; kymograph, 4 μm (horizontal) and 10 s (vertical). Dotted yellow lines point to the time point of the still in the kymograph.
(K) Percentage of total laser severing events resulting in either immediate microtubule regrowth at the site of photoablation, microtubule depolymerization to the seed or depolymerization followed by rescue along the lattice, in the presence of Rhodamine-tubulin alone or together with either 30 nM GFP-CLASP2α or 30 nM GFP-TOG2-S. Tubulin alone: n = 186 microtubules, 3 experiments: CLASP2α: n = 36 microtubules, 3 experiments; TOG2-S: n = 48 microtubules, 8 experiments.