Coordination between the microtubule and actin networks is essential for cell motility, neuronal growth cone guidance, and wound healing. Members of the CLASP (Cytoplasmic Linker-Associated Protein) family of proteins have been implicated in the cytoskeletal crosstalk between microtubules and actin networks, however, the molecular mechanisms underlying CLASPs role in cytoskeletal coordination are unclear. Here, we investigate CLASP2α’s crosslinking function with microtubules and F-actin. Our results demonstrate that CLASP2α crosslinks F-actin to the microtubule lattice in vitro. We find that the crosslinking ability is retained by L-TOG2-S, a minimal construct containing the TOG2 domain and serine-arginine rich region of CLASP2α. Furthermore, CLASP2α promotes the accumulation of multiple actin filaments along the microtubule, supporting up to 11 F-actin landing events on a single microtubule lattice region. CLASP2α also facilitates dynamic organization of polymerizing actin filaments templated by the microtubule network, with F-actin forming bridges between individual microtubules. Finally, we find that depletion of CLASPs in vascular smooth muscle cells results in disorganized actin fibers and reduced co-alignment of actin fibers with microtubules, suggesting that CLASP and microtubules contribute to higher-order actin structures. Taken together, our results indicate that CLASP2α can directly crosslink F-actin to microtubules, and that this microtubule-CLASP-actin interaction may influence overall cytoskeletal organization in cells.

Movie S1: F-actin landing on CLASP2-coated microtubules. Time-lapse of 6.5 μM F-actin and three-color merged time-lapse. F-actin time-lapse is merged with still microtubule image and average projection of CLASP2α -GFP signal. Video playback is 300 frames per second.

https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e22-05-0149/20221226/media/mc-e22-05-0149-s02.,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518515abb28bda92fbabe7b929571bd415190bf44d1790b7324fb90f184f91fab10e395327a1bef9725d0c99a358bbbe69c082a95f9d49957eb2932b2be9a6f328f5ec3ab1a013fda9142e0536569ad35575400f9456418f92e8ac902df641956f8cb7643d25499269fb7fa71ef86065ae2e530f48ca350750b7ed558b1eb025a0237441ba6af1f81bfc7d5937a1e960f9f6d77f3dca09

Movie S2: Dynamic actin filaments form bridges between microtubules over time. 30-minute timelapse merged images of Taxol microtubules and actin. Video playback is 25 frames per second.

https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e22-05-0149/20221226/media/mc-e22-05-0149-s03.,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518515abb28bda92fbabe7b929571bd415190bf44d1790b7324fb90f184f91fab10e395327a1bef9725d0c99a358bbbe69c082a95f9d49957eb2932b2be9a6f328f5ec3ab1a013fda9142e0536569ad35575400f9456418f92e8ac902df641956f8cb7643d25499269fb7fa71ef86165ae2e530f48ca72d06899339d6ee75d42d3cf936c6a1f86de0b10ce430987132a6a2881e9f3827a