Efimova, N. | Search bioRxiv

bioRxiv2026

Mechanism of branched actin assembly at microtubule tips downstream of Adenomatous Polyposis Coli (APC) protein

During cell migration, branched actin filaments nucleated by the Arp2/3 complex induce leading edge protrusions, whereas directionality of cell migration is controlled by microtubules. We showed previously that Adenomatous Polyposis Coli (APC) initiates branched actin assembly at microtubule tips, which can explain how microtubules control directional protrusion. Here, we investigate a link from APC to Arp2/3 complex activity. We show that protrusion-generating activity of APC resides in its N-terminal region containing the Armadillo Repeat Domain (ARD). Furthermore, Asef1/ARHGEF4, a Cdc42 GEF known to be activated by the ARD of APC, as well as Cdc42 itself and its effector N-WASP, an Arp2/3 complex activator, are all required for the assembly of branched actin filaments in neuronal growth cones and neurite outgrowth. As downregulation of Asef1, Cdc42 or N-WASP produces phenotypes similar to APC knockdown, we propose that microtubules regulate directional cell migration and neuron navigation by inducing local membrane protrusion through the APC - Asef1 - Cdc42 - N-WASP - Arp2/3 pathway. These findings bridge a significant gap in our knowledge of cell migration.

cell biology↗

bioRxiv2022

CLASP2 facilitates dynamic actin filament organization along the microtubule lattice

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 CLASP2s crosslinking function with microtubules and F-actin. Our results demonstrate that CLASP2 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 CLASP2. Furthermore, CLASP2 promotes the accumulation of multiple actin filaments along the microtubule, supporting up to 11 F-actin landing events on a single microtubule lattice region. CLASP2 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 CLASP2 can directly crosslink F-actin to microtubules, and that this microtubule-CLASP-actin interaction may influence overall cytoskeletal organization in cells.

cell biology↗