CCDC66 couples actin-microtubule crosslinking to KANK1-associated microtubule targeting and focal adhesion turnover

Cell migration requires actin-based protrusion to be coordinated with microtubule (MT)-dependent focal adhesion (FA) remodeling. Although KANK proteins link talin-containing FAs to cortical MT-capture machinery, how this machinery couples to force-bearing actin networks remains unclear. Here, we identify the ciliopathy-associated protein CCDC66 as an actin-MT crosslinker required for FA turnover and migration. CCDC66 depletion impaired collective, Transwell, and random migration, whereas CCDC66 overexpression enhanced wound closure and Matrigel invasion. Mechanistically, CCDC66 loss reduced MT targeting to peripheral FAs and shifted cells into a ROCK-dependent contractile state marked by impaired lamellipodial protrusion, stress fiber accumulation, enlarged long-lived adhesions, and increased RhoA-ROCK signaling. ROCK or formin inhibition suppressed this state, whereas Rac1 activation failed to restore productive protrusion, indicating that CCDC66 maintains the balance between protrusive and contractile actin organization. In vitro TIRF reconstitution demonstrated that purified CCDC66 directly crosslinks actin filaments and MTs. In cells, CCDC66 associated with KANK1, supported its peri-adhesion organization, and functioned with KANK1 in an overlapping migration pathway. TCGA analyses further revealed that a coordinated CCDC66/KANK/ROCK-associated module, but not CCDC66 expression alone, stratified outcome in chromophobe renal cell carcinoma. These findings reveal a non-ciliary role for CCDC66 in coupling actin–MT integration to FA turnover and contractile-state control, with relevance to developmental disease and cancer-associated motility.