<scp>KIF5B</scp> and Dynein‐Dependent Golgi Organization: Role in Adhesion‐Dependent Microtubule Acetylation

Cell-matrix adhesion regulates Golgi organization along microtubules (MTs), though how it couples with motor-driven Golgi positioning and function remains unclear. Our earlier work showed that loss of adhesion preferentially reduces Arf1 activation at the trans-Golgi (relative to cis-Golgi), possibly affecting dynein recruitment and could cause greater trans-Golgi disorganization along MTs. We now show that this differential Golgi disorganization leads to decreased MT acetylation, which recovers upon re-adhesion. Active Arf1 overexpression in non-adherent fibroblasts prevents Golgi disorganization and sustains MT acetylation, as also observed in T24 cancer cells. Using active-Arf1 pulldown, PLA and Co-immunoprecipitation studies, we further reveal that active Arf1 recruits KIF5B to the Golgi alongside dynein. siRNA-mediated knockdown (KD) of KIF5B or dynein disperses the Golgi into ministacks. In KD cells, loss of the juxtanuclear Golgi ribbon disrupts MT organization, which is also affected by altered MTOC positioning upon dynein KD. Notably, dispersed Golgi ministacks in single motor KDs maintain MT acetylation levels in both adherent and non-adherent cells. Dual KIF5B-dynein KD keeps the Golgi juxtanuclear but uniquely compact, supporting MT acetylation. This impairs Golgi-dependent trafficking and affects cell spreading, polarity and migration. Together, these reveal the Arf1-KIF5B-dynein crosstalk as a key regulator of adhesion-dependent Golgi organization and function.