Microtubule-associated motor proteins in adhesion-dependent regulation of Golgi organization, microtubule network, and cellular function

Abstract

Cell-matrix adhesion plays a vital role in maintaining the juxtanuclear ribbon organization of the Golgi apparatus along microtubules (MTs); however, how adhesion-dependent signalling is coupled to motor-driven Golgi positioning and function has remained unclear. Earlier studies from the lab show that adhesion regulates Golgi organization largely by activating the small GTPase Arf1. Active Arf1 promotes recruitment of the motor protein dynein, which is essential for Golgi ribbon integrity. Upon loss of adhesion, Arf1 activity declines differentially from the cis- and trans-Golgi compartments, leading to reduced dynein localization and triggering differential disorganization along MTs. The Golgi plays an active role in microtubule nucleation and stabilization. In this study, we show that upon loss of adhesion, microtubule stability, indicated by acetylated and detyrosinated tubulin, decreases in mouse fibroblasts and is gradually restored during re-adhesion. This effect may reflect reduced FAK-Rho signalling, which supports cortical microtubule stabilization, together with Golgi disorganization following adhesion loss. Active Arf1 preserves Golgi organization and microtubule acetylation in fibroblasts and in T24 bladder cancer cells upon loss of adhesion. Active Arf1 recruits kinesin-1 (KIF5B) to the Golgi, while knockdown of KIF5B or dynein disrupts the Golgi into ministacks retaining cis- and trans-Golgi elements. Dynein loss also mispositions the MTOC, thereby altering the organization of the microtubule network. Despite ribbon disorganization, ministacks in KIF5B or dynein-deficient cells maintain microtubule acetylation. Dual knockdown of both motors results in a compact Golgi apparatus near the MTOC, preserving the integrity of both cis- and trans-Golgi. These cells exhibit increased spreading, reduced aspect ratio, alterations in vesicle trafficking and cell migration, suggesting that Golgi morphology, governed by motor proteins, influences microtubule network organization and cell dynamics. Importantly, Golgi ministacks persist upon loss of adhesion in single motor knockdown cells and continue to regulate microtubule acetylation similarly to stable adherent cells. In evaluating adhesion-dependent Golgi organization, we highlight the Arf1-KIF5B-dynein axis as a key regulator of Golgi-driven tubulin acetylation and cell function in fibroblasts. We further extend our study to anchorage-independent cancer cells, which show variable Golgi organization. A screen from the lab revealed distinct Golgi phenotypes in paired lung (A549 and CaLu1) and breast cancer (MDA-MB231 and MCF7) cell lines- CaLu1 and MCF7 displaying disorganized Golgi, and A549 and MDA-MB231 showing organized Golgi. Based on these observations, we explore how differentially expressed motor proteins contribute to Golgi organization in these cancer cell lines.