Cell-matrix adhesion-dependent differential regulation of cis-medial vs trans-Golgi organisation

Abstract

The characteristic structure of the Golgi apparatus is made up of flattened cisternal stacks and conserved across eukaryotes. The Golgi plays a central role in protein processing and trafficking. It is a dynamic structure that undergoes a rapid reorganisation in response to different stimuli, including cell-matrix adhesion. In response to loss of adhesion, Golgi rapidly disorganises and is distributed throughout the cell while staying attached to the microtubule network. Adhesion-dependent Golgi disorganisation is seen to be controlled by Arf1 activation. In mouse fibroblasts, loss of adhesion triggers a drop (~50%) in Arf1 activation that causes the Golgi to disorganise but not fragment. The extent of this disorganisation is interestingly seen to be different for different Golgi compartments. While the trans-Golgi is seen to be more disorganised and distributed throughout the cell, the cis/medial Golgi disorganises much less. This difference seen in non-adherent cells we find is supported by the differential localisation of active Arf1 to these compartments. This could be mediated by the differential activation of Arf1 by GEFs. Non-adherent fibroblasts hence provide a unique setting to test and understand the role GEF-mediated Arf1 activation has in regulating Golgi organisation. Using Man II-GFP expressing non-adherent fibroblasts treated with increasing concentrations of Brefeldin-A (BFA) (inhibits BIG1/2 and GBF1) and Golgicide A (GCA) (inhibits GBF1 only), we test this regulation. This reveals comparable inhibition of Arf1 to cause a concentration-dependent increase in cis-Golgi fragmentation and ER fallback. A distinct difference in the kinetics of Arf1 inhibition at low vs high BFA/GCA concentrations we find could mediate this by regulating cis-Golgi localised GBF1. In agreement with this, on loss of adhesion, slower kinetics of Arf1 inhibition (~50% inactivation in 120min) causes the Golgi to disorganise. In contrast, a comparable but rapid inhibition of Arf1 (using BFA or GCA) causes the Golgi to instead fragment dramatically. This affects Golgi function reflected in altered cell surface glycosylation and delayed re-adherent cell spreading. Loss of adhesion also causes a distinct drop in microtubule acetylation which could be more an effect of Golgi disorganisation rather than a mediator of the same. These studies, using the distinct change in Golgi organisation and function on loss of adhesion, help provide a novel insight into the Arf1-mediated regulation of the Golgi.