Gßγ signaling regulates microtubule-dependent control of Golgi integrity.

ABSTRACT

Gßγ subunits regulate several non-canonical functions at distinct intracellular organelles. Previous studies have shown that Gßγ signaling at the Golgi is necessary to mediate vesicular protein transport function and to regulate mitotic Golgi fragmentation. Disruption of Golgi structure also occurs in response to microtubule depolymerizing agents, such as nocodazole. In this study, we use siRNA against Gß1/2 or specific Gγ subunits to deplete their expression, and show that their knockdown causes a significant reduction in nocodazole-induced Golgi fragmentation. We establish that knockdown of Gßγ or inhibition of Gßγ with gallein resulted in decreased activation of protein kinase D (PKD) in response to nocodazole treatment. We demonstrate that restricting the amount of free Gßγ available for signaling by either inhibiting Gαi activation using pertussis toxin or by knockdown of the non-GPCR GEF, Girdin/GIV protein, results in a substantial decrease in nocodazole-induced Golgi fragmentation and PKD phosphorylation. Our results also indicate that depletion of Gßγ or inhibition with gallein or pertussis toxin significantly reduces the microtubule disruption-dependent Golgi fragmentation phenotype observed in cells transfected with mutant SOD1, a major causative protein in familial amyotrophic lateral sclerosis (ALS). These results provide compelling evidence that Gßγ signaling is critical for the regulation of Golgi integrity.