A novel laser nanosurgery approach supports de novo Golgi biogenesis in mammalian cells.

ABSTRACT

The Golgi complex has a central role in the secretory pathway of all higher organisms. To explain the synthesis of its unique stacked structure in mammalian cells, two major models have been proposed. One suggests that it is synthesized de novo from the endoplasmic reticulum. The second model postulates a pre-existing Golgi template that serves as a scaffold for its biogenesis. To test these hypotheses directly, we have developed an approach in which we deplete the Golgi complex from living cells by laser nanosurgery, and subsequently analyze the 'Golgi-depleted' karyoplast using time-lapse and electron microscopy. We show that biosynthetic transport is blocked after Golgi depletion, but is restored 12 hours later. This recovery of secretory transport coincides with an ordered assembly of stacked Golgi structures, and we also observe the appearance of matrix proteins before that of Golgi enzymes. Functional experiments using RNA interference-mediated knockdown of GM130 further demonstrate the importance of the matrix during Golgi biogenesis. By contrast, the centrosome, which can also be removed by laser nanosurgery and is not reformed within the considered time frame, is not required for this process. Altogether, our data provide evidence that de novo Golgi biogenesis can occur in mammalian cells.