Membrane traffic in the early secretory pathway
The four steps involved in ER to Golgi transport in mammalian cells. (I): Biogenesis of COPII coated vesicles occurs at specialised ER exit sites of the ER.(II): COPII vesicles homotypically fuse to form larger vesicular tubular transport carriers (VTCs) that are transported to the Golgi complex along microtubules.(III): VTCs arrive at the Golgi complex and fuse to it todeliver their cargo. (IV): Transport machinery and misrouted proteins are return back to the ER by a distinct class of carriers.
The Pepperkok team develops novel approaches to investigate interactions between the endoplasmic reticulum and the Golgi complex.
Previous and current research
Transport between the endoplasmic reticulum (ER) and the Golgi complex in mammalian cells involves at least four basic steps (see figure). To warrant both the specificity of delivery of cargo and the maintenance of the unique protein and lipid compositions of the organelles involved, these four steps must be tightly regulated and coordinated at the molecular level. The specific questions we are presently addressing in this context are: 1) What are the mechanisms underlying the regulation of ER-exit sites’ biogenesis and function? 2) How are ER exit and microtubule-mediated ER to Golgi transport coupled at the molecular level? 3) What are the mechanisms of Golgi biogenesis? 4) Which are the molecules regulating recycling of Golgi resident proteins to the ER?
To investigate this, we develop computer-automated light microscopy approaches to directly visualise and quantify, in living cells, the kinetics of secretory and organelle markers simultaneously with vesicular coat molecules (COPI and COPII) and their regulators. We also use fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer measurements (FRET), together with mathematical modelling of the data in order to understand the mechanistic of the temporal and spatial regulation of the molecular interactions involved. Our combined data suggest that secretory cargo, lipids, and the microtubule motor-associated dynactin complex play a critical role in the stabilisation of the COPII vesicular coat complex to provide the time that is necessary for cargo selection and concentration at ER exit sites. In order to investigate the mechanisms of Golgi biogenesis we have developed an approach, using laser nanosurgery, to remove the entire Golgi complex from living cells and subsequently analyse the ‘Golgi-less’ karyoplast by time-lapse and electron microscopy. With this approach we are able to show that Golgi biogenesis in mammalian cells occurs de novo from ER derived membranes.
In order to identify putative molecules involved in this de novo Golgi biogenesis, we have developed and applied functional assays to assess the effect of knockins by cDNA over-expression and knockdowns by RNAi on processes such as constitutive protein transport, Golgi integrity, and function of vesicular coat complexes. To achieve the throughput that such genome-wide analyses require, we have developed a fully automated high-content screening microscopy platform, including sample preparation, image acquisition, and automated analysis of complex cellular phenotypes.We have applied this technology to genome-wide siRNA screens to identify and characterise comprehensively the genes and their underlying functional networks involved in secretory membrane traffic and Golgi integrity.
Future projects and goals
We will study the novel proteins that our screens identify as being involved in the early secretory pathway in further detail. An important question in this context will be if and how they participate in the temporal and spatial organisation of ER-exit sites and their function, and the biogenesis of the Golgi complex.