ayout 1 - EMBL Grenoble

EMBL Research at a Glance 2009
Rainer
Pepperkok
PhD 1992, University of
Kaiserslautern.
Postdoctoral work at
University of Geneva.
Lab head at the Imperial
Cancer Research Fund,
London.
Team leader at EMBL since
1998.
Membrane traffic in the early secretory pathway
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): 1) biogenesis of membrane bounded transport carriers
at specialised domains (ER-exit sites) of the ER; 2) microtubule mediated transport of the
carriers to the Golgi complex; 3) docking and fusion of the carriers with the Golgi complex; and
4) recycling of the transport machinery back to the ER. 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; and 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, in which we remove
by laser nanosurgery the entire Golgi complex from living cells and subsequently
analyse the “Golgi-less” karyoplast by time-lapse and electron microscopy.
With this approach we could 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 knock-ins 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
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 to
deliver their cargo. (IV): Transport machinery and
misrouted proteins are return back to the ER by a
distinct class of carriers.
We will study the novel proteins, which we revealed in our screens to be involved in the early secretory pathway, in further detail at the systems
level. 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.
Selected references
Simpson, J.C., Cetin, C., Erfle, H., Joggerst, B., Liebel, U., Ellenberg,
J. & Pepperkok, R. (2007). An RNAi screening platform to identify
secretion machinery in mammalian cells. J. Biotechnol., 129, 352-
365
Runz, H., Miura, K., Weiss, M. & Pepperkok, R. (2006). Sterols
regulate ER-export dynamics of secretory cargo protein ts-O5-G.
EMBO J., 25, 2953-2965
Forster, R., Weiss, M., Zimmermann, T., Reynaud, E.G., Verissimo,
F., Stephens, D.J. & Pepperkok, R. (2006). Secretory cargo regulates
the turnover of COPII subunits at single ER exit sites. Curr. Biol., 16,
173-179
Watson, P., Forster, R., Palmer, K.J., Pepperkok, R. & Stephens, D.J.
(2005). Coupling of ER exit to microtubules through direct interaction
of COPII with dynactin. Nat. Cell Biol., 7, 8-55
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