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Pepperkok Team

Advanced Light Microscopy Facility

EMBL 2000 Research Reports

We develop and run the Advanced Light Microscopy Facility (ALMF) at EMBL-Heidelberg.The goal of the ALMF is to provide advanced light microscopy and image analysis service for all of EMBL's scientific groups and visitors. We actively develop and maintain the equipment and software packages, teach and offer support to users. The facility also provides EU-funded short-term fellowships to carry out research projects that require microscopy equipment of the ALMF (European ALMF) and is not available at the visitor’s home institute.

now some of these software packages for the handling of images and quantitation of the motility of fluorescent structures in 3D multicolor time-lapse series. We have also developed simple software for fully automated execution and analysis of more complex image acquisition and analysis protocols running on the presently available hardware. This allows facilitated use of the systems and also increased throughput as it is typically required for large scale screening projects. These automated protocols include

Our research projects focus on how membrane traffic and organelle biogenesis in the early secretory pathway is regulated on the molecular level in space and time.

Fluorescence Recovery After Photobleaching (FRAP) and quantification of Fluorescence Resonance Energy Transfer (FRET) by acceptor or donor photobleaching. In collaboration with the group of Eric Karsenti we have started to develop a new screening method for

Team Leader: Rainer Pepperkok

Advanced Light Microscopy Facility (ALMF)

Postdoctoral fellows: Andreas Girod, Jeremy Simpson, David Stephens

(Andreas Girod, Jens Rietdorf, Timo Zimmermann)

Golgi complex) in vitro, exploiting the phenomenon of

Scientific assistant: Jens Rietdorfs, Timo Zimmermann*

In collaboration with industrial partners we develop and test, new light microscopy products and their applications to biological problems. After successful completion of this devlopment/evaluation phase the equipment is made available in the ALMF for the use by EMBL scientists and visitors.

PhD student: Rebecca Forster* Visitors: Gabriele Burger, Graziella Cappelletti, Susanna Castel, Winfried Denk, Eva Emig, Gregory Giannone, Maurice Hallett, Nicole Jenne, Leena Karhinen, Alla Karpova, Michael Keese, Martin Lowe, Carlos Luque, Torsten Muller, Veronika Neubrand, Elizabeth Pettit, Annette Pfennig, Lynne Roberts, Heiko Runz, Johannes Schmid, Ton Timmers, Mechthild Wagner, Stefan Wiemann, Stefan Wilms, Manuela Zaccolo

the measurement of the interaction of GFP-tagged proteins with complex structures (e.g. microtubules, GFP photo-activation. This screening method should allow us to rapidly identify from complete cDNA

In the past year we have started eight new such collaborations. The techniques currently available in the ALMF are: confocal laser scanning microscopy, twophoton excitation confocal laser scanning microscopy, spinning disk realtime-confocal microscopy, fast, multi-spectral 3D time-lapse microscopy, total internal reflection fluorescence microscopy (TIRF) and fluorescence correlation spectroscopy. Commercial software packages for the analysis, 3D reconstruction and deconvolution of multicolor image data are also available. We have developed and improved in our team 1

libraries novel proteins binding to the structures of interest. More information about the equipment and services of the ALMF is available at www.EMBL-heidelberg.de/ ExternalInfo/almf/index.html.

Membrane traffic in the early secretory pathway The aim of our scientific research is to understand the temporal and spatial molecular interactions of the key players in the early secretory pathway and how this contributes to regulated organelle biogenesis.

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EMBL 2000 Research Reports

Figure 1. Systematic localisation of novel proteins in cells. Novel cDNAs as they are identified and sequenced by the German Human cDNA Sequencing Consortium are GFPtagged at their C-terminal or N-terminal end, expressed and localised in living cells. More than 200 cDNAs have so far been localised. Examples of the different localisations obtained and their percentage of occurrence are shown.

simplified in vitro systems and it is now important to translate this information to an in vivo situation. To address this problem we have developed several GFP markers and light microscopy approaches to directly visualize in living cells the kinetics of ER to Golgi transport carriers simultaneously with the vesicular coat molecules and their regulators. Using such approach vesiculo-tubular transport clusters (VTCs, about 1 µm in size) rather than transport vesicles, could be identified as the major long range transport carriers of secretory cargo from the ER to the Golgi complex (Scales et al., 1997). We could show now that VTCs form in a COPII dependent manner from stable COPII coated ER exit sites (Stephens et al., 2000). Thereafter, COPII is replaced by COPI which is required for their transport along microtubules into the Golgi complex (Stephens et al., 2000). During this transport step COPI remains associated with the VTCs and is required for the establishment of a polarized distribution of anterograde (secretory) and retrograde (recycling) cargo within the VTC itself (Shima et al., 1999). It has remained an open question however, whether smaller (60nm) COPI vesicles or larger COPI coated retrograde VTCs mediate the recycling of the transport machinery from the Golgi to the ER (Stephens & Pepperkok, 2001).

Direct visualisation of ER-to- Golgi transport in living cells (David Stephens, Rebecca Forster) All transport vesicles known to date are coated with cytoplasmic coat protein complexes that function in the physical formation and movement of these carriers

as well as cargo selection at the donor membrane. Two vesicular coat complexes, COPI and COPII, are known to operate between the endoplasmic reticulum (ER) and the Golgi complex. COPII regulates ER export and COPI is involved in retrieval and recycling of material from post-ER membranes. Much of the data regarding these and other coat functions has been obtained from the study of yeast genetics and 2

Using these in vivo assays and ts-O45-G as transport marker we have started to investigate which factors are involved in the motility of the VTCs in ER to Golgi transport along microtubules. We have obtained evidence so far that COPI and Rab proteins are essential for the movement of VTCs along microtubules During the year we have established a regulated experimental system to study the transport from the ER to the Golgi complex of procollagen, a soluble secretory cargo that forms large (about 300 nm) rod-like structures upon folding in the ER. These structures are

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EMBL 2000 Research Reports

thought to be too large to be incorporated into a 60 nm transport vesicle. Therefore, a so far entirely unresolved question is how then procollagen is transported from the ER to the Golgi complex? Surprisingly, procollagen-GFP is seen to traffic primarily in small, distinct VTCs morphologically similar to those observed for membrane proteins or luminal GFP, and transport out of the ER of procollagen is blocked by inhibition of COPI and COPII. However, in contrast to luminal GFP or GFP-tagged secretory membrane proteins, procollagen-containing VTCs are not coated with COPI during transport to the Golgi complex and do not contain ERGIC-53 or ts-045-G. This suggests that procollagen is transported from the ER to the Golgi complex in morphologically similar but distinct carriers to those previously described. Altogether, this suggests that COPI is involved in the sorting of cargo at ER exit sites in mammalian cells.

and Golgi enzymes similar if not identical molecular mechanisms for ER recycling (Storrie et al., 2000).

identify new proteins with a functional role in the secretory pathway.

During the year we have addressed this problem in more details. Preliminary results suggest that rab6 needs to interact with rabkinesin 6 to exert its regulatory function on this recycling pathway. We have also identified a novel rab6 homologue that is ubiquitously expressed, localises to the Golgi complex but does not appear to interact with rabkinesin6. Presently, we investigate whether this novel rab6 homologue has a role in toxin transport to the ER.

One of them (2c18) is a soluble cytoplasmic protein that associates with the Golgi complex, is involved in transport from the trans Golgi network to the plasma membrane and its overexpression interferes with the association of TGN46 with the Golgi complex. Its expression is enhanced in cells of neuronal origin and in cultures of rat primary neurons it appears to be predominately associated with axons. In collaboration with the group of Carlos Dotti (Torino) we presently test the significance of these findings by studying the involvement of 2C18 in sorting events discriminating axonal from dentritic transport.

Golgi to ER retrograde transport of protein toxins

In collaboration with the group of Stefan Wiemann at the DKFZ in Heidelberg (Division of Molecular Genome Analysis) we have developed and tested a strategy which aims at the systematic identification of novel proteins important for structure and function in the secretory pathway (Simpson et al., 2000; Simpson et al., 2001). Novel full length human cDNAs cloned by the group at the DKFZ and sequenced by groups of the German cDNA sequencing consortium are tagged with DNA encoding the green fluorescent protein (GFP).Each of these cDNA molecules are then transfected into mammalian cells and their steady state localization is visualized in living cells. The results of a pilot study so far have revealed the respective proteins localized to many different cellular compartments. Importantly, about 25% of the cDNAs analyzed (200 in total) were identified to encode novel proteins localizing to organelles of the secretory pathway or structures associated with it (e.g. microtubules). Investigating their significance for various transport steps in the secretory pathway, using a well established transport marker (ts-O45-G), we were able to

(Andreas Girod, Jeremy Simpson) We use as Golgi to ER retrograde transport markers a specific class of protein toxins travelling the secretory pathway in reverse from the plasma membrane all the way to the ER. They bind to specific receptors on target cells, are then endocytosed and move via endosomes into the trans-Golgi network (TGN) and Golgi stacks, followed by complete retrograde transport to the ER. We showed earlier (Girod et al., 1999) that Shiga toxins travel from the Golgi to the ER in a COPI-independent but Rab6 dependent manner. In contrast transport of toxins equipped with a KDEL-like sequence at their c-terminal end, requires COPI but not Rab6. Recycling of Golgi enzymes to the ER appears to be also COPI independent, suggesting that Shiga toxins

Systematic localisation and functional characterisation of novel human proteins associated with the secretory pathway (Jeremy Simpson)

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Another of these proteins with a role in the secretory pathway that we analyzed in more details (22f21) is a microtubule associated protein that localizes also to the centrosomes. When overexpressed in cells it stabilizes microtubules, affects the integrity of the Golgi complex, and it inhibits the motility of ER to Golgi VTCs.

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Publications during the year Bastiaens, P.I. & Pepperkok, R. (2000). Observing proteins in their natural habitat: the living cell. Trends Biochem. Sci., 25, 631-637 Denzel, A., Otto, F., Girod, A., Pepperkok, R., Watson, R., Rosewell, I., Bergeron, J.J., Solari, R.C. & Owen, M.J. (2000). The p24 family member p23 is required for early embryonic development. Curr. Biol., 10, 55-58 Pepperkok, R., Hotz-Wagenblatt, A., Konig, N., Girod, A., Bossemeyer, D. & Kinzel, V. (2000). Intracellular distribution of mammalian protein kinase A catalytic subunit altered by conserved Asn2 deamidation. J. Cell Biol., 148, 715-726 Pepperkok, R., Whitney, J.A., Gomez, M. & Kreis, T.E. (2000). COPI vesicles accumulating in the presence of a GTP restricted Arf1 mutant are depleted of anterograde and retrograde cargo. J. Cell Sci., 113, 135-144 Pepperkok,R., Girod, A., Simpson, J., and Rietdorf, J. (2000). Immunofluorescence microscopy. In "Monoclonal Antibodies", Shepherd, P. & Dean, C. (eds.), Oxford Univ. Press, pp. 355-370 Rietdorf, J.& Zimmermann, T. (2000). 4Dmicroscopy: Exploring Time and space. Imaging and Microscopy, 2, 44-46 Simpson, J.C., Wellenreuther, R., Poustka, A., Pepperkok, R. & Wiemann, S. (2000). Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing. EMBO Reports, 1, 287-292

EMBL 2000 Research Reports

Sonnichsen, B., De Renzis, S., Nielsen, E., Rietdorf, J. & Zerial, M. (2000). Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11. J. Cell Biol., 149, 901-914 Stephens, D.J., Lin-Marq, N., Pagano, A., Pepperkok, R. & Paccaud, J.P. (2000). COPI-coated ER-to-Golgi transport complexes segregate from COPII in close proximity to ER exit sites. J. Cell Sci., 113, 2177-2185 Storrie, B., Pepperkok, R. & Nilsson, T. (2000). Breaking the COPI monopoly on Golgi recycling. J. Cell Biol., 10, 385-391

Other references Girod, A., Storrie, B., Simpson, J.C., Johannes, L., Goud, B., Roberts, L.M., Lord, J.M., Nilsson, T. & Pepperkok, R. (1999). Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum. Nat. Cell Biology, 1, 423430 Scales, S.J., Pepperkok, R. & Kreis, T.E. (1997). Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COPII and COPI. Cell, 90, 1137-1148 Shima, D.T., Scales, S.J., Kreis, T.E. & Pepperkok, R. (1999). Segregation of COPI-rich and anterogradecargo-rich domains in endoplasmic-reticulum-toGolgi transport complexes. Curr. Biol., 9, 821-824 Simpson, J.C., Neubrand, V.E., Wiemann, S. & Pepperkok, R. (2001). Illuminating the human genome. Histochem Cell Biol, 115, 23-29

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Stephens, D.J. & Pepperkok R. (2001). Illuminating the secretory pathway: when do we need vesicles? J. Cell Sci., 114, 1053-1059