Research interest
One focal point of our structural biology group is to reveal and manipulate the macromolecular organisation of cells under normal and pathogenic conditions at the nano-scale level. We use cryo-electron tomography of vitreous sections, currently the only method that can obtain molecular resolution of molecular machines in cells in a near-native situation. The tomograms contain a 3D map of the cellular proteome at about 4 nm resolution and we are just beginning to explore its potential by placing high effort on developing methods for our nanotechnology. The other central point of our group is to visualise gene products in cells by electron microscopy at the highest resolution with gold probes on cryo-sections.
Subcellular traffic of Mycobateria and its importance for vaccine development
In collaboration with the group of Michael Brenner (Harvard, Boston) we published last year that after prolonged infection in macrophages and dendritic cells, M. tuberculosis translocates from phago-lysosomes to the cytosol (Cell. 2007 Jun 29;129(7):1287-98). The BCG vaccine strain failed to translocate from the phago-lysosome. The translocation into the cytosol was totally unexpected and against textbook knowledge. This difference in localization might give an explanation for the ineffectiveness of BCG as a MHC class I / CD8 triggered vaccine. Looking further into the difference in localization can bring us closer to a better vaccine against tuberculosis but perhaps also against virus induced tumors such as HPV. The aim of our current work is to determine which regions of difference (RDs) between the M. tuberculosis and BCG genomes are important for translocation. In collaboration with the lab of Roland Brosch from Pasteur in Paris, we started with looking at BCG with a knock-in of the extended RD1 region (BCG::RD1), as this region is now known to encode the ESX-1 secretion system recently coined by one of our group members as a novel type VII secretion system (Abdallah AM et al., Type VII secretion. Nat Rev Microbiol. 2007;5(11):883-91). We found that this bacterium can be seen in the cytosol of the cell after 7 days of infection and conclude that the ESX-1 system (in a BCG background) is sufficient for translocation. In addition we are investigating ESX-5, another type VII secretion system from M. marinum (Bitter, VUmc Amsterdam). One of our next aims is to characterize the structural of ESX-1 secretion systems by cryo electron tomography. The proposed 3D-EM studies complement and expand upon on-going efforts to provide novel insights into the spatial organization of molecular machines within cells as outlined in the paragraph above. Currently we are also investigating by cryo-immunogold EM several different recombinant BCG based vaccine candidates. These are hypothesized to translocate from the phagosome to the cytosol using pore forming proteins from other bacteria, such as Listeriolysin from Listeria monocytogenes (Kaufmann, Berlin) and Perfringolysin O from Clostridium perfringens (Fulkerson, Aeras, Rockville USA).
Structure and conversion site of Prions
Prion diseases are caused by accumulation of an abnormally folded isoform (PrPSc) of the cellular prion protein (PrPC). The subcellular site where PrPSc is formed is still unclear. In collaboration with the lab of Stan Prusiner (UCSF, San Francisco) we have used quantitative cryo-immunogold electron microscopy to localise different populations of PrP on hippocampal sections from prion-infected mice. At a late subclinical stage of prion infection we detected a 6 fold overall increase in PrP levels in the stratum oriens. The biggest increase (24 fold) occurred on vesicles resembling early endocytic or recycling vesicles in small neurites in the neuropil, suggesting that these vesicles are important in PrPSc formation or pathology.
We are also performing cryo-electron tomography on prion preparations, in order to solve the 3D structure of prions. In collaboration with Jesus Requena (University of Santiago de Compostela, Spain) we have examined purified samples of the proteinase K-resistant core of hamster PrPSc, vitrified in the recently obtained FEI vitrobot. The preparations were found to contain 2 nm fibres, sometimes twisted together with other fibres. The data were consistent with a model in which individual fibres are comprised of ‘strings’ of monomers.
Single intestinal stem cells
The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. In collaboration with the lab of Hans Clevers (Hubrecht Institute, Utrecht) we have recently demonstrated the presence of approximately six cycling Lgr5+ve stem cells at the bottom of a small intestinal crypt (Barker N, et al., Nature. 2007;449(7165):1003-7). We have now established long-term culture conditions under which single crypts undergo multiple crypt fission events, whilst simultaneously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5+ve stem cells can also initiate these crypt-villus organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche.
Nanomachines in cells
Cryo electron tomography of vitreous sections is becoming an established technique to study the three-dimensional structure of molecular machines in situ. Specifically we have been looking at the 80S eukaryotic ribosome within the cell. Using high-resolution tomography of unfixed frozen hydrated cryo-sections the structure and relative orientation of the ribosome, in a cellular context, has been established by semi-automatic particle selection, volumetric alignment and averaging. With this template a ‘molecular atlas’ was constructed, mapping the ribosome over the entire cellular volume within a thin vitreous section of a cell (see image on this page). This study illustrates the possibilities of this nanotechnology towards visualizing cellular machinery in situ at macromolecular resolutions in large eukaryotic cells. Future challenges include visualizing individual conformations (imposed by drugs) of GFP tagged ribosomes and using volumetric classification algorithms based on maximum likelihood statistical approach. Our initial attempt at understanding the spatial arrangements of the ribosome in situ is exciting and hints at the possibilities of imaging the vast majority of other biological machines in which the cellular spatial arrangement and molecular interactions that still remain elusive. One specific example that we now address is the transmembrane protein conduit machinery, the SecY/Sec61 system, which is attached to the ribosome and responsible for translocating nascent proteins into the lumen of the endoplasmic reticulum. With new technologies being developed by us and others a resolution of 2.5 nm is approaching, allowing template matching the X-ray crystal structure with the ultimate goal to explain the structure of our averaged density maps.
Collaborators:
Jose Jesus Fernandez1, Bram Koster2, Henny Zandbergen3, Helmut Gnaegi4,
1 Centro Nacional de Biotechnologia-Conejo Superior de Investigaciones Cientificas, Madrid
2 Department of Molecular Cell Biology, Leiden University Medical Center, Leiden
3 Department of physic, Technical University Delft
4 Diatome Ltd, Biel, Switzerland
Curriculum vitae Peter Peters
Peters cv 2009
Postdoctoral fellows
Information on the activities for postdocs
Key publications
Most important publications Peter J. Peters 2003 - 2009
* Pierson J, Fernández JJ, Bos E, Amini S, Gnaegi H, Vos M, Bel B, Adolfsen F, Carrascosa JL, Peters PJ. Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box. J Struct Biol. 2009 Oct 12.
Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009 May 14;459(7244):262-5.
van der Flier LG, van Gijn ME, Hatzis P, Kujala P, Haegebarth A, Stange DE, Begthel H, van den Born M, Guryev V, Oving I, van Es JH, Barker N, Peters PJ, van de Wetering M, Clevers H. Transcription factor achaete scute-like 2 controls intestinal stem cell fate. Cell. 2009 Mar 6;136(5):903-12.
* Godsave SF, Wille H, Kujala P, Latawiec D, DeArmond SJ, Serban A, Prusiner SB, Peters PJ. Cryo-immunogold electron microscopy for prions: toward identification of a conversion site. J Neurosci. 2008 Nov 19;28(47):12489-99.
Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, Haegebarth A, Korving J, Begthel H, Peters PJ, Clevers H. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 2007 Oct 25;449(7165):1003-7.
* van der Wel N, Hava D, Houben D, Fluitsma D, van Zon M, Pierson J, Brenner M, Peters PJ. M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells. Cell. 2007 Jun 29;129(7):1287-98.
Touret N, Paroutis P, Terebiznik M, Harrison RE, Trombetta S, Pypaert M, Chow A, Jiang A, Shaw J, Yip C, Moore HP, van der Wel N, Houben D, Peters PJ, de Chastellier C, Mellman I, Grinstein S. Quantitative and dynamic assessment of the contribution of the ER to phagosome formation. Cell. 2005 Oct 7;123(1):157-70.
Baas AF, Kuipers J, van der Wel NN, Batlle E, Koerten HK, Peters PJ, Clevers HC. Complete polarization of single intestinal epithelial cells upon activation of LKB1 by STRAD. Cell. 2004 Feb 6;116(3):457-66.
Peters PJ, Mironov A Jr, Peretz D, van Donselaar E, Leclerc E, Erpel S, DeArmond SJ, Burton DR, Williamson RA, Vey M, Prusiner SB. Trafficking of prion proteins through a caveolae-mediated endosomal pathway. J Cell Biol. 2003 Aug 18;162(4):703-17.
* Mironov A Jr, Latawiec D, Wille H, Bouzamondo-Bernstein E, Legname G, Williamson RA, Burton D, DeArmond SJ, Prusiner SB, Peters PJ. Cytosolic prion protein in neurons. J Neurosci. 2003 Aug 6;23(18):7183-93
Most cited publications as first author
Peters PJ, Neefjes JJ, Oorschot V, Ploegh HL, Geuze HJ. Segregation of MHC class II molecules from MHC class I molecules in the Golgi complex for transport to lysosomal compartments. Nature. 1991 Feb 21;349(6311):669-76.
Peters PJ, Borst J, Oorschot V, Fukuda M, Krähenbühl O, Tschopp J, Slot JW, Geuze HJ. Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes. J Exp Med. 1991 May 1;173(5):1099-109.
Peters PJ, Hsu VW, Ooi CE, Finazzi D, Teal SB, Oorschot V, Donaldson JG, Klausner RD. Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments. J Cell Biol. 1995 Mar;128(6):1003-17.
More publications klick Curriculum vitae Peters cv 2009
More publications by Peter J. Peters on PubMed
Biographic sketch
Peters obtained his PhD from Utrecht University, where he analyzed the ultrastructure of MHC class II antigen processing and discovered the 'MHC class II compartment' (MIIC) (Peters PJ et al., Nature, 1991, Peters PJ et al., J Exp Med 1995) and studied exocytosis of cytotoxic mediators in T cells. He established that secretory granules are of lysosomal nature (Peters PJ et al., J Exp Med. 1991). Peters joined the group of Rick Klausner at the National Institute Health in Bethesda, USA and identified ARF6 as a regulator for endocytosis (Peters PJ et al., J Cell Biol. 1995). In 1998, he became Principal Investigator at the Netherlands Cancer Institute and was appointed as professor at the Free University of Amsterdam. His long-term focus is on understanding the molecular machinery and organization of molecular sorting within the endocytic membrane system of a cell (D'Souza-Schorey C et al., J Cell Biol. 1998, Peters PJ et al., Nature Cell Biol. 2002, van der Wel NN et al., Mol Biol Cell. 2003, Peters PJ et al., J Cell Biol. 2003 and Mironov A Jr et al, J Neurosci. 2003). Cryo immunogold-EM methods of aldehyde-fixed cells are the main techniques applied, allowing the subcellular detection of gene products at the highest resolution (Van der Wel N at al., Cell. 2007, Godsave SF et al., J Neurosci. 2008 and Peters PJ, Pierson J. Methods Cell Biol. 2008. Review). In addition, Peters’ lab is currently improving the ultrathin cryosectioning of native unfixed cells for high-resolution 3D cryo-electron tomography to visualize molecular machines in the context of organelles. Peters' lab consist of 9 scientists and 4 technicians.
Since 1998, Peters has been dean of postdoc affairs and built a ‘postdoc career development organization’ that facilitates young scientist making the right moves into their next career step by providing training in transferable skills and offering yearly 3 day retreats for 120 postdocs with workshops in topics including: Laboratory Leadership in Science, Mentoring and Being Mentored, Time Management, Project Management, Data Management, Getting Funded, Getting Published and Increasing Your Visibility, Understanding Technology Transfer, Setting Up Collaborations, Teaching and Course Design, Obtaining and Negotiating a Faculty Position, Staffing Your Laboratory.
The NKI-AVL ranked over the last years among the top 10 as the best place to work as postdoc outside the USA (the Scientist). Peters recently obtained 600.000 Euro from the Dutch government to professionalize the organization (www.pcdi.nl). With this new organization two successful retreats have been organized with an averaged rating of 8.5 (out of 10). Currently he seeks EU funding to initiate European wide collaborations with other research institutions. This initiative not only benefits postdocs but also the research institutes. The work received attention in: Cell. 2006;125(3):407 Aschwanden C. Learning to lead., Cell. 2006;124(3):445 Aschwanden C., Professionalizing the postdoctoral experience, The Scientist 2008; 22 (3): 53, The best places to work as postdoc and Nature 2007;445: 948 Griekspoor A., Torn between two ladders.
Co-workers
- Nicole van der Wel PhD Associate Staff Scientist
- Sue Godsave PhD Post-doc / EU project manager
- Pekka Kujala PhD Post-doc
- Musa Sani PhD Post-doc
- Cveta B. Tomova PhD Post-doc
- Abdallah M. Abdallah Post-doc (and at VUmc Amsterdam)
- Matthijn Vos PhD Post-doc (part-time) and at FEI company Eindhoven
- Diane Houben MSc Graduate student
- Jason Pierson MSc Graduate student
- Hans Jansen BSc Technical staff
- Maaike van Zon BSc Technical staff
- Karin de Punder MSc Technical staff
- Nico Ong Research assistant