Judith Klumperman, Ph.D.
Professor of Cell Biology
Head of the Cell Microscopy Center
Viola Oorschot, technician: immuno-electron microscopy (v.m.j.Oorschot@umcutrecht.nl)
Suzanne van Dijk, technician: immuno-electron microscopy (firstname.lastname@example.org)
Corlinda ten Brink, technician: (email@example.com)
Eline van Meel, Ph.D. student: (H.M.M.firstname.lastname@example.org)
Maaike Pols, Ph.D. student: (M.S.Polsemail@example.com)
Romain Galmes, Ph.D. student: (firstname.lastname@example.org)
Ann De Mazière, post-doc: trafficking of tumor antigens
I. Defining the machinery for lysosome biogenesis
Lysosomes are dynamic organelles containing a range of lumenal proteins involved in the degradation of cellular components. So far, more than ~45 human genetic disorders are related to lysosomal disfunctioning. A major cause is the perturbed delivery of lysosomal proteins from their site of synthesis to the lysosome. Multiple pathways are responsible for the transport of these proteins, of which the mannose-6-phosphate receptor (MPR) dependent pathway is the most well-characterized. Our studies aim at defining the poorly characterized MPR-independent pathways that concern the trafficking of the lysosomal membrane proteins LAMP-1, -2, and LAMP-3/CD63 (see movie). The techniques used for this project range from confocal live cell imaging, immuno-electron microscopy, molecular and biochemical techniques, and microscopy-based RNA interference screening. Movie showing intracellular trafficking in a HepG2 cell of the lysosomal membrane protein LAMP1 tagged to monomeric green fluorescent protein (gift from E. Dell'Angelica, UCLA, USA). Elapsed time 2 minutes, 200 ms interval.
Immunogold labeling of LAMP-1 in a multivesicular body of a HepG2 cell.
Defining the carriers involved in lysosome biogenesis
(Viola Oorschot, Corlinda ten Brink)
Little is known about the identity of the carriers that mediate the transport of LAMP-1,-2, and CD63. In order to identify the morphological and molecular characteristics of these carriers, we implemented a correlative live cell imaging/ immuno-electron microscopy (CLEM) approach in HepG2 and HeLa cells transfected with the mGFP-tagged LAMP-1 or CD63. We have investigated the movement of these proteins with live cell imaging at different time points after transfection and classified distinct types of carriers by CLEM.
Identifying the molecular machinery for lysosome biogenesis
(Vincent Schoonderwoert, in collaboration with Rainer Pepperkok, Holger Erfle, and Jeremy Simpson, EMBL, Heidelberg )
To isolate new candidate machinery proteins involved in lysosome biogenesis, we apply a high-throughput RNA interference screen with the human epithelial cell line HeLa using an automated microscope workstation. The readout for this fluorescence-based assay is the expression level of CD63 at the cell surface. Interesting candidate proteins are further characterized using siRNA knock-down and mutational experiments in combination with multimode live cell imaging and CLEM.
Endocytic pathways for lysosomal membrane proteins
Lysosomal membrane protein CD63 (also known as LAMP-3), is a member of the tetraspanin family, and can be found in the internal vesicles of late endosomes, lysosomes, and on the plasma membrane. CD63 can interact with MHCII, CD82, PI4K and Src and regulates several signalling pathways.CD63 is the only LAMP protein that is found in internal vesicles of late endosomes and previous studies have shown that a relatively large portion of CD63 transported to lysosomes via the plasma membrane, suggesting that this is an important pathway. Here we investigate the pathways by which CD63 can be endocytosed from the plasma membrane and is transported to endosomal compartments.
II. MPR-independent transport of lysosomal hydrolases
(Eline van Meel)
I-cell disease is a disorder characterized by intracellular deficiency of most lysosomal enzymes. This is due to severely reduced activity of the GlcNAc-1-phosphotransferase, which mediates the first step in the modification of lysosomal enzymes with mannose 6-phosphate residues, required for MPR transport. However in non-mesenchymal cells of I-cell disease patients, a subset of these enzymes still reaches the lysosome, pointing to the existence of MPR-independent transport of lysosomal hydrolases. This study focuses on the characterization of these MPR independent pathway(s), making use of live cell imaging and immuno-electron microscopy. Immunogold labeling of mannose 6-phosphate residues shows lysosomal enzymes in lysosomes of B lymphoblasts.
Griffiths G, Klumperman J. 2004. Membranes and organelles. Curr Opin Cell Biol. 16:341-342.
Junutula* J.R., A.M. De Mazière*, A.A. Peden, K.E. Ervin, R.J. Advani, S.M. van Dijk, J. Klumperman, and R.H. Scheller. Rab14 is involved in membrane trafficking between the Golgi complex and endosomes. Mol. Biol. Cell. 2004 May; 15(5): 2218-2229.
Sachse M., S. Urbé, V. Oorschot, G. Strous, and J. Klumperman. 2002. Bi-layered clathrin coats on endosomal vacuoles are involved in protein sorting towards lysosomes. Mol. Biol. Cell. 13:1313-1328.
Martinez-Menarguez, J.A., R. Prekeris, V. Oorschot, RH Scheller, JW Slot, HJ Geuze, and J. Klumperman. 2001. Peri-Golgi vesicles contain retrograde but not anterograde proteins consistent with the cisternal progression model of intra-Golgi transport. J Cell Biol. 155: 1213-1224.
de Wit, H., Y. Lichtenstein, R.B. Kelly, H.J. Geuze, J. Klumperman*, and P. van der Sluijs*. 2001. Rab4 regulates formation of synaptic-like microvesicles and constitutive recycling vesicles from early endosomes in PC12 cells. Mol Biol Cell. 12: 3703-3715. *with equal contributions.
Prekeris, R., J. Klumperman, and R.H. Scheller. 2000. A Rab11/Rip11 protein complex regulates apical membrane trafficking via recycling endosomes. Molecular Cell 6, 1437-1448.
Klumperman, J. 2000. Transport between ER and Golgi. [Review]. Current Opinion in Cell Biology 12:445-449.
Steegmaier, M., V. Oorschot, J. Klumperman, and R.H. Scheller. 2000. Syntaxin 17 is abundant in steroidogenic cells and implicated in smooth endoplasmic reticulum membrane dynamics. Mol. Biol. of the Cell. 11:2719-2731.
Prekeris, R., B. Yang, V. Oorschot, J. Klumperman, and R.H. Scheller. 1999. Differential roles of syntaxin 7 and syntaxin 8 in endosomal trafficking. Mol. Biol. of the Cell 10:3891-3908.
de Wit, H., Y. Lichtenstein, H.J. Geuze, R.B. Kelly, P. van der Sluijs and J. Klumperman. 1999. Synaptic vesicles form by budding from tubular extensions of sorting endosomes in PC12 cells. Mol. Biol. of the Cell 10: 4163-4176.
Advani, R.J., B. Yang, R. Prekeris, K.C. Lee, J. Klumperman, and R.H. Scheller. 1999. VAMP7 mediates vesicular transport from endosomes to lysosomes. J. Cell Biol.146:765-775.
Martinez-Menarguez, J., H.J. Geuze, J.W. Slot and J. Klumperman. Vesicular tubular clusters between ER and Golgi mediate concentration of soluble secretory proteins by exclusion from COPI-coated vesicles. Cell 98:81-90, 1999.
Chao, D., J.C. Hay, S. Winnick, R. Prekeris, J. Klumperman and R.H. Scheller. 1999. SNARE Membrane trafficking dynamics in vivo. J. Cell Biol. 144:869-881.
Klumperman, J., A. Schweizer, H. Clausen, B.L. Tang, W. Hong, V. Oorschot, and H.P. Hauri. 1998. The recycling pathway of protein ERGIC-53 and dynamics of the ER-Golgi intermediate compartment. J Cell Science 111:3411-3425.
Hay*, J.C., Klumperman*, J., V. Oorschot, M. Steegmaier, C.S. Kuo and R.H. Scheller. 1998. Localization, dynamics, and protein interactions reveal distinct roles for ER and Golgi SNAREs. J. Cell Biol. 141:1489-1502. *with equal contributions.
Klumperman, J., R. Kuliawat, J. Griffith, H.J. Geuze and P. Arvan. 1998. Mannose 6-phosphate receptors are sorted from immature secretory granules via AP-1, clathrin and syntaxin 6-positive vesicles. J. Cell Biol.141:359-371.
Dell’Angelica, E.C., J. Klumperman, W. Stoorvogel, and J.S. Bonaficino. 1998. Association of the AP-3 adaptor complex with clathrin. Science 280:431-434.
Bock, J., J. Klumperman, S. Davanger, and R.H. Scheller. 1997. Syntaxin 6 functions in trans-Golgi network vesicle trafficking. Mol. Biol of the Cell.8:1261-1271.
Klumperman, J., S. Spijker, J. van Minnen, H. Sharp-Baker, A.B. Smit, and W.P.M. Geraerts. 1996. Cell type specific sorting of neuropeptides: a mechanism to modulate peptide composition of large dense core vesicles. J. of Neuroscience 15:7930-7940.
Klumperman, J., A. Hille, T. Veenendaal, V. Oorschot, W. Stoorvogel, K. von Figura, and H.J. Geuze. 1993. Differences in the endosomal distributions of the two mannose 6-phosphate receptors. J. Cell Biol. 121:997-1010.
1985 - 1989 PhD study at the Laboratory for Electron Microscopy, University of Leiden, the Netherlands (Promotor: Prof. W. T. Daems) in collaboration with the Department for Biochemistry, University of Amsterdam (Promotor: Prof. J.M. Tager).
1989 - 1992 Post-doctoral fellowship at the Department of Cell Biology (Prof. H.J. Geuze), University Medical Center Utrecht (UMCU), the Netherlands.
1992 - 1997 Junior staff member (UD) at the Department of Molecular and Cellular Neurobiology, Free University Amsterdam, The Netherlands.
1997 - 1999 Junior staff member at the Department of Cell Biology, UMCU
1999 - 2001 Senior staff member at the Department of Cell Biology, UMCU
2001 - Full professor Cell Biology and Head of the Cell Microscopy Center, Department of Cell Biology, UMCU.
1999: Co-organiser of the international mini-symposium: ‘Cell Biology of Disease’. AZU-UMCU, Utrecht, The Netherlands.
2000: Co-organizer and chair of the mini-symposium ‘Membrane Traffic’ of the European Electron Microscopy congress EUREM 2000, Brno, Czech Republic, July 9-14, 2000.
2001: Co-organizer of and co-chairperson at the international symposium Molecular mechanisms in membrane transport. June 28-29, Utrecht, The Netherlands.
2001: Vice-president of the organizing committee of the annual meeting of the Dutch Society for Microscopy (NVvM). December 13-14, Papendal Conference Center, The Netherlands
2002: Member of the organizing committee of the 44th International Symposium of the Society for Histochemistry ‘Proteomics in situ: imaging proteins at work’. Vlissingen, 25-28 September 2002, The Netherlands.
2002: Co-organizer and chair of the mini-symposium ‘Organelle Biogenesis and Inheritance’ at the 42nd Annual Meeting of the American Society for Cell Biology (ASCB). 14-18 December 2002, San Francisco, USA.
2004: Member of the Scientific Advisory Board of the 2004 Electron Microscopical Society (EMS) Congress. Anticipated place: Jerusalem, Israel.
- revealing the organization of the many intracellular pathways, both biosynthetic and endocytic
- defining the regulatory mechanisms involved in intracellular protein transport by linking molecular to morphological information.
- define general concepts of protein transport in the cell and how these are adapted in specialized cells.
- diseases that are related to defects intracellular transport (Alzheimer, certain types of cancers)
- correlative live cell imaging and immuno-electron microscopy
- Dutch foundation for scientific research (NWO): ALW, ZonMW, FOM
- Genentech, Inc., San Francisco, USA.
- International foundation for Alzheimer Research ISAO (Internationale Stichting Alzheimer Onderzoek).