Projects
1. Munc13-4/rab27a complex in NK cell and CTL function
2. Functional analysis of FHL3 missense mutations in UNC13-D
3. Mechanism of centrosome-mediated polarized during CTL cytotoxicity
4. Mechanism of lytic granule maturation in cytotoxic lymphocytes
5. Regulation of endosome function during synaptic plasticity
6. Regulation of cilia dynamics during the cell cycle
7. Re-organization of cellular architecture during mitosis
Duration (of each of the projects)
6 or 9 months
Contact information
Dr. Peter van der Sluijs, T/EM 088-7557578/p.vandersluijs@umcutrecht.nl

Project 1
Munc13-4/rab27a complex in cytotoxic lymphocyte function
Mutations in RAB27a cause Griscelli syndrome type II (GS2). The disease is characterized by haemophagocytosis, and the inability of cytotoxic T cells and Natural Killer cells to release their lytic granule contents and kill virally infected and transdormed cells. We identified
Munc13-4 as a partner of rab27a and showed that the two proteins function in the granule exocytosis pathway. Mutations in MUNC13-4 cause Familal Haemophagocytic
Lymphohistiocytosis type 3 (FHL3) with symptoms that are related to, but distinct from those in GS2. In this project we propose to define the signaling pathways that lead from T cell receptor activation to rab27a and to characterize the function of the Munc13-4/rab27a complex in NK cells and CTLs.
Techniques
Molecular biology methods, lentiviral transduction, proteomics, advanced fluorescence techniques including live cell imaging, TIRF microscopy and FACS analysis.

Project 2
Functional analysis of missense mutations in UNC13-D
Mutations in UNC13-D cause Familal Haemophagocytic Lymphohistiocytosis type 3 (FHL3).
The autoimmune disease is characterized by haemophagocytosis, and the inability of cytotoxic lymphocytes to release their lytic granule contents and kill target cells. In this project we propose to characterize missense mutations of UNC13-D that have been found in FHL3 patients in UMC Utrecht. The approach will help to define the molecular requirents for munc13-4 in the degranulation pathway of cytotoxic lymphocytes.
Techniques
Molecular biology methods, lentiviral transduction, proteomics, advanced fluorescence techniques including live cell imaging, TIRF microscopy and FACS analysis.

Project 3
Mechanism of centrosome-mediated polarized during CTL cytotoxicity
CTLs that encounter a target cell undergo a series of precisely orchestrated architectural changes that ultimately lead to release of granule contents at the immune synapse and subsequent killing of virally infected or transformed cells. It was recently shown that the microtubule organizing center (MTOC) moves the secretory granules to the immune synapse and docks them on the plasma membrane in a process that appears to be related to the formation of cilia. The molecular mechanism that is responsible for centrosomemediated polarization of the secretory apparatus is not known. We recently identified p50, a novel centrosomal protein in haematopoietic cells and found that it interacts with proteins involved in degranulation. In this project we will define the mechanism how p50 functions in this novel polarization process.
Techniques
Molecular biology methods, lentiviral transduction, proteomics, advanced fluorescence techniques including live cell imaging, TIRF microscopy and FACS analysis.

Project 4
Mechanism of lytic granule maturation in cytotoxic lymphocytes
The lytic granule (also known as secretory lysosome) of cytotoxic lymphocytes is a heterogeneous organelle whose formation is dependent on influx of protein and lipid from the trans Golgi network and the endosomal system. Immuno EM analysis of CTLs and mast cells established three distinct forms of secretory lysosomes, likely reflecting different stages of maturation. Munc13-4 is required for the merger of rab11 recycling endosomes with rab27a and rab7-associated late endosomes to form a exocytic vesicle. The granule maturation pathway is essential for the execution of a normal CTL response. In this project we will elucidate the molecular mechanism that regulates the merger of recycling endosomes and late endosomal structures in lytic granule maturation.
Techniques
Molecular biology methods, lentiviral transduction, proteomics, advanced fluorescence techniques including live cell imaging, TIRF microscopy and FACS analysis.

Project 5
Regulation of endosome function during synaptic plasticity
The endosomal system in dendrites is essential for synaptic plasticity. The molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. We identified GRASP-1 as a key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. GRASP-1 regulates spine morphology, synaptic plasticity, and AMPA receptor recycling. GRASP-1 contains a caspase-3 cleavage site, of which we know little to nothing. The first aim is to determine if GRASP-1 is cleaved and if so during which activity of the brain. A second aim is to determine whether neuronal signaling regulate GRASP-1 expression and localization. Thirdly, we aim to find the endosomal receptor for GRAP-1. These experiments will be done in collaboration with the lab of Prof. C. Hoogenraad (Biology, UU)).
Techniques
Analysis of living cells with (dual label) fluorescence microscopy, Lentiviral transduction,
Fluorescence Activated Cell Sorting, Knock down in tissue culture cells with RNAi,
Recombinant DNA methods to prepare expression constructs, Protein methods like SDS-
PAGE, Western blot, immunoprecipitation

Project 6
Regulation of cilia dynamics during the cell cycle
Cilia are microtubule-based structures that protrude from the cell surface, and function as sensors for environmental cues that regulate cellular differentiation or division. In metazoans, ciliary signaling is important both during development and in the homeostasis controls of adult tissues. Cilia are dynamically regulated during cell cycle progression. They are present on interphase cells and usually resorbed before mitotic entry, to re-appear post-cytokinesis. It is not clear how this happens. We recently discovered a possible mechanism that involves regulation of polarized protein transport to the cilium. The aim of this project is to investigate regulation of protein transport to the cilium during the cell cycle with transfected GFP-tagged reporters using live cell imaging and single cell biochemistry.
Techniques
Analysis of living cells with (dual label) fluorescence microscopy, Lentiviral transduction,
Fluorescence Activated Cell Sorting, Knock down in tissue culture cells with RNAi,
Recombinant DNA methods to prepare expression constructs, Protein methods like SDS-
PAGE, Western blot, immunoprecipitation

Project 7
Re-organization of cellular architecture during mitosis
Many proteins controlling cellular organization are phosphorylated during mitosis. We are investigating the function of rab GTPases that play a critical role in membrane traffic and whose activity seems to be controlled by mitotic phosphorylation. In a genome wide screen, we discovered several rabs as novel targets for mitosis-specific phosphorylation.
We then selected one of these for further functional characterization of its phosphorylation. In expression experiments with GFP-tagged mutants, it was discovered that cytokinesis and cell division are impaired if this rab protein can not be phosphorylated. The aim of this project is to investigate the function of rab phosphorylation in dividing cells using live cell imaging and single cell biochemistry.
Techniques
Analysis of living cells with (dual label) fluorescence microscopy, Lentiviral transduction,
Fluorescence Activated Cell Sorting, Knock down in tissue culture cells with RNAi,
Recombinant DNA methods to prepare expression constructs, Protein methods like SDS-
PAGE, Western blot, immunoprecipitation
Recent papers from the group
Elstak ED, Neeft M, Nehme NT, Voortman J, Cheung M, Goodarzifard M, Gerritsen HC, van Bergen En
Henegouwen PM, Callebaut I, de Saint Basile G, van der Sluijs P. Blood. 2011 Jun 21. [Epub ahead of print]
Elstak ED, Te Loo M, Tesselaar K, van Kerkhof P, Loeffen J, Grivas D, Hennekam E, Boelens JJ, Hoogerbrugge
PM, van der Sluijs P, van Gijn ME, van de Corput L. Pediatr Blood Cancer. 2011 Jul 13. doi:
10.1002/pbc.23253. [Epub ahead of print]
Elstak E, de Jong A, van der Sluijs P. J Immunol Methods. 2011 Feb 28;365(1-2):58-66.
Grigoriev I*, Yu KL*, Martinez-Sanchez E*, Serra Marques AA, Keijzer N, Demmers J, Peränen J, Pasterkamp
RJ, van der Sluijs P, Hoogenraad CC, and Akhmanova A. 2011. Rab6 and Rab8 cooperate in controlling docking and fusion of exocytotic carriers. Curr. Biol. 21:967-974 (*equal contributions).
Hoogenraad CC, Popa I, Futai K, Sanchez-Martinez E, Wulf PS, van Vlijmen T, Dortland BR, Oorschot V, Govers
R, Monti M, Heck AJR, Sheng M, Klumperman J, Rehmann H, Jaarsma D, Kapitein LC, and van der Sluijs P.
2010. Neuron specific Rab4 effector GRASP-1 coordinates membrane specialization and maturation of recycling endosomes. PLoS Biol. 8: e1000283.
Kloer DP, Rojas R, Ivan V, Moriyama K, van Vlijmen T, Ghirlando R, van der Sluijs P, Hurley JH and Bonifacino
JS. 2009. Assembly of the biogenesis of lysosome-related organelles complex-3 (BLOC-3) and its interaction with Rab9. J. Biol. Chem. 285: 7794-8004. van Vlijmen* T, Rojas* R, Mardones G, Mohammed S, Heck AJR, Raposo G, van der Sluijs P and Bonifacino JS.
2008. Regulation of retromer recruitment to endosomes by sequential action of rab5 and rab7. (* denotes equal contributions). J. Cell Biol.183: 513-526.
Neeft M, Wieffer M, de Jong AS, Negroiu G, Metz CH, van Loon A, Griffith J, Krijgsveld J, Wulffraat N, Koch H,
Heck AJ, Brose N, Kleijmeer M, van der Sluijs P. Mol Biol Cell. 2005 Feb;16(2):731-41.