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Connecting peripheral membrane transport and endoplasmic-reticulum stress

Tuesday 28 July 2015

Connecting peripheral membrane transport and endoplasmic-reticulum stress

Dr. Peter van der Sluijs / Prof. Ineke Braakman

Project
We identified Munc18-2 as a possible partner of Munc13-4 in its function in granule exocytosis. Mutations in MUNC13-4 cause Familal Haemophagocytic Lymphohistiocytosis type 3 (FHL3), in which cytotoxic T cells and Natural Killer cells fail to release their lytic granule contents and kill virally infected and transformed cells. Cells depleted of Munc18-2 are viable, as are cells overexpressing a membrane protein in the secretory pathway, but we found the combination to be lethal. ThIs is reminiscent of a 2013 report (Johnson) that connected peripheral membrane transport (rab27) to endoplasmic reticulum (ER) protein folding stress.

In this project we propose to establish the connection of ER (protein folding) stress with regulated exocytosis from lytic granules.

Techniques
Molecular biology methods, qPCR, luciferase reporter assays, lentiviral transduction (of B cells), advanced fluorescence techniques including live cell imaging, TIRF microscopy and FACS analysis, radioactive protein folding assays.


Duration
6 or 9 months

References
Johnson JL, Napolitano G, Monfregola J, Rocca CJ, Cherqui S, Catz SD. 2013. Upregulation of the Rab27a-dependent trafficking and secretory mechanisms improves lysosomal transport, alleviates endoplasmic reticulum stress, and reduces lysosome overload in cystinosis. Mol Cell Biol. 33:158-174.

van der Sluijs P, Zibouche M and van Kerkhof P. 2013. Late steps in secretory lysosome exocytosis in cytotoxic lymphocytes, Front Immunol 18(4): 359
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. 2011. The munc13-4-rab27 complex is specifically required for tethering secretory lysosomes at the plasma membrane. Blood. 118: 1570-1578.
Elstak E, de Jong A, van der Sluijs P. 2011. A platform for complementation and characterization of familial haemophagocytic lymphohistiocytosis 3 mutations. J Immunol Methods. 365:58-66.



Contact
Dr. Peter van der Sluijs, p.vandersluijs@umcutrecht.nl, tel. 088 75 575 78