Re-organization of cellular architecture during mitosis
Contact
Dr. Peter van der Sluijs, Department of Cell Biology, UMC Utrecht, p.vandersluijs@umcutrecht.nl,
088-7557580
Background
Many proteins controlling membrane traffic and organelle integrity are phosphorylated
during mitosis and it is thought that this triggers downregulation of transport and
architectural reorganization of intracellular compartments. The activity of the mitotic
kinase cdk1 is required for the inhibition of intracellular transport pathways at the onset
of mitosis in higher eukaryotes. Since rab proteins are critically involved in membrane
transport, their activity might be controlled by mitotic phosphorylation, as we orginally
showed for rab4a. To address this hypothesis, we undertook a bioinformatic screen for
conserved S/T-P sites in rab proteins as these residues are invariably present in the
consensus motif for substrates of proline-directed kinases. We identified rab1b, rab7b,
rab8a and rab40 as novel targets for mitosis-specific phosphorylation by cdk1. We then
selected rab8a to investigate the function of phosphorylation. Rab8 is required for
membrane delivery from endosomes to the plasma membrane and it serves an important
role in the biogenesis of cilia. We created GFP-rab8a and the GFP-tagged rab8aS181A
mutant that can not be phosphorylated by cdk1. In preliminary live imaging experiments
we discovered that expression of the mutant gave rise to multinucleated cells and
abnormal cytokinesis.
Objective
The aim of this project is to investigate the function of rab8a phosphorylation in dividing
cells using live cell imaging.
Methods
- Recombinant DNA methods to prepare expression constructs
- Protein methods like SDS-PAGE, Western blot, immunoprecipitation
- Cell culture and transfections
- Knock down in tissue culture cells with RNAi
- Analysis of living cells with (dual label) fluorescence microscopy
- Fluorescence Activated Cell Sorting
Selected papers from the group
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., in press
- 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 C, van Loon A, Krijgsveld J, Griffith J, Wulffraat N, Koch H,
Heck A, Brose N, Kleijmeer M, and van der Sluijs P. 2005. Munc13-4 is an effector of rab27a and controls
secretion of lysosomes in haematopoietic cells. Mol. Biol. Cell 15, 731-741.