Project 1. Platelet tether formation in real time (6-9 months rotation) 
Blood platelets are a-nucleated discoid cells, 2-5 um diameter large, that derive from the megakaryocyte in the bone marrow. Platelets circulate in a non-adhesive state and are committed to be removed within 7-10 days of circulation. Circulating platelets are crucial for the maintenance of the haemostatic balance. Upon vascular injury they rapidly interact with components of the damaged vessel wall, i.e. collagen, von Willebrand factor, which is a prerequisite for the arrest of bleeding. Upon adhesion to these subendothelial components, platelets become activated, change shape, and release the contents of their storage granules, enabling progression and stabilization of platelet-substrate (spreading) and platelet-platelet interaction (aggregation). To study platelet adhesive responses under flow we make use of a perfusion model, by which whole blood is drawn over immobilized vessel wall components under different shear conditions. Using this model we have established that under high-shear conditions platelets roll and tether on immobilized vWF, before becoming irreversibly attached. Platelet rolling and tethering is the result of transiently formed discrete adhesion points (DAP). DAPs contain high numbers of GPIb, the receptor for VWF. DAPs are also the starting point of long membrane tethers that are pulled out of the platelet body. These membrane tethers can be severalmicrometerslong. Possibly due to shear stress tethers severe and travel further in circulation as microparticles. Analysis by reflection interference contrast microscopy (RICM) has also shown that tethers can retract. Little is known on the mechanism of their formation and the composition of these membrane tethers.
Central theme of the rotation will be the application of live-cell imaging techniques to visualize platelet tethering and cell spreading under high shear conditions using state of the art imaging equipment available at the Cell Microscopy Center.
Questions to address: * Is passive shear stress the solely force that drives the formation of these membranes?
* Are microtubules and/or motor proteins involved in tether formation?
* What is the composition of these membrane tethers?
Techniques: Platelet flow studies, Live Cell Imaging, Immunofluorescence, Confocal Microscopy, Immuno Electron Microscopy.
Duration: 6-9 months
Equipment: Delta Vision
Zeiss CSLM 510 Live cell imaging station with 32 PMT meta-detector
Zeiss UltraView Live Cell Imager
JEOL Electron Microscopes
Info: Dr. Harry F. G. Heijnen, Cell Microscopy Center, UMCU, Phone: +31.88.755.7654 / Fax +31.88.755.5418 email: h.f.g.heijnen@umcutrecht.nl
Project 2. Autophagy in dendritic cells (6-9 months master rotation) Dendritic cells (DCs) are specialized antigen-presenting cells that continuously scavenge for pathogens in peripheral tissues and their immediate surroundings. DCs capture foreign antigens, process them to small peptides and subsequently load them onto MHC class II molecules in the endosomal/lysosomal compartment (MIIC). During this process the dendritic cell matures, and the peptide-bound class II molecules are transported to the cell surface to be presented to T cells. During this maturation program the MIIC undergoes profound structural changes: internal membranes within the MIIC fuse with the limiting membrane, and MIICs fuse with each other to form a large tubular network. In vitro maturation of dendritic cells and starvation induces autophage-like double membrane structures, and expression of LC3, a marker for autophagy. Little is known on the regulatory role of autophagy in antigen presentation. Autophagy in dendritic cells may play a role in MHC class II based presentation of viral and/or self antigens.
Aim:
To further elucidate the role of autophagy in antigen presentation in DCs, the master will use the DC cell line D1 and live cell imaging techniques. DCs will be exposed to LPS under normal and starvation conditions, and the transformation of MIICs will be studied in real time using fluorescent loading of MIICs. Formation of autophagosomes will be studied after transfection with a GFP-LC3 construct.
Techniques: Cell culture, molecular biology, confocal microscopy, live cell imaging, immuno electronmicroscopy.
Equipment: Delta Vision, Zeiss CSLM 510, Zeiss Ultra Viewer, JEOL Electron Microscopes
Schedule: Month 1 & 2 1) Learn the basic principles of cell culturing, immuno fluorescence, confocal microscopy and live cell imaging.
2) Start loading experiments and transfections of GFP- LC3 constructs into 2 cell lines
Month 2–5 1) Continuation of the above mentioned points until finished.
2) Experiments with GFP transfected D1 cells will be performed. Induction of autophagy, serum starvation and rapamycin treatment.
3) Investigate the role of microtubules in autophagy. If time permits the role of nitric oxide in autophagy will be investigated as well.
Month 5 & 6
Finishing up experiments and writing of the report.
Info: Dr. Harry F. G. Heijnen, Cell Microscopy Center, UMCU, Phone: +31.88.755.7654 / Fax +31.88.755.5418 email: h.f.g.heijnen@umcutrecht.nl