Biomolecules and nanostructures
The Optical Sciences group studies the interaction of light and matter. Our current focus is on detection and sensing/imaging with an emphasis on the development of integrated photonics. We are part of Twente University's Department of Science and Technology and member of the MESA+ institute.
A Search for the Driving Force in White Blood Cell AdhesionVisualizing LFA-1 clustering dynamics in living cells
Many viruses affect our immune system. For instance the HIV virus, it uses an antigen presenting cell (which recognizes pathogens in the body) as a Trojan horse, to infect many T-cells1. The viruses are very small and the initial steps they take to enter our immune system are on the molecular level. To control illnesses like AIDS, there's the need to fully understand the immune response. And processes on the molecular level play an important role in its functioning.
It is known that leukocyte (white blood cell) adhesion is very important in the immune system: it's a dynamical process which ensures that the leukocytes can 'walk and talk around'. Leukocytes can patrol throughout the vascular system of the body and they can form stable cell-cell contacts due to the adhesion processes. A stable cell-cell contact in important in cell arrest (stop patrolling), in cell migration trough the tissue and in information exchange between leukocytes. Stable leukocyte adhesion is especially accomplished by the adhesion molecule LFA-1 (Leukocyte Function-associated Antigen 1), which is integrated in the cell membrane of leukocytes. These molecules stick to counterpart molecules, called ICAMs (InterCellular Adhesion Molecule), which are present in the cell membrane of leukocytes and other cell types. From former research it is known that LFA-1 mediated adhesion is regulated by conformational changes of LFA-1, as well as by alteration of the spatial distribution on the cell membrane2.
A big question remains, how do these conformational changes and clustering relate to each other in time? On what timescales do these clusters form and disappear and how do they do that? In other words, what is the dynamics of these clusters? These are the questions we like to answer in this project. With the answers, we hope to get a better understanding of the 'walking and talking' of leukocytes in the immune system.
We use a wide field EPI/TIRF microscope setup with an intensified Pentamax CCD camera to investigate the dynamics of fluorescently labeled LFA-1 on stretched THP-1 cells (a cell line from human acute monocytic leukemia cells). To be able to follow moving labeled molecules in the membrane, a fast mechanical shutter has been placed in the excitation path. A TS2/4 antibody - Alexa647 dye conjugate was used to label the LFA-1 proteins in the membrane. Labeling concentrations were kept sufficiently low (0.1µg/ml) to be able to visualize individual clusters and to suppress antibody crosslinking. Recently, we visualized moving clusters containing active LFA-1 on stretched THP-1 cells. We are currently further improving the experimental conditions and developing tracking analysis software to quantify cluster dynamics.
This project is multi disciplinary: the biological part is carried out by Suzanne van Helden, a PhD student at the Tumor Immunology Lab at the NCMLS, Nijmegen. The physics part is carried out here, by Gert-Jan Bakker, a PhD student at the Applied Optics group. Wide Field microscopy experiments will also be carried out in Leiden, in cooperation with the Biophysics group of Prof. Dr. Thomas Schmidt. The project is funded by FOM.
1 Gijtenbeek, T.B. et al., 2001, Cell, 100:587
2 Y. Kooyk, C.G. Figdor, 2000. Curr. Op. Cell Biol., 12:542
Shortcut NCMLS: http://www.ncmls.kun.nl/
Shortcut Biophysics Group Leiden: http://www2.physics.leidenuniv.nl/bfintern//