Peroxisome research: the need for Fluorescence Activated Cell Sorting (FACS)
10 / 2008 - 12 / 2013
Eukaryotic cells are characterized by the presence of specific compartments, the organelles. The advantage of this compartmentalization is apparently that segregation of sets of enzymes from the cytosol or the creation of unique microenvironments with specific (bio) chemical properties can increase the efficiency of certain cellular processes. As a consequence, the cell had to evolve highly complex regulatory mechanisms for the generation and maintenance of these compartments, a process termed organelle homeostasis. One of the important modes to achieve this is to precisely control the abundance and function of the organelles. In the research group Molecular Cell Biology (formerly Eukaryotic Microbiology) the principles of peroxisome function and homeostasis are studied for over two decades now. Peroxisomes are ubiquitous organelles that were discovered rather late (1954). The functional diversity of the organelles is unprecedented. So far, more than 50 biochemical pathways have been characterized in peroxisomes. Their importance is probably best illustrated by the existence of inherited peroxisome diseases like Zellweger syndrome, which are associated with major neurological impairments that may result in an early death. With the development of fluorescent protein tags (e.g. DsRed, GFP and derivatives thereof) that allow in vivo marking of specific cell compartments, including peroxisomes, novel fluorescence microscopy approaches (confocal laser scanning microscopy, life cell imaging) have become available that have led to ground breaking novel insights in peroxisome research. These methods allow visualizing the organelles in all aspects of their development, turnover and dynamics and have been instrumental to open new approaches in e.g. organelle trafficking and inheritance. To facilitate massive screening of mutants affected in organelle biogenesis/degradation and dynamics, Fluorescence Activated Cell Sorting (FACS) methods are indispensable. However, the available FACS facilities in Dutch laboratories are not available for screening of yeast cultures because of the risk of unwanted contaminations in mammalian cell samples. Moreover, recent improvements in FACS technologies resulted in the development of systems having very sensitive detectors, containing four different laser lines and allowing sorting of single cells to agar or microtiter plates. We aim to use innovative FACS-based methods for sorting of cells on the basis of multiple fluorescent characteristics. This approach is novel in peroxisome research.