In eukaryotic cells, a network of organelles and membranes forms a highly organized and dynamic endomembrane system. The precise trafficking of plasma membrane (PM) proteins within this system is crucial for their proper function, ultimately influencing plant growth and development.
MitTally (Mitochondria Tally marks)
Under construction
PSB researchers are actively involved in generating tools to allow real-time visualization of plants under conditions that disturb specific processes. Examples are the use of microfluidics setups to monitor the effect of chemical compounds as well as the effect of altered temperature, on cellular dynamics.
Dynamic imaging of fast-occurring processes is hampered by the fact that the signal/noise ratio of a fluorescent image is inversely related to the exposure time. The exposure time in turn limits the imaging speed and thereby the temporal resolution. We are increasing the temporal resolution of live-cell imaging plant samples by modulating the temperature of the samples in situ on the microscope stage as lowering the temperature slows down biological processes.
Proximity biotinylation uses a promiscuous biotin ligase, which causes biotinylation of proteins in the vicinity of the bait. These biotinylated proteins can be identified using mass spectrometry without the need to maintain the protein-protein interactions during the purification. This tool is therefore especially suited for interactions between cytosolic and transmembrane proteins. We are designing protocols to adequately perform proximity biotinylation in plants.
To gain insight into protein-protein interactions that occur in planta, we expanded the interaction assay toolkit. We have developed Knocksideways in plants (KSP) as an imaging-based protein-protein interaction assays to visualize binary as well as higher-order interactions by. KSP can be compared to an intracellular Co-IP experiment. The tool uses the ability of rapamycin to alter the localization of a bait protein and its interactors via the heterodimerization of FKBP and FRB domains.
Description
The PSB features 5 confocal microscopes, ranging from standard devices to more advanced ones. Depending on your experiment, one device will suit your need more than another. Below are some guidelines for choosing the most suitable confocal.
A main criteria is speed.
Whenever speed is crucial than the spinning disc is the best choice. Speed can be in time: imaging of fast events (below 1 sec), in space: large regions to be obtained by stitching and Z-stack, or a combination with multiple colours and Z-stack.
