Confocal spinning disk microscope with FRAP and TIRF

• Subject Area: Basic Research in Biology and Medicine
• Term: Funded in 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 461336323

For the investigation of subcellular structures and processes, high-resolution microscopy of fluorescently labelled proteins is an essential method. For its studies of dynamic plasma membrane-associated processes, my recently established research group for Nanophysiology at the Technische Universität Kaiserslautern (TUK) is particularly dependent on high-resolution live cell imaging to be able to follow the involved proteins over time. Changes in plasma membrane-bound proteins significantly influence how our cells interact with their environment. The type and number of receptor proteins incorporated in the plasma membrane determines, for example, which signaling pathways are activated and to what extent. For the survival of our organism it is essential that our cells react dynamically to their constantly changing environment. One way to achieve this is the targeted adaptation of their surface proteome via endocytosis. In this process, the plasma membrane around selected surface proteins invaginates with the help of a sophisticated protein machinery to form a vesicle that transports its cargo into the cell. This mechanism allows, for example, to remove adhesion proteins from the surface and thus to modulate cell migration or to internalize postsynaptic glutamate receptors and thus enable synaptic plasticity. We are investigating how individual surface proteins are selected for endocytosis with the help of specific adaptor proteins and what physiological consequences defects in their internalization have, be it for the functioning of our brain or for cell migration. Cell migration requires that cells repeatedly form new focal adhesions as anchor points at their advancing end, which then have to be disassembled again as the cell moves on. Endocytosis is a possible process to remove adhesion proteins from the membrane, but how exactly the controlled disassembly of the complex focal adhesions takes place is not yet understood. In recent years, we have successfully used high-resolution live cell microscopy in a variety of ways to study the process of endocytosis and the regulation of focal adhesions in detail. In order to continue our studies at the TUK successfully, we are applying for a confocal spinning disk microscope together with Prof. Kins and Prof. Storchová, who are also studying dynamic subcellular processes, as this type of microscopy is particularly well suited for both fast and long-lasting multi-colour live cell microscopy. The spinning disk microscope needs to include extensions for FRAP (fluorescence recovery after photobleaching), TIRF (total internal reflection) and super resolution microscopy, in order to resolve the nanometer-scale subcellular structures we are investigating sufficiently well and also to study the dynamics of their components.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Konfokales Spinning Disk Mikroskop mit FRAP und TIRF
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Rheinland-Pfälzische Technische Universität Kaiserslautern-Landau
• Leader: Professorin Dr. Tanja Maritzen