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Project

Superresolutie fluorescentie microscopie gebaseerde technieken voor de studie van signaaltransductie.

To transmit a message from one to another, correct communication is crucial. To obtain correct communication, it is important that both transmitter and receiver of the signal are present at the correct time at the correct location. In a cell this communication is taken care of by signal transduction pathways. They consist of a highly branched network of proteins and messenger molecules, integrating signals to a final work plan guiding the cell in its functions mediating both short- and long-term responses. Therefore, proper distribution in time and space of the components of these networks is crucial for cell behaviour and fate. Single molecule fluorescence microscopy has been proven an excellent tool to study distributions and behaviour of single molecules in time and space providing detection of heterogeneities otherwise lost in ensemble averaging.

The objectives of this dissertation were to expand and use fluorescence microscopy tools to study signal transduction in mammalian cultured cells at a high spatial resolution as well as on a single molecule level with as a case study, the EGFR/ MAPK signal transduction pathway.

The results are divided in two parts. The first part, 'Fluorescent probes for microscopy', explores the utility of single emission band optical highlighting with LSSmOrange (chapter 5). This study shows that LSSmOrange can be photoconverted in living cells with 'blue' light irradiation. Since minimal binding to endogenous proteins was observed, LSSmOrange is suitable for optical highlighting incellulo. In addition, highlighting experiments were easily expanded to multicolour imaging due to an invariable emission band. Furthermore, confined photoconversion of LSSmOrange was achieved in three dimensions with a femtosecond laser pulse via a two-photon process.

The second part, 'Single molecule fluorescence microscopy applied to signal transduction', consists of chapters 6-8. These chapters deals with single molecule methods to study signal transduction. Protein-protein interactions (PPIs) form the basis of cellular processes regulating cell fate and can be extremely transient. In chapter 6, an approach to detect these short-lived transient interactions on the plasma membrane in living cells was developed. This approach is based on selective detection of interacting single molecules. This was possible due to the difference in mobility between proteins on the membrane and in the cytosol, and therefore decrease in motion blur of the fluorescence signal. The proof of concept was performed with a model system based on modular protein domains interacting with short linear peptide motifs. Interacting molecules are detected and localized. By accumulation of all localizations, an interaction map at the nanoscale was drawn. This approach was then used to image the interaction of several cytosolic proteins involved in the EGF signalling pathway with the plasma membrane. Interaction maps of Grb2, PLC-γ and c-Raf show that these molecules interact with different compartments at the plasma membrane, illustrating the role of membrane heterogeneity in the spatial regulation of cell signalling.

In chapter 7, we use this method to investigate the role of receptor distribution in clathrin coated pits on the membrane and on vesicles in the cytosol. Clathrin mediated endocytosis (CME) serves to downregulate membrane receptors but several studies show involvement of this compartmentalization in signalling. Downstream molecules interact with the receptor during signal transduction. To observe the role of EGFR enriched pits and vesicles due to CME, we recorded c-Raf interactions throughout the cell together with the EGFR or clathrin distribution. An analysis method to correlate the receptor distribution with the c-Raf interactions was developed. No increased number of interacting c-Raf molecules was observed located on clathrin coated pits (CCPs) or cytosolic vesicles (CCVs) suggesting that neither on the basal plasma membrane or in the cytosol, EGFR accumulation in CCPs and CCVs played a role as a platform where c-Raf molecules are efficiently binding and activated.

 In the last chapter (chapter 8), molecular mobility is linked with the cellular response. A cell is a complex entity and response to a trigger can be inhomogeneous between cells.  This response is regulated on the molecular level. Proteins such as receptors on the plasma membrane move around and this movement is linked to molecular interactions and the protein environment. To analyze the molecular dynamics in relation to the response, a system was developed to perform functional imaging during aquisition for SPT analysis and applied to the EGFR in its heterogeneous Ca2+ response upon a trigger with submaximal concentration of EGF. Different diffusion properties of the EGFR were revealed in responding and non-responding cells which is thought to reflect the shift in monomer–dimer equilibrium of the EGFR.

Date:1 Jan 2013 →  14 Mar 2017
Keywords:Superresolutie fluorescentie Microscopie, Signaaltransductie
Disciplines:Biochemistry and metabolism, Medical biochemistry and metabolism, Systems biology
Project type:PhD project