Ultrastructural aspects of endosomal signalling

Communication between cells is based on signal perception at the cell surface, followed by intracellular signal transduction, and finally the generation of biological responses. Here, we will use modern genetics combined with molecular cell biology and high-resolution microscopy for investigating the spatial and temporal properties of intracellular signal transduction from one type of cell surface receptor to its associated intracellular pathway (i.e., signal transduction from the activated epidermal growth factor receptor (EGFR) to the mitogen-activated protein kinase pathway (MAPK)). In order to evoke an appropriate biological response, MAPK-signalling requires specification. Specification can be achieved by the use of scaffold proteins that organise pathway components into signalling complexes. Interestingly, different scaffold complexes were identified to reside at the plasma membrane and on specific intracellular membrane-bound compartments, so-called endosomes. However, little is known about the compartmentalisation of signalling. Therefore, it is of high interest to investigate how plasma membrane activated receptors are diverted from early endosomes and routed to late endosomes, where they partition into different membrane domains and meet scaffold complexes. These signalling complexes will be visualised at the plasma membrane and on endosomes. Vice versa we will analyse the impact of MAPK-signalling on the organisation of endosomal compartments at the ultrastructural level. To summarise, on the basis of high-resolution imaging methods we will generate a "subcellular activity map" of one highly important signalling pathway. Such a map could serve as a diagnostic blue print for normal signalling, and could therefore shed new light on defective signalling processes, as occuring, for example, in cancer.

A prerequisite for communication between cells is perception of signals at the cell surface through specific receptor molecules. This is followed by signal transfer within the cell, and finally by evoking appropriate biological responses to these signals. One important representative of growth factor receptors is the so-called epidermal growth factor receptor (EGFR) necessary for regulating cellular proliferation and differentiation. Notably, hyperactivation and overexpression of EGFR occurs in many types of cancer and correlates with poor clinical outcome. Transduction of cellular signalling events through the EGFR activates signalling pathways that stimulate many of the properties associated with cancer cells: proliferation, migration, stromal invasion and tumour neovascularisation. Activation of the EGFR not only results in signalling but also in uptake of the active receptor into cells and their transport through structurally and biochemically distinct membrane compartments. Over the past years multiple lines of evidence have, thus, established that certain kinds of intracellular membrane vesicles, so- called `endosomes` play a key role in regulating EGFR signalling. Our research aims at a better understanding of the multiple in vivo functions of a particular signalling module locating at specialised endosomal membrane domains of cells from vertebrates but also, for example, fruit flies. This signalling module or complex consists of an endosomal adaptor protein, called p14 and its interaction partners. We found that p14-bearing endosomal vesicles are responsible of specific spatial and temporal fine tuning of ERK activation that is essential for timing cell cycle progression. Furthermore, it was shown that signalling from endosomes generates a biological output that differs from the output generated by signalling from the cell surface. In line with this, we found that also the correct positioning of signalling endosomes within the cell body is crucial for proper signalling quality, including accurate duration of the signaling process. We further addressed the simple, but so far unresolved question, namely, what `signalling endosomes` are. In particular, we characterised signalling endosomes and their subdomains in detail with respect to their dynamic ultrastructural and biochemical properties in relevant cell types of model organisms. For this we used genetically modified murine cell cultures, devoid of the above mentioned adaptor protein 14 or one of its interaction partners, namely MEK1. One major task for answering these questions was to set-up optimised high-resolution imaging methods such as cryo-electron microscopy and live-cell video microscopy to be used under complex experimental conditions. By doing so, we produced a detailed "subcellular map" with high spatial and temporal resolution of key components of one highly important signalling pathway (i.e., the MEK/ERK/MAPK signalling pathway).