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In living cells, internal and external signals are continuously transformed to functional changes. To determine which actions should be taken, information should be efficiently communicated. Therefore, proteins which contain a broad variety of protein domains by which they bind to multiple signalling components are crucial. Postsynaptic density protein, Discs-large, Zona occludens 1 (PDZ) domains are one of the most common protein domains represented in the human sequenced genome and are present in organisms as diverse as bacteria, yeast, plants, invertebrates and vertebrates. They have been shown to play important roles in assembly of various signalling complexes. In 2000 the PDZ protein syntenin-2 (S2) was identified. Interestingly, it was shown that the PDZ domains of S2 bind with nuclear phosphatidylinositol 4,5-bisphosphate (PIP2) and that S2 self-associates in a yeast two-hybrid-assay. Consequently, one can hypothesize that this PDZ protein plays an important role in cell signalling. Therefore, the challenge of this project was to characterise the influence of PIP2 on the nuclear binding behaviour of S2 in living cells. Using previously described S2 PIP2 mutants, we showed that nuclear PIP2 plays an important role in the distribution of eYFP-S2-deltaN (S2 deleted for the first 94 amino acids and fused with eYFP) among three diffusing groups i.e. a fraction which is (almost) freely diffusing, a fraction that is interacting with practically immobile structures, possibly chromatin, and an immobile fraction which indicates long-term binding. More specifically, we showed that nuclear PIP2 favours the enrichment of eYFP-S2-deltaN in the slower diffusing and immobile fraction. Furthermore, our work also clearly indicates that PIP2-independent interactions must exists and we showed by Fluorescence Cross-Correlation Spectroscopy (FCCS) that the self-association of S2 is PIP2-independent. However, further experiments are necessary to shed light on the slow diffusing components of S2-deltaN and these will further clarify S2-deltaN nuclear functions. Recently, A. Hubert (Laboratory for Signal Integration in Cell Fate Decision, Prof. P. Zimmermann, KU Leuven) obtained an extensive list with candidate binding partners of S2 in a proteomic study. In preliminary FCCS experiments, performed to confirm the interaction of three chosen candidates with S2 in living cells, we only observed for the transcription elongation factor A (SII)-like 3 (TCEAL3) a low affinity interaction. In conclusion, cellular differences in PIP2 concentration can influence S2 binding behaviour. All together, this fits well with the speculated function of S2 in nuclear signalling. Finally, we studied a close homologue of S2 with a 70% sequence identity in the PDZ tandem, termed syntenin-1 (S1). In contrast to S2, many binding partners are identified for S1. In addition, it was shown recently that S1 is important in the biogenesis of intraluminal vesicles (ILVs) and exosomes and the segregation of signalling cargo to these vesicles. Density-gradient centrifugation and immune-fractionation suggested that syndecan, S1 and Alix co-accumulate in a distinctive set of exosomes which is consistent with the suspected heterogeneity of exosomes. We showed that the single-molecule approach of Two-Colour Coincidence Detection (TCCD) makes it an excellent tool to study the protein distribution between individual exosomes. To our knowledge, this was the first time that TCCD was used in the study of exosomes. Using TCCD, we confirmed that most but not all S1 accumulates in CD63-positive exosomes. Interestingly, if specific antibody staining of exosomal proteins is optimised, TCCD could become a useful tool to study exosomal samples of patients in the future.

academic collection --- 543.42 <043> --- 547.96 <043> --- Spectrum analysis. Spectroscopy. Spectrography. Spectrometry. Spectrophotometry. Fluorescence analysis--Dissertaties --- Proteins--Dissertaties --- Theses --- 547.96 <043> Proteins--Dissertaties