Analyse von Markerproteinen für Plasmamembran-abgeleitete Mikrovesikel

by Leon Kaiser
Doctoral thesis
Date of Examination:2023-07-12
Date of issue:2023-07-05
Advisor:Prof. Dr. Annalen Bleckmann
Referee:Prof. Dr. Annalen Bleckmann
Referee:Prof. Dr. Hassan Dihazi
Persistent Address: http://dx.doi.org/10.53846/goediss-9978

 

 

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Abstract

English

Cells are known to secrete extracellular vesicles (EVs) into their microenvironment. EVs act as mediators of intercellular communication, can travel long distances, and are involved in various ways in the progression and metastasis of malignant cells in the context of tumor diseases. Exosomes (Exo) (50-150 nm), which originate from an endosomal sorting process, are distinguished from microvesicles (MV) (100-1000 nm) that directly bud off from the plasma membrane. While the biogenesis of Exo is mainly dependent on the syndecan-syntenin-Alix signaling pathway and various ESCRT proteins, the mechanisms of MV budding are still poorly understood. At the beginning of this study, the research group had results from a stable isotope labeling with amino acids in cell culture (SILAC) analysis, in which the protein expression in MV and Exo from SK-BR-3 breast cancer cells was quantitatively compared. Assuming that proteins specifically expressed in MV could be relevant to their biogenesis, the aim of this study was to validate seven potential MV marker proteins identified in the SILAC analysis and analyze their impact on MV release. The MV-specific expression of the candidate proteins was examined using immunoblots in various malignant and one benign cell line. The analyses identified KIF4A and PRC1 as new marker proteins specifically expressed in MV and not in Exo. In the second part of the study, both proteins were investigated for their function in EV release. After siRNA-mediated knockdown of the proteins in SK-BR-3 cells, the size and number of secreted EVs were quantified using nanoparticle tracking analysis (NTA). In this regard, no direct effect was observed on MV, but surprisingly, the KIF4A knockdown led to a decreased number of Exo. In summary, this study successfully identified and validated two new MV-specific proteins, KIF4A and PRC1. This makes them useful markers for distinguishing MV from the significantly smaller Exo. Further investigations are needed to understand the influence of both proteins on EV release. Only through a more comprehensive understanding of EV biogenesis can we fully exploit the therapeutically applicable potential of EVs in the future and contribute to the treatment of malignant cancer diseases.
Keywords: ev; extracellular vesicles; microvesicles; exosomes; biogenesis; proteins; kif4; prc1; cytoskeleton; breast cancer; cancer; exosomes
 
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