Highly modular protein micropatterning sheds light on the role of clathrin-mediated endocytosis for the quantitative analysis of protein-protein interactions in live cells

Protein micropatterning is a powerful tool for spatial arrangement of transmembrane and intracellular proteins in living cells. The restriction of one interaction partner (the bait, e.g., the receptor) in regular micropatterns within the plasma membrane and the monitoring of the lateral distribution of the bait’s interaction partner (the prey, e.g., the cytosolic downstream molecule) enables the in-depth examination of protein-protein interactions in a live cell context. This study reports on potential pitfalls and difficulties in data interpretation based on the enrichment of clathrin, which is a protein essential for clathrin-mediated receptor endocytosis. Using a highly modular micropatterning approach based on large-area micro-contact printing and streptavidin-biotin-mediated surface functionalization, clathrin was found to form internalization hotspots within the patterned areas, which, potentially, leads to unspecific bait/prey protein co-recruitment. We discuss the consequences of clathrin-coated pit formation on the quantitative analysis of relevant protein-protein interactions, describe controls and strategies to prevent the misinterpretation of data, and show that the use of DNA-based linker systems can lead to the improvement of the technical platform

Multi-level suppression of receptor-PI3K-mTORC1 by fatty acid synthase inhibitors is crucial for their efficacy against ovarian cancer cells

Receptor-PI3K-mTORC1 signaling and fatty acid synthase (FASN)-regulated lipid biosynthesis harbor numerous drug targets and are molecularly connected. We hypothesize that unraveling the mechanisms of pathway cross-talk will be useful for designing novel co-targeting strategies for ovarian cancer (OC). The impact of receptor-PI3K-mTORC1 onto FASN is already well-characterized. However, reverse actions–from FASN towards receptor-PI3K-mTORC1–are still elusive. We show that FASN-blockade impairs receptor-PI3K-mTORC1 signaling at multiple levels. Thin-layer chromatography and MALDI-MS/MS reveals that FASN-inhibitors (C75, G28UCM) augment polyunsaturated fatty acids and diminish signaling lipids diacylglycerol (DAG) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) in OC cells (SKOV3, OVCAR-3, A2780, HOC-7). Western blotting and micropatterning demonstrate that FASN-blockers impair phosphorylation/expression of EGF-receptor/ERBB/HER and decrease GRB2–EGF-receptor recruitment leading to PI3K-AKT suppression. FASN-inhibitors activate stress response-genes HIF-1α-REDD1 (RTP801/DIG2/DDIT4) and AMPKα causing mTORC1- and S6-repression. We conclude that FASN-inhibitor-mediated blockade of receptor-PI3K-mTORC1 occurs due to a number of distinct but cooperating processes. Moreover, decrease of PI3K-mTORC1 abolishes cross-repression of MEK-ERK causing ERK activation. Consequently, the MEK-inhibitor selumetinib/AZD6244, in contrast to the PI3K/mTOR-inhibitor dactolisib/NVP-BEZ235, increases growth inhibition when given together with a FASN-blocker. We are the first to provide deep insight on how FASN-inhibition blocks ERBB-PI3K-mTORC1 activity at multiple molecular levels. Moreover, our data encourage therapeutic approaches using FASN-antagonists together with MEK-ERK-inhibitors

Illustrating the effect of viscoelastic additives on cavitation and turbulence with X-ray imaging

The effect of viscoelastic additives on the topology and dynamics of the two-phase flow arising within an axisymmetric orifice with a flow path constriction along its main axis has been investigated employing high-flux synchrotron radiation. X-ray Phase Contrast Imaging (XPCI) has been conducted to visualise the cavitating flow of different types of diesel fuel within the orifice. An additised blend containing Quaternary Ammonium Salt (QAS) additives with a concentration of 500 ppm has been comparatively examined against a pure (base) diesel compound. A high-flux, 12 keV X-ray beam has been utilised to obtain time resolved radiographs depicting the vapour extent within the orifice from two views (side and top) with reference to its main axis. Different test cases have been examined for both fuel types and for a range of flow conditions characterised by Reynolds number of 35500 and cavitation numbers (CN) lying in the range 3.0–7.7. It has been established that the behaviour of viscoelastic micelles in the regions of shear flow is not consistent depending on the cavitation regimes encountered. Namely, viscoelastic effects enhance vortical (string) cavitation, whereas hinder cloud cavitation. Furthermore, the use of additised fuel has been demonstrated to suppress the level of turbulence within the orifice