Browsing by Subject "endocytosis"
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(2022)Clathrin-mediated endocytosis is the most common pathway by which cells internalize cargoes from the membrane. It is a critical process in cell communication, development, and homeostasis. In order to study endocytic dynamics, it is critical that one can clearly distinguish receptors that have entered the cell from those which remain on the cell membrane. Current techniques for investigating endocytosis rely on removing membrane-bound components with harsh treatments which may interfere with cell physiology, and often depend on antibodies which are not widely available and - even when they are - may give unreliable signals and may affect receptor behavior and internalization rates. Additionally, a large portion of studies on clathrin-mediated endocytosis have been done on a single receptor, the transferrin receptor. Here we have developed a new assay which resolves the above issues through use of a novel protein probe. This fusion protein will allow us to resolve the issues with current endocytic assays mentioned above, and in theory can be used to study any membrane receptor which is endocytosed. Our preliminary results show that we can use our protein to effectively track endocytosed receptors without interference from signal of receptors remaining on the cell membrane. This shows that our protein may be a powerful tool for studying endocytosis across a wide variety of membrane-bound receptors.
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(2020)Intersectins (ITSNs) are important scaffold and adaptor proteins that play an important role in various cellular processes such as endocytosis. Although we know a lot about their function, there is little information on the regulation of these proteins. On the other hand, microRNAs have been shown to have an extensive function in regulating numerous genes in animals and their dysfunction is credited for down regulation of many proteins. In this study, I demonstrate that microRNAs are potential regulators of ITSNs in HEK293 cells and human neuronal cell cultures. In this study, I cloned 3’UTRs of different isoforms of intersectins (ITSNs) and microRNAs to the expression vectors to express them in cells. I then transfected HEK293T or neuronal stem cell line (HEL47.2) with the constructed vectors and used various methods to analyse the effect of microRNAs on the expression of ITSNs. The main methods I used were dual-luciferase assay, reverse transcription quantitative PCR and western blotting, human neuronal stem cell culturing and lentiviral transduction. My results demonstrate that there were two microRNAs that stood out from other and had a significant downregulation of ITSNs mRNA levels in HEK293T cells. Those were miR-124 and miR-19. However, in the human neuronal cell line I did not observe a significant alteration of the ITSNs transcript level. Additionally, I suggest that the given microRNAs regulate protein levels by promoting the decay of the ITSN transcripts. However, more studies are needed to show a stronger causative effect of microRNAs on ITSNs. Subsequent studies should also look at how multiple microRNAs can influence gene expression cooperatively.
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