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Daniel Wustner, Ph.D - "Quantitative fluorescence imaging of sterol transport through the endocytic pathway"

Associate Professor at the Department of Biochemistry and Molecular Biology (BMB) University of Southern Denmark, Odense, Denmark
When Feb 15, 2017
from 12:00 PM to 01:30 PM
Where Tigem Auditorium Vesuvius
Contact Name
Contact Phone 081-19230659
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Curriculum Vitae

Abstract

Cholesterol is an indispensable molecule in cellular membranes. Its abundance in cells is tightly regulated, both at the metabolic and transcriptional level. Sterols, such as cholesterol, move by vesicular and non-vesicular processes between the plasma membrane (PM) and intracellular sites. However, the molecular mechanisms underlying both transport modes are only partly understood. Here, I will discuss fluorescence-based methods for direct observation and quantitative analysis of sterol transport between PM, recycling endosomes (REs), endoplasmic reticulum (ER) and late endosomes/lysosomes (LE/LYSs) in living cells.  I will discuss how to use and image intrinsically fluorescent and dye-tagged derivatives of cholesterol and its esters, both by wide field and by multiphoton microscopy. I will also discuss recently introduced fluorescent oxysterols, and how their transport differs from that of cholesterol. In the second part, a concrete example of the coupling of vesicular and non-vesicular sterol transport will be presented based on the lysosomal trafficking disorder Niemann Pick type C2 disease. Quantitative imaging of endo-lysosomal sterol trapping in disease fibroblasts and sterol remobilization by internalized Niemann Pick protein C2 (NPC2) will be discussed. In this context, I will explain how we use advanced image analysis tools not only to quantify co-trafficking of sterol binding proteins and their ligands but also to determine the impact of sterol content of LE/LYSs on the mobility and intracellular distribution of these organelles. The discussed methods can be of use in studying the tight coupling of endo-lysosomal sterol trafficking, signalling and organelle dynamics.   

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