Loos 2015 Abstract MiPschool Cape Town 2015
|SR-SIM, FRET and photo-activation: imaging autophagy and cell death in cell biology.|
Loos B (2015)
Event: MiPschool Cape Town 2015
Impaired autophagic machinery is implicated in a number of human pathologies such as neuro-degeneration and cancer. A common denominator in these diseases is a dysregulation of autophagy, which has been linked to a change in the susceptibility to undergo cell death. Autophagy is a major protein degradation pathway, which is used to degrade long lived cytoplasmic proteins on the one hand, and to responde to metabolic perturbations or toxicity on the other. Although we have progressed in understanding the molecular machinery and regulation of the autophagic pathway, many unanswered questions remain. How does a change in autophagic flux connect to cell death modalities? Why can both increased and decreased autophagic flux lead to cell death onset? Which are the key parameters that report on the intracellular metabolic state, and how can they quantified sensitively and dynamically? Due to the central role of autophagy in controlling protein degradation and cell death susceptibility, reliable and measurable data points are required that allow to quantify the function (and dysfunction) of organelles associated with the autophagic pathway. By using in vitro model systems, a comnbination of fluorescence microscopy techniques and various fluorescenent constructs, we report on on a highly heterogeneous morphological distribution of both autophagosomes and lysosomes, indicating the dynamic membrane turnover process during autophagosome and lysosome formation. Moreover, a morphologically highly complex fusion zone and mitochondrial network is observed. Furthermore we highlight how these morphological changes can be quantified and expressed organized as single cell pool size data. Our data indicate a previously unknown complexity of membrane morphology and dynamics of these organelles. Importantly, this study highlights the strength of fluorescence resonance energy transfer (FRET), photocativation and super-resolution structured illumination microscopy (SR-SIM) in deriving numerical data that may allow the quantification, prediction and modelling of organelle fusion behaviour and mitochondrial network function especially applicable in neurodegenerative disorders that are characterized by dysfunctional protein clearance.
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