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Measuring Endoplasmic Reticulum Viscosity in Cell Models of α1-Antitrypsin Deficiency
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A4637 - Measuring Endoplasmic Reticulum Viscosity in Cell Models of α1-Antitrypsin Deficiency
Author Block: J. Chambers1, M. Kubankova2, R. Huber3, P. Bond4, S. Marciniak1, M. Kuimova2; 1Department of Medicine, University of Cambridge, Cambridge, United Kingdom, 2Department of Chemistry, Imperial College, London, United Kingdom, 3Bioinformatics Institute, Agency for Science, Technology and Research, Singapore, United Kingdom, 4Bioinformatics Institute (BII), Bioinformatics Institute (BII), Singapore, United Kingdom.
Alpha-1-antitrypsin is a serine protease inhibitor produced in the liver that is responsible for the regulation of pulmonary inflammation. The commonest pathogenic gene mutation yields Z-α1-antitrypsin, which has a propensity to self-associate forming protein polymers that accumulate in the endoplasmic reticulum (ER). We have shown that accumulation of Z-α1-antitrypsin polymers leads to a poorly understood phenomenon known as ER-overload, which is accompanied by fragmentation of the tubular ER network into inclusion-like reservoirs. We investigated the effect of polymer accumulation and ER fragmentation on organelle crowdedness and viscosity, biophysical parameters that have the potential to influence ER-protein folding and cellular fitness. Here, we describe a novel technique, Rotor-based organelle viscosity imaging (ROVI), whereby fluorescent molecular rotors are directed to intracellular compartments of live cells for the real time measurement of microviscosity. The application of ROVI in specific organelles revealed significant heterogeneity of microviscosity throughout the cell. In the ER lumen, ROVI visualised novel biophysical effects of Z-α1-antitrypsin expression, providing new insight into the pathogenic mechanisms associated with ER-overload in α1-antitrypsin deficiency.
Author Block: J. Chambers1, M. Kubankova2, R. Huber3, P. Bond4, S. Marciniak1, M. Kuimova2; 1Department of Medicine, University of Cambridge, Cambridge, United Kingdom, 2Department of Chemistry, Imperial College, London, United Kingdom, 3Bioinformatics Institute, Agency for Science, Technology and Research, Singapore, United Kingdom, 4Bioinformatics Institute (BII), Bioinformatics Institute (BII), Singapore, United Kingdom.
Alpha-1-antitrypsin is a serine protease inhibitor produced in the liver that is responsible for the regulation of pulmonary inflammation. The commonest pathogenic gene mutation yields Z-α1-antitrypsin, which has a propensity to self-associate forming protein polymers that accumulate in the endoplasmic reticulum (ER). We have shown that accumulation of Z-α1-antitrypsin polymers leads to a poorly understood phenomenon known as ER-overload, which is accompanied by fragmentation of the tubular ER network into inclusion-like reservoirs. We investigated the effect of polymer accumulation and ER fragmentation on organelle crowdedness and viscosity, biophysical parameters that have the potential to influence ER-protein folding and cellular fitness. Here, we describe a novel technique, Rotor-based organelle viscosity imaging (ROVI), whereby fluorescent molecular rotors are directed to intracellular compartments of live cells for the real time measurement of microviscosity. The application of ROVI in specific organelles revealed significant heterogeneity of microviscosity throughout the cell. In the ER lumen, ROVI visualised novel biophysical effects of Z-α1-antitrypsin expression, providing new insight into the pathogenic mechanisms associated with ER-overload in α1-antitrypsin deficiency.
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