Abstract
FRAP (Fluorescence Recovery After Photobleaching) is probably the most direct method to investigate the dynamics of molecules in living cells. Here, we describe FRAP to quantify membrane fluidity in C. elegans. Using FRAP, we have shown that cold, glucose and exogenous saturated fatty acids can decrease the fluidity of cellular membranes in certain mutants.
Keywords: FRAP, Membrane fluidity, Prenylated GFP, C. elegans, Fatty acid, PAQR-2
Background
Biological membranes, defining features of all cells, are primarily composed of phospholipids (Van Meer et al., 2008). The fatty acid species present in the phospholipid bilayer greatly influence its properties. For example, a high saturated fatty acid content increases membrane rigidity while a high unsaturated fatty acid content promotes fluidity (Pilon, 2016). The fatty acid composition of cellular membranes often shows clear correlations with the composition of dietary fats, which can be incorporated directly into phospholipids (Abbott et al., 2012; Dancy et al., 2015). However, considering that wide variations in diets exist, cells must have regulatory mechanisms that monitor and adjust membrane composition and achieve the desired membrane properties, such as fluidity, curvature, thickness, etc. Using FRAP on worms that express a prenylated GFP in intestinal cells, we have previously shown that mutants lacking PAQR-2, a homolog of the mammalian adiponectin receptors, have reduced membrane fluidity upon cultivation in the presence of glucose or on diets rich in saturated fatty acids (Svensk et al., 2016; Devkota et al., 2017). During FRAP, fluorescent molecules in a specified region are photobleached using a high-power laser and subsequent recovery of the bleached region is recorded and quantified (Reits and Neefjes, 2001). Here we describe a detailed FRAP protocol to study the fluidity of membranes in C. elegans.
Materials and Reagents
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Acknowledgments
We thank the Center for Cellular Imaging at University of Gothenburg for microscopy support. This work was funded by Vetenskaprådet, Cancerfonden, Carl Trygger Stiftelsen, Diabetesfonden and Kungliga Vetenskaps och Vitterhets-Samhället. The authors declare no conflicts of interest or competing interests.
References
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