Multiplexed measurement of protein diffusion in Caenorhabditis elegans embryos with SPIM-FCS

作者：Struntz Philipp^*; Weiss Matthias

来源：Journal of Physics D: Applied Physics , 2016, 49(4): 044002.

DOI：10.1088/0022-3727/49/4/044002

• SPIM
• FCS
• diffusion
• Caenorhabditis elegans
• fluctuation microscopy
• light sheet microscopy
• peripheral membrane protein

Quantifying the diffusion behavior of proteins in different environments, e.g. on cellular membranes, is a key step in uncovering the vital action of protein networks in living organisms. While several established techniques for local diffusion measurements exist, the life sciences are currently in need of a multiplexed, i.e. spatially parallelized, data acquisition that allows for obtaining diffusion maps with high spatiotemporal resolution. Following this demand, the combination of camera-based single-plane illumination microscopy (SPIM) and fluorescence correlation spectroscopy (FCS) has recently emerged as a promising approach. So far, SPIM-FCS has mainly been used to assess the diffusion of soluble particles and proteins in vitro and in culture cells, but due to a particularly low photobleaching and -toxicity the method is also well applicable to developmental organisms. Here, we have probed the performance of SPIM-FCS on an established developmental model organism, the small nematode Caenorhabditis elegans. In particular, we have quantified the diffusion of the peripheral membrane protein PLC1 delta 1 in the embryo's cytoplasm and on the plasma membrane. As a result, we were able to derive diffusion maps of PLC1 delta 1 in both compartments in multiple individuals, showing the spatially varying diffusion coefficients across the embryo. Our data also report on the dissociation kinetics of PLC1 delta 1 from the plasma membrane, hence underlining that SPIM-FCS can be used to explore key features of peripheral membrane proteins in fragile developmental model organisms.

• 出版日期2016-2-3