Applying optical tweezers with TIRF microscopy to quantify physical interactions between organelles in the plant endomembrane system

Ellie Fletcher, Benji C. Bateman*, Stanley W Botchway, Andrew Ward, Imogen A Sparkes*

*Corresponding author for this work

Research output: Contribution to journalProtocol

Abstract

Plant organelles are associated with each other through tethering proteins at membrane contact sites (MCS). Methods such as total internal reflection fluorescence (TIRF) optical tweezers allow us to probe organelle interactions in live plant cells. Optical tweezers (focused infrared laser beams) can trap organelles that have a different refractive index to their surrounding medium (cytosol), whilst TIRF allows us to simultaneously image behaviors of organelles in the thin region of cortical cytoplasm. However, few MCS tethering proteins have so far been identified and tested in a quantitative manner. Automated routines (such as setting trapping laser power and controlling the stage speed and distance) mean we can quantify organelle interactions in a repeatable and reproducible manner. Here we outline a series of protocols which describe laser calibrations required to collect robust data sets, generation of fluorescent plant material (Nicotiana tabacum, tobacco), how to set up an automated organelle trapping routine, and how to quantify organelle interactions (particularly organelle interactions with the endoplasmic reticulum). TIRF-optical tweezers enable quantitative testing of putative tethering proteins to reveal their role in plant organelle associations at MCS.
Original languageEnglish
Article numbere854
JournalCurrent Protocols
Volume3
Issue number8
DOIs
Publication statusPublished - 9 Aug 2023

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