Single-molecule fluorescence imaging to quantify membrane protein dynamics and oligomerization in living plant cells View Full Text


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

2015-12

AUTHORS

Xiaohua Wang, Xiaojuan Li, Xin Deng, Doan-Trung Luu, Christophe Maurel, Jinxing Lin

ABSTRACT

Measuring the mobility and interactions of proteins is key to understanding cellular signaling mechanisms; however, quantitative analysis of protein dynamics in living plant cells remains a major challenge. Here we describe an automated, single-molecule protocol based on total internal reflection fluorescence microscopy (TIRFM) imaging that allows protein tracking and subunit counting in living plant cells. This protocol uses TIRFM to image transgenic plant tissues expressing fluorescently tagged proteins that are localized to the plasma membrane. Next, a tracking algorithm quantifies dynamic changes in fluorescent protein motion types, temporary particle displacement and protein photobleaching steps. This protocol allows researchers to study the kinetic characteristics of heterogeneously distributed proteins. The approach has potential applications for studies of protein dynamics and subunit stoichiometry for a wide variety of plasma membrane and intracellular proteins in living plant cells and other biological specimens visualized by TIRFM or other fluorescence imaging techniques. The whole protocol can be completed in 5-6 h. More... »

PAGES

2054-2063

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/nprot.2015.132

DOI

http://dx.doi.org/10.1038/nprot.2015.132

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1048041440

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/26584445


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