Dynamics and Endocytosis of Flot1 in Arabidopsis Require CPI1 Function

Abstract

Membrane microdomains are nano-scale domains (10–200 nm) enriched in sterols and sphingolipids. They have many important biological functions, including vesicle transport, endocytosis, and pathogen invasion. A previous study reported that the membrane microdomain-associated protein Flotillin1 (Flot1) was involved in plant development in Arabidopsis thaliana; however, whether sterols affect the plant immunity conveyed by Flot1 is unknown. Here, we showed that the root length in sterol-deficient cyclopropylsterol isomerase 1 (cpi1-1) mutants expressing Flot1 was significantly shorter than in control seedlings. The cotyledon epidermal cells in cpi1-1 mutants expressing Flot1 were smaller than in controls. Moreover, variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) and single-particle tracking (SPT) analysis demonstrated that the long-distance Flot1-GFP movement was decreased significantly in cpi1-1 mutants compared with the control seedlings. Meanwhile, the value of the diffusion coefficient Ĝ was dramatically decreased in cpi1-1 mutants after flagelin22 (flg22) treatment compared with the control seedlings, indicating that sterols affect the lateral mobility of Flot1-GFP within the plasma membrane. Importantly, using confocal microscopy, we determined that the endocytosis of Flot1-GFP was decreased in cpi1-1 mutants, which was confirmed by fluorescence cross spectroscopy (FCS) analysis. Hence, these results demonstrate that sterol composition plays a critical role in the plant defense responses of Flot1.

Membrane nanodomains and transport functions in plant

“…How plant SPFH proteins are targeted to membranes and are recruited into nanodomains remains largely unknown. However, wild-type (WT) plants treated with the sterol-depleting agent methyl-β-cyclodextrin (mβCD) as well as the sterol biosynthesis mutant cyclopropylsterol isomerase 1 (cpi1-1) display altered intracellular dynamics of Flotillin 1 (Flot1; Li et al, 2012 ; Cao et al, 2020 ). In addition, flotillins possess putative sterol binding motifs named CRAC/CARC, which may participate in the recruitment of flotillins in sterol-enriched domains, although experimental evidences are still needed ( Daněk et al, 2016 ).…”

Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis

“…However, this approach relies on a non-native plant cargo in order to examine the evolutionarily unique process of plant CME. Additionally, researchers have made use of transient expression of CME-related proteins and cargos in tobacco leaf pavement cells as a rapid experimental alternative to generation of novel plant lines (Bandmann et al, 2012;Cao et al, 2020;Gadeyne et al, 2014;Leborgne-Castel et al, 2008;Mbengue et al, 2016). However, it is important to consider that examination of a protein of interest relies on its overexpression, and there appears to be differences in the vesicle trafficking machinery/processing between Arabidopsis and tobacco (Langhans et al, 2011).…”

Single-Molecule Imaging in Living Plant Cells: A Methodological Review

“…TIRFM was first achieved in plant single-molecule imaging in 2011, when Lin’s group first detected and studied single-molecule PIP2;1 and found that it was distributed heterogeneously [ 16 ]. Since then, TIRFM has been widely used to study biological processes occurring on or near cell membranes, such as cell signaling and cytoskeleton assembly [ 83 , 84 ]. However, because of the principle of TIRFM, most studies in living plant cells have been limited to membrane or near-membrane studies.…”