Spatiotemporal Dynamics of the BRI1 Receptor and its Regulation by Membrane Microdomains in Living Arabidopsis Cells

Abstract

The major brassinosteroid (BR) receptor of Arabidopsis BRASSINOSTEROID INSENSITIVE1 (BRI1) plays fundamental roles in BR signaling, but the molecular mechanisms underlying the effects of BR on BRI1 internalization and assembly state remain unclear. Here, we applied variable angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy to analyze the dynamics of GFP-tagged BRI1. We found that, in response to BR, the degree of co-localization of BRI1-GFP with AtFlot1-mCherry increased, and especially BR stimulated the membrane microdomain-associated pathway of BRI1 internalization. We also verified these observations in endocytosis-defective chc2-1 mutants and the AtFlot1 amiRNA 15-5 lines. Furthermore, examination of the phosphorylation status of bri1-EMS-suppressor 1 and measurement of BR-responsive gene expression revealed that membrane microdomains affect BR signaling. These results suggest that BR promotes the partitioning of BRI1 into functional membrane microdomains to activate BR signaling.

Visualization of BRI1 and SERK3/BAK1 Nanoclusters in Arabidopsis Roots

“…Further analysis employing a one-dimensional Gaussian fit of the fluorescence intensity over the cross section of the PM suggests that most of the observed mobility in the elongation zone is due to replenishment from internal receptor pools (S7 Fig). In a similar study by Wang and coworkers [19], BRI1-GFP was expressed under its native promoter in Col 0 background and a diffusion coefficient of 8.8x10 -3 μm 2 *s -1 was found, which is in similar range to our observations in meristem cells. Taken together, our VAEM and FRAP data suggest that both BRI1 and SERK3/BAK1 receptors are distributed in PM nanoclusters, which are largely immobile and contain only a limited number of BRI1 and SERK3/BAK1 receptors.…”

“…This is conjunction with the work of Wang and coworkers in which a diffusion coefficient of BRI1-GFP of (8.8 ± 0.6) 10 −3 μm 2 *s -1 was found [19]. As in the case of EGFR, animal receptor nanoclusters are thought to be confined by the cortical actin filament network and cholesterol rich domains [43].…”

“…In the elongation zone of Arabidopsis root cells, BRI1-GFP and SERK3/BAK1-GFP showed a slow diffusion (below 0.1 μm 2 *s -1 ) and a mobile fraction of 35 and 60% for BRI1 and SERK3/BAK1, respectively. These values were at the lower end of 0.1–1.0 μm 2 *s -1 , reported for free lateral diffusion of a PM receptor [15, 19] and of control measurement (Fig 2). In meristem cells, the diffusion coefficients were even lower, rendering both receptors virtually immobile (Table 3 and S6 Fig).…”

“…Recently, the plant receptor kinase BRI1 was found to localize in nanoclusters or membrane microdomains in the PM [19]. Upon BR stimulation, an increased colocalization of BRI1-GFP with a membrane microdomain marker protein (AtFlot1-mCherry) was observed.…”

Arabidopsis SYT1 maintains stability of cortical endoplasmic reticulum networks and VAP27-1-enriched endoplasmic reticulum–plasma membrane contact sites

“…The ER marker RFP–HDEL and cyan fluorescent protein (CFP)–HDEL (Lee et al , 2013; Wang et al , 2014), the Golgi marker sialyltransferase (ST)–RFP (Renna et al , 2005; Schoberer et al , 2010), the early endosome marker CLC–mCherry (Wang et al , 2013; Wang et al , 2015), the microtubule marker MBD-MAP4 (microtubule-binding domain of microtubule-associated protein 4)–DsRed (Marc et al , 1998; Granger and Cyr, 2001), and the actin maker ABD2–mCherry (Voigt et al , 2005) were described in the indicated reports.…”