The Arabidopsis receptor kinase STRUBBELIG undergoes clathrin-dependent endocytosis

Abstract

Signaling mediated by cell surface receptor kinases is central to the coordination of growth patterns during organogenesis. Receptor kinase signaling is in part controlled through endocytosis and subcellular distribution of the respective receptor kinase. For the majority of plant cell surface receptors, the underlying trafficking mechanisms are not characterized. In Arabidopsis, tissue morphogenesis requires the atypical receptor kinase STRUBBELIG (SUB). Here, we studied the endocytic mechanism of SUB. Our data revealed that a functional SUB–enhanced green fluorescent protein (EGFP) fusion is ubiquitinated in vivo. We further showed that plasma membrane-bound SUB:EGFP becomes internalized in a clathrin-dependent fashion. We also found that SUB:EGFP associates with the trans-Golgi network and accumulates in multivesicular bodies and the vacuole. Co-immunoprecipitation experiments revealed that SUB:EGFP and clathrin are present within the same protein complex. Our genetic analysis showed that SUB and CLATHRIN HEAVY CHAIN (CHC) 2 regulate root hair patterning. By contrast, genetic reduction of CHC activity ameliorates the floral defects of sub mutants. Taken together, the data indicate that SUB undergoes clathrin-mediated endocytosis, that this process does not rely on stimulation of SUB signaling by an exogenous agent, and that SUB genetically interacts with clathrin-dependent pathways in a tissue-specific manner.

Protocol for rapid clearing and staining of fixed Arabidopsis ovules for improved imaging by confocal laser scanning microscopy

“…Confocal laser scanning microscopy of tissue expressing GFP-based reporters was done using standard procedures, as described earlier [70, 71]. Confocal laser scanning microscopy of ovules stained with SR2200 and TO-PRO-3 iodide was performed on an upright Leica TCS SP8 X WLL2 HyVolution 2 (Leica Microsystems) equipped with GaAsP (HyD) detectors and a 63× glycerol objective (HC PL APO CS2 63×/1.30 GLYC, CORR CS2).…”

Arabidopsis O-fucosyltransferase SPINDLY regulates root hair patterning independently of gibberellin signaling

“…8 ). The atypical receptor-like kinase SCRAMBLED (SCM) plays an important role in signaling from the cortex to the epidermis and further on to WER in this context ( Gao et al, 2019 ; Kwak et al, 2005 ). Further experiments targeting the function, localization or turnover of SCM might help determine how SPY participates in cell-to-cell communication at this stage, or alternatively in upstream signaling events in the cortex.…”

Arabidopsis O-fucosyltransferase SPINDLY regulates root hair patterning independently of gibberellin signalling

“…Monitoring the expression of WER by using a transcriptional reporter suggests that the patterning defect in spy-22 is established already early on during epidermal cell fate determination, potentially due to defects in cortex development or cell-to cell communication between cortex and epidermis, as these processes regulate cell type specific WER expression levels. The atypical receptor-like kinase SCRAMBLED (SCR) plays an important role in signalling from the cortex to the epidermis and further on to WER in this context (Gao et al 2019; Kwak et al 2005). Further experiments targeting the function, localization or turn-over of SCR might help determining how SPY participates in cell-to-cell communication at this stage, or alternatively in upstream signalling events in the cortex.…”