A Membrane Microdomain-Associated Protein, Arabidopsis Flot1, Is Involved in a Clathrin-Independent Endocytic Pathway and Is Required for Seedling Development

Abstract

Endocytosis is essential for the maintenance of protein and lipid compositions in the plasma membrane and for the acquisition of materials from the extracellular space. Clathrin-dependent and -independent endocytic processes are well established in yeast and animals; however, endocytic pathways involved in cargo internalization and intracellular trafficking remain to be fully elucidated for plants. Here, we used transgenic green fluorescent protein-flotillin1 (GFP-Flot1) Arabidopsis thaliana plants in combination with confocal microscopy analysis and transmission electron microscopy immunogold labeling to study the spatial and dynamic aspects of GFP-Flot1-positive vesicle formation. Vesicle size, as outlined by the gold particles, was ;100 nm, which is larger than the 30-nm size of clathrin-coated vesicles. GFP-Flot1 also did not colocalize with clathrin light chainmOrange. Variable-angle total internal reflection fluorescence microscopy also revealed that the dynamic behavior of GFPFlot1-positive puncta was different from that of clathrin light chain-mOrange puncta. Furthermore, disruption of membrane microdomains caused a significant alteration in the dynamics of Flot1-positive puncta. Analysis of artificial microRNA Flot1 transgenic Arabidopsis lines established that a reduction in Flot1 transcript levels gave rise to a reduction in shoot and root meristem size plus retardation in seedling growth. Taken together, these findings support the hypothesis that, in plant cells, Flot1 is involved in a clathrin-independent endocytic pathway and functions in seedling development.

Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice

“…Because DRMs include sphingolipid-and sterol-enriched PM microdomains (Cacas et al, 2012), these results suggest that the decrease in 2-hydroxy sphingolipids by knockdown of Os-FAHs decreased the amount of DRMs, and especially PM microdomains. We also found that rice PIP1s, members of a family of PM microdomain proteins (Hachez et al, 2013), were not present in the DRM fraction of OsFAH1/2-KD, although Os-CHC, which does not localize in PM microdomains (Li et al, 2012), was detected, supporting the idea that PM microdomains would decrease in the DRM fraction of OsFAH1/ 2-KD. In addition, in vivo visualization of PM order using di-4-ANEPPDHQ demonstrated that the heterogeneity of the rice cell PM was lower in OsFAH1/2-KD1 than in the wild type.…”

“…Observation of PM microdomain proteins by immunoelectron microscopy or stimulated emission-depletion microscopy has shown that the diameter of PM microdomains in plant cells is <100 nm (Raffaele et al, 2009;Li et al, 2012;Demir et al, 2013). In this study, PM microdomains themselves were observed as highly ordered puncta by the combination of di-4-ANEPPDHQ and TIRFM and were ;250 nm in diameter.…”

“…Interestingly, a similar result was obtained when Os-Rbohs were detected by an a-StRbohB antibody ( Figure 5B). The accumulation of rice plasma membrane intrinsic protein 1s (Os-PIP1s), an aquaporin that localizes to PM microdomains (Hachez et al, 2013), was confirmed only in the DRM fraction of the wild type, whereas clathrin heavy chain (CHC), which is considered not to localize to PM microdomains (Li et al, 2012), was detected not only in the non-DRM fraction but also in the DRM fraction of both the wild type and OsFAH1/2-KD1 ( Figure 5B). In addition, levels of both proteins gradually decreased during chitin treatment ( Figure 5B).…”

“…These results indicate that Os-Rac1 and Os-Rbohs are specific PM microdomain proteins that respond to chitin elicitor and that PM microdomains are necessary for their localization and dynamics in the PM. Moreover, Os-RACK1 and Os-HSP70, which interact with Os-Rac1 (Thao et al, 2007;Nakashima et al, 2008), and Os-flotillin, a PM microdomain protein (Li et al, 2012), were detected in only DRM fraction of the wild type (Supplemental Figure 10). On the other hand, Os-CEBiP, a chitin receptor, was present in most fractions of both the wild type and OsFAH1/2-KD1 and declined with chitin treatment ( Figure 5B; Supplemental Figure 10).…”

Clathrin-dependent endocytosis is required for immunity mediated by pattern recognition receptor kinases

“…Both clathrindependent and -independent internalization mechanisms have been described in plants (23,24). Because clathrin has been shown to function in BRI1 endocytosis (9), we addressed genetically whether clathrin is required for the internalization of PRRs.…”