Cellular requirements for PIN polar cargo clustering in Arabidopsis thaliana

Abstract

Cell and tissue polarization is fundamental for plant growth and morphogenesis. The polar, cellular localization of Arabidopsis PIN-FORMED (PIN) proteins is crucial for their function in directional auxin transport. The clustering of PIN polar cargoes within the plasma membrane has been proposed to be important for the maintenance of their polar distribution. However, the more detailed features of PIN clusters and the cellular requirements of cargo clustering remain unclear. Here, we characterized PIN clusters in detail by means of multiple advanced microscopy and quantification methods, such as 3D quantitative imaging or freeze-fracture replica labeling. The size and aggregation types of PIN clusters were determined by electron microscopy at the nanometer level at different polar domains and at different developmental stages, revealing a strong preference for clustering at the polar domains. Pharmacological and genetic studies revealed that PIN clusters depend on phosphoinositol pathways, cytoskeletal structures and specific cell-wall components as well as connections between the cell wall and the plasma membrane. This study identifies the role of different cellular processes and structures in polar cargo clustering and provides initial mechanistic insight into the maintenance of polarity in plants and other systems.

Salicylic acid regulates PIN2 auxin transporter hyperclustering and root gravitropic growth via Remorin‐dependent lipid nanodomain organisation in Arabidopsis thaliana

“…However, SA treatment markedly promoted the segmentation of PIN2-GFP with enhanced interval distances between clusters (Figs 3a, S2a,c). According to the definition in a previous study of the PIN2 clustering pattern (Kleine-Vehn et al, 2011) and recent findings (Li et al, 2020), we here found that SA-triggered hyperclustering of PIN2 predominantly exhibited as tiny (31%) and small islands (61%) (Figs 3a, S2c). Interestingly, REM1.2 exhibited a similar hyperclustering pattern after SA treatment (Fig.…”

Auxin and Root Gravitropism: Addressing Basic Cellular Processes by Exploiting a Defined Growth Response

“…Microtubule organization and cell wall integrity might also participate in PIN clustering control; this relationship however remains to be elaborated in more detail [147]. This is also the case when it comes to elucidating PIN-PIP crosstalk in control of clustering.…”

“…Furthermore, polar proteins show higher tendency of clustering when compared to non-polar plasma membrane proteins. Thus, variations in clustering could affect protein diffusion as a means to regulate their polar distribution [147]. However, how is such PIN clustering and intramembrane mobility controlled?…”

“…However, how is such PIN clustering and intramembrane mobility controlled? Sterol content of membranes, which is long known to impact on PIN polarity [102,148,149], has been suggested to participate [147] ( Figure 2C). Sterols are a key factor in determining lipid raft/nanodomain organization in membranes by affecting formation of liquid-ordered (L o ) phase areas versus less tightly packed liquid-disordered (L d ) phase areas [150].…”

“…Mutant analysis and pharmacological interference revealed an immanent impact of PIP signaling and distribution on the clustering of PIN2 at the plasma membrane. In particular, plasma membrane-resident PIP5K as well as Phospholipase C (PLC) activity appear to be involved, promoting PIN2 clustering and consequently influencing its polar localization [147]. Stimulus-induced variations in PIP5K or PLC expression or activity thus appear to contribute to PIN regulation, representing yet another mode of dynamic regulation of PIN distribution in, for example, gravistimulated roots.…”