Armadillo-repeat kinesin1 interacts with Arabidopsis atlastin RHD3 to move ER with plus-end of microtubules

Abstract

In living cells, dynamics of the endoplasmic reticulum (ER) are driven by the cytoskeleton motor machinery as well as the action of ER-shaping proteins such as atlastin GTPases including RHD3 in Arabidopsis. It is not known if the two systems interplay, and, if so, how they do. Here we report the identification of ARK1 (Armadillo-Repeat Kinesin1) via a genetic screen for enhancers of the rhd3 mutant phenotype. In addition to defects in microtubule dynamics, ER organization is also defective in mutants lacking a functional ARK1. In growing root hair cells, ARK1 comets predominantly localize on the growing-end of microtubules and partially overlap with RHD3 in the cortex of the subapical region. ARK1 co-moves with RHD3 during tip growth of root hair cells. We show that there is a functional interdependence between ARK1 and RHD3. ARK1 physically interacts with RHD3 via its armadillo domain (ARM). In leaf epidermal cells where a polygonal ER network can be resolved, ARK1, but not ARK1ΔARM, moves together with RHD3 to pull an ER tubule toward another and stays with the newly formed 3-way junction of the ER for a while. We conclude that ARK1 acts together with RHD3 to move the ER on microtubules to generate a fine ER network.

ROOT HAIR DEFECTIVE3 Is a Receptor for Selective Autophagy of the Endoplasmic Reticulum in Arabidopsis

“…All mutant seeds, including characterized rhd3-8 (SALK_025215) ( Sun et al, 2020b ) and atg5-1 (SAIL_129_B07) ( Le Bars et al, 2014 ), were obtained from the Arabidopsis Biological Resource Center and then genotyped and propagated. Col.0 ( 35Spro:YFP-TMC ) and rhd3-8 ( RHD3pro:YFP-RHD3 ) transgenic line was generated as previously described ( Sun and Zheng, 2018 ; Sun et al, 2020b ). rhd3-8 ( 35Spro:YFP-TMC ) and atg5-1 ( 35Spro:YFP-TMC ) plants were created by crossing rhd3-8 and atg5-1 mutants with Col.0 ( 35Spro:YFP-TMC ), respectively.…”

“…For bimolecular fluorescence complementation (BiFC) assay, RHD3, RHD3(ΔAIM1), ATG8e, and p24 were cloned into the 3-in-1 BiFC vector ( Sun et al, 2020b ) with the AQUA cloning method ( Beyer et al, 2015 ). Then, agrobacteria containing different constructs were infiltrated into Nicotiana benthamiana leaves with OD 600 = 0.01.…”

“…Co-immunoprecipitation (Co-IP) was performed as previously described ( Sun et al, 2020b ). The infiltrated N. benthamiana leaves were grinded with the extraction buffer [50 mM Tris–HCl pH 7.5, 150 mM NaCl, 10% (v/v) glycerol, and 0.5% IGEPAL ® CA-630 (#I8896, Sigma), 1/100 volume of Protease Inhibitor Cocktail (#P9599, Sigma)].…”

Plant multiscale networks: charting plant connectivity by multi-level analysis and imaging techniques

“…Tubular ER, the organelle with the largest membrane surface area, extends and branches throughout the cytoplasm forming various MCSs and interacting with multiple organelles, including the PM, chloroplasts, mitochondria, Golgi, vacuoles, and endosomes (Figure 2E) (Stefano and Brandizzi, 2018;Wu et al, 2018). A recent study found that ARMADILLO-REPEAT KINESIN1 acts together with ROOT HAIR DEFECTIVE3 to move the ER on microtubules to generate a sophisticated ER network (Sun et al, 2020). This study provided genetic evidence and revealed the molecular mechanism for the interaction between the cytoskeleton and the ER in plant cells.…”

A novel plant actin-microtubule bridging complex regulates cytoskeletal and ER structure at ER-PM contact sites

“…It is known that the ER dynamics in plants is also regulated by microtubules. 21,22 As the NET3C-KLCR1-IQD2 complex can mediate the connection between actin filaments and microtubules, this indicates plants may have a specific mechanism for enabling actin-microtubule and ER interactions. Furthermore, enhanced association of ER and the cytoskeleton is found when NET3C-KLCR-IQD2 were overexpressed (Figures 3C, 3K, and 3L), which is in agreement with our conclusion that these proteins have the capability to mediate the link between ER and the cytoskeletal network ( Figures 3E and 3F).…”