CESA TRAFFICKING INHIBITOR Inhibits Cellulose Deposition and Interferes with the Trafficking of Cellulose Synthase Complexes and Their Associated Proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1

Abstract

Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls.

Two Complementary Mechanisms Underpin Cell Wall Patterning during Xylem Vessel Development

“…The 3xYFP-CSI1/POM2 can be seen as fluorescent foci that track together with the CSCs at the cell cortex along linear trajectories during primary wall synthesis (Worden et al, 2015). After induction of VND7, we observed a clear change in the cellular distribution of the 3xYFP-CSI1/POM2.…”

“…To investigate how the CSI1/POM2 behaves during the transition from primary to secondary wall synthesis, we crossed plants expressing a functional, native promoter-driven triple (3x) YFP translational fusion with CSI1/POM2 (Worden et al, 2015) into the VND7-inducible Arabidopsis line. The 3xYFP-CSI1/POM2 can be seen as fluorescent foci that track together with the CSCs at the cell cortex along linear trajectories during primary wall synthesis (Worden et al, 2015).…”

“…To generate multiple marker lines in the VND7 background, we crossed seeds of pom2-4 and native promoter-driven triple (3x)YFP-CSI1/POM2 (Worden et al, 2015) into the VND7-inducible Arabidopsis line proCaMV35s::VND7::VP16::GR (Yamaguchi et al, 2010). The F3 progeny was used for analysis.…”

Arabidopsis thalianaKORRIGAN1 protein: N-glycan modification, localization, and function in cellulose biosynthesis and osmotic stress responses

“…By contrast, CesA-trafficking inhibitor CESTRIN promoted extensive colocalization of both KOR1 and CSCs with TGN marker SYP61 in walled cells of intact tissue. 18 Such induced colocalization was observed also by either isoxaben or osmotic stress treatment. 19 NaCl/osmotic stress induces transient depolymerization and repolymerization of microtubules, which is essential for stress acclimation 20,21 and triggered by tubulin kinase PHS1.…”

Plant cortical microtubule dynamics and cell division plane orientation

“…From the experimental side, it will be interesting to promote anisotropic cell growth inhibition, where expected outcome of such experiment will be a large number of anticlinal-vertical/periclinal division instead of the normal anticlinal-horizontal division. Based on existing genetic tools (Scheible et al, 2001;Harris et al, 2012;Worden et al, 2015), we can now perform such anisotropic growth inhibition experiments.…”