Delivery Of Cellulose Synthase Complexes Research Articles

The TRAPPIII subunit, Trs85, has a dual role in the trafficking of cellulose synthase complexes in Arabidopsis.

Plant cell walls are essential for defining plant growth and development, providing structural support to the main body and responding to abiotic and biotic cues. Cellulose, the main structural polymer of plant cell walls, is synthesized at the plasma membrane by cellulose synthase complexes (CSCs). The construction and transport of CSCs to and from the plasma membrane is poorly understood but is known to rely on the coordinated activity of cellulose synthase-interactive protein 1 (CSI1), a key regulator of CSC trafficking. In this study, we found that Trs85, a TRAPPIII complex subunit, interacted with CSI1 in vitro. Using functional genetics and live-cell imaging, we have shown that trs85-1 mutants have reduced cellulose content, stimulated CSC delivery, an increased population of static CSCs and deficient clathrin-mediated endocytosis in the primary cell wall. Overall, our findings suggest that Trs85 has a dual role in the trafficking of CSCs, by negatively regulating the exocytosis and clathrin-mediated endocytosis of CSCs.

TRANVIA (TVA) facilitates cellulose synthase trafficking and delivery to the plasma membrane

Cellulose is synthesized at the plasma membrane by cellulose synthase (CESA) complexes (CSCs), which are assembled in the Golgi and secreted to the plasma membrane through the trans-Golgi network (TGN) compartment. However, the molecular mechanisms that guide CSCs through the secretory system and deliver them to the plasma membrane are poorly understood. Here, we identified an uncharacterized gene, TRANVIA (TVA), that is transcriptionally coregulated with the CESA genes required for primary cell wall synthesis. The tva mutant exhibits enhanced sensitivity to cellulose synthesis inhibitors; reduced cellulose content; and defective dynamics, density, and secretion of CSCs to the plasma membrane as compared to wild type. TVA is a plant-specific protein of unknown function that is detected in at least two different intracellular compartments: organelles labeled by markers for the TGN and smaller compartments that deliver CSCs to the plasma membrane. Together, our data suggest that TVA promotes trafficking of CSCs to the plasma membrane by facilitating exit from the TGN and/or interaction of CSC secretory vesicles with the plasma membrane.

Disruption of Very-Long-Chain-Fatty Acid Synthesis Has an Impact on the Dynamics of Cellulose Synthase in Arabidopsis thaliana.

In higher plants, cellulose is synthesized by membrane-spanning large protein complexes named cellulose synthase complexes (CSCs). In this study, the Arabidopsis PASTICCINO2 (PAS2) was identified as an interacting partner of cellulose synthases. PAS2 was previously characterized as the plant 3-hydroxy-acyl-CoA dehydratase, an ER membrane-localized dehydratase that is essential for very-long-chain-fatty acid (VLCFA) elongation. The pas2-1 mutants show defective cell elongation and reduction in cellulose content in both etiolated hypocotyls and light-grown roots. Although disruption of VLCFA synthesis by a genetic alteration had a reduction in VLCFA in both etiolated hypocotyls and light-grown roots, it had a differential effect on cellulose content in the two systems, suggesting the threshold level of VLCFA for efficient cellulose synthesis may be different in the two biological systems. pas2-1 had a reduction in both CSC delivery rate and CSC velocity at the PM in etiolated hypocotyls. Interestingly, Golgi but not post-Golgi endomembrane structures exhibited a severe defect in motility. Experiments using pharmacological perturbation of VLCFA content in etiolated hypocotyls strongly indicate a novel function of PAS2 in the regulation of CSC and Golgi motility. Through a combination of genetic, biochemical and cell biology studies, our study demonstrated that PAS2 as a multifunction protein has an important role in the regulation of cellulose biosynthesis in Arabidopsis hypocotyl.

Myosins XI Are Involved in Exocytosis of Cellulose Synthase Complexes.

In plants, cellulose is synthesized at the cell surface by plasma membrane (PM)-localized cellulose synthase (CESA) complexes (CSCs). The molecular and cellular mechanisms that underpin delivery of CSCs to the PM, however, are poorly understood. Cortical microtubules have been shown to interact with CESA-containing compartments and mark the site for CSC delivery, but are not required for the delivery itself. Here, we demonstrate that myosin XI and the actin cytoskeleton mediate CSC delivery to the PM by coordinating the exocytosis of CESA-containing compartments. Measurement of cellulose content indicated that cellulose biosynthesis was significantly reduced in a myosin xik xi1 xi2 triple-knockout mutant. By combining genetic and pharmacological disruption of myosin activity with quantitative live-cell imaging, we observed decreased abundance of PM-localized CSCs and reduced delivery rate of CSCs in myosin-deficient cells. These phenotypes correlated with a significant increase in failed vesicle secretion events at the PM as well as an abnormal accumulation of CESA-containing compartments at the cell cortex. Through high-resolution spatiotemporal assays of cortical vesicle behavior, we identified defects in CSC vesicle tethering and fusion at the PM. Furthermore, disruption of myosin activity reduced the delivery of several other secretory markers to the PM and reduced constitutive and receptor-mediated endocytosis. These findings reveal a previously undescribed role for myosin in vesicle secretion and cellulose production at the cytoskeleton-PM-cell wall nexus.

CSI1, PATROL1, and exocyst complex cooperate in delivery of cellulose synthase complexes to the plasma membrane

Cellulose synthesis occurs exclusively at the plasma membrane by cellulose synthase complexes (CSCs). Therefore, delivery of CSCs to discrete sites at the plasma membrane is critical for cellulose synthesis. Despite their significance, the delivery of CSCs is poorly understood. Here we used proteomics approaches, functional genetics, and live cell imaging to show that the de novo secretion of CSCs is mediated by cooperation among cellulose synthase interactive 1 (CSI1), the plant-specific protein PATROL1, and exocyst complex in Arabidopsis thaliana We propose that CSI1 plays a role in marking the docking site, which allows CSCs-containing vesicles access to the plasma membrane through its interaction with microtubules. PATROL1 assists in exocytosis by its interaction with multiple components, including CSI1, CSCs, and exocyst subunits. Both PATROL1 and the exocyst complex determine the rate of delivery of CSCs to the plasma membrane. By monitoring the exocyst complex, PATROL1, CSI1, and CSCs dynamics in real time, we present a timeline of events for exocytosis of CSCs. Our findings provide unique insights into the evolution of exocytosis in eukaryotes.

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.

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.

Spatio‐temporal analysis of cellulose synthesis during cell plate formation in Arabidopsis

During cytokinesis a new crosswall is rapidly laid down. This process involves the formation at the cell equator of a tubulo-vesicular membrane network (TVN). This TVN evolves into a tubular network (TN) and a planar fenestrated sheet, which extends at its periphery before fusing to the mother cell wall. The role of cell wall polymers in cell plate assembly is poorly understood. We used specific stains and GFP-labelled cellulose synthases (CESAs) to show that cellulose, as well as three distinct CESAs, accumulated in the cell plate already at the TVN stage. This early presence suggests that cellulose is extruded into the tubular membrane structures of the TVN. Co-localisation studies using GFP-CESAs suggest the delivery of cellulose synthase complexes (CSCs) to the cell plate via phragmoplast-associated vesicles. In the more mature TN part of the cell plate, we observed delivery of GFP-CESA from doughnut-shaped organelles, presumably Golgi bodies. During the conversion of the TN into a planar fenestrated sheet, the GFP-CESA density diminished, whereas GFP-CESA levels remained high in the TVN zone at the periphery of the expanding cell plate. We observed retrieval of GFP-CESA in clathrin-containing structures from the central zone of the cell plate and from the plasma membrane of the mother cell, which may contribute to the recycling of CESAs to the peripheral growth zone of the cell plate. These observations, together with mutant phenotypes of cellulose-deficient mutants and pharmacological experiments, suggest a key role for cellulose synthesis already at early stages of cell plate assembly.