Endosidin20 does not affect cellulose synthase complex transport from ER to the Golgi

ABSTRACT

Cellulose, as the main component of the plant cell wall, is synthesized by plasma membrane-embedded cellulose synthase (CESA) complexes (CSCs). We recently reported a new CESA inhibitor named Endosidin20 (ES20) that targets the catalytic site of CESA6 in Arabidopsis (Arabidopsis thaliana). We found that inhibiting CESA catalytic activity by ES20 treatment reduces the motility of CSC at the plasma membrane and reduces the delivery of CSC to the plasma membrane. We also found that ES20 treatment causes an increased abundance of CSC at the Golgi. Through further investigation, here we show that inhibiting CESA catalytic activity by ES20 treatment does not interfere with the transport of CSC from endoplasmic reticulum (ER) to the Golgi, indicating that inhibiting CESA catalytic activity reduces efficient CSC exit from Golgi. We also show that ES20 affects CSC trafficking without interfering with the trafficking of other cargo proteins in the secretory pathway and does not disturb the cellular localization of typical organelle marker proteins. In combination with our recent findings, our results show that inhibiting CESA catalytic activity by short-term ES20 treatment affects CSC exit from Golgi and CSC post-Golgi transport but does not affect CSC transport from ER to the Golgi.

Plant cell walls are the physical barrier that provides tough and flexible environment for the cells to keep the normal cell shape and protect the cells from adverse stresses. Plant cell walls are also the main biomass on earth and are important sources of food supply, clothing, industry material, and biofuel. Cellulose is a major component of the plant cell wall that consists of β(1,4)-D-glucan chains and is synthesized by rosette-structured cellulose synthase (CESA) complex (CSCs) at the plasma membrane.^1-3 The composition of CSCs varies depending on the type of cell wall. The CSCs of the primary cell wall are consisted of CESA1, 3, and 6/6 like proteins, whereas the CESA4, 7, and 8 form the CSCs to synthesize the secondary cell wall with 1:1:1 molar ratio in each CSC.^4,5 Freeze-fracture electron microscopy analysis reveals that CSCs are localized to the Golgi, Golgi-derived vesicles, and the PM, indicating that CSCs are transported through the vesicle trafficking pathway.⁶ Live cell imaging of CSCs using functional fluorescence-tagged CESA provides further compelling evidence that CSCs are localized to the Golgi/trans-Golgi network (TGN), small CESA-containing compartments (SmaCCs)/microtubule-associated cellulose synthase compartments (MASCs), and PM.^7-10 PM-localized CSCs show bidirectional motility, and the velocity is related to the cellulose polymerization process.^7-9,11

Precise CSC delivery to the PM requires the coordinated function of multiple cellular machineries including microtubules, actin, and general exocytosis machinery.^7-10,12-18 CSCs in the Golgi and SmaCCs require actin cytoskeleton for long- and short-distance cellular transport.^8,9,13 SmaCCs that are close to the PM interact with microtubules and CELLULOSE SYNTHASE INTERACTIVE 1 (CSI1) for targeted delivery to the PM.^9,12,19 CSCs also require the conserved exocyst complex, PATROL1, actin, and myosin XI for tethering and fusion to the PM.^10,13,18 There are also new cellular components, such as STELLO and SHOU4, that regulate CSC delivery to the PM through molecular mechanisms that require further investigation.^20,21

Endosidin20 (ES20) is a small molecule that was recently found to target the catalytic domain of CESA in Arabidopsis.²² Inhibiting CESA catalytic activity by ES20 treatment of seedlings reduces not only the motility of CSC at the plasma membrane but also the delivery of CSC to the plasma membrane and increases the abundance of CSC at the Golgi.²² Increased abundance of CSC at the Golgi could result from increased CSC delivery from ER to the Golgi, reduced CSC exit from the Golgi, or both. To test this, we analyzed the dynamics of YFP-CESA6/CSC transport from the ER to the Golgi using fluorescence recovery after photobleaching (FRAP) analysis. We treated YFP-CESA6 seedlings with 0.1% DMSO or 6 μM ES20 for 1 h in the presence of a low concentration of latrunculin B (2 μM) to immobilize Golgi, followed by photobleaching of individual Golgi stacks and time-lapse imaging to examine fluorescence recovery. We found that about 30% of the photobleached YFP-CESA6 fluorescence intensity could be recovered from both DMSO- and ES20-treated seedlings within 5 min, but the rate of fluorescence recovery was not significantly different between the two treatments (Figure 1). Increased YFP-CESA6 fluorescence intensity at the Golgi and normal delivery rate from ER to the Golgi indicate that ES20 likely affects CSC transport out of Golgi but not for transport from ER to the Golgi.

Figure 1.

ES20 does not affect the delivery of CSCs from ER to the Golgi in root epidermal cells. Five days of YFP-CESA6 seedlings were immerged in liquid ½ MS medium with 0.1% DMSO or 6 μM ES20 for 1 h in the presence of latrunculin B (2 μM). Root elongation zone was used for image collection. (a) Representative images of Golgi-localized YFP-CESA6 during a FRAP assay. (b) Quantification of the relative recovery of CSCs at Golgi at different time points during FRAP assay. Data represent mean ± SE (n = 12 from 12 seedlings). Scale bars: 5 μm.

In order to test the specificity of ES20 in inhibiting CSC trafficking, we tested the effect of ES20 on two other typical cargo proteins in the secretory pathway and different organelle markers. Brassinosteroid-Insensitive 1 (BRI1) brassinosteroid receptor²³ and some PIN-FORMED (PIN) auxin transporters²⁴ are cargo proteins of constitutive plant exocytosis during normal plant growth,^25,26 like that of CSC.¹⁸ We found that 2 h of 6 μM ES20 treatment did not affect the localization of PIN2 nor BRI1 (Figure 2), indicating that it does not disrupt general exocytosis in plants. ES20 also did not disrupt the localization of two other plasma membrane proteins, ROP6 and PIP2a, or general marker proteins for ER (GFP-HDEL), Golgi (YFP-Got1p), and trans-Golgi Network/Early Endosomes (TGN/EEs) (VHA-a1-GFP) (Figure 2). Normal localization of cargo proteins and organelle marker proteins indicates that ES20 is not a general membrane trafficking inhibitor.

Figure 2.

ES20 does not disrupt general endomembrane system of plants. Representative images of cellular localization of different organelle marker proteins in 5-day-old transgenic plants after treatment with 0.1% DMSO or 6 μM ES20 for 2 h. PIN2-GFP and BRI1-GFP were used as plant exocytosis cargo proteins. GFP-ROP6 and GFP-PIP2a were used as PM marker proteins. GFP-HDEL was used as an ER marker protein. YFP-Got1p (Golgi transporter 1 protein) was used as a Golgi marker protein. VHA-a1-GFP (v-type proton ATPase subunit a1) was used as a TGN/EE marker protein. Scale bars: 10 μm.

It was found recently that mutations in amino acids at the catalytic site of CESA cause different defects in CSC subcellular transport,²⁷ indicating that the catalytic activity of CESA influences CSC transport. Our analysis of CSC transport using CESA catalytic activity inhibitor ES20 treatment also shows that CESA catalytic activity influences CSC subcellular transport. Future detailed analysis of CSC trafficking dynamics using fluorescence-tagged CESA carrying different mutations at the catalytic site will allow a better understanding of how the catalytic activity is integrated with CSC trafficking.

Materials and methods

To test the effect of ES20 on exocytic transport and cellular localization of proteins in different organelles, transgenic plants expressing fluorescence-tagged PIN2, BRI1, PIP2a, HDEL, VHA-a1, ROP6, and GOT1p were used.^28-34 To test CSC trafficking dynamics from ER to Golgi in FRAP assay, YFP-CESA6 was used.²² Seeds for plants that were used for live-cell imaging were sequentially sterilized with 50% bleach and 75% ethanol. After washing with sterilized water, seeds were sowed on ½-strength Murashige and Skoog (MS) media with 1% sucrose and 0.8% agar at pH 5.8. The plants were grown under continuous light of 130 μmol m⁻² s⁻¹ intensity illuminated by Philips F25T8/TL841 25 watt bulb at 22°C. The images were collected using a Zeiss 710 laser scanning confocal microscope equipped with a 40X/1.2 NA water objective. For imaging GFP-tagged proteins, the 488-nm laser line was used as an excitation source and emission light at 493–598 nm was collected. For imaging YFP-tagged proteins, the 514-nm laser line was used as an excitation source and the emission light at 519–621 nm was collected.

For FRAP experiments, images were collected using a Zeiss Observer Z.1 microscope, equipped with a Yokogawa CSU-X1 head and a 100X/1.46 NA PlanApo objective (Zeiss), and a 515-nm laser line was set to 100% power with 3 ms/scan. Five-day-old light-grown YFP-CESA6 seedlings were treated with 0.1% DMSO or 6 μM ES20 for 1 h in the presence of a low concentration of latrunculin B (2 μM) to immobilize Golgi and time-lapse images were collected at the cortical cytoplasm (about 0.4 µm below the PM) at root elongation zone with 5-s intervals for 121 frames. Photobleaching of a small region (7.1 µm²) was performed after the fourth frame, and recovery measured for 10 min. An area (7.1 µm²) within the bleached region was used for analyses. To measure the fluorescence intensity, the integrated fluorescence at the selected region at different time points was calculated by subtracting the background fluorescence outside of the cell with the same size of the area. The relative fluorescence of different time points was calculated by dividing the integrated fluorescence of different time points by integrated fluorescence before photobleaching.

Funding Statement

This work was supported by the Purdue University.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.