However, how biochar and arbuscular mycorrhizal fungi (AMF) can regulate crop growth and denitrification in soils with different phosphorus (P) supplies to influence N2O emission remains largely unknown.Promoting crop growth and regulating denitrification process are two main ways to reduce soil N2O emission.Here, an eight-week greenhouse and one-year field experiments biochar and/or AMF (only in greenhouse experiment) additions under low and high P environments were conducted to characterize the effects on wheat (Triticum aestivum L.) growth and N2O emissions (by 32-95%). Biochar and/or AMF addition exhibited either no or negative effects on wheat biomass and nutrient content in greenhouse, and biochar addition promoted wheat yield only under high P environment in field. However, biochar and/or AMF addition decreased soil N2O emissions by 24-93% and 32% in greenhouse and field experiments, respectively. This decrease was associated mainly with the diminished abundance of N2O-producing denitrifiers  and the increased abundance of N2O-consuming denitrifiers , and also with the increased wheat nutrient content, yield and leaf Mn concentration.With low P supply, AMF addition decreased leaf Mn concentration (indicates carboxylate-releasing P-acquisition strategies), whereas biochar addition increased leaf Mn concentration, suggesting biochar and AMF addition regulated root morphological and physiological traits to capture P. Compared with low P supply, the high P significantly promoted wheat growth (by 16-34%), nutrient content (by 33-218%) and yield (by 33-41%), but suppressed soil N2O emissions via manipulating plant nutrient acquisition and soil denitrification.These findings suggest that strengthening the plant-soil-microbe interactions can mitigate soil N Thus, linking plant nutrient-acquisition strategy and soil microbial community (composition and activity) can improve our understanding of mechanisms effective in suppressing N2O emissions under different management practices.As a significant greenhouse gas, nitrous oxide  pro2O emissions are influenced strongly by soil conditions . The N2O:N2 product ratio in denitrification was correlated negatively with soil pH, labile C availability, and soil water-filled pore space, and positively with soil NO3- content  in soil for N2O producers, thus decreasing N2O emissions (nirK type denitrifiers (that produce N2O) and increase the abundance of nosZ type (that consume N2O), thus hampering denitrification  benefit their host plant regarding nutrient uptake  from soil . AMF inomissions . AMF maymissions  and soilmissions , and incmissions . To datefication .2O production by modifying the plant growth and abundance of microorganisms associated with the N cycle. Plants usually increase root/shoot ratio, specific root length and carboxylate exudation and decrease root tissue density to enhance their capacity to acquire P in P-limited environments  AMF may increase crop growth and inhibit key denitrification microorganisms to decrease soil N2O emissions at the low P supply, whereas at the high P supply, AMF may inhibit wheat growth and diminish the mitigating effect on N2O emission; and (iii) biochar can suppress N2O emission via directly inhibiting the key denitrification genes, and via directly influence wheat growth or indirectly modifying the effect of AMF on wheat growth at differential P supply.Wheat is one of the most widely cultivated crops in the world and is highly sensitive to soil P availability , with AM22.12.1.1-1 organic matter, 870 mg kg-1 N, 2.9 mg kg-1 Olsen P, 156 mg kg-1 available K, and had pH value 7.8 (soil:water ratio was 1:5). The soil was air dried, sieved (2 mm) and sterilized by radiation with 60Co γ-rays at 10 kGy to eliminate indigenous AMF.A calcareous loamy soil (0-20 cm) was collected from Shangzhuang experimental station  of China Agricultural University. The soil contained 17.8 g kg-1 soil as KH2PO4, denoted hereafter as P40 and P300, respectively), two mycorrhizal (AMF) treatments (with and without inoculation with Rhizophagus irregularis) and two biochar (BC) rates (with and without maize straw BC). There were five replicates per treatment. To achieve the same soil K addition in the two P treatments, K2SO4 was supplied at 327 mg K kg-1 soil in the P40 treatment. Furthermore, the mineral nutrients were added to all treatments (per kg soil) as follows: 200 mg of N (as KNO3), 50 mg of Mg (as MgSO4), 5 mg of Zn (as ZnSO4), and 2 mg of Cu (as CuSO4). The nutrients were uniformly mixed in soil before placing the soil in plastic pots . The glasshouse temperature range was 15–20°C with 10–12 h daylight throughout the wheat growth period.A greenhouse experiment was conducted with two phosphorus rates  and was mixed thoroughly.Biochar was derived from maize straw residue at 450°C, and was provided by Mingchen Sanitation Equipment Co. LTD, Shandong Province, China. Biochar contained (per kg) 9.48 g total N, 1.34 g total P and 530 g carbon; pH was 9.7. Biochar was added at 3.5 g kgRhizophagus irregularis was propagated on wheat plants growing in a 1:5 mixture (w/w) of river sand and zeolite in a greenhouse for 4 months. The inoculum used in the present study included substrate containing spores (150 g-1 potting substrate), mycelium and fine-root segments. The inoculum was added at 20 g kg-1 soil in the AMF treatment. The 10 mL of microbial wash filtrate from unsterilized soil was added to all pots to minimize differences in microbial communities among treatments (The AMF eatments .Wheat seeds (JiMai 22) were surface sterilized by stirring in 10% (v/v) hydrogen peroxide for 10 min and in 70% (v/v) ethanol for 3 min, followed by rinsing at least five times in deionized water. Six uniformly germinated seeds were sown into each pot and were thinned after emergence to four seedlings similar in size. The pots were watered daily and weighed every 2 days to adjust soil moisture to 18-20% (w/w).2.1.22O emissions. The study site is located in the Shunyi District, Beijing. The soil contained 9.68 g kg-1 organic matter,1.32 g kg-1 N, 6.48 mg kg-1 Olsen P, 73.6 mg kg-1 available K in 0-20 cm soil depth and a pH of 8.05. The field plots were conducted with a completely randomized block design with four treatments: BC0_P0 , BC0_P+ (52 kg hm-2 P), BC+_P0 (9 t hm-2 biochar), BC+_P+ . Before sowing of winter wheat in 2020, the amount of biochar and P in four treatments were supplied every year. Sixteen plots were arranged with four replicates, and every plot size was 180 m2 (6 m × 30 m).A one-year field experiment with four-treatment was designed to test the effects of biochar and P addition mitigate N-2 (P+) (Ca(H2PO4)2), K 124 kg.hm-2 (K2SO4), N 112 kg hm-2 (KNO3), respectively. N fertilizer was uniformly applied at a 1:1 ratio according to topdressing (15 March 2021). Each treatment was irrigated after fertilization.In field experiment, wheat (JiMai 22) was sown on 12 October 2020 and harvested on 6 June 2021. The P fertilizer input amount of each treatment was 0 (P0) and 52 kg P hm2.22.2.1-1 KNO3 was added in each pot, and the soil moisture adjusted to 30% (w/w), which was beneficial to denitrification process but had little effect on wheat growth during a week of sampling N2O. After 24 hours, N2O samples were collected from each pot by using a PVC cylindrical chamber (diameter of 16 cm and height of 50 cm), with a base having a 5 cm groove to which water was added to seal the system during sample collection. The gas in the chamber was sampled starting at 9:00 and then again starting at 15:00 each day for a week; during this period, the soil moisture was kept at 30% (w/w) by adding water. The four 50 mL gas samples were extracted by plastic syringes at 0, 10, 20 and 30 min in each of the two daily sampling periods.Seven weeks after sowing, nutrients solution with 200 mg kg2.2.22O samples were collected by using a PVC box chamber (50cm×50cm×50cm) with a thickness of 1.5 mm and a base (50 cm × 50 cm × 15 cm). The gas was collected twice a week after fertilization, irrigation, rain and snow before the booting stage. Afterwards, the collection frequency decreased to once every 15 days during the fallow period. Gas samples were taken between 09:00 to 11:00 am. During this period, the four 200 mL gas samples were extracted by plastic syringes at 0, 10, 20 and 30 min for each plot.N2.2.32O was done by gas chromatography  with an electron capture detector (ECD) at 350°C (-2 h-1) of N2O were calculated according to The measurement of Nat 350°C . The houF= ρ×(V/A)×(ΔC/Δt)×(273/(273+T))2O under standard conditions, V is the chamber volume (m3), A is the area covered by chamber (m2), ΔC/Δt is the change in N2O concentration in the chamber over time, and T is the chamber air temperature.where ρ is the density of N2O emission  was calculated as follows:The cumulative Ni+Fi+1)/2 ×24×10-3×TE= was the nosZ and (nirK+nirS) gene copy numbers raito. nosZ associated with N2O consumption in the process of denitrification, and (nirK+nirS) was significantly positively correlated with the denitrification rates , shoots were cut at the soil surface, and roots were extracted from the soil. Shoots were rinsed in distilled water and then oven-dried at 75°C for 72 h, weighed and ground to fine powder. Nitrogen and phosphorus concentrations in shoots were determined after digestion with a mixture of 5 mL of concentrated Hrategies  and was Roots were washed under running water and were scanned at the resolution of 400 dpi (EPSON 1680) . Root images were analyzed using a WinRHIZO image system (WinRHIZOPro2004b)  to calculate root length and diameter . After s3--N and NH4+-N were extracted with 0.01 mol L-1 CaCl2 solution and determined by a AA3 flow analyzer . Soil available P was measured by the Olsen method. Soil organic carbon was analyzed by wet digestion with 10% w/w H3PO4.The soil samples were collected on the same day as the gas samples. The soil was sieved (2 mm) and separated into two subsamples: one was kept at -80°C for quantitative PCR assay and the other one was stored at 4°C for determining C, P and N content. Soil NOnirS, nirK) and nitrous oxide reductase (nosZ) were determined. DNA was extracted using a Fast DNA Spin Kit for Soils  from 0.5 g of soil. The quantitative polymerase chain reaction (q-PCR) amplification primers for nirK, nirS and nosZ genes and the reaction conditions are shown in In order to test the effects of P, BC and AMF on denitrification, the copy numbers of the key genes of copper nitrite reductases  and was kept at -80°C, and then used to measure the functional genes of denitrification.At wheat maturity, wheat yield was measured in each treatment plot. Soil samples (0–20 cm) were collected when soil N2.42O emission, gene abundance, root traits and soil properties (P ≤0.05) at low or high P supply. Post-hoc Tukey HSD tests were performed to determine significant differences. If the two-way interaction AMF× BC was significant, the complete data was shown; if not, only the significant main effects were shown. Significant differences between the AMF and no AMF inoculation at each P supply or between BC and no BC addition at each P supply were based on the t-test (P ≤0.05). Pearson’s correlation analysis was used to test the relationships among the N2O emission, abundance of genes, root traits and soil properties. The random forest model in R studio software was used to analyze the comprehensive effects of genes, root traits and soil properties on the soil N2O emissions.Two-way ANOVA was performed to test for effects of AMF and biochar addition on N2O emission and gene abundance (P ≤0.05) in the field experiment. Post-hoc Tukey HSD tests were performed to determine significant differences. Pearson’s correlation analysis was used to test the relationships among the yield, N2O emission and gene abundance.Two-way ANOVA was performed to test for effects of P and biochar addition on yield, N33.1The interaction AMF × BC was not significant for shoot biomass and shoot N and P contents Table 1.3.2Root biomass and root tissue density were significantly influenced by the interaction AMF×BC at P40 and P300 Table 1.The AMF×BC interaction was a significant source of variation for total root length and specific root length at P40 only Table 1.Leaf Mn concentration was significantly influenced by the interaction AMF×BC at P300 only Table 1.3.3The mycorrhizal colonization was significantly influenced by the AMF × BC interaction Table 1.3.42O emissions was significantly influenced by the AMF×BC interaction at P40 only  in, respectively, the AMF, BC and the combined AMF and BC treatments  with biochar addition and mycorrhizal inoculation. Compared to the low P supply, the high P significantly suppressed soil N2O emissions by 50-95%  with biochar addition or mycorrhizal inoculation, but abundance of nirS gene increased by 19% with biochar addition at P300 compared to the no biochar treatment (nirK gene at P300 only (nirK gene copy number decreased significantly (by 35% to 59%) in the other treatments  in the other treatments. at the high P supply, the number of nosZ gene copies increased significantly  (The sZ genes Table 1.nosZ/(nirK+nirS) ratio was significantly influenced by the interaction AMF×BC at P40 and P300 (nosZ/(nirK+nirS) ratio increased significantly (by 115% to 256% at P40 and 78% to 102% at P300) in the other treatments (nosZ/(nirK+nirS) ratio at high P level was 4.06 to 7.56 folds higher than at low P level  ratio were significantly influenced by the BC×P interaction. Compared to the BC0P0 treatment, the nosZ gene and nosZ/(nirK+nirS) ratio increased significantly by 43% and 226% in the BC+P+ treatment. The nosZ gene and nosZ/(nirK+nirS) ratio increased significantly by 35% and 172% with P addition compared to no P addition treatment  ratio, soil NO3–N, NH4+-N, SOC and AMF colonization were correlated negatively with N2O emission. At the high P supply, the nosZ abundance and nosZ/(nirK+nirS) ratio were correlated negatively with N2O emission, whereas shoot growth, root morphology, nirK copy number and soil NO3–N were correlated positively with N2O emission  ratio  ratio showed a negative correlation with N2O emission. Root growth was correlated with N2O positively, and shoot growth and AMF colonization were correlated negatively. The mature leaf Mn content was correlated positively with N2O. Additionally, Olsen P and soil NH4+-N showed a negative correlation with N2O  ratio, soil NH4+-N, nirS abundance and mature leaf Mn concentration were the significant factors associated with N2O emission. Shoot P and N content, root/shoot ratio, and nosZ copy numbers were the additional significant factors influencing N2O emission (Most important variables to influence soil N2O emissions (32-95%). However, biochar and/or AMF addition reduced soil N2O emissions by 24-93% and 32%, respectively, in greenhouse and field experiments. This decrease was associated mainly with the diminished abundance of nirK and nirS (17-59%) and the increased nosZ (35-65%) in the greenhouse and field experiments, respectively.In summary, the addition of biochar and/or AMF had a negative effect on the total root length for both P40 and P300. The AMF addition decreased leaf Mn concentration, whereas biochar addition increased leaf Mn concentration at P40, suggesting biochar and AMF addition promote P uptake. Compared with P40, the high P significantly promoted wheat growth (16-34%), nutrient content (33-218%) and yield (33-41%), but inhibited soil N44.1Kennedia species that the AMF inoculation reduced exudation of organic carboxylates up to 50%  in denitrification were increased by 34% to 1340% at the high P supply, which result in the nosZ/(nirK+nirS) ratio at the high P supply was 2.72 to 7.56 times greater than that at the low P supply , nirS, leaf Mn concentration and stem P content were closely related to soil Olsen P across all the treatments  ratio compared to the low P supply. The soil N2O emissions was mitigate after AMF and/or biochar addition regardless of P supply in greenhouse or in field experiments, but the effect of under high P supply which might be attributed mainly to the decreased copy numbers of genes associated with N2O production (nirK and nirS) and the increased copy number of the nosZ gene associated with N2O consumption in the process of denitrification. Our findings highlight that strong interaction among plant, soil microbiome and soil properties in regulating denitrification.The eight-week greenhouse and one-year field experiments verified that the high P supply suppressed soil NThe original contributions presented in the study are included in the article/ZH: conceptualization, writing-original draft, review & editing. ZD: data analysis. SH: data analysis. AZ: conceptualization and writing-review & editing. All authors contributed to the article and approved the submitted version.


Through reconstitution and epithelial cell biology experiments, we show that Arf6-mediated recruitment of the lipid kinase PIP5K1C rapidly converts phosphatidylinositol 4-phosphate (PI(4)P) to PIP2, driving exocyst recruitment and membrane tethering. These results provide a molecular mechanism of exocyst-mediated tethering and a unique functional requirement for phosphoinositide signaling on late-stage vesicles in the vicinity of the plasma membrane.Polarized trafficking is necessary for the development of eukaryotes and is regulated by a conserved molecular machinery. Late steps of cargo delivery are mediated by the exocyst complex, which integrates lipid and protein components to tether vesicles for plasma membrane fusion. However, the molecular mechanisms of this process are poorly defined. Here, we reconstitute functional octameric human exocyst, demonstrating the basis for holocomplex coalescence and biochemically stable subcomplexes. We determine that each subcomplex independently binds to phosphatidylinositol 4,5-bisphosphate P  •Complete reconstitution and subunit connectivity of the human exocyst complex•2 in trans by each subcomplex enables membrane tetheringBinding to PIP•PI(4)P to PIP2 conversion is sufficient for exocyst recruitment and tethering•in vitroArf6 controls phosphoinositide conversion by PIP5K1C in cells and  2 by PIP5K1C is controlled by the small GTPase Arf6, and the conversion is minimally sufficient for exocyst-mediated tethering.How are vesicles tethered to the plasma membrane during polarized membrane trafficking? Through complete reconstitution of the mammalian exocyst complex, Maib and Murray show that phosphoinositide conversion of PI(4)P into PIP These structures are interconnected, with logistics facilitated by both vesicular transport mechanisms and direct membrane contact sites. Strikingly, the specialization of organelles and their distinct identities are maintained, which are results of continuous sorting and exchange of phospholipid and protein content. In the polarized trafficking pathway, this sorting includes polarity determinants destined to the apical or basal domains of epithelia, integrins, and metalloproteases to the leading edge of invasive cancers and the transport of receptors to post-synaptic densities in neurons.,,Orchestration of polarized trafficking requires a highly conserved trafficking machinery. Early screens for factors responsible for polarized secretion in yeast identified the components of the exocyst complex and its regulatory machinery, including small GTPases.2 is enriched and is important for cargo delivery and fusion.,,-4 phosphate is established on vesicles originating from both the Golgi and recycling endosomes.2.,2 along the route to the plasma membrane.Drosophila.2-positive trafficking vesicles are tethered to the plasma membrane.Phosphoinositides are a family of phospholipids that are signposts for membrane identity.2.,in vivo colocalization of exocyst subunits,2 binding site, although the function of this duplication is unknown.The exocyst is a multi-subunit tethering complexThe interconnectivity of exocyst subunits enables a potential for regulatory modification through disassembly of the holocomplex. Indeed, live-cell imaging of endogenously tagged exocyst subunits suggests that although the subcomplexes are recruited to vesicles independently, they arrive at the plasma membrane together.2 and that the holocomplex is able to tether two opposing PIP2-positive membranes. Using cryo-electron tomography, we directly visualize this exocyst-dependent tethering of liposomes and determine a regular tethering distance consistent with the known dimensions of the exocyst complex. Next, we determined how exocyst function is linked to PIP2. In vitro reconstitution of phosphoinositide conversion by PIP5K1C is sufficient to drive both exocyst recruitment and membrane tethering. Using endogenous knockin cell lines, we observe exocyst colocalization with PIP5K1C at dynamic subdomains of the plasma membrane. Finally, we demonstrate that PI(4)P to PIP2 conversion is driven by Arf6 activation both in vitro and in cells. Therefore, we propose a model in which phosphoinositide conversion, mediated by Arf6 and PIP5K1C, regulates exocyst-dependent membrane tethering.Here, we reconstitute the complete human exocyst complex from its individual subunits to address the mechanism by which it tethers vesicles to the plasma membrane during polarized trafficking. We find that the complete octamer readily associates from its separate subunits and is suborganized into two distinct subcomplexes with differing modes of assembly. Furthermore, we show that each subcomplex independently binds to PIPTo understand the mechanism of exocyst holocomplex assembly and tethering, we reconstituted the human exocyst complex using baculovirus-mediated insect cell expression .44 We coin vitro from small GTPases. Moreover, the human exocyst efficiently self-assembles in a hierarchal manner, evolutionarily conserved from yeast P.2 in membranes chosen to mimic the plasma membrane. We formed supported-lipid bilayers on silica beads with liposomes composed of 85% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine, and 5% phosphoinositide . The exocyst and Exoc1/Exoc7-containing subcomplexes are unambiguously recruited selectively to PIP2 and not PI(4)P-containing membranes at non-saturating conditions (100 nM complex). Moreover, by harnessing the assembly hierarchy to generate exocyst subcomplexes, we explicitly demonstrate the requirement for Exoc1 or Exoc7 subunits for membrane binding P, questions the potential role of avidity in membrane binding by the holocomplex.To determine the selectivity of exocyst complexes to phosphoinositides, we measured the binding of purified, fluorescently tagged holo- and sub-complexes for PI(4)P or PIPnositide A.50,51 Bositide ,B. To staositide ,C P2. By systematic imaging and analysis, we determined that both subcomplexes-1 and -2 were recruited to membranes with similar strength   P2. These results, together with phosphoinositide selectivity P2-containing membranes.To quantitate the independence and modality of lipid binding, we determined the relative affinities of subcomplexes-1, -2, and the holocomplex for PIP38.5 nM) D and 2E.< 0.01%) F. Intereectivity  and the ectivity , suggest2-containing POPC membrane-coated beads  to PIPed beads G, as preed beads B. Next, osomes.2 H. Indiviethering I. These ethering J.2-containing POPC liposomes extruded to 100 nm and mixed with exocyst. Samples were plunge frozen, micrographs acquired, and tomograms reconstructed to visualize the samples in three dimensions, including both lipid membranes and proteinaceous material corresponding to the exocyst holocomplex. In tomograms, we observed protein density, dispersed in regions devoid of liposomes. In regions with liposomes, we observed protein density on the membrane and tethering Pethering . By manu,,2 in a modified assay for exocyst recruitment P-positive membrane-coated beads. Following conversion until a saturating time point, we evaluated the fluorescence intensity of tethered liposomes P2 by PIP5K1C mediates in vitro exocyst-dependent tethering. This suggests that late-stage phosphoinositide conversion, on some vesicles destined to the plasma membrane, could drive tethering during polarized trafficking.In polarized trafficking, phosphoinositide dynamics are required for delivery of vesicles prior to the engagement of fusion machinery. A key factor in these dynamics is the endosomal recruitment of the phosphatidylinositol-5 kinase PIP5K1C.ruitment A.52 We mruitment B. After ruitment B. In thiembranes D–2F and iposomes C and 4D.,2.To validate these findings in a cellular system, we used genome edited knockin cells from mouse mammary epithelia (NMuMG) with sfGFP at the C terminus of Exoc4.2 at intracellular membranes is catalyzed by the Arf6-mediated recruitment of PIP5K1C,2 generation and subsequent exocyst-mediated tethering. To observe cellular phosphoinositides, we harnessed a staining approach using purified phosphoinositide-binding probes, allowing us to visualize lipid populations in fixed cells P by SidC staining (2 and E-cadherin in PIP5K1C expression conditions (The conversion of PI(4)P into PIPed cells A and S3Bstaining A and S3Bnditions B. This m2, we used expression of constitutively active or inactive Arf6 to disrupt GTPase cycling .,,2 (2 (2.To address Arf6-mediated regulation of the conversion of PI(4)P to PIPely).,,2 B. Moreov).,,2 .,,2 Ppression B. Moreov2, assessed by the recruitment of RFP-PLCδ-PH. As expected, we observed relatively slow recruitment of this probe to Arf6-T27N membranes, reflecting basal activity of the lipid kinase P together with Arf6. We conjugated either Arf6-T27N or Arf6-Q67L to membrane-coated beads harboring the high-affinity TrisNTA-DODA lipid via N-terminal 6xHis tags.d kinase E. In con kinase ,, which s2-positive internal membranes. This result demonstrates that the exocyst is linked to Arf6 in an activity-dependent manner. Furthermore, we observe the exocyst at Arf6-WT containing vesicles in the direct vicinity of the plasma membrane P2-containing membranes, opens up a myriad of regulatory mechanisms to control vesicle tethering in vivo. Indeed, other late-stage vesicle delivery events may be independent of phosphoinositide conversion. In such events, it is possible that the exocyst complex binds to PIP2 with one of its binding interfaces at the plasma membrane, while being targeted to the vesicle through other mechanisms, such as GTPase or SNARE binding.,,,Our data support a model whereby exocyst-mediated selectivity and tethering results in part from the coordination of intracellular PIP2 membranes to each other suggests a physical opposition of the two lipid binding regions of the holocomplex. However, in current structures of the yeast exocyst complex,,2 binding regions of the yeast exocyst may be redundant, as vesicle tethering still occurs if either of them is deleted.,2 binding regions for the polarized trafficking of E-cadherin to cellular junctionsThe direct observation that the exocyst holocomplex tethers two distinct PIP2 on intracellular trafficking vesicles must be tightly regulated and places a requirement for the generation of PIP2 downstream of endosome exit at the strictest of vicinity to the plasma membrane. Indeed, PIP2 is present on internal membranes regulating distinct trafficking events.,2 generation and suggest a holistic model for exocyst-mediated tethering in polarized trafficking.The generation of minor pools of PIP2 binding of each exocyst subcomplex is paralleled by Ral GTPase-mediated regulation, exerted through Ral binding independently to both Exoc2 in subcomplex-1 and Exoc8 in subcomplex-2.,2 binding may be specifically relevant to these processes. Furthermore, the complete assembly model of the human exocyst and its separate subcomplexes provide new insights into regulatory pathways such as ULK1- and TBK1-mediated phosphorylation of Exoc7 and Exoc8, respectively.,,,,Indeed, further layers of regulatory complexity influence the composition of the exocyst holocomplex. The independent PIP,Finally, the role of small GTPases in exocyst-dependent trafficking remains to be clarified. In particular, the activation and interconnections of Rab-family GTPases to Arf6dhmurray@dundee.ac.uk).Please contact the lead contact, Dr. David H Murray, for additional information and requests for resources and reagents . Each was subcloned into FlexiBAC SF9 expression plasmids as GST-HRV3C, 6xHis-MBP-HRV3C, and 6xHis-HRV3C-mNeonGreen fusions, where HRV3C is a protease cleavage site, by standard molecular biology approaches.6 cells/ml were co-transfected by the linearised Defbac viral backbone and the plasmids encoding for exocyst subunits or PIP5K1C using ESCORT-IV (Sigma). After 5 days, cells were checked for signs of viral transfection and 50-200 μl of supernatant of this P1 virus were used to infect 50 ml of insect cells to generate P2 virus. 5 days after infection, this P2 virus was again evaluated for virus production .Exocyst subunits and PIP5K1C were expressed in SF9 cells cultured in Insect-XPRESS (Lonza) by baculovirus mediated expression. Baculovirus was produced using the FlexiBac methods.6 SF9 cells/ml were grown in 24 well round bottom plates (Qiagen) and infected with baculovirus of the indicated single subunits. After 2 days of infection, the cells were pelleted at 500xg for 10 min, the supernatant discarded and the pellet frozen in liquid nitrogen. Upon use, the pellet was thawed and resuspended in 2 ml of standard buffer , sonicated, and clarified at 4 °C. 25 μl of glutathione agarose beads (MRC-PPU Reagents and Services) were added and incubated for 1-2 h at 4 °C. Beads were isolated by centrifugation and washed three times in standard buffer using MobiSpin Column F (MoBiTec GmbH). The washed beads were resuspended in 50 μl 1x SDS loading buffer, boiled at 95 °C, and separated by SDS-PAGE. Gels were stained with Quick Coomassie Stain (Generon) and scanned.For identification of exocyst subunit interactions, SF9 cells at 1x10Exocyst complexes and PIP5K1C were expressed using the SF9 insect cell system.To express exocyst Subcomplex-2, cells were coinfected with N-terminal 6xHis-mNeonGreen tagged Exoc6 and N-terminal 6xHis-MBP tagged Exoc5 at 0.8 relative MOI. Separately, untagged Exoc7 and untagged Exoc8 were coexpressed at 1.2 relative MOI. After two days of infection, the cultures were mixed, pelleted and frozen in liquid nitrogen.To express exocyst holocomplex, cells were coinfected with untagged Exoc1 and untagged Exoc2, and separately with untagged Exoc3 and untagged Exoc4. Separately, cells were coinfected with N-terminal 6xHis-MBP tagged Exoc5 at 0.8 relative MOI, and N-terminal 6xHis-mNeonGreen tagged Exoc6. Finally, a final separate culture was infected with untagged Exoc7 and untagged Exoc8 at 1.2 relative MOI. After two days of infection, each of the 4 cultures was combined, pelleted and frozen in liquid nitrogen. In our hands, the MOI ratio of tagged subunit was of critical importance for generation of stoichiometric complexes.To express PIP5K1C, cells were infected with N-terminal 6xHis-MBP tagged PIP5K1C for two days, pelleted and frozen in liquid nitrogen.2HPO4. Cell suspensions were lysed by Dounce homogenization followed by clarification through centrifugation. Cleared lysate was filtered through 0.45 μm membranes and bound to preequilibrated Amylose Fast Flow resin (MRC-PPU Reagents and Services). After washing, protein/proteins complexes were cleaved by addition of recombinant HRV-3C protease for at least 3 hours. Following cleavage and centrifugation, the supernatant was concentrated to using Vivaspin concentrators (Cytiva) and injected onto a 24 ml Superose 6 Increase 10/300 GL (or Superdex200 10/300 GL for Exoc1:mNeonGreen-Exoc4 and Exoc5:mNeonGreen-Exoc6:Exoc7) equilibrated in standard buffer. Peak fractions were analyzed by SDS-PAGE, pooled and concentrated and either used immediately in experiments, or aliquoted and frozen in liquid nitrogen and stored at -80 °C. Samples were sent to the mass spectrometry facility  for quantitative peptide analysis.To purify the exocyst complexes, all pellets were thawed on ice and resuspended to 50ml final volume in standard buffer with the addition of protease inhibitors and Benzonase (Sigma). PIP5K1C was resuspended in buffer containing 20 mM HEPES, 400 mM NaCl, 0.5 mM TCEP, 50 mM Na600=0.8, whereupon temperature was lowered to 18 °C for 10-12 h. Cells were pelleted resuspended in standard buffer, lysed, and clarified. Protein was purified by Ni affinity chromatography using 5 ml His-Trap HP column (Cytiva) followed by anion exchange on a 5ml Capto-Q column (Cytiva). Peak fractions were pooled and purified by size exclusion chromatography using a Superdex 200 16/60 pg column (Cytiva). All proteins were aliquoted, frozen in liquid nitrogen and stored at -80 °C.Small GTPases and lipid probes were expressed in BL21 bacterial cells using standard approaches. All constructs were grown in LB containing 1.75 w/vol % lactose and antibiotic at 37 °C to ODProtein concentrations for purified exocyst complexes and PIP5K1C were determined using a Pierce™ Coomassie (Bradford) Protein Assay Kit across several dilutions using systematically and independently generated standard curves each time. Protein concentrations for purified Arf6 were determined by measuring absorbance at 280 nm using a Nanodrop (Thermofisher) and calculated extinction coefficients.2O before being stained with 0.75% uranyl formate for approximately 30s. Excess stain was removed by blotting, and samples were imaged using a JEOL 2200FS 200 kV with inline omega filter on a Gatan Ultrascan 4k 15 μm pixel camera at 30,000x magnification. Images were obtained at a defocus of -0.5-2.0 μm. Particles were manually selected using Boxer implemented in EMAN2.Carbon Film, 400 Mesh copper grids (Electron Microscopy Sciences) were glow discharged and exocyst holocomplex (1.9 μM) was applied for 10 minutes and blotted away with Whatman paper. Grids where washed twice with ddH2 (Echelon Biosciences) were produced by mixing 85 mol % 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10 mol % phosphatidylserine (POPS) with 5 mol % of the respective phosphatidylinositol together with 0.1% Atto647N-DOPE (ATTO-TEC) or Rhodamine-DOPE. Control liposomes without phosphoinositides contain 90 mol% POPC, 10 mol% POPS and 0.1% Atto647N-DOPE (ATTO-TEC) or Rhodamine-DOPE. The mixtures were evaporated under nitrogen flow and dried overnight in a vacuum. Dried lipids were resuspended in buffer containing 20 mM HEPES, 150 mM NaCl, and 0.5 mM TCEP at 37 °C and subjected to 6 cycles of freeze thawing in liquid nitrogen. Where samples contained 0.5% Tris-NTA-DODA2 was added during the resuspension step. Liposomes were extruded to 100 nm and aliquoted, frozen in liquid nitrogen and stored at -20 °C.All lipids were obtained from Avanti Polar Lipids unless otherwise stated. Liposomes containing either PI(4)P (Echelon Biosciences) or PIP,Membrane-coated beadsTo assess lipid binding, beads were added to uncoated μ-Slide 8 well chambers (Ibidi) and protein added and mixed by pipetting at the indicated final concentrations. Lipid binding kinetics were allowed to equilibrate for 30 min at room temperature before imaging at the transverse plane.Confocal images were acquired using a Leica SP8 Confocal Microscope with a Leica HC PL APO CS2 63x/1.40 Oil objective at 0.75 base zoom with 1024x1024 pixels scan.2, 0.5 mM EGTA, 200 μg/mL β-casein, 20 mM BME, and 20 mM glucose) in the presence or absence of 1 mM ATP. Samples were applied to an imaging chamber and protein was added to a final concentration of 100 nM. Immediately before acquisition, reconstituted PIP5K1C was added to the beads at a final concentration of 150 nM. The reaction was monitored at 0.2 Hz using a Leica SP8 Confocal Microscope with a Leica HC PL APO CS2 63x/1.40 Oil objective at 0.75 base zoom with 1024x1024 pixel scan.Membrane-coated beads containing 0.1% Atto647N-DOPE (ATTO-TEC), 5% PI(4)P and 10% POPS in 84.9% POPC were prepared as described above and resuspended in 100 μl kinase buffer (20 mM HEPES (pH 7.0), 150 mM NaCl, 5 mM MgCl2 were added to the beads together with PIP5K1C for 30 min while rotation at RT before imaging.For membrane tethering assays, liposomes containing 5% PIPFor Arf6 mediated phosphoinositide conversion, 1 μM purified 6xHis-Arf6-T27N/Q67L was added to membrane-coated beads containing 0.5% Tris-NTA-DODA,2 and protein A coated 5 nm gold fiducials (Electron Microscopy Sciences) for 3 minutes and to a final protein concentration of 1 μM. Samples were applied to glow-discharged holey carbon film grids (Quantifoil) and plunge frozen into liquid ethane using a Vitrobot (ThermoFisher). Tilt series were collected on a JEOL CRYO ARM 300 (Jeol Ltd) electron microscope operated at 300 keV and equipped with in-column omega energy filter and DE-64 direct electron detector  operated in electron counting mode. Data acquisition was controlled by SerialEMExocyst holocomplex was mixed with liposomes composed of 95 mol% POPC and 5 mol% PIP2 using a Zeiss LSM880 Airyscan microscope with a 60x oil objective NA1.4.NMuMG cells (ATCC) and NMuMG genome-modified cellsStaining protocols for phosphoinositides using purified SidC-GFP and GFP-PLCδ-PH were adopted from Hammond et al. 2019. For staining with purified SidC-GFP, cells were fixed with 37 °C prewarmed PBS with 4% PFA for 20 min, followed by quenching in 50 mM NH4Cl for 20 min. Fixed cells were permeabilised with 20 μM digitonin in 1X PIPES buffer for 5 min at room temperature and washed. Fixed and permeabilised cells were blocked with 5% goat serum in 50 mM NH4Cl containing PIPES buffer in the presence of 1 μM purified SidC-GFP for 1 h at room temperature. Cells were washed in PIPES buffer and stained with mouse anti-GM130 (BD Biosciences 610822) for 1 h, washed and stained with secondary antibodies coupled to Alexa 647 for 45 min, washed and postfix in PBS with 2% PFA for 10 min. Samples were washed in 50 mM NH4Cl, mounted using Prolong Gold (ThermoFisher) and imaged using an LSM880 Airyscan microscope with a 60x, NA1.4 oil objective.For staining with purified GFP-PLCδ-PH, cells were fixed with prewarmed 37C 4% PFA in PBS for 20min, followed by quenching in 50 mM NH4Cl for 20 min. From now on all steps were carried out on ice and with all solutions chilled to 4 °C. Fixed cells were permeabilised with 0.5% saponin in PIPES buffer with 5% goat serum and 2 μM purified GFP-PLCδ-PH for 1 h. Fixed and permeabilised cells were washed with PIPES buffer and stained with mouse anti-E-cadherin  in 5% goat serum and 0.1% saponin containing PIPES buffer for 1 h washed and stained with secondary antibodies coupled to Alexa 647 for 45 min. Ice cold PBS with 2% PFA was added for 10 min before transferring the slides to room temperature and letting them adjust for another 10 min. Samples were rinsed in 50 mM NH4Cl, mounted, and imaged as above described.600=0.8, whereupon temperature was lowered to 18 °C for 10-12 h. Cells were pelleted, resuspended in standard buffer, lysed, and clarified. Sample was then filtered through a 0.22 μm filter and bound to GSH agarose beads (MRC-PPU Reagents and Services) for 1 h at 4 °C. Beads were washed, and protein was eluted with 20 μM glutathione. Eluted protein was further purified by size exclusion chromatography using a Superdex 200 16/60 pg column. Peak fractions were pooled and snap frozen in liquid nitrogen. Equal amounts of purified protein were added to 50 μl GSH agarose for 1 h at 4 °C, washed and resuspended in standard buffer. Purified PIP5K1C was added to a final concentration of 400 nM and incubated at 4 °C under constant rotation for 1 h. Beads were washed, resuspended in SDS-loading buffer and analysed by SDS page, followed by Coomassie staining.BL21 cells transformed with GST alone or GST-Arf6-Q67L/T27N were grown in LB containing 1.75% lactose and antibiotic at 37 °C to ODMembrane coated bead assays were analysed using a custom written ImageJ script. The signal from the Atto647N-DOPE or Rhodamine-DOPE was used to segment each bead individually, thereby creating a mask for the circumference for bead. The mask was used in the GFP/mNeonGreen channel to measure the recruitment of the fluorescent probes to each bead. Statistical details, including n numbers and repeats can be found in the corresponding figure legends. Statistics for determination of relative binding constants were acquired in GraphPad9 as single-site model, probability by Aoike Information Criterion <0.01%.The amount of signal overlap from the cell-based imaging approaches was determined using ImageJ. Overlap between the different channels was quantified using the automated segmentation plugin Squassh


FBW2 loss of function does not lead to strong growth or developmental defects, it significantly increases RNA-silencing activity. Interestingly, under conditions in which small-RNA accumulation is affected, the failure to degrade AGO1 in fbw2 mutants becomes more deleterious for the plant. Accordingly, the non-degradable AGO1 protein assembles high-molecular-weight complexes and binds illegitimate small RNA, leading to off-target cleavage. Therefore, control of AGO1 homeostasis by FBW2 plays an important role in quality control of RNA silencing.RNA silencing is a conserved mechanism in eukaryotes involved in development and defense against viruses. In plants, ARGONAUTE1 (AGO1) protein plays a central role in both microRNA- and small interfering RNA-directed silencing, and its expression is regulated at multiple levels. Here, we report that the F-box protein FBW2 assembles an SCF complex that selectively targets for proteolysis AGO1 when it is unloaded and mutated. Although   •FBW2 assembles an SCF complex targeting specifically empty AGO1•FBW2 mutation leads to AGO1 accumulation and increased RNA silencing activity•fbw2 results in AGO1 illegitimate loadingImpaired sRNA accumulation in •FBW2-mediated AGO1 proteolysis avoids off-target cleavage Argonaute proteins, main effectors of RNA silencing, remain stable only when they are loaded with small RNA. Hacquard et al. show that the E3 ubiquitin ligase component FBW2 triggers empty AGO1 degradation in Arabidopsis. Interfering with this mechanism leads to AGO1 spurious loading of illegitimate small RNA and off-target cleavage. MIR genes and act by post-transcriptionally repressing the levels of mRNA to which they are partly complementary. The second category encompasses so-called siRNA, which are processed from long double-stranded RNA arising from a variety of sources  and act as repressor of expression, either transcriptionally or post-transcriptionally, by mediating RNA cleavage and/or translational repression.In eukaryotes, RNA silencing is crucial for development and plays major roles in response to the environment, including pathogens, as well as in the control of transposable elements. This pathway involves processing of double-stranded (ds)RNA by the RNase III enzyme Dicer into small RNA (sRNA) of 21 to 24 nucleotides in length . These sArabidopsis thaliana (hereafter referred to as Arabidopsis) genome encodes 10 Argonaute paralogs  proteins P0 from poleroviruses encode F-box proteins that hijack the host S phase kinase-associated protein 1 (SKP1)-cullin 1 (CUL1)-F-box protein (SCF) ubiquitin-protein ligase (E3) to promote AGO1 degradation . P0 trigpty Ago1 . Moreovepty Ago1 .SQUINT (SQN), encoding a cyclophilin-40 chaperone acting as a positive regulator of AGO1 activity , encoding a subunit of the polycomb repressor complex 2 (PRC2) and that this regulation may be important for AGO1 protein homeostasis when plants are exposed to UV radiation  proteins in yeast two-hybrid interactions and was initially identified by a genetic suppressor screen of a null allele of activity . While fack loop , overexpin level . Loss of protein . Interesadiation .in planta, FBW2 does not induce the degradation of all AGOs equally, but preferentially targets AGO1. Our results indicate that FBW2 plays a critical role to maintain AGO1 proteostasis by preferentially degrading its unloaded form. Interestingly, in mutant plants lacking FBW2 and which are impaired in sRNA accumulation, stabilized AGO1 further worsens their phenotype. Hence, we show that the non-degradable AGO1 protein assembles high molecular complexes and binds illegitimate sRNA, leading to the cleavage of different target genes. Our studies identify a mechanism to avoid AGO1 spurious loading of sRNA, which could conditionally become detrimental for cells.In the present work, we show that, Nicotiana benthamiana (N. benthamiana) leaves, and the level of each AGO protein was assessed with the Flag antibody -inducible promoter XVE , in wildociation , ago1-57nwinding  and ago1pression   was enriched in the microsomal fraction, whereas the cytosolic enzyme UDP-glucose pyrophosphorylase (UGPase) was absent yet enriched in the soluble fraction  and membrane-bound  forms . We therfraction D. Whereain planta. We first examined the subcellular localization of both proteins. The coding sequence of FBW2 was fused to the Venus fluorescent protein at its N terminus and put under the control of its own promoter (pFBW2:Venus-FBW2) and co-expressed with cyan fluorescent protein (CFP)-AGO1 in N. benthamiana leaves. Confocal imaging revealed the co-localization of both proteins in the cytosol  was tested. For this, we used a patch assay in which a peat RNA . In this and P19 , a brigh and P19 A, left. xpressed A, right,xpressed B, suggesago1-27 mutation totally impaired S-PTGS, whereas it only decreased IR-PTGS -PTGS was tested because S-PTGS is more sensitive to small perturbation in AGO1 activity than IR-PTGS. Indeed, the hypomorphic  IR-PTGS . Therefoline Hc1 . This li n = 96; C. This i mutants B. It is  mutants . Analysi mutants C, which mutation C. This rmutation .AGO1 and FBW2 with or without a construct carrying the inverse repeat of GFFG, which is known to produce functional siRNA , and the elution fractions were analyzed by western blot , whereas in hyl1-2 and hyl1-2 fbw2-4 miR159 was barely detected in any fraction, probably because of its impaired synthesis   A and 5B.fbw2-4 mutation, we performed deep-sequencing analyses on total sRNA and AGO1-associated sRNA in Col-0 and five different mutant backgrounds  (To get more insights into the identity of sRNA in the context of the mutants) . AGO1 prmutants) .hen1 and hyl1 mutant backgrounds  genes  genes . HoweverS) genes D and S6BS) genes D. We theS) genes , allowinS) genes A. In ourS) genes , and smaS) genes , correspS) genes B. We obsAlthough the function of AGO proteins and their bound sRNA have been extensively studied in various biological processes across several organisms , their rDrosophila (Ago1) and mammals (Ago2)  . Since t of AGO1 , whereast siRNAs . Interesed Iruka . Thus, Ied Iruka .ago1-27, leading to an increased degradation rate of the AGO1-27 protein by FBW2, would be subjected to a similar regulation. While the ago1-27 hypomorphic allele has normal AGO1 slicer activity, it is defective in translational repression  complexes  FBW2 mediates AGO1 degradation and which type of ubiquitin chains are involved. Finally, the physiological context of this proteolytic mechanism remains to be elucidated, but specific stress conditions involving AGO1-RISC reprogramming may shed light on it.pascal.genschik@ibmp-cnrs.unistra.fr).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Pascal Genschik  were used in this study. The following Arabidopsis mutants, ago1-27 (ago1-42 (ago1-57 (fbw2-1 (fbw2-4 (SALK_144548), hen1-6 (SALK_090960), hyl1-2 (SALK_064863) and sqn-1 -AGO1) was generated by PCR amplification from the Arabidopsis AGO1 cDNA with the oligonucleotide primers listed in The Flag-NtAGO1 construct (pENTRY(221)-FlagNtAGO1) was amplified using primers listed in The 3HA-FBW2 construct was generated by PCR amplification from the FBW2 cDNA with the oligonucleotide primers listed in The 35S:FBW2 construct was generated by PCR amplification from the FBW2 cDNA with the oligonucleotide primers listed in FBW2 promoter, the Venus and FBW2 coding sequences were amplified by PCR using primers listed in FBW2 promoter (pFBW2:Venus-FBW2).For microscopy analysis, we generated the pFBW2:Venus-FBW2 construct. The FBW2 genomic sequence containing the attB sites was recombined into pDONR 221 plasmids (Invitrogen) and then transferred into the binary vector pGWB415 (The FBW2 genomic construct (pENTRY(221)-iFBW2) was amplified from genomic DNA by PCR amplification using primers listed  pGWB415  by GatewThe pUBQ:RFP-iFBW2  construct was obtained by Gateway LR recombination (Invitrogen) using pENTRY(221)-iFBW2 described earlier and the binary vector pUBN-RFP  by GatewN. benthamiana leaves, Agrobacterium cells (GV3101 Pmp90 or C58C1) harboring the constructs of interest were grown overnight at 28°C in 10mL LB medium supplemented with antibiotics, resuspended in 10mM MgCl2 supplemented with 200mM acetosyringone at an OD of 0.3 per construct (unless otherwise specified), and incubated for 1 h at room temperature before being pressure infiltrated into leaves of 4 week-old plants. Unless otherwise specified, all agro-infiltration assays were conducted in presence of P19. Plants were maintained in growth chambers under 16 h light and 8 h dark photoperiod with a constant temperature of 22°C. Sampling and observations were performed 72 h after agro-infiltration.For transient expression in in vitro culture conditions, Arabidopsis seeds were surface-sterilized using ethanol and plated on MS agar . The seeds were then stratified for 2 days at 4°C in the dark and then transferred in 16h-light/8h-dark  growth chamber, under fluorescent light (Osram Biolux 58W/965). Unless otherwise specified, seedlings were transferred in liquid MS  and acclimated for 24 h prior to chemical treatments.T-DNA transformation of Arabidopsis plants was performed using the floral dip method . For in For P0-myc and XVE:3HA-FBW2 induction during plant growth, MS-agar plates were supplemented with 10μM β-estradiol, while for mock treatment, an equal amount of DMSO was used. Plates were then handled as indicated above, and seedlings were harvested at 7 to 8 days or as indicated after sowing for protein content analysis or 9 to 10 days after sowing for aerial and root growth measurements. For kinetic induction of XVE:3HA-FBW2, seedlings were grown as indicated above for 8 to 12 days, then transferred into liquid MS medium  +1% sucrose in sterile conditions. Liquid MS medium was then replaced with either MS + DMSO (mock) or MS + 10μM β-estradiol.ago1.27 Arabidopsis lines were grown for 8 days on MS-agar plates then transferred into liquid MS medium +20μM MLN4924 for 20 h before harvesting.For IP-MS experiments, Col-0, FBW2OE and FBW2OE/GUS activity was quantified using crude extracts from plant leaves and monitoring the quantity of 4-methylumbelliferone products generated from the substrate 4-methylumbelliferyl-β-D-glucuronide (Duchefa) on a fluorometer (Thermo Scientific fluoroskan ascent) .Confocal microscopy was performed on a LEICA TCS SP8 laser scanning microscope (Leica Microsystem) using the objective HCX APO CS 20× magnification with a numeric aperture of 0,7 without immersion. Usual excitation/detection-range parameters for CFP and Venus were 458 nm/465–510 nm and 514 nm/600–630 nm, respectively and emissions were collected using system hybrid (Hyd) detectors.For immunoprecipitation of HA-FBW2, 1g of frozen plant material (10 day-old seedlings) ground to a fine powder with a mortar and pestle, resuspended in 3 volumes of IP Extraction Buffer  and incubated for 30 min at 8 rpm in the cold room. Insoluble material was removed by centrifugation . Identical amounts of crude extracts were incubated with 25μL anti-HA magnetic beads (Pierce Anti-HA magnetic Beads) (pre-washed three times in IP Extraction buffer) for 3 h at 8 rpm at room temperature. Immune complexes were washed three times in the IP extraction buffer. Elution of the immunoprecipitated proteins was performed by adding 30μL of glycine  to the magnetic beads and transfer to a solution containing 10μL Tris HCl 1M pH 11. Before analysis on SDS-PAGE gels, 4X Laemmli loading buffer was added to a final concentration of 1X to the samples and then denatured for 5 min at 95°C.For immunoprecipitation of endogenous AGO1, 500mg of frozen tissues (from 7 day-old seedlings) was ground to a fine powder with a mortar and pestle, resuspended in 3 volumes of crude extract buffer , and incubated for 20 min at 8 rpm in the cold room. Insoluble material was removed by centrifugation . Identical amounts of crude extracts were incubated with prebound @AGO1 (5μg) PureProteome Protein A magnetic beads  for 2 h at 7 rpm in the cold room. Immune complexes were washed four times in the crude extract buffer, and purified sRNA was eluted from the beads in Tri-Reagent (Sigma-Aldrich) following the manufacturer’s instructions.For each IP, 1g of seedlings was ground in liquid nitrogen for 10 min in 3 mL of ice-cold lysis buffer . The crosslinked protein extract was quenched 2 min with glycine to a final concentration of 200mM. The cleared supernatants were divided in two affinity purifications, incubated with magnetic microbeads coupled to HA antibodies , and complexes were eluted in 100 μL of pre-warmed elution buffer (Miltenyi). Co-IP experiments were performed in two independent biological replicates with two different transgenic lines (FBW2OE and FBW2OE/ago1.27). Each biological replicate was divided into two affinity-purification replicates. In parallel control IPs were carried out with HA antibodies in Col-0.6. Up to 10 of the most intense multiply charged ions (≥2) were selected for HCD fragmentation with a normalized collision energy set at 27, at 17.5K resolution, with a maximum injection time at 100ms and AGC set at 1 × 103. A dynamic exclusion time of 10 s was applied during the peak selection process. Raw files were finally transformed into mgf files using Proteome Discoverer software .Eluted proteins were digested with sequencing-grade trypsin . Each sample was further analyzed by nanoLC-MS/MS on a QExactive + mass spectrometer coupled to an EASY-nanoLC-1000 . Proteins were precipitated overnight with 5 volumes of cold 0.1 M ammonium acetate in 100% methanol. After washing twice the protein pellets with cold 0.1 M ammonium acetate in 80% methanol, proteins were further resuspended in 50 mM ammonium bicarbonate. Proteins were reduced  and alkylated . After a quenching step (5 mM dithiothreitol), proteins were digested overnight with 150ng of sequencing-grade porcine trypsin . The resulting vacuum-dried peptides were resuspended in water containing 0.1% (v/v) formic acid (solvent A). The peptide mixtures (500ng) were analyzed using an Easy-nanoLC-1000 system coupled to a Q-Exactive Plus mass spectrometer  operating in positive mode with a nanoelectrospray source. 5μL of each sample were loaded on a C-18 precolumn  at 800 bars in solvent A. After desalting and concentration, the pre-column was switched online with the analytical C18 analytical column  equilibrated in solvent A: solvent B . Peptides were eluted at a flow rate of 300 nL/min using a gradient from 5% B to 20% B in 120 min, from 20% B to 32% B in 15min, from 32% B to 95% B in 1min and 95% B to 95% B during 24min. The Q-Exactive Plus was operated in data-dependent acquisition mode (DDA) with Xcalibur software (Thermo-Fisher Scientific). Survey MS scans were acquired at a resolution of 70K at 200 m/z (mass range 350–1250), with a maximum injection time at 100ms and an automatic gain control (AGC) set at 3 × 10Arabidopsis thaliana with a decoy strategy (27.282 forward protein sequences). Peptides and proteins were identified with Mascot algorithm  and data were further imported into Proline v2.0 software (http://proline.profiproteomics.fr/). Proteins were validated on Mascot pretty rank equal to 1, and 1% FDR on both peptide spectrum matches (PSM score) and protein sets (Protein Set score). The total number of MS/MS fragmentation spectra was used to quantify each protein from at least six independent biological and affinity replicates. After a DEseq2 normalization of the data matrix, the spectral count values were submitted to a negative-binomial test using an edgeR GLM regression through R (R v3.2.5). For each identified protein, an adjusted p-value (adjp) corrected by Benjamini–Hochberg was calculated, as well as a protein fold-change (FC). The results are presented in a Volcano plot using protein log2 fold changes and their corresponding adjusted (-log10adjp) to highlight upregulated and downregulated proteins.Data were searched against the TAIRv10 fasta protein sequences from Proteins were extracted in pre-heated (95°C) 2X Laemmli sample buffer, quantified using amido-black staining  and 10 tThe crude cell extracts were prepared from 7 day-old seedlings that were ground with mortar and pestle in an ice-cold buffer containing 50mM HEPES pH 7.6, 30mM KCl, 5mM MgCl2, 5mM EGTA pH 8.0 and 250mM sucrose supplemented with freshly added 1mM DTT and Protease Inhibitor cocktail (Roche). After centrifugation at 1,000 x g for 5 min at 4°C the resulting supernatant represents the total extract . The microsomal (Micro) and cytoplasmic (Cyto) fractions were collected by centrifugation of soluble cell extract in a TLA-110 rotor (Beckman Coulter ultracentrifuge) at 100,000 x g for 30 min at 4°C. For protein analysis, fractions were precipitated with methanol/chloroform and protein pellets were dissolved in 1x Laemmli buffer.About 800mg seedlings of the indicated genotypes were extracted in 2.5 Volumes of 50mM Tris-HCl pH7.5, 150mM NaCl, 5mM MgCl2, 10% glycerol, 0.1% IGEPAL, 5mM DTT, 10μM MG132, 1X cOmplete™ Protease Inhibitor Cocktail (Roche) and left to mix on carousel for 30 min. Extracts were centrifuged 10 min at 4400 rpm in Falcon tubes, filtered through Miracloth, and filtered through a 0,2μm Minisart RC 4 syringe filter. Crude extracts were calibrated to 1.9μg/μL using the amidoblack method, and 500μL were injected in 4 separate loops. Separation was performed sequentially on a Superdex 200 10/300 increase column on an AKTA Pure system with the following settings: 500μL/min, fraction volume of 250μL collected from 7.25mL to 13mL. For proteins, half of the fractions was precipitated in 2 volumes of absolute ethanol at 4°C for 48 h. Samples were centrifuged at maximum speed for 30 min at 4°C, pellets were resuspended in 2x Laemmli buffer and treated at 95°C for 5 min. Denatured samples were separated in 7.5% acrylamide SDS PAGE gels and treated as indicated in the immunoblot section. Input samples were collected from the crude extract and analyzed separately following the same method. For RNA, half the fraction (every two fraction) was mixed with 300μL tri-Reagent and extracted as indicated. Precipitated RNA was resuspended in a final concentration of 60% formamide, 5mM EDTA, 0.05% bromophenol blue-0.05% xylene cyanol, heated a 95°C for 5 min, and separated by electrophoresis on a 15% polyacrylamide (19:1 acrylamide:bisacrylamide), 8M Urea, 0.5× TBE gel at 15 watts. Electroblotting, crosslinking and hybridization was performed as standard. Input samples were collected from the original plant material and analyzed separately following the same method.For quantitative RT-PCR (qPCR), 1μg of total RNA extracted in Tri-Reagent according to the manufacturer’s instruction was treated with DNaseI (Fisher Scientific) and reverse transcribed with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem). PCR was performed using gene specific primers see  in a totFor sRNA analysis, RNA was extracted in Tri-Reagent according to the manufacturer’s protocol, and aqueous phase was left in 1 volume of isopropanol over-night at −20°C, precipitated 30 min at 16000g (4°C). Pellets were rinsed in 1mL 70% ethanol and centrifuged an additional 10 min. Dry RNA pellets were resuspended in 60% deionised formamide. RNA gel blot analyses of low molecular weight RNA were performed with 10μg of total RNA. Low molecular weight RNAs were resuspended in a final concentration of 60% formamide, 5mM EDTA, 0.05% bromophenol blue- 0.05% xylene cyanol, heated a 95°C for 5 min, and separated by electrophoresis on 15% polyacrylamide gels (19:1 acrylamide:bisacrylamide) 8 M Urea, 0.5× TBE gel. Separated RNA species were electroblotted on Hybond-NX (Amersham) membrane and fixed by carbodiimide-mediated cross-linking. DNA oligonucleotides complementary to miR403, miR168, miR159 and U6 RNA see  were 5′ hyl1-2/fbw2-4 crude extract was verified by immunoblot before proceeding with labeling. Precipitated RNA was resuspended in 10μL of ultrapure water and 5μL was [γ-32P]ATP labeled by T4 polynucleotide kinase (Thermo Fisher Scientific) for 35 min at 37°C in buffer B. Labeled RNA was Tri-Reagent extracted as indicated, the aqueous phase filtered through a G25 MicroSpin column  and the flowthrough precipitated in 75% isopropanol with 40μg of glycogen overnight at −20°C. A control reaction with 90ng of siR255 (21-nt) RNA oligo was treated in an identical fashion. Labeled RNA was resuspended in a final concentration of 60% formamide-5 mM EDTA-0.05% bromophenol blue-0.05% xylene cyanol, heated a 95°C for 5 min, and separated by electrophoresis on a 15% polyacrylamide (19:1 acrylamide:bisacrylamide), 8M urea, 0.5×TBE gel at 15 watts for 230 min. The gel was wrapped in plastic and the signal was detected using FUJI medical x-ray films.AGO1 immunoprecipitation were performed as indicated, from 400mg of 2 week-old seedlings of the indicated genotypes. Purified RNA was eluted from the beads in tri-Reagent, as indicated, and RNA was precipitated overnight at −20°C in 50% isopropanol and 40μg of glycogen. Stabilization of AGO1 in the hyl1-2, hyl1-2 fbw2-4, hen1-6, hen1-6 fbw2-4 and fbw2-4 seedlings grown on MS-agar plates using Tri-Reagent according to the manufacturer’s instruction. For AGO1-loaded sRNA samples, IPs were performed as described above from 500mg of 1-week-old Arabidopsis seedlings grown on MS-agar plates. AGO1-loaded sRNA were then extracted by adding Tri-Reagent directly on the magnetic beads and extraction of RNA was then performed according to the manufacturer’s instructions.Total RNA samples were extracted from 1-week-old Col-0, Small RNA libraries were constructed using Real-Seq-AC kit  according to manufacturer’s instructions with 500ng as starting material. Libraries were sequenced using Illumina Next-Seq 500 technology at University of Delaware . Parallel analysis of RNA end (PARE) libraries were constructed following the previously published protocol  and usinwww.arabidopsis.org/download/) and its corresponding TAIR10 BLAST sets for all the features, using Bowtie2  and 12 PARE libraries were constructed. For sRNA libraries, we trimmed adapters and low-quality reads using Cutadapt v2.9 software , retaini Bowtie2 . Differe Bowtie2  and all  Bowtie2  packages Bowtie2 . To achi Bowtie2  and the https://rsb.info.nih.gov/ij/). Ten plants per line were used.Analysis of leaf area and lateral roots. For leaf area analysis, seedlings were grown in soil. The seeds were stratified in water and darkness for 48 h. Plastic pots (7x7x7cm) were filled with 62g of soil (Hawita Fruhstorfer erde) and watered with tap water to reach a relative humidity of 2.2gH20/gsoil (RWC 69%). Three seeds were sown in the middle of the pot. The pots were covered with a plastic foil to maintain the humidity level and placed in a 16h-light (21°C) and 8h-darkness (18°C) regime. At 4 days after stratification (DAS), the plastic film was removed, and at 5 DAS plants were thinned out to keep one plant per pot. The pots were watered every 2 to 3 days and maintained at a relative soil humidity of 2.2gH20/gsoil. Plant size at 22 DAS was determined by dissecting every leaf and placing it from oldest to youngest on a petri dish with 1% agar. The plates were photographed and the leaf area was measured with ImageJ v1.45 . At least ten seedlings per line were analyzed.For GFP fluorescence quantification A, GFP fl


Ribosomal protein S6 kinase 1 (S6K1) is a serine–threonine kinase that has two main isoforms: p70S6K (70-kDa isoform) and p85S6K (85-kDa isoform). p70S6K, with its upstream mammalian target of rapamycin (mTOR), has been shown to be involved in learning and memory and participate in the pathophysiology of Alzheimer’s disease (AD). However, the function of p85S6K has long been neglected due to its high similarity to p70S6k. The role of p85S6K in learning and memory is still largely unknown.We fractionated the postsynaptic densities to illustrate the differential distribution of p85S6K and p70S6K. Coimmunoprecipitation was performed to unveil interactions between p85S6K and the GluA1 subunit of AMPA receptor. The roles of p85S6K in synaptic targeting of GluA1 and learning and memory were evaluated by specific knockdown or overexpression of p85S6K followed by a broad range of methodologies including immunofluorescence, Western blot, in situ proximity ligation assay, morphological staining and behavioral examination. Further, the expression level of p85S6K was measured in brains from AD patients and AD model mice.p85S6K, but not p70S6K, was enriched in the postsynaptic densities. Moreover, knockdown of p85S6K resulted in defective spatial and recognition memory. In addition, p85S6K could interact with the GluA1 subunit of AMPA receptor through synapse-associated protein 97 and A-kinase anchoring protein 79/150. Mechanistic studies demonstrated that p85S6K could directly phosphorylate GluA1 at Ser845 and increase the amount of GluA1 in synapses, thus sustaining synaptic function and spine densities. Moreover, p85S6K was found to be specifically decreased in the synaptosomal compartment in the brains of AD patients and AD mice. Overexpression of p85S6K ameliorated the synaptic deficits and cognitive impairment in transgenic AD model mice.These results strongly imply a significant role for p85S6K in maintaining synaptic and cognitive function by interacting with GluA1. The findings provide an insight into the rational targeting of p85S6K as a therapeutic potential for AD.The online version contains supplementary material available at 10.1186/s40035-022-00334-w. Ribosomal protein S6 kinase 1 (S6K1) belongs to the AGC kinase family, which is a group of kinases related to protein kinase A (PKA), cGMP-dependent protein kinase/protein kinase G and protein kinase C, whose members are serine–threonine kinases and share some structural features. S6K1 has two main isoforms transcribed from a single gene: p70S6K (70-kDa isoform) and p85S6K (85-kDa isoform) , 3. Compp70S6K, together with its upstream mTOR, has received attention as a key modulator in memory and Alzheimer’s disease (AD) –12, the Wild-type (WT) C57BL/6J male mice and 24-h newborn Sprague–Dawley (SD) rats were purchased from SLAC Laboratory Animal Co. Ltd. . APP/PS1 (APPswe and PSEN1dE9 mutation) male mice with C57BL/6J background were from the Nanjing Biomedical Research Institute of Nanjing University .  5×FAD male mice were from the Jackson Laboratory . Heterozygous GluA1-mutated transgenic mice in which Ser845 of GluA1 is mutated to Ala (S845A)  were breed to obtain homozygous ones. All animal procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine.www.brainbank.nl). Written informed consents for a brain autopsy and the use of the material and clinical information for research purposes had been obtained by The Netherlands Brain Bank. Detailed information of the brain donors is described in Additional file Postmortem temporal cortex samples from AD patients and non-demented controls were obtained from The Netherlands Brain Bank, Netherlands Institute for Neuroscience, Amsterdam  in PcDNA3.1 were purchased from SunBio Technology  and verified by DNA sequencing. Lentiviruses expressing p70S6K, p85S6K or Flag-p85S6KT421A were from OBiO Technology . Adeno-associated viruses  expressing p70S6K, p85S6K, or shRNA for S6K1 (target sequence: CCTTTCAGACCGGAGGAAA for mice), and lentiviruses expressing S6K1 shRNA (target sequence: GCACCTGCGTATGAATCTA for rats) and corresponding controls were from SunBio Technology. The control sequence for shRNA was TTCTCCGAACGTGTCACGT. All AAVs were with the synapsin-1 promoter.2. Cells were transfected with plasmids using Lipofectamine 3000  according to the manufacture’s instruction, and processed 48 h after transfection.HEK293 cells  were maintained with DMEM containing 50 U/ml penicillin and streptomycin, and 10% fetal bovine serum, in a humidified incubator at 37 °C with 5% CO6 cells/dish or onto a 6-well plate coated with poly-L-lysine  at a density of 1 × 106 cells/well. The neurons were used for experiments between culture days 18 and 24. The neurons were infected on day 9 with lentiviruses. For specific knockdown of p85S6K, lentiviruses expressing S6K1 shRNA and p70S6K were administered simultaneously in a 4:1 viral particle ratio. The control neurons were infected with corresponding control AAVs.Primary hippocampal neurons were obtained from the hippocampus of newborn SD rats at 24 h after birth as previously described . NeuronsThe mice were anesthetized by inhalation of 2% isoflurane. To ensure better coverage of the whole hippocampus, each hippocampus was given two injections at positions stereotaxically defined as (1) 1.95 mm posterior to the bregma, 1.0 mm lateral to the midline and 2.2 mm ventral to the skull surface, and (2) 2.9 mm posterior to the bregma, 3.0 mm lateral to the midline, and 3.2 mm ventral to the skull surface. One microliter of AAV particles was slowly injected over 10 min and then left for 10 min to facilitate diffusion. The needle was slowly raised over a 2-min period. For specific knockdown of p85S6K, AVVs expressing S6K1 shRNA and p70S6K were co-administered in a 2.5:1 viral particle ratio. The control mice were injected with corresponding control AAVs. Subsequent experiments were carried out 2 months after viral injection.The pool of PSDs was prepared as previously described . HippocaTrypsin treatment of synaptosomes was performed as previously described . BrieflyCultured cells were lysed in Pierce™ IP lysis buffer (Thermo Fisher Scientific) with a protease inhibitor cocktail (APExBIO Technology). Hippocampi from mice were dissected, homogenized, and solubilized at 4 °C for 1 h in IP lysis buffer with protease inhibitors. The PSD-1 pellet from PSD fractionation was also lysed in Pierce™ IP lysis buffer. Five hundred micrograms of protein of the lysates were added with 1 μg antibody (mouse anti-Flag (M2) , mouse anti-c-Myc (9E10) , mouse anti-GluA1 , and rabbit anti-p70S6K ) and incubated at 4 °C overnight. Next, the samples were incubated with protein A/G agarose resin  with rotation for 2 h. Proteins were eluted from the resin with 2 × SDS sample buffer and then subjected to SDS-PAGE and western blotting analysis. For multiple blotting, eluates were split equally and subjected to SDS-PAGE separately.The cultured neurons were incubated with rabbit anti-GluA1 N-terminus  and then fixed in 2% paraformaldehyde. The cells were blocked and permeabilized with PBS containing 10% normal goat serum and 1% Triton X-100 for 2 h at room temperature. Then, the slices were stained with mouse anti-PSD95  overnight at 4 °C and subsequently stained with Alexa 488-conjugated goat anti-rabbit  and Alexa 633-conjugated goat anti-mouse antibodies . Images were captured using a Leica TCS SP8 laser confocal microscope  and colocalization of GluA1 and PSD95 was analyzed using ImageJ software . For staining of endogenous S6K1 or exogenous p70S6K and p85S6K in neurons and HEK293 cells, the cells were fixed first. After permeabilization and blocking, the cells were then incubated with anti-p70S6K or anti-Flag antibody.2+/Mg2+ containing 0.1% BSA at 4 °C. Cells were then solubilized in lysis buffer . Thirty microliters of each lysate were used to determine the total GluA1, and the remaining lysate was incubated with streptavidin beads (Thermo Fisher Scientific) to detect surface GluA1 using rabbit anti-GluA1 antibody .Surface proteins of primary hippocampal neurons were biotinylated with 1 mg/ml sulfo-NHS-LC-biotin (Thermo Fisher Scientific) according to the manufacturer’s instructions. The unbound biotin was washed away by PBS/CaCells were solubilized as above. Mouse cortex and hippocampal tissues stored in liquid nitrogen were thawed on ice and lysed in RIPA buffer containing 10 mM Tris–Cl (pH 8), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, and 1 mM PMSF and a protease inhibitor and phosphatase inhibitor cocktail (APExBIO Technology). The extracts were centrifuged at 12,000 g for 20 min at 4 °C. Equal amounts of proteins were subjected to SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore Sigma). The following primary antibodies were used: rabbit anti-p70S6K phosphorylated at Thr389 , rabbit anti-p70S6K , rabbit anti-S6 , rabbit anti-S6 phosphorylated at Ser235 , mouse anti-SAP97 , mouse anti-AKAP79 , mouse anti-AKAP150 , rabbit anti-GluA1 phosphorylated at Ser845 , rabbit anti-GluA1 , rabbit anti-GluA2 , rabbit anti-GluA3 , mouse anti-synaptophysin , mouse anti-PSD95 , rabbit anti-Flag , rabbit anti-Myc , rabbit anti-PKA catalytic subunit α  and rabbit anti-GAPDH . Secondary antibodies were IRDye goat anti-rabbit or -mouse IgG (H + L) . The bands were visualized and analyzed by Li-Cor Odyssey Fc Image Studio (Li-Cor Biosciences). Immunofluorescent intensity values were normalized to the corresponding total protein or GAPDH. For proteins with similar molecular weights to be blotted with antibodies of the same species of origin, the lysates were split equally and subjected to SDS-PAGE separately.The brain slices were prepared according to previous descriptions with modifications . The ane2 and 5% CO2) artificial cerebrospinal fluid . Transverse hippocampal slices of 300-μm thickness were cut and allowed to recover for at least 2 h in 30 °C ACSF. Then chemical long-term potentiation (LTP) was induced in the hippocampal slices using MgCl2-free ACSF containing100 nM rolipram, 50 µM forskolin and 100 µM picrotoxin for 16 min, and then immediately fixed in 4% paraformaldehyde. Then the PLA was performed with Duolink in situ PLA Kit using rabbit anti-GluA1  and mouse anti-synaptophysin  antibodies following the manufacturer's instructions. Images were captured using the same settings by a Leica TCS SP8 laser confocal microscope with a 63 × oil objective. Three z stacks (1 μm) of 1024 × 1024 pixels from ∼2 to 3 μm below the slice surface were acquired in the CA1 region for each slice. PLA puncta in all images were identified using the same settings by ImageJ. The dendrites at > 15 µm away from the soma were selected for analysis of PLA puncta.SYNPLA was used to detect synaptic insertion of GluA1-containing AMPA receptors as previously described with mild modifications , 27. Dor2 and a protease inhibitor cocktail) supplemented with or without 1 mM ATP. After incubation at 30 °C for 30 min, the reaction was terminated by 2 × SDS sample buffer and then subjected to SDS-PAGE and western blotting analysis.The GST-GluA1 C-terminus expressed in HEK293 cells was pulled down by glutathione agarose (Santa Cruz Biotechnology), and mixed with Flag-p85S6K immunoprecipitated from HEK293 cell extracts in phosphorylation assay buffer  following the manufacturer's instructions. Brain tissue sections of 100-μm thickness were cut, and dehydrated in sequential rinses of 50%, 75%, 95% and 100% ethanol and cleared in xylene. The staining was viewed under a Leica DFC320 microscope. ImageJ software was used to analyze the number of spines and the total dendritic length. Distal dendrites at > 50 µm away from the soma were selected for measurement of spine density.Male WT mice of 2 to 3 months old were used for behavioral assessment involving p85S6K knockdown. Male APP/PS1, S845A and WT male mice of 7 months old were used for behavioral assessment involving p85S6K overexpression. Behavioral tests were performed 2 months after AAV injection.Open field test was performed to assess the motor activity and anxiety behavior. The mouse was placed in the center of an open field apparatus (30 cm × 30 cm × 30 cm) and allowed to freely explore it for 10 min. The movement of the mouse was recorded and analyzed by a video tracking system .The MWM procedure was performed as previously described with mild modifications , 29. TheThe NOR procedure was used to investigate non-spatial memory performed as previously described with mild modifications . The mict-test . Comparisons among 3 or more groups were performed using one-way, two-way or three-way ANOVA followed by post-hoc analysis where appropriate . Data that are not normally distributed were analyzed with the non-parametric two-tailed Mann–Whitney test between two groups . Pearson correlation was used to assess the correlations of p85S6K level with Braak stage and colocalization of GluA1 and PSD95. P < 0.05 was considered statistically significant.Data are presented as the means ± standard error of the mean (SEM). The data were analyzed by GraphPad Prism 8.0. The comparison between two groups was conducted using unpaired two-tail Student’s First, we examined whether the distribution of p85S6K is different from that of p70S6K. Exogenous expression of p85S6K and p70S6K demonstrated that they were expressed similarly in cytosol and nucleus in HEK293 cells  of human AD brains to further explore whether it was affected in AD. p85S6K was significantly decreased in the P2 fraction of the postmortem AD brains compared with non-demented control brains Fig. : Fig. S7Next, we investigated whether p85S6K could ameliorate the cognitive deficits in AD mice. As  5×FAD mice exhibit higher anxiolytic behavior , we usedAs overexpression of p85S6K also showed beneficial effects in WT mice, we overexpressed p85S6K in S845A mice, in which phosphorylation of GluA1 at Ser845 was deficient due to its mutation to alanine. In these mice, p85S6K overexpression had no effect on spatial learning and memory  by p85S6K. Elevated surface CP-AMPARs have been shown to facilitate LTP and improve hippocampus-dependent spatial learning [AMPA receptors mediate a majority of fast excitatory synaptic transmission and are important for synaptic plasticity and memory , 58. Thelearning . The explearning , 60. Exalearning , 61, 62.The expression of p70S6K is reported to be elevated in the whole homogenates of AD brains . Most stSimilar to p70S6K, mTOR is the defined upstream regulator of p85S6K. mTOR signaling has been shown to be implicated in various brain functions , 10. TheThe present study further showed that the reduction of p85S6K did not parallel PSD95 in AD brains. Measurements of PSD95 in AD have produced contradictory results in both animal and human brains . Some stBesides AMPA receptors, NMDA receptors are also located in PSDs and play important roles in cognition . The funIn summary, the present study illustrates the interaction between p85S6K and GluA1 and the regulatory role of p85S6K in synaptic function and cognition, which can advance our understanding of modulation of GluA1-containing AMPA receptors. Furthermore, we found pathological alterations of p85S6K in AD. The present study provides a new mechanism of the role for S6K1 in cognition and uncovers p85S6K-GluA1 signaling in memory modulation.Additional file 1. Table S1: Information of brain donors in this study. Fig. S1: The expression pattern of S6K1 in HEK293 cells and neurons. Fig. S2: The quantification of PSD95 and synaptophysin in fractions and the validation of p70S6K antibodies. Fig. S3: Knockdown of p85S6K efficiently reduces p85S6K in PSD-1 and does not affect the locomotor activity and anxiety-like behavior. Fig. S4: p85S6K cannot be immunoprecipitated with GluA1 with little expression of AKAP79 in HEK293 cells. Fig. S5: p85S6K does not interfere with the interaction between GluA1 and PKA. Fig. S6: The immunoblots of p85S6K/p70S6K and phosphorylated form in remaining human temporal cortex samples related to Fig. Fig. S7: p85S6K expression in P2 pellets of cortex of APP/PS1 mice. Fig. S8: p85S6K expression is decreased in PSD-1 of cortex of  5×FAD mice. Fig. S9: PSD95 expression is not altered in AD brains. Fig. S10: Overexpression of p85S6K does not affect the locomotor activity and anxiety-like behavior. Fig. S11: Upregulation of p85S6K does not enhance the spatial learning and spine density in S845A mice. Fig. S12: The NLS predicted in p85S6K by www.csbio.sjtu.edu.cn/bioinf/INSP/. Fig. S13: The expression of GluA1 and its phosphorylation at Ser845 are reduced in P2 pellets from fractionation of cortex of 7-month old  5×FAD and 9-month old APP/PS1 mice.


Herein, well-defined hierarchical CoSeO3‧2H2O nanoflowers assembled using 10 nm-thick nanosheets are successfully synthesized via a facile one-step hydrothermal method. When used as anodic materials for LIBs, the CoSeO3‧2H2O nanoflowers exhibit a considerably high discharge capacity of 1064.1 mAh g−1 at a current density of 0.1 A g−1. In addition, the obtained anode possesses good rate capability and cycling stability. Owing to the superior electrochemical properties, the CoSeO3‧2H2O nanoflowers would serve as promising anodic materials for high-performance LIBs.As novel anodic materials for lithium-ion batteries (LIBs), transitional metal selenites can transform into metal oxide/selenide heterostructures in the first cycle, which helps to enhance the Li T−1) ,6. Thus,2 can form an electric field in the nanocrystal, so they possess much lower ion-diffusion resistance and accordingly exhibit outstanding high-rate capability and good cycle stability [In the last few years, transitional metal selenides have received considerable attention for their rich redox active sites, high electronic conductivity, and large theoretical capacities, thus leading to superior electrochemical performance. Accordingly, transitional metal selenides are promising anodes of LIBs ,8,9,10. tability . Therefo3‧H2O nanosheets, which could exhibit reversible capacities of 1100 and 515 mAh g−1 at 3 and 10 A g−1 after 1000 cycles, respectively [3 porous microspheres were shown to be capable of delivering a high reversible capacity of 709 mAh g−1 after 1400 cycles at a current density of 3 A g−1 [3‧2H2O, has a different crystal structure from other cobalt selenites [3‧2H2O with a unique morphology would be obtained by rational design, and accordingly, the electrochemical performance would be improved. However, the synthesis and Li+ storage properties of CoSeO3‧2H2O have not been reported.Nevertheless, cobalt selenites still suffer from pulverization due to the large volume change during the charge and discharge processes, leading to rapid capacity decay . It is cectively ; anhydro 3 A g−1 . It is welenites ,20,21. T+ transport [3‧2H2O nanostructures to achieve the goals of high capacity and long life.Recently, 3D hierarchical nanostructures assembled from low-dimensional building blocks have attracted tremendous attention in the field of LIBs . As is kransport . Hence, 3‧2H2O nanoflowers for the first time. The CoSeO3‧2H2O nanoflowers were assembled using nanosheets with thickness of ~10 nm. Owing to the unique structure, the obtained CoSeO3‧2H2O nanoflowers exhibited high specific capacity, superior rate capability, and excellent cycling stability.Herein, we report a simple and effective one-step hydrothermal method for the synthesis of hierarchical CoSeO3COOH)2‧4H2O (0.2491 g) and Na2SeO3‧5H2O (0.1315 g) were dissolved into a mixed solvent of 10 mL deionized water and 5 mL ammonium hydroxide (80%) and stirred for 10 min. Then, the obtained solution was transferred into an autoclave and heated at 150 °C for 12 h. After the reaction, the product was washed with deionized water and ethanol. After dried at 80 °C for 10 h, hierarchical CoSeO3‧2H2O nanoflowers were obtained. All the chemicals were purchased from Shanghai Aladdin Bio-Chem Technology Co. Ltd. and used without purification. Co using Cu Kα radiation. The morphology, nanostructure, and composition of the sample were characterized using field emission scanning electron microscopy , transmission electron microscopy , and high-resolution TEM .3‧2H2O nanoflowers (70 wt%), carbon black (20 wt%), and polyvinylidene fluoride  were mixed into N-methyl-2-pyrrolidone to form a slurry, which was then uniformly coated on copper foils and dried at 120 °C for 12 h in vacuum to generate working electrodes. The loading amount of the electrode was about 2 mg cm−2. 1 mol L−1 LiPF6 dissolved in ethylene carbonate (EC) and diethyl carbonate (DC) (1:1 by volume) was employed as the electrolyte. Li foil and polypropylene membrane were chosen as the counter electrode and separator, respectively. Then, the CR2025 button batteries were assembled in an argon-filled glovebox. The LAND CT2001A battery tester  was used to evaluate the electrochemical properties of the obtained anode.Hierarchical CoSeO3‧2H2O with space group of P21/n(14) and lattice parameters of a = 6.5151 Å, b = 8.8253 Å, and c = 7.6404 Å (JCPSD No. 52-0215). The diffraction peaks at 15.48°, 25.71°, 29.60°, 32.73°, 35.81°, 37.83°, 40.95°, and 52.78° can be indexed to the (011), (012), (210), (031), (12−2), (113), (040), and (313) facets, respectively. Clearly, no other crystal phases  can be found, which reveals the high purity of the sample. The XRD pattern of the sample is shown in 3‧2H2O. As shown in the high-magnification FESEM image (3‧2H2O nanoflowers are made up of numerous ultrathin nanosheets with thicknesses of ~10 nm (marked by the red arrows). In the synthesis, CoSeO3‧2H2O nuclei prefer to grow into ultrathin nanosheets owing to their anisotropic crystal characteristics. Thus, in the following growth process, to minimize the surface energies, these primary nanosheets have a strong tendency to interconnect with each other and form into 3D hierarchical nanoflowers with obviously open structure. This special hierarchical structure can accelerate the diffusion of Li+, provide more areas for the contact between electrode and electrolyte, and alleviate the volume changes during the charge/discharge processes, thus leading to enhanced electrochemical performance [The morphologies of the products were analyzed using FESEM, and the results are shown in EM image c, the Coformance ,25.3‧2H2O nanoflowers were studied using TEM analysis. The different magnification TEM images  at a current density of 0.1 A g−1. The initial discharge and charge capacities are 1064.1 and 897.3 mAh g−1, respectively, and the coulombic efficiency and the irreversible capacity of the first cycle is 84.3% and 166.8 mAh g−1. The capacity loss may be attributed to the formation of Li2O by intercalated Li+ and SEI film owing to the decomposition of electrolyte during the first cycle on the electrode surface [3‧2H2O anode.The electrochemical properties of the obtained CoSeO surface . In the 3‧2H2O electrode was studied by progressively increasing the current densities from 0.1 to 0.2, 0.5, 1.0, and 2 A g−1. As shown in 3‧2H2O nanoflowers gradually decreases from 1058.9 to 858.1, 816.7, 765.5 and 678.6 mAh g−1 when increasing the current density from 0.1 to 0.2, 0.5, 1.0 and 2.0 A g−1, respectively. Importantly, the discharge capacity is still as high as 678.6 mAh g−1 as the current density is increased to 2.0 A g−1. Then, the discharge capacity can return to 762.7 mAh g−1 as the current density is reduced to 1 A g−1. The above results suggest the excellent structural stability at high current density and superior rate performance of the anode.The rate capability of the CoSeO3‧2H2O electrode was also evaluated at a constant current density of 0.5 A g−1 for 180 cycles, and the result is shown in −1 after 180 cycles. Meanwhile, the high coulombic efficiency of 98% can be obtained in the subsequent cycles after the first few cycles, which further indicates the excellent cyclic stability of the CoSeO3‧2H2O electrode. 3‧2H2O electrode after the cyclic stability test. Obviously, the hierarchical structure of the sample can be maintained even after long and intensive battery operation, suggesting the excellent electrochemical stability. In addition, the discharge capacity of the electrode is slightly increased from the first to the fiftieth cycle and then slowly decreased during the following cycles, which may be attributed to the gradual electrolyte penetration and the reversible growth of the electrochemistry active polymeric gel-like film by the activated electrolyte degradation [3‧2H2O nanoflowers exhibit better electrochemical performance ):Based on some important literature ,18,36, C+-ions storage mechanism of CoSeO3‧2H2O, some analysis methods, such as in situ EIS, in situ XRD, ex suit XPS, ex suit TEM, and so on, can be further conducted [3‧2H2O with other morphologies, heteroatoms doped CoSeO3‧2H2O, and CoSeO3‧2H2O@C composites.In order to better comprehend the Lionducted ,37. On tonducted ; furtheronducted ,40. Henc3‧2H2O nanoflowers were successfully synthesized using an effective and simple hydrothermal method for the first time. When used as the anode materials for LIBs, the CoSeO3‧2H2O electrodes exhibited excellent Li+ storage properties, which can be attributed to the hierarchical flower-like architecture and the open structure. The present method is expected to be extended to the synthesis of other metal selenites with unique morphologies and improved properties.In summary, CoSeO