S-acylation of a non-secreted peptide controls plant immunity via secreted-peptide signal activation.

Small peptides modulate multiple processes in plant cells, but their regulation by post-translational modification remains unclear. ROT4 (ROTUNDIFOLIA4) belongs to a family of Arabidopsis non-secreted small peptides, but knowledge on its molecular function and how it is regulated is limited. Here, we find that ROT4 is S-acylated in plant cells. S-acylation is an important form of protein lipidation, yet so far it has not been reported to regulate small peptides in plants. We show that this modification is essential for the plasma membrane association of ROT4. Overexpression of S-acylated ROT4 results in a dramatic increase in immune gene expression. S-acylation of ROT4 enhances its interaction with BSK5 (BRASSINOSTEROID-SIGNALING KINASE 5) to block the association between BSK5 and PEPR1 (PEP RECEPTOR1), a receptor kinase for secreted plant elicitor peptides (PEPs), thereby activating immune signaling. Phenotype analysis indicates that S-acylation is necessary for ROT4 functions in pathogen resistance, PEP response, and the regulation of development. Collectively, our work reveals an important role for S-acylation in the cross-talk of non-secreted and secreted peptide signaling in plant immunity.

Modeling evolution of cell morphology under stretching.

The mechanical stimulation of cells from the surrounding environment is an important life activity. However, there has been a lack of dynamically quantitative analysis of cell morphology under mechanical stimulations. A theoretical model for the evolution of cell morphology under stretching is proposed by analyzing the transformation between cell deformation energy and adhesion energy. The images of the evolution show that detachment of the adhesion membrane or re-adhesion of the cell, causes a decrease or increase of the adhesion area, respectively. In addition to the stretching amplitude and velocity, we found that the duration of the stretching stage and the no stretching stage can affect the cell morphology during cyclic stretching. The theoretical model explains some counter-intuitive experimental results and provides many quantitative insights into understanding the physical mechanisms of cell adhesion under stretching. This study can provide a useful guide to control cell adhesion under mechanical stimulations.

Live-cell imaging analysis on the anti-apoptotic function of the Bcl-xL transmembrane carboxyl terminal domain.

The Transmembrane Carboxyl Terminal Domain (TMD) of some Bcl-2 family proteins has been demonstrated to play a key role in modulating apoptosis. We here ustilzed live-cell fluorescence imaging to evaluate how the Bcl-xL TMD (XT) regulate apoptosis. Cell viability assay revealed that XT had strong anti-apoptotic ability similarly to the full-length Bcl-xL. Fluorescence images of living cells co-expressing CFP-XT and Bad-YFP or YFP-Bax revealed that XT recruited Bad to mitochondria but prevented Bax translocation to mitochondria, and also significantly suppressed Bad/Bax-mediated apoptosis, indicating that XT prevents the pro-apoptotic function of Bad and Bax. Fluorescence Resonance Energy Transfer (FRET) analyses determined that XT directly interacted with Bad and Bax, and deletion of XT completely eliminated the mitochondrial localization and homo-oligomerization of Bcl-xL. Fluorescence images of living cells co-expressing CFP-XT and YFP-Bax revealed that XT significantly prevented mitochondrial Bax oligomerization, resulting in cytosolic Bax distribution. Collectively, XT is necessary for the mitochondrial localization and anti-apoptotic capacity of Bcl-xL, and XT, similarly to the full-length Bcl-xL, forms homo-oligomers on mitochondria to directly interact with Bad and Bax to inhibit their apoptotic functions.

Im-SCC-FRET: improved single-cell-based calibration of a FRET system.

We recently developed a SCC-FRET (single-cell-based calibration of a FRET system) method to quantify spectral crosstalk correction parameters (ß and δ) and system calibration parameters (G and k) of a Förster resonance energy transfer (FRET) system by imaging a single cell expressing a standard FRET plasmid with known FRET efficiency (E) and donor-acceptor concentration ratio (RC) (Liu et al., Opt. Express30, 29063 (2022)10.1364/OE.459861). Here we improved the SCC-FRET method (named as Im-SCC-FRET) to simultaneously obtain ß, δ, G, k and the acceptor-to-donor extinction coefficient ratio (ε A ε D), which is a key parameter to calculate the acceptor-centric FRET efficiency (EA), of a FRET system when the range of ß and δ values is set as 0-1. In Im-SCC-FRET, the target function is changed from the sum of absolute values to the sum of squares according to the least squares method, and the initial value of ß and δ estimated by the integral but not the maximum value spectral overlap between fluorophore and filter. Compared with SCC-FRET, the experimental results demonstrate that Im-SCC-FRET can obtain more accurate and stable results for ß, δ, G, and k, and add the ratio ε A ε D, which is necessary for the FRET hybrid assay. Im-SCC-FRET reduces the complexity of experiment preparation and opens up a promising avenue for developing an intelligent FRET correction system.

Suramin, an antiparasitic drug, stimulates adipocyte differentiation and promotes adipogenesis.

BACKGROUND: Previous studies demonstrated that mast cells with their degranulated component heparin are the major endogenous factors that stimulate preadipocyte differentiation and promote fascial adipogenesis, and this effect is related to the structure of heparin. Regarding the structural and physiological properties of the negatively charged polymers, hexasulfonated suramin, a centuries-old medicine that is still used for treating African trypanosomiasis and onchocerciasis, is assumed to be a heparin-related analog or heparinoid. This investigation aims to elucidate the influence of suramin on the adipogenesis. METHODS: To assess the influence exerted by suramin on adipogenic differentiation of primary white adipocytes in rats, this exploration was conducted both in vitro and in vivo. Moreover, it was attempted to explore the role played by the sulfonic acid groups present in suramin in mediating this adipogenic process. RESULTS: Suramin demonstrated a dose- and time-dependent propensity to stimulate the adipogenic differentiation of rat preadipocytes isolated from the superficial fascia tissue and from adult adipose tissue. This stimulation was concomitant with a notable upregulation in expression levels of pivotal adipogenic factors as the adipocyte differentiation process unfolded. Intraperitoneal injection of suramin into rats slightly increased adipogenesis in the superficial fascia and in the epididymal and inguinal fat depots. PPADS, NF023, and NF449 are suramin analogs respectively containing 2, 6, and 8 sulfonic acid groups, among which the last two moderately promoted lipid droplet formation and adipocyte differentiation. The number and position of sulfonate groups may be related to the adipogenic effect of suramin. CONCLUSIONS: Suramin emerges as a noteworthy pharmaceutical agent with the unique capability to significantly induce adipocyte differentiation, thereby fostering adipogenesis.

Tea consumption and risk of lung diseases: a two­sample Mendelian randomization study.

BACKGROUND: Numerous studies have reported the association between tea intake and lung diseases. However, the probable relationship between tea consumption on lung diseases still remain controversial and it is unclear whether these findings are due to reverse causality or confounding factor. METHODS: In order to systematically investigate the causal connection between tea intake on respiratory system disorders, we employed a two-sample Mendelian randomized (MR) study. Genetic instruments for tea intake were identified from a genome-wide association study (GWAS) involving 447,385 individuals. Data on lung diseases were collected from a variety of publicly available genome-wide association studies. The main method used for MR analysis is the inverse variance weighting (IVW) method. To ensure the accuracy of the findings, further sensitivity analysis was conducted. RESULTS: The IVW method in our MR analysis revealed no evidence to support a causal relationship between tea intake and lung diseases (IPF: OR = 0.997, 95% CI = 0.994-1.000, p = 0.065; Lung cancer: OR = 1.003, 95% CI = 0.998-1.008, P = 0.261; COPD: OR = 1.001, 95% CI = 0.993-1.006, p = 0.552; acute bronchitis: OR = 0.919, 95% CI = 0.536-1.576, p = 0.759; tuberculosis: OR = 1.002, 95% CI = 0.998-1.008, p = 0.301; pneumonia: OR = 0.789, 95% CI = 0.583-1.068, p = 0.125). The reliability of the results was further demonstrated by four additional MR analysis techniques and additional sensitivity testing. CONCLUSION: We found no evidence of a link between tea intake on lung diseases in our MR results based on genetic information.

Mitofusin 2 but not mitofusin 1 mediates Bcl-XL-induced mitochondrial aggregation.

Bcl-2 family proteins, as central players of the apoptotic program, participate in regulation of the mitochondrial network. Here, a quantitative live-cell fluorescence resonance energy transfer (FRET) two-hybrid assay was used to confirm the homo-/hetero-oligomerization of mitofusins 2 and 1 (MFN2 and MFN1), and also demonstrate the binding of MFN2 to MFN1 with 1:1 stoichiometry. A FRET two-hybrid assay for living cells co-expressing CFP-labeled Bcl-XL (an anti-apoptotic Bcl-2 family protein encoded by BCL2L1) and YFP-labeled MFN2 or MFN1 demonstrated the binding of MFN2 or MFN1 to Bcl-XL with 1:1 stoichiometry. Neither MFN2 nor MFN1 bound with monomeric Bax in healthy cells, but both MFN2 and MFN1 bind to punctate Bax (pro-apoptotic Bcl-2 family protein) during apoptosis. Oligomerized Bak (also known as BAK1; a pro-apoptotic Bcl-2 family protein) only associated with MFN1 but not MFN2. Moreover, co-expression of Bcl-XL with MFN2 or MFN1 had the same anti-apoptotic effect as the expression of Bcl-XL alone to staurosporine-induced apoptosis, indicating the Bcl-XL has its full anti-apoptotic ability when complexed with MFN2 or MFN1. However, knockdown of MFN2 but not MFN1 reduced mitochondrial aggregation induced by overexpression of Bcl-XL, indicating that MFN2 but not MFN1 mediates Bcl-XL-induced mitochondrial aggregation.

Bioorthogonal Light-Up Fluorescent Probe Enables Wash-Free Real-Time Dynamic Monitoring of Cellular Glucose Uptake.

As a significant energy source for living systems, the aberrant cellular glucose uptake is seriously implicated in numerous metabolic diseases. Unfortunately, current shortage of robust tools leaves the limitation to understand its precise biology. Herein we presented a bioorthogonal light-up fluorescent probe consist of two reagents, Glu-HT-Me+AzGlu2, for rapidly responsive (within 25 min), highly specific and sensitive (20-folds enhancement) detection of live-cell glucose uptake based on arylphosphine-induced a-PET effect and Staudinger ligation. Especially, taking the advantage of wash-free characteristic, the probe displayed the real-time dynamic monitoring of cellular glucose uptake. Furthermore, it was successfully capable of not only differentiating cancer cells from normal cells, but also allowing evaluation of anticancer/glycolysis/transport mediated glucose flux. Importantly, it was employed to monitor the fluctuations of glucose uptake in a doxycycline-inducible K-rasG12 V expression oncogenic cell system, implying its potential as a valuable tool to explore glucose uptake biology.

BID- and BAX-mediated mitochondrial pathway dominates A-1331852-induced apoptosis in senescent A549 cells.

Recovered senescent tumor cells harbor higher migration and invasion potential, owing to which they play a crucial role in tumor recurrence and drug resistance. The aim of this study was to explore the ability of BH3 mimetics in clearing senescent A549 cells and elucidate their underlying killing mechanism. Doxorubicin-induced cell senescence was determined using augmented senescence-associated beta-galactosidase (SA-ß-Gal) staining and increased P16 expression. CCK-8 and crystal violet staining demonstrated that A-1331852, BH3 mimetic, could kill senescent tumor cells without affecting the proliferating cells. A-1331852 induced caspase-dependent senescent cell death accompanied by nuclear concentration, decreased mitochondrial membrane potential, and cleavage of poly (ADP-ribose) polymerase. Most importantly, A-1331852 upregulated the expression of BID and BAX indicating their role in mediating A-1331852-induced apoptosis in senescent A549 cells. The results of fluorescence resonance energy transfer showed that A-1331852 loosened or even released the binding between BCL-xL and tBID, releasing tBID. In addition, A-1331852 also dissociated the binding between BCL-xL and BAX, eventually leading to BAX oligomerization in the mitochondria, and resulting in apoptosis via the mitochondrial pathway. In conclusion, our data demonstrate for the first time that A-1331852 promotes apoptosis of senescent A549 cells by influencing the interaction between BCL-xL and tBID and that between BCL-xL and BAX.

BAK plays a key role in A-1331852-induced apoptosis in senescent chondrocytes.

Osteoarthritis occurs when the number of senescent chondrocytes in the joints reaches an intolerable level. The purpose of our study was to explore the therapeutic effect and mechanism of action of A-1331852 in osteoarthritis. Doxorubicin and etoposide were used to induce cell senescence as determined by the cessation of cell proliferation, augmented senescence-associated beta-galactosidase (SA-ß-Gal) staining, and increased p53 expression levels. The CCK-8 cytotoxicity assay and SA-ß-Gal staining demonstrated that Bcl-xL inhibitors could selectively remove senescent chondrocytes without damaging healthy chondrocytes. A-1331852 induced caspase-dependent death of senescent chondrocytes with decreased mitochondrial membrane potential, nuclear concentration, plasma membrane rupture, and PARP cleavage. Most importantly, A-1331852 upregulated BAK expression levels, indicating that BAK plays a key role in the A-1331852-induced apoptosis of senescent chondrocytes. Live-cell fluorescence resonance energy transfer showed that A-1331852 detached the binding of Bcl-xL to BAK and promoted the oligomerization of BAK on the mitochondrial membrane. In conclusion, this study provides the first evidence that A-1331852 selectively promotes apoptosis in senescent chondrocytes by interfering with the interaction between Bcl-xL and BAK.

High-Efficiency Capture of Cells by Softening Cell Membrane.

The capture of circulating tumor cells (CTCs) by nanostructured substrate surface is a useful method for early diagnosis of cancer. At present, most methods used to improve the cell capture efficiency are based on changing substrate surface properties. However, there are still some gaps between these methods and practical applications. Here, a method is presented for improving cell capture efficiency from a different perspective, that is, changing the properties of the cells. Concretely, the mechanical properties of the cell membrane are changed by adding Cytochalasin D to soften the cell membrane. Furthermore, a corresponding theoretical model is proposed to explain the experimental results. It is found that cell softening can reduce the resistance of cell adhesion, which makes the adhesion ability stronger. The high-efficiency capture of cells by softening the cell membrane provides a potential method to improve the detection performance of CTCs.

Optical section structured illumination-based Förster resonance energy transfer imaging.

Förster resonance energy transfer (FRET) microscopy is an important tool suitable for studying molecular interactions in living cells. Optical section structured illumination microscopy (OS-SIM), like confocal microscopy, has about 200 nm spatial resolution. In this report, we performed quantitative 3-cube FRET imaging in OS-SIM mode and widefield microscopy (WF) mode, respectively, for living cells expressing FRET constructs consisting of Cerulean (C, donor) and Venus (V, acceptor). OS-SIM images exhibited higher resolution than WF images. Four spectral crosstalk coefficients measured under OS-SIM mode are consistent with those measured under WF mode. Similarly, the system calibration factors G and k measured under OS-SIM mode were consistent with those measured under WF mode. The measured FRET efficiency (E) values of C32V and C17V as well as C5V constructs, standard FRET plasmids, in living Hela cells were EC32VOSF=0.32±0.02,EC17VOSF=0.38±0.02 , and EC5VOSF=0.45±0.03 , and the measured acceptor-to-donor concentration ratios ( Rc ) were RC32VOSF=1.07±0.03 , RC17VOSF=1.09±0.03 , and RC5VOSF=1.02±0.04 , consistent with the reported values. Collectively, our data demonstrates that OS-SIM can be integrated into FRET microscopy to build an OS-SIM-FRET with confocal microscopy-like resolution.

SCC-FRET: single-cell-based calibration of a FRET system.

Reliable measurements of calibration parameters are crucial for quantitative three-cube Förster resonance energy transfer (FRET) measurements. Here we have developed a single-cell-based calibration method (SCC-FRET), which can simultaneously obtain spectral crosstalk correction parameters (ß and δ) and calibration parameters (G and k) of a quantitative FRET system by imaging a cell expressing one kind of standard FRET plasmid with a known FRET efficiency (E) and the donor-to-acceptor concentration ratio (RC). We performed the SCC-FRET method on a three-cube FRET microscopy for the cells expressing C5V, and obtained ß = 0.150 ± 0.000, δ = 0.610 ± 0.000, G = 2.840 ± 0.065, and k = 0.847 ± 0.013. These parameters were used to measure the E and RC values of C17V and C32V constructs in living cells and obtained EC17V = 0.382 ± 0.010 and EC32V = 0.311 ± 0.007, RC17V = 1.010 ± 0.023 and RC32V = 1.050 ± 0.022, consistent with the reported values, demonstrating the effectiveness of the the SCC-FRET method. We also performed the SCC-FRET method for the cells with different S/N levels (S/N > 10, 10 > S/N > 3, 3 > S/N > 1, respectively), and obtained consistent system calibration parameters under different S/N levels, indicating excellent robustness. SCC-FRET requires only imaging a cell expressing one kind of standard FRET plasmid for measuring all calibration parameters under identical imaging conditions, rendering the SCC-FRET method extremely convenient, accurate, and robust. The SCC-FRET provides strong support for expanding the biological application of quantitative FRET analysis in living cells.

An ESIPT-Dependent AIE Fluorophore Based on HBT Derivative: Substituent Positional Impact on Aggregated Luminescence and its Application for Hydrogen Peroxide Detection.

Aiming to develop the facile organic fluorophore possessing excited state intramolecular proton transfer (ESIPT) and aggregation-induced emission (AIE), we designed and synthesized two isomers with different linkage site between hydroxyl of 2-(2-hydroxyphenyl) benzothiazole (HBT) and a benzothiazole substituent (para position refers to p-BHBT and ortho position refers to o-BHBT). Fluorescence emission properties of p-BHBT and o-BHBT in THF/water mixtures with different water volume fractions indicated an opposite luminescence in aggregates, in which p-BHBT showed an ESIPT-dependent AIE properties while o-BHBT displayed ESIPT effect and aggregation-caused quenching (ACQ) qualities. A possible mechanism for molecular actions to illustrate the aggregating luminescence alteration of these two isomers had been proposed and verified by theoretical and experimental studies. More importantly, Probe-1, generated from dual ESIPT-AIE fluorophore p-BHBT, was successfully used as a ratiometric fluorescent chemosensor for highly selective (above 15-fold over other ROS) and sensitive (69-fold fluorescence enhancement with 0.22 µM of detection limit) detection of hydrogen peroxide in aqueous solution and living cells, respectively.

A static force model to analyze the nuclear deformation on cell adhesion to vertical nanostructures.

Vertical nanostructures have been found to induce the deformation of the nuclear envelope during cell adhesion. However, there has been a lack of quantitative analysis of the influence of nanostructures morphology on the degree of nuclear deformation. Here, a theoretical model was proposed to investigate the mechanism of nuclear deformation by analyzing the mechanical force balance. Based on the established model, we analyzed the effects of the morphology of the nanopillar array on nuclear deformation and gave the quantitative relationship of the deformation depth of the nucleus with the pitch and radius of nanopillars. Our theoretical results seem to show broad agreements with experimental observations, which implies that the work can provide useful guidance to the design of nanostructures for biomedical applications.

A ratiometric ESIPT probe based on 2-aza-Cope rearrangement for rapid and selective detection of formaldehyde in living cells.

Formaldehyde (FA) is a crucial reactive signaling molecule participating in epigenetic and metabolic pathways. However, abnormally elevated levels of FA are implicated in various diseases spanning from tumors to neurodegenerative disorders. Despite being highly selective for FA, current 2-aza-Cope-based fluorescent probes leave room for improvement because their relatively slow reaction kinetics (1-9 hours response time) hinder their capability to track transient biological FA. Herein, we present a ratiometric fluorescent probe, FormAFP, based on excited state intramolecular photon transfer (ESIPT) for rapid (within 10 min), selective (above 70-fold over other RCS) and sensitive (240-times fluorescence enhancement with 66 nM detection limit) detection of FA via 2-aza-Cope rearrangement. The probe also displayed a fast response (<20 min) to both exogenous and endogenous FA in living cells. Besides, FormAFP was capable of monitoring FA released by folate degradation in living MCF7 cells. More importantly, FormAFP successfully detected fluctuations of endogenous FA levels in oxidative stress stimulation, demonstrating its potential as an ideal tool to explore FA biology.

Automated ExEm-spFRET Microscope.

Excitation­emission-spectral unmixing-based fluorescence resonance energy transfer (ExEm-spFRET) microscopy exhibits excellent robustness in living cells. We here develop an automatic ExEm-spFRET microscope with 3.04 s of time resolution for a quantitative FRET imaging. The user-friendly interface software has been designed to operate in two modes: administrator and user. Automatic background recognition, subtraction, and cell segmentation were integrated into the software, which enables FRET calibration or measurement in a one-click operation manner. In administrator mode, both correction factors and spectral fingerprints are only calibrated periodically for a stable system. In user mode, quantitative ExEm-spFRET imaging is directly implemented for FRET samples. We implemented quantitative ExEm-spFRET imaging for living cells expressing different tandem constructs (C80Y, C40Y, C10Y, and C4Y, respectively) and obtained consistent results for at least 3 months, demonstrating the stability of our microscope. Next, we investigated Bcl-xL-Bad interaction by using ExEm-spFRET imaging and FRET two-hybrid assay and found that the Bcl-xL-Bad complexes exist mainly in Bad-Bcl-xL trimers in healthy cells and Bad-Bcl-xL2 trimers in apoptotic cells. We also performed time-lapse FRET imaging on our system for living cells expressing Yellow Cameleon 3.6 (YC3.6) to monitor ionomycin-induced rapid extracellular Ca2+ influx with a time interval of 5 s for total 250 s.

Evaluating the inhibitory priority of Bcl-xL to Bad, tBid and Bax by using live-cell imaging assay.

Molecular regulatory network among the B cell leukemia-2 (Bcl-2) family proteins is a research hotspot on apoptosis. The inhibitory priority of anti-apoptotic Bcl-2 family proteins (such as Bcl-xL) to pro-apoptotic Bcl-2 family proteins (such as Bad, tBid and Bax) determines the outcome of their interactions. Based on over-expression model system, we here evaluate the inhibitory priority of Bcl-xL to Bad, tBid and Bax by using live-cell imaging assay on cell viability. Fluorescence images of living cells co-expressing CFP-Bcl-xL and YFP-Bad or YFP-tBid or YFP-Bax showed that Bcl-xL markedly inhibited Bad/tBid/Bax-mediated apoptosis, revealing that Bcl-xL inhibits the proapoptotic function of Bad, tBid and Bax. In the case of equimolar co-expression of Bad and CFP-Bcl-xL, the inhibition of Bcl-xL on tBid/Bax mediate-apoptosis was completely relieved. Moreover, co-expression of tBid-P2A-CFP-Bcl-xL significantly relieved the inhibition of Bcl-xL on the pro-apoptotic ability Bax, suggesting that Bcl-xL preferentially inhibits the pro-apoptotic ability of Bad over tBid, subsequently to Bax.

Measuring calibration factors by imaging a dish of cells expressing different tandem constructs plasmids.

Three-cube Förster resonance energy transfer (FRET) method is the most extensively applied approach for live-cell FRET quantification. Reliable measurements of calibration factors are crucial for quantitative FRET measurement. We here proposed a modified TA-G method (termed as mTA-G) to simultaneously obtain the FRET-sensitized quenching transition factor (G) and extinction coefficients ratio (γ) between donor and acceptor. mTA-G method includes four steps: (1) predetermining the ratio ranges of the sensitized emission of acceptor (FC ) to the donor excitation and donor channel image (IDD [(DA])) for all FRET plasmids; (2) culturing the cells which express every FRET plasmid in one dish respectively; (3) distinguishing and marking the cells expressing different FRET plasmids by detecting their FC /IDD (DA) values; (4) linearly fitting FC /IAA (DA) (acceptor excitation and acceptor channel image) to IDD (DA)/IAA (DA) for different kinds of cells. We implemented mTA-G method by imaging tandem constructs cells with different FRET efficiency cultured in one dish on different days, and obtained consistent G and γ values. mTA-G method not only circumvents switchover of different culture dishes but also keep the constant imaging conditions, exhibiting excellent robustness, and thus will expands the biological applications of quantitative FRET analysis in living cells.

A plant-unique ESCRT component, FYVE4, regulates multivesicular endosome biogenesis and plant growth.

During evolution, land plants generated unique proteins that participate in endosomal sorting and multivesicular endosome (MVE) biogenesis, many of them with specific phosphoinositide-binding capabilities. Nonetheless, the function of most plant phosphoinositide-binding proteins in endosomal trafficking remains elusive. Here, we analysed several Arabidopsis mutants lacking predicted phosphoinositide-binding proteins and first identified fyve4-1 as a mutant with a hypersensitive response to high-boron conditions and defects in degradative vacuolar sorting of membrane proteins such as the borate exporter BOR1-GFP. FYVE4 encodes a plant-unique, FYVE domain-containing protein that interacts with SNF7, a core component of ESCRT-III (Endosomal Sorting Complex Required for Transport III). FYVE4 affects the membrane association of the late-acting ESCRT components SNF7 and VPS4, and modulates the formation of intraluminal vesicles (ILVs) inside MVEs. The critical function of FYVE4 in the ESCRT pathway was further demonstrated by the strong genetic interactions with SNF7B and LIP5. Although the fyve4-1, snf7b and lip5 single mutants were viable, the fyve4-1 snf7b and fyve4-1 lip5 double mutants were seedling lethal, with strong defects in MVE biogenesis and vacuolar sorting of ubiquitinated membrane proteins. Taken together, we identified FYVE4 as a novel plant endosomal regulator, which functions in ESCRTing pathway to regulate MVE biogenesis.