Cytoskeleton disruption and plasma membrane damage determine methuosis of normal and malignant cells

Cytoskeleton disruption and plasma membrane damage determine methuosis of normal and malignant cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cytoskeleton disruption and plasma membrane damage determine methuosis of normal and malignant cells Xiuge Gao, Bin Dong, Jing Xiao, Junqi Wang, Xinhao Song, Hui Ji, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5422638/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Methuosis represents a novel cell death modality characterized by catastrophic cytoplasmic vacuolization in normal and malignant cells. However, the critical role and the underlying mechanism of cytoskeleton and plasma membrane damage in methuotic cells are largely unknown. Herein, maduramicin-treated myocardial cells (H9c2) and indole chalcone-exposed glioma cells (U251) were used as methuosis model to uncover this secret. We found that cytoskeleton protein F-actin, α-tubulin, β-tubulin and filamin A/B were disrupted in a reversible-dependent manner. In addition, RhoA-ROCK1 signaling pathway mediated cytoskeleton disruption in methuotic cells. Excessive cytoplasmic vacuolization triggered cellular plasma membrane damage and the release of DAMPs, including LDH, ATP and CRT. Furthermore, at the end phase of methuotic cells, plasma membrane was damaged independent of pore-forming protein p-MLKL and GSDMD. Endosomal sorting complex required for transport (ESCRT)-Ⅲ especially its subunit CHMP3 and CHMP5 negatively regulated excessive vacuolization-induced plasma membrane damage in cells undergoing methuosis. In conclusion, for the first time, the critical role and potential mechanism of cytoskeleton and plasma membrane damage in methuotic cells are known, which would facilitate the employment of methuosis in life science and pharmacology. Biological sciences/Drug discovery/Toxicology Biological sciences/Cell biology/Cell death Methuosis Cytoskeleton Membrane damage DAMPs ESCRT-Ⅲ Maduramicin MOMIPP Cell death Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Unlike the well-known programmed cell death types such as apoptosis, necroptosis, autophagic cell death and ferroptosis, a relatively novel cell death methuosis (drink to death) coined by Dr. Maltese was firstly defined in glioma cells, in which excessive cytoplasmic vacuolization due to macropinocytosis persists until cellular plasma membrane rupture [ 1 , 2 ] . Since the first recognition of methuosis, a large amount of malignant cells have been demonstrated sensitive to methuosis, which represents an alternative anticancer strategy [ 3 ] . Intriguingly, therapy-resistant cancer cells such as prostate cancer and breast cancer cells [ 4 , 5 ] are vulnerable to methuosis initial genes and pharmacological chemicals, proposing a novel solution to overcome resistant cancer cells. In addition, methuosis acts as an emerging mechanism in drug toxicology area because that normal cells such as myocardial cells have been demonstrated to undergo methuotic cell death in a context-dependent manner [ 6 , 7 ] . At the same time, recent critical findings shown that anti-parasite drugs exert pharmacological activities through the induction of methuosis in nematode model [ 8 ] . That means methuosis can be induced in a broad range of cells, including normal cells, malignant cells as well as parasite cells [ 9 ] . However, the cytoplasm process and the core mechanism of cells undergoing methuosis remain to be explored. Concerning that methuosis is characterized by catastrophic cytoplasmic vacuolization, the origin and trafficking of methuotic vacuoles have been extensively investigated [ 10 , 11 , 12 ] . In the early stage of methuosis, macropinocytosis is triggered by genetic mutation or pharmacologic chemicals which facilitate generation of lamellipodia, small size vacuoles (pinososome) containing extracellular fluid enter cytoplasm. In the middle phase of methuosis, pinososomes merge with each other to form larger vacuoles. At the terminal stage of methuosis, large number of phase-lucent giant vacuoles occupy almost the cytoplasm and ultimately result in cellular membrane rupture [ 13 ] . Although the phenotype process of methuosis is clear, how the numerous cytoplasmic vacuoles affect cytoskeleton and cellular plasma membrane are largely unknown. A recent study demonstrates that chemical substitution at 2-indolyl position of methuosis inducer 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (MOMIPP) switches the mode of cytotoxicity from methuosis to microtubule disruption [ 14 ] . Given that cytoskeleton supports cellular structure integrity, playing a key role in maintaining cells survival. If cytoskeleton is changed or damaged, cells will be driven to death under some contexts. For instance, in rotenone-induced necroptotic neuronal cells, actin cytoskeleton cofilin is altered and degraded, thereby leading to neurotoxicity [ 15 ] . Similarly, actin cytoskeleton has been demonstrated to determine apoptosis process due to the upregulation of cleavage of actin and gelsolin in mammalian system [ 16 ] . Therefore, we hypothesized that excessive cytoplasmic vacuoles might disrupt cytoskeleton in methuotic cells. In addition to cytoskeleton, plasma membrane functions the last protective barrier to avoid entering of extracellular harmful materials or releasing of intracellular substances. As a result, cellular membrane rupture is the hallmark of all known cell death modalities. Plasma membrane damage occurs in alternative forms, including pore-formation, membrane deformation and membrane alteration [ 17 ] . For instance, mixed lineage kinase domain-like (MLKL) is a necessary executioner of necroptosis in which MLKL inserts into plasma membrane and results in cellular rupture; besides, cells undergoing pyroptosis requires the formation of balloon-like membrane protrusions which further contributes to plasma membrane rupture; for cells ferroptosis, lipid peroxidation due to imbalance of antioxidant enzymes results in plasma membrane permeabilization and further rupture [ 18 ] . But for methuosis, there are no available data on the damage mode of plasma membrane, and the exact membrane damage mechanism of methuosis is also unknown. Given that large giant cytoplasmic vacuoles continuously accumulate until eventual loss of plasma membrane integrity [ 19 ] . Cells undergoing methuosis may suffer from distinctive membrane damage which are differ from the known programmed cell death models. Herein, we propose a hypothesis that catastrophic cytoplasmic vacuoles in methuotic cells damage plasma membrane in an alternative way. Hence, the understanding the form of plasma membrane damage of methuotic cells will facilitate the development of preventive strategy to maintain cells survival. In this study, the classic methuosis model induced by indole-based chalcone in glioma G251 cells, and a novel methuosis model induced by maduramicin in myocardial H9c2 cells were used to elucidate the role and underlying mechanism of cytoskeleton and plasma membrane damage in methuosis. Intriguingly, for the first time, cytoskeleton proteins include F-actin, tubulin and filamin A/B were disrupted through the inhibition of RhoA-ROCK1 signaling pathway, plasma membrane was damaged with certain DAMPs leakage in pore-forming proteins- independent manner, and membrane repair system ESCRT-Ⅲ acted as a negatively regulator of methuosis are illuminated in current study. These findings will benefit for the exploiting of cytoskeleton and plasma membrane to promote or prevent methuosis in cancer therapy and drug toxicology. RESULTS Excessive cytoplasmic vacuolization provokes cytoskeleton disruption in methuotic cells To understand the role of cytoskeleton in cells undergoing methuosis, first for microfilament, F-actin was stained with phalloidin. In maduramicin-treated H9c2 cells methuosis model, compared to drug-untreated H9c2 cells, in which F-actin was neatly arranged, H9c2 cells exposed to 1 µM maduramicin for 24 h shown that thickened actin fibers and cortical actin, along with several fluorescent bright spots (Fig. 1 A). When maduramicin-treated H9c2 cells for 48 h, cell boundaries were more distinct, stress fibers were reduced, and cells lacked stress fibers, showing smooth edges and a loss of pseudopods (Fig. 1 A). Furthermore, in MOMIPP-induced U251 cells methuosis model, after 2.5 µM MOMIPP exposed for 12 h, U251 cells became to thickened actin fibers, increased cortical actin, and disorganized microfilaments with pseudopod disappearance (Fig. 1 B). When MOMIPP treatment extended for 24 h, stress fibers were decreased and some fluorescent foci were generated in U251 cells (Fig. 1 B). However, in the absence of MOMIPP, U251 cells exhibited that clearly F-actin arrangement with a pronounced aggregation of actin stress fibers and conspicuous pseudopodia. In addition to F-actin, as important parts of microfilament, filamin A, filamin B and α-actinin-1 were also assayed by performing western blotting. As shown in Fig. 1 C and 1 D, filamin A and filamin B were significantly decreased in H9c2 and U251 cells after methuosis-inducing drugs exposed for 48 h and 72 h (Fig. 1 E), but not at other time points, while the expression of α-actinin-1 was not significantly changed after maduramicin and MOMIPP exposure, suggesting that the reduction of filamin A and filamin B may contribute to microfilament disorder-mediated methuosis. Moreover, other critical cytoskeleton proteins, α-tubulin and β-tubulin, were examined using immunofluorescence staining. As shown in Fig. 1 F and Fig. 1 G, compared to cells in absence of drugs, α-tubulin and β-tubulin were reorganized and formed microtubule bundle which surrounded partial cytoplasmic vacuoles induced by maduramicin or MOMIPP. However, H9c2 and U251 cells in negative control group exhibited a well-developed array of microtubules radiating from the juxtanuclear microtubule organizing center (Fig. 1 F and 1 G). Taken together, cytoskeletal microfilament and tubulin in H9c2 cells and U251 cells are disrupted during methuosis. Cytoskeleton disruption in methuosis is reversed by blocking cytoplasmic vacuolization Given that cytoplasmic vacuolization is reversible when methuosis stimulus is removed [ 6 , 10 ] , we next investigated whether drug-induced cytoskeleton damage is reversible in methuotic H9c2 and U251 cells. As shown in Fig. 1 H, F-actin of H9c2 cell treated by maduramicin was altered in disorder as above described, however, after the absence of maduramicin from 4 h to 48 h, F-actin arrangement in H9c2 cells gradually restored to normal pattern. Similar to H9c2 cells, F-actin configuration was disrupted after MOMIPP exposure to U251 cells, while the removal of MOMIPP for 48 h reversed F-actin disruption gradually. Moreover, bafilomycin A1, an inhibitor of methuosis that targets V-ATPase [ 20 ] , was used to inhibit the generation of vacuolization. In H9c2 cells, bafilomycin A1 alone treatment had no visible effects on F-actin arrangement and distribution, but eliminated maduramicin-induced thickened actin fibers and cortical actin, along with several fluorescent bright spots (Fig. 2 A). Unlike H9c2 cells, in U251 cells, bafilomycin A1 alone decreased pseudopodia on cell surface and altered arrangement of F-actin, however, MOMIPP-triggered disturbance of F-actin was largely reversed by bafilomycin A1 although pseudopodia disappearance (Fig. 2 B). Intriguingly, bafilomycin A1 could not reverse cytochalasin D-induced severe cytoskeleton damage in H9c2 cells and U251 cells (Fig. 2 A and 2 B). Next, the effect of bafilomycin A1 on filamin A and filamin B after methuosis-inducing drugs exposure were evaluated in H9c2 and U251 cells. Our results showed that while drugs significantly decreased the expression of filamin A and filamin B, bafilomycin A1 significantly restored their levels to drugs-untreated cells (Fig. 2 C and 2 D). Further immunofluorescence examination indicated that the arrangement of filamin A and filamin B was disrupted by cytoplasmic vacuoles accumulation followed by methuosis-inducing drugs treatment but was normalized after bafilomycin A1 pretreatment (Fig. 2 E- 2 H). These findings together indicate that cytoskeletal disruption due to excessive cytoplasmic vacuolization is reversible in methuotic cells, and the inhibition of cytoplasmic vacuolization can restore cytoskeleton integrity. RhoA-ROCK1 inhibition mediates cytoskeleton disruption in methuosis To explore the mechanism underlying the disruption of cytoskeleton in methuotic cells, RhoA-ROCK1 signaling pathway which is pivotal for stress fiber formation, was focused in this study. RhoA, a key regulator of cytoskeleton and cellular shape, controls the assembly of contractile actin-myosin filaments. Upon RhoA binds to ROCK1, facilitating stress fiber formation by promoting actin polymerization as well as the inhibiting of actin depolymerization [ 21 ] . Furthermore, the activated ROCK1 phosphorylates myosin light chain (MLC) and activates myosin Mg 2+ -ATPase, which in turn leads to the binding of myosin with F-actin and the formation of actomyosin stress fibers essential for cell contraction [ 22 ] . As depicted in Fig. 3 A and 3 C, RhoA was significantly downregulated in H9c2 cells treated by maduramicin for 48 h and 72 h, ROCK1 expression was significantly decreased at 12 h-72 h, and p-MLC expression was notably decreased at 12 h-72 h. In U251 cells, the expression of RhoA was significantly decreased at 24 h to 72 h after MOMIPP exposure, and ROCK1 was markedly reduced at 48 h-72 h, but the expression of p-MLC was not significantly changed after MOMIPP treatment (Fig. 3 B and 3 C). These results suggest that RhoA-ROCK1 pathway was inhibited by maduramicin and MOMIPP in methuotic cells. To further elucidate the role of the RhoA-ROCK1 cascade in microfilament disruption, calpeptin, a RhoA agonist, was employed to observe the alteration of microfilament by phalloidin staining. As shown in Fig. 3 D, in maduramicin-induced H9c2 cells methuosis model, compared to cells in control group, calpeptin alone treatment enhanced the density of microfilament in alignment, maduramicin disrupted F-actin in varying degrees, however, calpeptin pretreatment restored F-actin arrangement even if maduramicin exposure. Similarly, in MOMIPP-induced glioma U251 cells methuosis model, compared to control group, calpeptin alone increased the density of F-actin with the accumulation of fluorescent faculae on the cellular surface, MOMIPP-treated cells markedly decreased F-actin expression with disrupted structure, but calpeptin pretreatment reversed F-actin distribution and arrangement (Fig. 3 E). Collectively, these findings indicate that the inhibition of RhoA-ROCK1 signaling pathway contributes to cytoskeletal microfilament disruption in drug-induced methuosis. Excessive cytoplasmic vacuolization results in plasma membrane damage and DAMPs release in methuosis Cellular plasma membrane damage plays a critical role in cell death. However, how plasma membrane is ruptured remains unknown in cells undergoing methuosis. To assess plasma membrane damage mode, lactate dehydrogenase (LDH) was firstly determined in this study. As shown in Fig. 4 A, with the prolongation of maduramicin or MOMIPP treatment, LDH levels in the supernatant of H9c2 cells and U251 cells were significantly increased at 12 h or 24 h, 48 h and 72 h, respectively. Moreover, Hoechst 33342/PI staining demonstrated that plasma membrane was damaged in H9c2 cells and U251 cells undergoing drug-induced methuosis due to the number of PI-positive cells was increased from 24 h to 72 h (Fig. 4 D- 4 E). Additionally, pore-forming proteins p-MLKL and GSDMD-N, which are essential for plasma membrane damage, were detected in drug-induced methuotic cells. As shown in Fig. 4 F- 4 G and 4 I, MLKL, p-MLKL, GSDMD and GSDMD-N expression in H9c2 cells were not changed after maduramicin exposed for 12 h to 72 h, similarly, the expression of MLKL, p-MLKL, GSDMD and GSDMD-N was not altered by MOMIPP treatment for 12 h to 72 h, indicating that cell membrane damage during methuosis is independent of canonical pore-forming proteins. Concerning that plasma membrane damage is followed by the release of cellular damage associated molecular patterns (DAMPs) such as calreticulin (CRT) and ATP, which trigger inflammatory response [ 23 ] . We further investigated the release of DAMPs from damaged methuotic cells. Normally, CRT localizes on the endoplasmic reticulum, but translocates to the outer cellular membrane surface under some injury conditions. Results indicated that maduramicin increased the expression of CRT in plasma membrane of H9c2 cells after pharmacological exposure for 48 h and 72 h (Fig. 4 H and 4 I). Furthermore, CRT expression in MOMIPP-treated U251 cells was also elevated slightly at 48 h and 72 h (the final phase of methuosis) (Fig. 4 H and 4 I). At the same time, immunofluorescence assay exhibited that the expression of CRT was enhanced in cytoplasm as well as on cellular membrane surface of H9c2 cells and U251 cells, compared to the control group (Fig. 4 J and 4 K). In addition, ATP levels of cytoplasm and the cultured supernatant were measured after drugs treated methuotic cells. As shown in Fig. 4 B, upon exposure of maduramicin and MOMIPP for 24 h to 72 h, intracellular ATP levels were significantly decreased in H9c2 cells and U251 cells, compared to cells in the control group. In contrast, the extracellular ATP levels were significantly increased after maduramicin treatment for 48 h and 72 h or MOMIPP exposure for 24 h to 72 h (Fig. 4 C). In conclusion, these above findings demonstrate pore-forming protein-independent plasma membrane damage and DAMPs release mediate drug-induced methuosis. Inhibition of cytoplasmic vacuolization impedes plasma membrane damage To further elucidate the relationship between cytoplasmic vacuolization and plasma membrane damage, bafilomycin A1 was employed to inhibit the generation of cytoplasmic vacuoles. As depicted in Fig. 5 A, compared to negative control group, bafilomycin A1 alone did not induce significantly LDH release from H9c2 cells and U251 cells, maduramicin and MOMIPP both triggered significant release of LDH (P < 0.01), however, bafilomycin A1 pretreatment reduced LDH release from maduramicin- or MOMIPP-treated cells. At the same time, PI-positive H9c2 cells induced by maduramicin were decreased by bafilomycin A1 pretreatment, similarly, MOMIPP-induced PI-positive U251 cells were reduced after bafilomycin A1 pre-exposure (Fig. 5 D- 5 E). In addition, the effect of vacuolization inhibition by bafilomycin A1 on DAMPs released from methuotic cells was also evaluated herein. As depicted in Fig. 5 F- 5 H, compared to the CRT expression in negative control group, bafilomycin A1 alone did not change CRT expression in H9c2 cells and U251 cells, and maduramicin- and MOMIPP-induced the increase of CRT expression was inhibited by bafilomycin A1 pretreatment. Moreover, immunofluorescence analysis exhibited that maduramicin-triggered enhancement of CRT expression and cytoplasmic distribution in H9c2 cells was markedly inhibited by bafilomycin A1 (Fig. 5 I), the same findings were observed in MOMIPP-treated U251 cells after bafilomycin A1 pretreatment (Fig. 5 J). Furthermore, compared to negative control group, bafilomycin A1 did not change intracellular ATP levels of H9c2 cells and U251 cells, but significantly attenuated maduramicin- and MOMIPP-induced the decrease of intracellular ATP (Fig. 5 B). Similarly, for extracellular ATP, maduramicin and MOMIPP both significantly increased ATP release from H9c2 cells and U251 cells, respectively, but bafilomycin A1 pretreatment decreased extracellular ATP to baseline level (Fig. 5 C). Based on these above results, excessive cytoplasmic vacuolization induced by maduramicin or MOMIPP mediates final plasma membrane damage in methuotic cells. ESCRT-III membrane repair system negatively controls methuosis Under normal condition, cells are able to maintain plasma membrane integrity by activating self-repair system upon suffer from injury. The ESCRT-III repair mechinary has recently been shown as an important membrane repair mechanism for inhibiting necroptosis [ 24 ] , ferroptosis [ 25 ] , and pyroptosis [ 26 ] . Herein, the role of ESCRT-III system was investigated in the determinant of methuotic cell death. As shown in Fig. 6 A and 6 C, compared to negative control group, the subunits of ESCRT-III complex such as CHMP2B was significantly elevated in plasma membrane of H9c2 cells after maduramicin treatment for 24 h to 72 h, CHMP3 and CHMP5 were significantly elevated after maduramicin treatment for 48 h and 72 h, whereas CHMP4B expression was significantly downregulated at 24 h but not other time points. In U251 cells, compared to the control group, the expression of CHMP2B were significantly decreased after MOMIPP exposed for 48 h but not 12 h, 24 h and 72 h (Fig. 6 B- 6 C), the expression of CHMP4B was also significantly reduced by MOMIPP treatment for 48 h-72 h. In contrast, the expression of CHMP3 was significantly increased after MOMIPP treatment for 24 h to 72 h (Fig. 6 B- 6 C). However, CHMP5 expression was not changed even MOMIPP exposed for 12 h to 72 h (Fig. 6 B- 6 C). Furthermore, to understand the role of ESCRT-III system in methuotic cells, siRNA was used to knockdown CHMP3 and CHMP5 in H9c2 cells and CHMP3 in U251 cells, followed by cell viability, LDH release and Hoechst 33342/PI staining assays. As illustrated in Fig. 6 E and 6 F, siRNA-mediated knockdown of CHMP3 and CHMP5 augmented drug-induced cell death of H9c2 cells compared to maduramicin treatment individually. Similarly, compared to MOMIPP group, knockdown of CHMP3 significantly suppressed cell viability of U251 cells (Fig. 6 G). Moreover, LDH release from H9c2 cells and U251 cells after maduramicin or MOMIPP was significantly elevated when knockdown CHMP3, but knockdown CHMP5 did not affect maduramicin-induced LDH release from H9c2 cells (Fig. 6 H- 6 I). In addition, compared to negative control group, maduramicin or MOMIPP alone exposure increased the number of PI-positive cells, knockdown CHMP3 and CHMP5 markedly elevated the number of maduramicin-induced PI-positive H9c2 cells, and knockdown CHMP3 also heightened the number of MOMIPP-triggered PI-positive U251 cells (Fig. 6 J- 6 L). Collectively, ESCRT-III-dependent membrane repair system participates in drug-induced methuosis of H9c2 and U251 cells, and CHMP3 could be exploited as a potential negative regulator of methuosis. DISCUSSION Cytoskeleton together with integral plasma membrane support cellular shape, biological functions and protect cells from extracellular dangers. Cells undergoing death might suffer varying degrees of cytoskeleton damage and plasma membrane rupture. As a well-known cell death model, apoptosis, has been demonstrated that at least three components of cytoskeleton (keratins, polymerized actin and acto-myosin) involve in several steps of apoptotic death [ 27 ] . Recently, a previously uncharacterized cell death form has been coined disulfidptosis, which depends on the vulnerability of actin cytoskeleton to disulfide stress, making this unique cell death form distinct from apoptosis and ferroptosis [ 28 ] . Apart from cytoskeleton, plasma membrane rupture is the final phase of different cell death modalities, including apoptosis, necroptosis, pyroptosis, ferroptosis and other forms [ 29 ] . Although pore-forming proteins such as MLKL and GSDMD are critical to destroy plasma membrane in cells suffering necroptosis and pyroptosis, whereas the plasma membrane damage mode of methuosis remains unclear. Given that aberrant accumulation of macropinososome-derived cytoplasmic vacuoles is the hallmark of methuosis, it is naturally to hypothesize that cytoskeleton and plasma membrane damage might drive cells to methuosis. We herein for the first time reveal that the reversible disruption of cytoskeleton especially microfilament and tubulin, and pore-forming proteins-independent membrane rupture and ESCRT-Ⅲ membrane repair system participate in methuotic cell death. Concerning that large number of cytoplasmic vacuoles occupy almost area of cytoplasm in methuotic cells, how would the cytoskeleton be altered attracted our considerable interests. In this study, our findings indicate that microfilament protein F-actin distribution and arrangement were disordered in drug-treated H9c2 cells and U251 cells, additionally, actin stress fibers were reduced with the loss of pseudopods. Similar with these findings, recombinant human lactoferrin disrupts F-actin cytoskeleton organization and induces apoptosis in triple-negative breast cancer MDA-MB-231 cells [ 30 ] . In addition, other microfilament filamin A and filamin B were also suppressed by methuosis-inducing drugs. However, α-tubulin and β-tubulin seemed gather around the nucleus of methuotic cells. These findings suggest cytoskeleton in methuotic cells were disrupted by methuosis-inducing drugs. Studies have shown that knockdown of filamin A promotes cells death [ 31 , 32 ] . It could be concluded that cytoskeleton maintains cellular architecture at the early phase of methuosis, with the prolongation of cytoplasmic vacuoles dilation, cytoskeleton would be further disrupted until cellular final collapse. In other drugs-induced cytotoxicity model, cinnamic acid induces F-actin and tubulin disorganization and apoptotic cell death of human melanoma cells [ 2 ] , similarly, rotenone exposure results in actin cytoskeleton degradation, followed by programmed necroptosis which contributes to neurodegeneration [ 15 ] , indicating that disruption of cytoskeleton mediates varied cell death modalities. Furthermore, the underlying mechanisms of cytoskeleton disruption caused by methuosis-inducing drugs are also elucidated in this study. RhoA binds to its substrate ROCK, and activates ROCK phosphorylates myosin light chain phosphatase (MLCP) and inhibits MLCP activity, increasing the level of MLC phosphorylation and promoting stress fiber formation [ 21 ] . In the present study, we found that the stress fibers were reduced and the expression level of RhoA-ROCK-p-MLC was significantly decreased during cells undergoing methuosis. When RhoA agonist was exploited to activate RhoA, the destruction of stress fibers in methuotic cells could be markedly prevented, suggesting that the RhoA-ROCK signaling pathway involves in drugs-induced methuosis. Similar with maduramicin, another ionophore drug salinomycin has been demonstrated to suppress growth of pancreatic cancer through the inhibition of actin stress fibers due to RhoA downregulation [ 33 ] . RhoA-mediated cytoskeleton reorganization also contributes to microglia inflammatory reactivity during neuroinflammation [ 34 ] , suggesting the critical role of RhoA-related signaling pathways in cell death. Together with our findings, the manipulation of cytoskeleton might function as a potential strategy to reverse or delay drugs-induced methuosis. Membrane rupture represents the final phase of non-apoptotic cell death forms, there is no doubt that methuotic cells would suffer plasma membrane damage until the end of cellular life. However, the defined membrane rupture form as well as the potential mechanism of methuotic cells have not been characterized. In programmed cell death forms, plasma membrane rupture depends on pore-forming protein MLKL in necroptotic cells but is mediated by non-selective gasdermin D (GSDMD) pores when cells undergoing pyroptosis [ 35 , 36 ] . As a consequence, a variety of damage-associated molecular patterns (DAMPs) release and elicit an immune response. Cellular membrane rupture has been thought to be passive process for a long time, intriguingly, it is recently demonstrated as an actively event in which cell-surface ninjurin-1 (NINJ1) protein aggregates to mediate membrane rupture during toxin-induced death, necroptosis, pyroptosis, ferroptosis and secondary necrosis of apoptotic cells [ 37 , 38 ] . In this study, excessive cytoplasmic vacuoles-derived membrane rupture in methuotic cells is independent of MLKL and GSDMD which forms large pores in the plasma membrane, suggesting alternative membrane damage mechanism remains in methuosis. During this study, we attempted to elucidate the relationship between NINJ1 and methuosis, unfortunately, we were fail to perform native-page to detect the expression of this novel membrane damage protein NINJ1 in methuotic cells, requiring additional efforts to examine that possibility. Although it is not clear the exact plasma membrane damage form of methuosis, we further to explore what DAMPs release from methuotic cells. Intracellular components such as HMGB1, LDH, HSP, CRT and other proteins release through the damaged membrane will alert neighbouring cells injury and elicit immune response [ 23 ] . For instance, mouse embryonic fibroblasts (MEFs) treated by ML162 (ferroptosis inducer) or methylnitronitrosoguanidine (MNNG, parthanatos inducer) for inducing necrotic cell death, thereby triggering the release of HMGB1 and HSP90 [ 39 ] . In present study, DAMPs such as LDH, ATP and calreticulin were released from H9c2 cells and U251 cells after methuosis inducers treatment, suggesting that methuosis may act as an inflammatory cell death modality which is distinct from apoptosis. When cytoplasmic vacuoles generation was inhibited by bafilomycin A1, DAMPs released from methuotic cells were largely prevented, concluding that excessive cytoplasmic vacuoles drive membrane damage and DAMPs release during methuosis. However, to date, there are no relevant reports on the detailed DAMPs types released from methuotic cells. Our early study found that pro-inflammatory cytokines TNF-α and IL-8 release from maduramicin-treated myocardial cells [ 40 ] . Unfortunately, the spectrum of DAMPs released from methuotic cells is not clear in this study, proteomics and metabolomics technology could be exploited to reveal large scale of DAMPs from methuosis in further studies. Although plasma membrane injury involved in methuosis is independent of pore-forming proteins, ESCRT-III-dependent membrane repair machinery in programmed cell death forms (necroptosis, pyroptosis and ferroptosis) regulates maduramicin and MOMIPP-induced methuosis [ 41 ] . Given that ESCRT-III acts a critical membrane repair system to allow cells undergoing death switch to survival with integral membrane, we investigated the role of ESCRT-III in methuosis and found the activation of ESCRT-III subunits especially CHMP3 and CHMP5 are potential negative regulators against methuosis. Genetic inhibition of the ESCRT-III machinery highly increased methuosis rates and DAMPs release in H9c2 cells and U251 cells after drugs exposure. Similarly, in immortalized mouse BMDMs (iBMDMs) and HeLa cells, CHMP3 plays a central role in preventing pyroptosis from the activation of GSDMD [ 42 ] . In addition to CHMP3, other subunits of ESCRT-Ⅲ CHMP4B and CHMP2A are engaged to protect HT-29 cells, L929 cells and RIPK3-2Fv-NIH 3T3 cells from necroptotic death [ 24 ] . Intriguingly, a recent study found that other ESCRT-Ⅲ components CHMP5 and CHMP6 are demonstrated as critical factors which confer human cancer cells (PANC1 and HepG2) to limit lipid peroxidation-derived ferroptosis in vitro and in vivo [ 43 ] . Notably, as a complex, ESCRT-Ⅲ consists of twelve different subunits which involve in multiple biological procedures, including formation of vesicles for the processing of ubiquitin-tagged proteins, membrane repair, nuclear membrane reorganization and viral outgrowth [ 44 ] , eliciting a more complicated scientific questions that what conditions decide the regulation of ESCRT-Ⅲ components in a variety of cell death forms. These results suggest that the precise role of ESCRT-Ⅲ subunits determining the prolongation of cells survival require more experiments to elucidate in future studies. Our findings on the pivotal role of ESCRT-Ⅲ component CHMP3 and CHMP5 to antagonize methuosis may facilitate novel regulation strategy in methuotic cell death. In the end, even though our results reveal the role and potential mechanisms of cytoskeleton and plasma membrane damage involved in drugs-induced methuosis in normal and malignant cells, there are still several limitations for this study. First, the exact mechanisms underlying cytoskeleton disruption due to excessive cytoplasmic vacuolization remain unknown in methuosis. Second, pore-forming protein-independent plasma membrane damage mode of methuotic cells is still unclear, requiring more defining morphological characterization by performing fluorescence staining assay. Third, although some of the released DAMPs from drugs-induced methuotic cells are known in present study, a full scale of DAMPs remains to be explored in further studies. Last, the precise regulation mechanism of ESCRT-Ⅲ components during methuosis merits further investigation in vitro and in vivo due to its novelty in cell death field. The answers to these aforementioned scientific questions would greatly accelerate the known of methuosis. In conclusion, our study firstly ascertains the role of cytoskeleton disruption and plasma membrane damage, as well as their underlying mechanisms during chemical drugs-induced methusis (Fig. 7 ). Cytoskeleton such as F-actin and tubulin disruption based on RhoA-ROCK1 in methuotic myocardial cells or glioma cells is reversible. Targeting inhibition of the generation of excessive cytoplasmic vacuoles could impede cytoskeleton damage during methuosis. Furthermore, plasma membrane damage in MLKL and GSDMD-independent manner mediates drugs-induced methuosis, along with the release of several DAMPs, which may elicit immune response in vivo. In addition, ESCRT-Ⅲ components CHMP3 and CHMP5-mediated membrane repair involves in drugs-induced methuosis, suggesting that the regulation of ESCRT-Ⅲ represents a novel strategy to interfere methuotic death in life science. METHODS Cell lines and cell culture The rat myocardial cell line H9c2 was obtained from the American Type Culture Collection (ATCC, USA). The human glioblastoma cell line U251 was obtained from the National Biomedical Cell-Line Resource, NSTI-BMCR, http://cellresource.cn ). H9c2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, L110KJ, BasalMedia), and U251 cells were cultured in Minimum Essential Medium (MEM, L510KJ, BasalMedia), both media supplemented with 10% Fetal Bovine Serum (FBS, FS301, TransGen Biotech), 100 U/mL penicillin and 100 µg/mL streptomycin (SV30010, HyClone). The cells were incubated in a humidified atmosphere of 5% CO 2 at 37°C using an incubator (Thermo Fisher Scientific). Phalloidin staining Cells were seeded in 6-well plates (Corning) at a density of 1 × 10 5 cells per well and incubated for 24 h to allow adherence. To determine the effects of maduramicin or MOMIPP on the F-actin arrangement, H9c2 cells were treated by 0.1% dimethyl sulfoxide (DMSO, D8418, Sigma-Aldrich, negative control group) or 1 µM maduramicin (Mad, obtained from China Institute of Veterinary Drug Control) for 24 h and 48 h, whereas U251 cells were exposed to 0.1% DMSO (negative control group) or 2.5 µM MOMIPP (T33467, TargetMol) for 12 h and 24 h. Similarly, to explore whether drugs-induced alteration of cytoskeleton was reversible, H9c2 cells were exposed to 0.1% DMSO, maduramicin (1 µM, 48 h) and maduramicin (1 µM, 48 h) followed by drug withdraw for 4 h, 12 h, 24 h and 48 h, U251 cells were treated by 0.1% DMSO, MOMIPP (2.5 µM, 24 h) and MOMIPP (2.5 µM, 24 h) followed by drug withdraw for 4 h, 12 h, 24 h and 48 h. In addition, to understand the effect of bafilomycin A1 on the microfilament change, H9c2 cells were pretreated with 1 nM bafilomycin A1 (Baf A1, S1413, Selleck) for 1 h prior to the treatment with 0.1% DMSO or 1 µM maduramicin for 48 h. U251 cells underwent a pretreatment with 100 nM bafilomycin A1 for 1 h, followed by the treatment with 0.1% DMSO (negative control group) or 2.5 µM MOMIPP for additional 24 h. The obtained H9c2 and U251 cells were then fixed with 4% paraformaldehyde (BL539A, Biosharp) in phosphate-buffered saline (PBS) for 15 min, and were washed three times with PBS, and permeabilized with 0.2% Triton X-100 (P0096, Beyotime) in PBS for 5 min at room temperature. After three additional washing with PBS, the obtained cells were incubated with CoraLite 488-Phalloidin dye (PF00001, Proteintech) for 20 min at room temperature. Finally, the cells were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, C1005, Beyotime) for 5 min and were visualized using an inverted fluorescence microscope (EVOS FL Auto 2, Thermo Fisher Scientific). Immunofluorescence examination H9c2 and U251 cells were seeded at a density of 1 × 10 4 cells per well in 12-well plates (Corning) and were cultured for 24 h. Subsequently, H9c2 cells were treated by 0.1% DMSO (negative control group) or 1 µM maduramicin for 48 h, whereas U251 cells were exposed to 0.1% DMSO (negative control group) or 2.5 µM MOMIPP for 24 h. Cells were fixed with 4% paraformaldehyde at room temperature for 12 min, followed by washing with PBS. Permeabilization was performed using 0.2% Triton X-100 in PBS for 5 min at room temperature, after which the cells were washed again with PBS. Cells were then blocked with 5% bovine serum albumin (BSA, BL736A, Biosharp) for 1 h at room temperature before overnight incubation with primary antibodies against filamin A (sc-17749, 1:200, Santa Cruz), filamin B (TD13572, 1:50, Abmart), α-tubulin (MS00237, 1:200, Abmart), β-tubulin (M2005, 1:100, Abmart) and calreticulin (CRT, DF3139, 1:100, Affinity) at 4°C. On the next day, H9c2 cells and U251 cells were washed three times with PBS containing 0.1% Tween-20 (PBST), and then incubated with a secondary antibody (SA00013-1, SA00013-2, SA00013-3 or SA00013-4, Proteintech) for 1 h at room temperature in the dark. After a final washing with PBST, nuclei of H9c2 cells and U251 cells were counterstained with DAPI (C1005, Beyotime), and photographs were taken using an inverted fluorescence microscope (EVOS FL Auto 2, Thermo Fisher Scientific). Western blotting To explore the effects of maduramicin and MOMIPP on the expression of target proteins, including filamin A, filamin B, α-actinin, RhoA, ROCK1, p-MLC, β-tubulin, calreticulin (CRT), MLKL, p-MLKL, GSDMD, GSDMD-N, CHMP2B, CHMP3, CHMP4B and CHMP5, H9c2 cells and U251 cells were exposed to maduramicin (1 µM) and MOMIPP (2.5 µM) for 12 h, 24 h, 48 h, and 72 h, respectively. To determine the effect of cytoplasmic vacuolization inhibitor bafilomycin A1 on the expression of target proteins on filamin A, filamin B, and calreticulin, H9c2 cells and U251 cells were treated by 0.1% DMSO (negative control), bafilomycin A1 (1 nM or 100 nM) for 1 h, maduramicin (1 µM) for 48 h or MOMIPP (2.5 µM) for 24 h, and were pretreated by bafilomycin A1 (1 nM or 100 nM) for 1 h followed by maduramicin (1 µM) for 48 h or MOMIPP (2.5 µM) for 24 h, respectively. Subsequently, cells were collected and lysed using a rapid cell lysis buffer to extract total proteins (abs9229, absin) or membrane proteins (A10008, Abmart) according to the manufacturer's protocol. Protein concentrations were measured using a bicinchoninic acid (BCA) protein assay kit (PC0020, Solarbio). Equal aliquots of protein samples were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto nitrocellulose (NC) membranes. The obtained membranes were blocked with skimmed milk for 2 h at room temperature, and were incubated with primary antibodies overnight at 4°C. Primary antibodies filamin A (sc-17749, 1:2000, Santa Cruz), filamin B (TD13572, 1:1500, Abmart), α-actinin1 (sc-17829, 1:2000, Santa Cruz), RhoA (SC-418, 1:1500, Santa Cruz), ROCK1 (sc-17794, 1:1500, Santa Cruz), p-MLC (TA5443, 1:1500, Abmart), β-tubulin (M20005, 1:5000, Abmart), calreticulin (DF3139, 1:1500, Affinity), MLKL (DF-7412, 1:1500, Affinity), p-MLKL (AF-7420, 1:1500, Affinity), GSDMD/GSDMD-N (AF-4012, 1:1500, Affinity), CHMP2B (T510002S, 1:1500, Abmart), CHMP3 (T58143, 1:2000, Abmart), CHMP4B (13683-1-AP, 1:1000, Proteintech), CHMP5 (MG138543, 1:2000, Abmart), Na + /K + -ATPase (sc-48345, 1:2000, Santa Cruz) were used in this section. The next day, the membranes were washed three times with Tris-buffered saline containing Tween-20 (TBST), and were further incubated with secondary antibody (m21003, Abmart) for 1 h at room temperature. Followed three additional washing with TBST, protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit (362-8ES, YEASEN) and were imaged with an automated imaging system (Tanon). Gray values of target proteins were quantified utilizing ImageJ software (1.46 r version by NIH, USA). LDH release assay H9c2 and U251 cells were seeded in 12-well plates at a density of 1 × 10 4 cells per well and were incubated for 24 h. Subsequently, H9c2 cells were treated with 0.1% DMSO (negative control group) or 1 µM maduramicin, and U251 cells were exposed to 0.1% DMSO (negative control group) or 2.5 µM MOMIPP at intervals of 12 h, 24 h, 48 h, and 72 h. Additionally, to determine the effect of cytoplasmic vacuolization inhibitor bafilomycin A1 on the release of LDH, H9c2 cells were exposed to 0.1% DMSO, 1 nM bafilomycin A1 for 1 h, 1 µM maduramicin for 48 h, 1 nM bafilomycin A1 for 1 h prior to maduramicin (1 µM) for 48 h, similarly, U251 cells were treated by 0.1% DMSO, 100 nM bafilomycin A1 for 1 h, 2.5 µM MOMIPP for 24 h, 100 nM bafilomycin A1 for 1 h prior to MOMIPP (2.5 µM) for 24 h. After that, cell culture supernatants were collected and were centrifuged at 500 × g for 5 min to remove pellet debris. Furthermore, lactate dehydrogenase (LDH) release was quantified using an LDH detection kit (A020, Nanjing Jiancheng Bioengineering Institute) according to the manufacture’s protocol and previous study [ 7 ] . Hoechst 33342/PI staining H9c2 and U251 cells were seeded in 12-well plates at a density of 1 × 10 4 cells per well and were incubated for 24 h. H9c2 cells were treated with 0.1% DMSO (negative control group) or 1 µM maduramicin for 24 h, 48 h, and 72 h, and U251 cells were treated by 0.1% DMSO (negative control group) or 2.5 µM MOMIPP for 24 h, 48 h, and 72 h. In addition, to determine the effect of bafilomycin A1 on the membrane integrity, H9c2 cells were exposed to 0.1% DMSO, 1 nM bafilomycin A1 for 1 h, 1 µM maduramicin for 48 h, 1 nM bafilomycin A1 for 1 h prior to maduramicin (1 µM) for 48 h, simiarly, U251 cells were treated by 0.1% DMSO, 100 nM bafilomycin A1 for 1 h, 2.5 µM MOMIPP for 24 h, 100 nM bafilomycin A1 for 1 h prior to MOMIPP (2.5 µM) for 24 h. Then, Hoechst 33342 solution, propidium iodide (PI) solution, and cell staining buffer were prepared at a 1:1:100 ratio. After that, 500 µL of this mixed Hoechst 33342/PI staining solution was added to each well and was incubated on ice for 20–30 min. The stained cells were subsequently observed and imaged using an inverted fluorescence microscope (EVOS FL Auto 2, Thermo Fisher Scientific). Intracellular ATP assay H9c2 cells and U251 cells were seeded in 6-well plates at a density of 1 × 10 5 cells per well and were incubated for 24 h to allow 60% ~ 70% confluence. To assess the effects of maduramicin and MOMIPP on the level of intracellular ATP, H9c2 cells were treated with 0.1% DMSO (0 h), 1 µM maduramicin for 24 h, 48 h and 72 h, and U251 cells were exposed to 0.1% DMSO (0 h), 2.5 µM MOMIPP for 24 h, 48 h and 72 h. In addition, to determine the effect of bafilomycin A1 on intracellular ATP level, H9c2 cells were treated by 0.1% DMSO, 1 nM bafilomycin A1 for 1 h, 1 µM maduramicin for 48 h, 1 nM bafilomycin A1 for 1 h prior to maduramicin (1 µM) for 48 h, similarly, U251 cells were treated by 0.1% DMSO, 100 nM bafilomycin A1 for 1 h, 2.5 µM MOMIPP for 24 h, 100 nM bafilomycin A1 for 1 h prior to MOMIPP (2.5 µM) for 24 h. After that, intracellular ATP was measured using the commercial ATP assay kit (S0027, Beyotime) according to the manufacturer’s instructions. Subsequently, H9c2 and U251 cells were collected and lysed using a rapid cell lysis buffer (abs9229, absin). Protein concentrations were measured using a bicinchoninic acid (BCA) protein assay kit (PC0020, Solarbio). After the addition of an equal volume of rLuciferase/Luciferin reagent, luminescence was measured as readout for ATP levels by using a luminescence system (Snyergy H1, BioTek). Extracellular ATP assay To determine the levels of extracellular ATP, H9c2 cells and U251 cells were treated as same as aforementioned drugs exposure procedure in section of 2.7. Then, cell supernatants were collected and were centrifuged at 500 × g for 5 min to remove pellet debris. After that, ATP was measured using the Promega's ENLITEN® ATP assay system (FF2000, Promega) according to the manufacturer’s instructions by manipulating a luminometer (Snyergy H1, BioTek). siRNA transfection siRNAs targeting rat CHMP3 and CHMP5, and siRNAs targeting human CHMP3 were designed and synthesized by Tsingke biotechnology Co.,Ltd., with their sequences provided in Table S1 . H9c2 cells were seeded in 6-well plates at a density of 5 × 10 4 cells per well and were cultured to reach approximately 50% confluence, at which point siRNA transfection was initiated. Additionally, U251 cells were seeded in 6-well plates at a density of 5 × 10 4 cells per well and allowed to grow to approximately 50% confluence prior to siRNA transfection. The green fluorescence- labeled siRNAs (50 nM) targeted CHMP3 or CHMP5 were mixed with 200 µL of jetPRIME buffer (100100046, Polyplus), followed by the addition of 4 µL jetPRIME transfection reagent. The mixture was incubated for 10–15 min to form the transfection mixture, which was then added to the cells. The cell culture plates were gentle shook to ensure homogeneous distribution of the mixture. Transfection efficiency was determined using an inverted fluorescence microscope (EVOS FL Auto 2, Thermo Fisher Scientific) after targeting siRNAs were transfected for 6 h, and siRNAs-transfected cells were further cultured for 24 h to obtain adequate transfection efficiency. H9c2 cells and U251 cells with successfully transfection efficiency were used for further analysis of cellular activity, LDH release and membrane integrity as described. CCK-8 assay siRNAs-transfected H9c2 cells or U251 cells were seeded in 96-well plates at a density of 3 × 10 3 cells per well and were cultured until 80% confluence. H9c2 cells were then treated with 0.1% DMSO (negative control) and 1 µM maduramicin for 48 h, and U251 cells were treated with 0.1% DMSO (negative control) and 2.5 µM MOMIPP for 24 h. Subsequently, 10 µL of CCK-8 (BS350A, Biosharp) reagent was added to each well and the cell culture plates were incubated for 1–2 h at 37°C. The optical density (OD) at 450 nm was detected by using a multifunctional microplate reader (Snyergy H1, BioTek), and cell viability for each group was calculated. Statistical analysis Data were shown as mean ± standard deviation (SD). GraphPad (version 9.5) was used to analyze these obtained data from at least three independent experiments. Statistical differences were evaluated by performing one-way ANOVA or unpaired Student’s t-test. A level of P < 0.05 was considered as statistically significant. Declarations COMPETING INTERESTS The authors declare that no competing financial interests. FUNDING This study was supported by the grants from National Natural Science Foundation of China (No:31902326), China Postdoctoral Science Foundation (No: 2018M642271), Jiangsu Province Postdoctoral Research Foundation (No: 2019K166) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). AUTHOR CONTRIBUTIONS Bin Dong : Investigation, Writing-Original Draft, Validation. Jing Xiao : Methodology, Investigation, Writing-Original Draft. Junqi Wang : Methodology, Visualization. Xinhao Song : Resources, Writing-Review and Editing. Hui Ji : Resources, Validation, Software. Jiurong Peng : Resources, Methodology. Xinru Weng : Methodology, Validation. Dawei Guo : Writing-Review and Editing. Shanxiang Jiang : Conceptualization, Supervision, Writing-Review and Editing, Funding acquisition. Xiuge Gao : Conceptualization, Supervision, Writing-Review and Editing, Funding acquisition. 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Escrt-dependent membrane repair negatively regulates pyroptosis downstream of gsdmd activation. Science 2018, 362(6417): 956-+. Dai EY, Meng LJ, Kang R, Wang XF, Tang DL. Escrt-iii-dependent membrane repair blocks ferroptosis. Biochem Biophys Res Commun 2020, 522(2): 415–421. Campsteijn C, Vietri M, Stenmark H. Novel escrt functions in cell biology: Spiraling out of control? Curr Opin Cell Biol 2016, 41: 1–8. Additional Declarations (Not answered) Supplementary Files SupplementalMaterials.rar Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5422638","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":377778902,"identity":"ff7051d1-9a4f-4c57-936f-b27a30949b82","order_by":0,"name":"Xiuge Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYPACGwbGBhDNRpRqZhCRRrqWw1AOMVoMjvcffvGj4nwec/sZA4YPZYcZ+Gc3ENBy5jCbZc+Z28WMPTkGjDPOHWaQuHMAvxbJGclsBrxttxMbG3IMmHnbDjMYSCQQ0DL/MZvh37ZziY39bwyY/xKjhV+Cmfkxb9uBxMYZQFsYidLCk2zGLHMmGajlWcHBnnPpPBI3CGhhYz/4+OObCrvEjf3JGx/8KLOW459BQAtIlwSINGxgYDgApHkIqgcC5g8gUp4YpaNgFIyCUTAyAQCNUkOs4ze+1AAAAABJRU5ErkJggg==","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Xiuge","middleName":"","lastName":"Gao","suffix":""},{"id":377778903,"identity":"7b364842-c73b-4a59-9e77-9397bf13dd59","order_by":1,"name":"Bin Dong","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Dong","suffix":""},{"id":377778904,"identity":"6fe8f175-3699-431d-b764-892741f2dfa7","order_by":2,"name":"Jing Xiao","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Xiao","suffix":""},{"id":377778905,"identity":"7961acdd-d98f-431b-b46a-1525f80da6fd","order_by":3,"name":"Junqi Wang","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Junqi","middleName":"","lastName":"Wang","suffix":""},{"id":377778906,"identity":"8e56620b-0b45-40a9-85ec-d6e8b76220df","order_by":4,"name":"Xinhao Song","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xinhao","middleName":"","lastName":"Song","suffix":""},{"id":377778907,"identity":"d6fdb226-5950-437c-a10e-e3d007d2a4ab","order_by":5,"name":"Hui Ji","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Ji","suffix":""},{"id":377778908,"identity":"1a271829-d07f-4f08-8b03-36f374176bfd","order_by":6,"name":"Jiurong Peng","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jiurong","middleName":"","lastName":"Peng","suffix":""},{"id":377778909,"identity":"a584c65d-804e-4c58-8e0a-9afdb5eb830d","order_by":7,"name":"Xinru Weng","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xinru","middleName":"","lastName":"Weng","suffix":""},{"id":377778910,"identity":"31a54f39-e843-407c-af15-983be83c58d3","order_by":8,"name":"Dawei Guo","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Dawei","middleName":"","lastName":"Guo","suffix":""},{"id":377778911,"identity":"5ee20659-d441-462d-aa57-9c8cdfc64588","order_by":9,"name":"Shanxiang Jiang","email":"","orcid":"https://orcid.org/0000-0002-4799-6314","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shanxiang","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2024-11-09 15:30:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5422638/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5422638/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71106951,"identity":"c2166c68-3f4a-44bb-b1cb-1a72a6d40e30","added_by":"auto","created_at":"2024-12-11 08:07:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1242085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExcessive cytoplasmic vacuolization results in cytoskeleton disruption in methuosis. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) H9c2 cells were treated with either vehicle control (0.1% DMSO) or 1 μM maduramicin for durations ranging from 24 h to 48 h for further phalloidin staining (scale bars = 40 µm). (\u003cstrong\u003eB\u003c/strong\u003e) Similarly, U251 cells were exposed to vehicle control (0.1% DMSO) or 2.5 μM MOMIPP over the same time periods, followed by phalloidin staining assay (scale bars = 20 µm). (\u003cstrong\u003eC, D\u003c/strong\u003e) Cytoskeleton protein bands of filamin A, filamin B, and α-actinin-1 in H9c2 cells and U251 cells were determined by performing western blotting (WB) after the treatment with either vehicle control or maduramicin and MOMIPP at various time intervals (12 h, 24 h, 48 h, and 72 h), respectively. (\u003cstrong\u003eE\u003c/strong\u003e) Quantitative data of target proteins from triplicate WB assays, and the data are presented as mean ± standard deviation (SD), n = 3. Significance levels were denoted as\u003csup\u003e *\u003c/sup\u003efor P \u0026lt; 0.05 and \u003csup\u003e**\u003c/sup\u003efor P \u0026lt; 0.01. (\u003cstrong\u003eF, G\u003c/strong\u003e) Immunofluorescence staining of α-tubulin and β-tubulin, along with phase-contrast microscopy, was conducted on H9c2 cells treated by 1 μM maduramicin for 48 h or vehicle control (0.1% DMSO), as well as on U251 cells treated by 2.5 μM MOMIPP for 24 h or vehicle control (0.1% DMSO) (scale bars = 40 µm or 20 µm). (\u003cstrong\u003eH\u003c/strong\u003e) H9c2 cells and U251 cells were pretreated by maduramicin (1 μM, 48 h) or MOMIPP (2.5 μM) for 24 h, respectively, and followed by drug withdraw for different time intervals (4 h, 12 h, 24 h, and 48 h) and further phalloidin staining (scale bars = 20 µm or 40 µm).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5422638/v1/d36750d7fc2080cffa5c6071.png"},{"id":71105781,"identity":"c4322f38-7b42-4fe9-ad37-e99086608948","added_by":"auto","created_at":"2024-12-11 07:51:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1110483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytoskeleton disruption in methuosis is reversed by blocking cytoplasmic vacuolization. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eH9c2 cells were subjected to vehicle control (0.1% DMSO), bafilomycin A1 (Baf A1, 1 nM) for 1 h, maduramicin (Mad, 1 μM) for 48 h, or a sequential regimen of Baf A1 (1 nM) for 1 h followed by Mad (1 μM) for 48 h or cytochalasin D (Cyto D, 3 μM) for 15 min, after which cells were stained with phalloidin (scale bars = 20 µm). (\u003cstrong\u003eB\u003c/strong\u003e) U251 cells were exposed to vehicle control (0.1% DMSO), Baf A1 (100 nM) for 1 h, MOMIPP (2.5 μM) for 24 h, or a combination of Baf A1 (100 nM) for 1 h followed by MOMIPP (2.5 μM) for 24 h or cytochalasin D (Cyto D, 3 μM) for 15 min, and also followed by phalloidin staining (scale bars = 20 µm). (\u003cstrong\u003eC\u003c/strong\u003e) Western blotting was carried out to analyze the expression of filamin A and filamin B in H9c2 cells and U251 cells which were treated by vehicle control (0.1% DMSO), Baf A1 (1 nM or 100 nM) for 1 h, Mad (1 μM) for 48 h or MOMIPP (2.5 μM) for 48 h, and the combination of Baf A1 (1 nM or 100 nM) pretreatment for 1 h and Mad (1 μM) for 48 h or MOMIPP (2.5 μM) for 48 h. (\u003cstrong\u003eD\u003c/strong\u003e) Quantitative results of filamin A and filamin B expression of H9c2 cells and U251 cells in triplicate, data are shown as mean ± standard deviation (SD), \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05 and \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01. (\u003cstrong\u003eE-H\u003c/strong\u003e) Immunofluorescence staining of filamin A and filamin B in H9c2 cells and U251 cells were performed with the same treatments above mentioned regimen in western blotting assay (scale bars = 40 µm or 20 µm).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5422638/v1/a1c988d4339912f0a3e054ca.png"},{"id":71105783,"identity":"8d43b5af-e46f-49a9-a864-058afc07c236","added_by":"auto","created_at":"2024-12-11 07:51:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":715074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRhoA-ROCK1 inhibition mediates cytoskeletal disruption in methuosis. \u003c/strong\u003e(\u003cstrong\u003eA, B\u003c/strong\u003e) Western blotting was conducted to detect the expression of RhoA, ROCK1, and p-MLC in H9c2 and U251 cells at multiple time points (12 h, 24 h, 48 h, and 72 h) after treatment with either vehicle control (0.1% DMSO) and Mad (1 μM) or MOMIPP (2.5 μM), respectively. (\u003cstrong\u003eC\u003c/strong\u003e) Quantitative results of RhoA, ROCK1, and p-MLC expression of H9c2 cells and U251 cells, data in triplicate are shown as mean ± standard deviation (SD), \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05 and \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01. (\u003cstrong\u003eD, E\u003c/strong\u003e) For fluorescence examination, H9c2 cells and U251 cells were treated by vehicle control (0.1% DMSO), calpeptin (100 μM) for 1 h, Mad (1 μM) for 48 h or MOMIPP (2.5 μM) for 24 h, and the combination of calpeptin (100 μM) pretreatment for 1 h and Mad (1 μM) for 48 h or MOMIPP (2.5 μM) for 24 h, respectively, followed by phalloidin staining (scale bars = 40 µm or 20 µm).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5422638/v1/caadd7ef9640036ecc676247.png"},{"id":71106753,"identity":"783aa4bd-c6ab-4e6a-9af0-c660fef5e8f0","added_by":"auto","created_at":"2024-12-11 07:59:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":808699,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExcessive cytoplasmic vacuolization results in plasma membrane damage in methuosis. \u003c/strong\u003eLactate dehydrogenase (LDH) release was detected after Mad (1 μM) or MOMIPP (2.5 μM) exposed to H9c2 cells and U251 cells for 12 h, 24 h, 48 h and 72 h (\u003cstrong\u003eA\u003c/strong\u003e), intracellular ATP and extracellular ATP levels of H9c2 cells and U251 cells treated by Mad (1 μM) or MOMIPP (2.5 μM) for 24 h, 48 h and 72 h, respectively (\u003cstrong\u003eB, C\u003c/strong\u003e), compared with negative control (0.1% DMSO), data in triplicate or in quadruplicate are shown as mean ± standard deviation (SD), **P\u0026lt;0.01, compared to negative control. (\u003cstrong\u003eD, E\u003c/strong\u003e) H9c2 cells were treated with vehicle control (0.1% DMSO) or 1 μM maduramicin for 24 h to 72 h, and U251 cells were exposed to vehicle control (0.1% DMSO) or 2.5 μM MOMIPP for 24 h to 72 h, followed by Hoechst 33342/PI staining. (\u003cstrong\u003eF-H\u003c/strong\u003e) The expression of p-MLKL, MLKL, GSDMD, GSDMD-N, and CRT was analyzed by performing western blotting in H9c2 cells and U251 cells treated by vehicle control (0.1% DMSO), Mad (1 μM) or MOMIPP (2.5 μM) at 12 h, 24 h, 48 h and 72 h. (\u003cstrong\u003eI\u003c/strong\u003e) Quantitative data of p-MLKL/MLKL, GSDMD-N/GSDMD and CRT expression of H9c2 cells and U251 cells, data in triplicate are shown as mean ± standard deviation (SD), \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05 and \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01. (\u003cstrong\u003eJ, K\u003c/strong\u003e) Immunofluorescence staining of CRT in H9c2 cells and U251 cells after treatment by Mad (1 μM) for 48 h and MOMIPP (2.5 μM) for 48 h, and DAPI staining for nucleus (scale bars = 20 µm or 40 µm).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5422638/v1/1113377311663a63a1ee3ac8.png"},{"id":71105782,"identity":"80396a2a-18c5-44d1-b5c5-c4d4b0cd21ab","added_by":"auto","created_at":"2024-12-11 07:51:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":767826,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of cytoplasmic vacuolization prevents plasma membrane damage. \u003c/strong\u003eH9c2 and U251 cells were treated with vehicle control (0.1% DMSO), Baf A1 (1 nM for H9c2 and 100 nM for U251), Mad (1 μM), MOMIPP (2.5 μM) or a pretreatment with Baf A1 followed by Mad (1 μM) for 48 h (H9c2) or MOMIPP (2.5 μM) for 24 h (U251). Thereafter, lactate dehydrogenase (LDH) release (\u003cstrong\u003eA\u003c/strong\u003e), intracellular ATP levels (\u003cstrong\u003eB\u003c/strong\u003e) and extracellular ATP levels (\u003cstrong\u003eC\u003c/strong\u003e) were determined accordingly at least in triplicate, data are shown as mean ± standard deviation (SD), \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, compared to negative control. (\u003cstrong\u003eD, E\u003c/strong\u003e) With the same drug exposure regimen, H9c2 and U251 cells were stained with Hoechst 33342/PI to assess plasma membrane integrity. (\u003cstrong\u003eF, G, H\u003c/strong\u003e) Western blotting assay of CRT expression in H9c2 cells and U251 cells in H9c2 cells and U251 cells after treatment by negative control (0.1% DMSO), Baf A1 (1 nM for H9c2 cells, and 100 nM for U251 cells) for 1 h, Mad (1 μM) for 48 h, MOMIPP (2.5 μM) for 48 h, and Baf A1 pre-exposure for 1 h followed by the treatment of Mad (1 μM) for 48 h or MOMIPP (2.5 μM) for 48 h,, quantitative data of CRT expression are shown as bar graph, data from triplicate experiments are shown as mean ± standard deviation (SD), \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, compared to negative control or Mad/MOMIPP treatment. (\u003cstrong\u003eI, J\u003c/strong\u003e) Immunofluorescence staining of CRT in H9c2 cells and U251 cells after the same treatment regimen with above WB experiment, and DAPI staining for nucleus (scale bars = 20 µm or 40 µm).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5422638/v1/6cd73f79bde6f54aa3b79603.png"},{"id":71106755,"identity":"2a59e173-6ec4-4590-9969-f77d0e23602b","added_by":"auto","created_at":"2024-12-11 07:59:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":791513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eESCRT-Ⅲ membrane repair system negatively control methuosis. \u003c/strong\u003eH9c2 cells (\u003cstrong\u003eA\u003c/strong\u003e) and U251 cells (\u003cstrong\u003eB\u003c/strong\u003e) were treated by negative control (0.1% DMSO), Mad (1 μM) or MOMIPP (2.5 μM) for 12 h, 24 h, 48 h and 72 h, respectively, followed by western blotting analysis of ESCRT-III subunits CHMP2B, CHMP3, CHMP4B, and CHMP5. (\u003cstrong\u003eC\u003c/strong\u003e) Quantitative data of western blotting bands from triplicate assays, data are shown as mean ± standard deviation (SD), \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, compared with negative control. (\u003cstrong\u003eD\u003c/strong\u003e) siRNA targeting the ESCRT-III subunits CHMP3 and CHMP5 was transfected into H9c2 cells, and siRNA targeting CHMP3 was transfected into U251 cells using the transfection reagent jetPRIME, followed by western blotting assay of CHMP3 and CHMP5. (\u003cstrong\u003eE-G\u003c/strong\u003e) CCK-8 assay was conducted to assess the effect of siRNA-mediated knockdown of CHMP3 and CHMP5 on cell viability upon drugs exposure for 24 h or 48 h (n = 6), \u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01 compared to negative control or Mad/MOMIPP alone treatment. (H, I) LDH release assay was performed in H9c2 cells and U251 cells treated by Mad or MOMIPP after siRNA knockdown of CHMP3 or/and CHMP5 (n = 3). \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01, compared to negative control or Mad/MOMIPP alone treatment. (J-L) Hoechst 33342/PI staining was carried out to determine the effect of siRNA knockdown of CHMP3 or CHMP5 on membrane integrity after Mad (48 h) or MOMIPP (24 h) exposure.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5422638/v1/7879b93b12847a311a4c3c2b.png"},{"id":71105786,"identity":"e1e79f02-4316-4f62-bea1-ea4d37f3d485","added_by":"auto","created_at":"2024-12-11 07:51:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2064381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of cytokeleton and plasma membrane damage in methuosis\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5422638/v1/35d441c3b8d035d335e90d43.png"},{"id":71108198,"identity":"cc44ba8e-36b2-48bd-8e98-469ce0f8efc6","added_by":"auto","created_at":"2024-12-11 08:23:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8941220,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5422638/v1/3e56a9cf-32a3-494d-a580-d84cd88bcf2f.pdf"},{"id":71105788,"identity":"9997dd35-825a-4a77-8842-6c1acc72785f","added_by":"auto","created_at":"2024-12-11 07:51:01","extension":"rar","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":30852268,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterials.rar","url":"https://assets-eu.researchsquare.com/files/rs-5422638/v1/994370ef24cce9a6bf14ce18.rar"}],"financialInterests":"(Not answered)","formattedTitle":"Cytoskeleton disruption and plasma membrane damage determine methuosis of normal and malignant cells","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eUnlike the well-known programmed cell death types such as apoptosis, necroptosis, autophagic cell death and ferroptosis, a relatively novel cell death methuosis (drink to death) coined by Dr. Maltese was firstly defined in glioma cells, in which excessive cytoplasmic vacuolization due to macropinocytosis persists until cellular plasma membrane rupture \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Since the first recognition of methuosis, a large amount of malignant cells have been demonstrated sensitive to methuosis, which represents an alternative anticancer strategy \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Intriguingly, therapy-resistant cancer cells such as prostate cancer and breast cancer cells \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e are vulnerable to methuosis initial genes and pharmacological chemicals, proposing a novel solution to overcome resistant cancer cells. In addition, methuosis acts as an emerging mechanism in drug toxicology area because that normal cells such as myocardial cells have been demonstrated to undergo methuotic cell death in a context-dependent manner \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. At the same time, recent critical findings shown that anti-parasite drugs exert pharmacological activities through the induction of methuosis in nematode model \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. That means methuosis can be induced in a broad range of cells, including normal cells, malignant cells as well as parasite cells \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. However, the cytoplasm process and the core mechanism of cells undergoing methuosis remain to be explored.\u003c/p\u003e \u003cp\u003eConcerning that methuosis is characterized by catastrophic cytoplasmic vacuolization, the origin and trafficking of methuotic vacuoles have been extensively investigated \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. In the early stage of methuosis, macropinocytosis is triggered by genetic mutation or pharmacologic chemicals which facilitate generation of lamellipodia, small size vacuoles (pinososome) containing extracellular fluid enter cytoplasm. In the middle phase of methuosis, pinososomes merge with each other to form larger vacuoles. At the terminal stage of methuosis, large number of phase-lucent giant vacuoles occupy almost the cytoplasm and ultimately result in cellular membrane rupture \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Although the phenotype process of methuosis is clear, how the numerous cytoplasmic vacuoles affect cytoskeleton and cellular plasma membrane are largely unknown. A recent study demonstrates that chemical substitution at 2-indolyl position of methuosis inducer 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (MOMIPP) switches the mode of cytotoxicity from methuosis to microtubule disruption \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Given that cytoskeleton supports cellular structure integrity, playing a key role in maintaining cells survival. If cytoskeleton is changed or damaged, cells will be driven to death under some contexts. For instance, in rotenone-induced necroptotic neuronal cells, actin cytoskeleton cofilin is altered and degraded, thereby leading to neurotoxicity \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Similarly, actin cytoskeleton has been demonstrated to determine apoptosis process due to the upregulation of cleavage of actin and gelsolin in mammalian system \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Therefore, we hypothesized that excessive cytoplasmic vacuoles might disrupt cytoskeleton in methuotic cells.\u003c/p\u003e \u003cp\u003eIn addition to cytoskeleton, plasma membrane functions the last protective barrier to avoid entering of extracellular harmful materials or releasing of intracellular substances. As a result, cellular membrane rupture is the hallmark of all known cell death modalities. Plasma membrane damage occurs in alternative forms, including pore-formation, membrane deformation and membrane alteration \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. For instance, mixed lineage kinase domain-like (MLKL) is a necessary executioner of necroptosis in which MLKL inserts into plasma membrane and results in cellular rupture; besides, cells undergoing pyroptosis requires the formation of balloon-like membrane protrusions which further contributes to plasma membrane rupture; for cells ferroptosis, lipid peroxidation due to imbalance of antioxidant enzymes results in plasma membrane permeabilization and further rupture \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. But for methuosis, there are no available data on the damage mode of plasma membrane, and the exact membrane damage mechanism of methuosis is also unknown. Given that large giant cytoplasmic vacuoles continuously accumulate until eventual loss of plasma membrane integrity \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Cells undergoing methuosis may suffer from distinctive membrane damage which are differ from the known programmed cell death models. Herein, we propose a hypothesis that catastrophic cytoplasmic vacuoles in methuotic cells damage plasma membrane in an alternative way. Hence, the understanding the form of plasma membrane damage of methuotic cells will facilitate the development of preventive strategy to maintain cells survival.\u003c/p\u003e \u003cp\u003eIn this study, the classic methuosis model induced by indole-based chalcone in glioma G251 cells, and a novel methuosis model induced by maduramicin in myocardial H9c2 cells were used to elucidate the role and underlying mechanism of cytoskeleton and plasma membrane damage in methuosis. Intriguingly, for the first time, cytoskeleton proteins include F-actin, tubulin and filamin A/B were disrupted through the inhibition of RhoA-ROCK1 signaling pathway, plasma membrane was damaged with certain DAMPs leakage in pore-forming proteins- independent manner, and membrane repair system ESCRT-Ⅲ acted as a negatively regulator of methuosis are illuminated in current study. These findings will benefit for the exploiting of cytoskeleton and plasma membrane to promote or prevent methuosis in cancer therapy and drug toxicology.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExcessive cytoplasmic vacuolization provokes cytoskeleton disruption in methuotic cells\u003c/h2\u003e \u003cp\u003eTo understand the role of cytoskeleton in cells undergoing methuosis, first for microfilament, F-actin was stained with phalloidin. In maduramicin-treated H9c2 cells methuosis model, compared to drug-untreated H9c2 cells, in which F-actin was neatly arranged, H9c2 cells exposed to 1 \u0026micro;M maduramicin for 24 h shown that thickened actin fibers and cortical actin, along with several fluorescent bright spots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). When maduramicin-treated H9c2 cells for 48 h, cell boundaries were more distinct, stress fibers were reduced, and cells lacked stress fibers, showing smooth edges and a loss of pseudopods (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Furthermore, in MOMIPP-induced U251 cells methuosis model, after 2.5 \u0026micro;M MOMIPP exposed for 12 h, U251 cells became to thickened actin fibers, increased cortical actin, and disorganized microfilaments with pseudopod disappearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). When MOMIPP treatment extended for 24 h, stress fibers were decreased and some fluorescent foci were generated in U251 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). However, in the absence of MOMIPP, U251 cells exhibited that clearly F-actin arrangement with a pronounced aggregation of actin stress fibers and conspicuous pseudopodia. In addition to F-actin, as important parts of microfilament, filamin A, filamin B and α-actinin-1 were also assayed by performing western blotting. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, filamin A and filamin B were significantly decreased in H9c2 and U251 cells after methuosis-inducing drugs exposed for 48 h and 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), but not at other time points, while the expression of α-actinin-1 was not significantly changed after maduramicin and MOMIPP exposure, suggesting that the reduction of filamin A and filamin B may contribute to microfilament disorder-mediated methuosis. Moreover, other critical cytoskeleton proteins, α-tubulin and β-tubulin, were examined using immunofluorescence staining. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, compared to cells in absence of drugs, α-tubulin and β-tubulin were reorganized and formed microtubule bundle which surrounded partial cytoplasmic vacuoles induced by maduramicin or MOMIPP. However, H9c2 and U251 cells in negative control group exhibited a well-developed array of microtubules radiating from the juxtanuclear microtubule organizing center (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Taken together, cytoskeletal microfilament and tubulin in H9c2 cells and U251 cells are disrupted during methuosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCytoskeleton disruption in methuosis is reversed by blocking cytoplasmic vacuolization\u003c/h3\u003e\n\u003cp\u003eGiven that cytoplasmic vacuolization is reversible when methuosis stimulus is removed \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, we next investigated whether drug-induced cytoskeleton damage is reversible in methuotic H9c2 and U251 cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, F-actin of H9c2 cell treated by maduramicin was altered in disorder as above described, however, after the absence of maduramicin from 4 h to 48 h, F-actin arrangement in H9c2 cells gradually restored to normal pattern. Similar to H9c2 cells, F-actin configuration was disrupted after MOMIPP exposure to U251 cells, while the removal of MOMIPP for 48 h reversed F-actin disruption gradually. Moreover, bafilomycin A1, an inhibitor of methuosis that targets V-ATPase \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, was used to inhibit the generation of vacuolization. In H9c2 cells, bafilomycin A1 alone treatment had no visible effects on F-actin arrangement and distribution, but eliminated maduramicin-induced thickened actin fibers and cortical actin, along with several fluorescent bright spots (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Unlike H9c2 cells, in U251 cells, bafilomycin A1 alone decreased pseudopodia on cell surface and altered arrangement of F-actin, however, MOMIPP-triggered disturbance of F-actin was largely reversed by bafilomycin A1 although pseudopodia disappearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Intriguingly, bafilomycin A1 could not reverse cytochalasin D-induced severe cytoskeleton damage in H9c2 cells and U251 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Next, the effect of bafilomycin A1 on filamin A and filamin B after methuosis-inducing drugs exposure were evaluated in H9c2 and U251 cells. Our results showed that while drugs significantly decreased the expression of filamin A and filamin B, bafilomycin A1 significantly restored their levels to drugs-untreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Further immunofluorescence examination indicated that the arrangement of filamin A and filamin B was disrupted by cytoplasmic vacuoles accumulation followed by methuosis-inducing drugs treatment but was normalized after bafilomycin A1 pretreatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). These findings together indicate that cytoskeletal disruption due to excessive cytoplasmic vacuolization is reversible in methuotic cells, and the inhibition of cytoplasmic vacuolization can restore cytoskeleton integrity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eRhoA-ROCK1 inhibition mediates cytoskeleton disruption in methuosis\u003c/h3\u003e\n\u003cp\u003eTo explore the mechanism underlying the disruption of cytoskeleton in methuotic cells, RhoA-ROCK1 signaling pathway which is pivotal for stress fiber formation, was focused in this study. RhoA, a key regulator of cytoskeleton and cellular shape, controls the assembly of contractile actin-myosin filaments. Upon RhoA binds to ROCK1, facilitating stress fiber formation by promoting actin polymerization as well as the inhibiting of actin depolymerization \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Furthermore, the activated ROCK1 phosphorylates myosin light chain (MLC) and activates myosin Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase, which in turn leads to the binding of myosin with F-actin and the formation of actomyosin stress fibers essential for cell contraction \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, RhoA was significantly downregulated in H9c2 cells treated by maduramicin for 48 h and 72 h, ROCK1 expression was significantly decreased at 12 h-72 h, and p-MLC expression was notably decreased at 12 h-72 h. In U251 cells, the expression of RhoA was significantly decreased at 24 h to 72 h after MOMIPP exposure, and ROCK1 was markedly reduced at 48 h-72 h, but the expression of p-MLC was not significantly changed after MOMIPP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These results suggest that RhoA-ROCK1 pathway was inhibited by maduramicin and MOMIPP in methuotic cells. To further elucidate the role of the RhoA-ROCK1 cascade in microfilament disruption, calpeptin, a RhoA agonist, was employed to observe the alteration of microfilament by phalloidin staining. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, in maduramicin-induced H9c2 cells methuosis model, compared to cells in control group, calpeptin alone treatment enhanced the density of microfilament in alignment, maduramicin disrupted F-actin in varying degrees, however, calpeptin pretreatment restored F-actin arrangement even if maduramicin exposure. Similarly, in MOMIPP-induced glioma U251 cells methuosis model, compared to control group, calpeptin alone increased the density of F-actin with the accumulation of fluorescent faculae on the cellular surface, MOMIPP-treated cells markedly decreased F-actin expression with disrupted structure, but calpeptin pretreatment reversed F-actin distribution and arrangement (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Collectively, these findings indicate that the inhibition of RhoA-ROCK1 signaling pathway contributes to cytoskeletal microfilament disruption in drug-induced methuosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eExcessive cytoplasmic vacuolization results in plasma membrane damage and DAMPs release in methuosis\u003c/h3\u003e\n\u003cp\u003eCellular plasma membrane damage plays a critical role in cell death. However, how plasma membrane is ruptured remains unknown in cells undergoing methuosis. To assess plasma membrane damage mode, lactate dehydrogenase (LDH) was firstly determined in this study. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, with the prolongation of maduramicin or MOMIPP treatment, LDH levels in the supernatant of H9c2 cells and U251 cells were significantly increased at 12 h or 24 h, 48 h and 72 h, respectively. Moreover, Hoechst 33342/PI staining demonstrated that plasma membrane was damaged in H9c2 cells and U251 cells undergoing drug-induced methuosis due to the number of PI-positive cells was increased from 24 h to 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Additionally, pore-forming proteins p-MLKL and GSDMD-N, which are essential for plasma membrane damage, were detected in drug-induced methuotic cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, MLKL, p-MLKL, GSDMD and GSDMD-N expression in H9c2 cells were not changed after maduramicin exposed for 12 h to 72 h, similarly, the expression of MLKL, p-MLKL, GSDMD and GSDMD-N was not altered by MOMIPP treatment for 12 h to 72 h, indicating that cell membrane damage during methuosis is independent of canonical pore-forming proteins. Concerning that plasma membrane damage is followed by the release of cellular damage associated molecular patterns (DAMPs) such as calreticulin (CRT) and ATP, which trigger inflammatory response \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. We further investigated the release of DAMPs from damaged methuotic cells. Normally, CRT localizes on the endoplasmic reticulum, but translocates to the outer cellular membrane surface under some injury conditions. Results indicated that maduramicin increased the expression of CRT in plasma membrane of H9c2 cells after pharmacological exposure for 48 h and 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Furthermore, CRT expression in MOMIPP-treated U251 cells was also elevated slightly at 48 h and 72 h (the final phase of methuosis) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). At the same time, immunofluorescence assay exhibited that the expression of CRT was enhanced in cytoplasm as well as on cellular membrane surface of H9c2 cells and U251 cells, compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). In addition, ATP levels of cytoplasm and the cultured supernatant were measured after drugs treated methuotic cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, upon exposure of maduramicin and MOMIPP for 24 h to 72 h, intracellular ATP levels were significantly decreased in H9c2 cells and U251 cells, compared to cells in the control group. In contrast, the extracellular ATP levels were significantly increased after maduramicin treatment for 48 h and 72 h or MOMIPP exposure for 24 h to 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In conclusion, these above findings demonstrate pore-forming protein-independent plasma membrane damage and DAMPs release mediate drug-induced methuosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eInhibition of cytoplasmic vacuolization impedes plasma membrane damage\u003c/h3\u003e\n\u003cp\u003eTo further elucidate the relationship between cytoplasmic vacuolization and plasma membrane damage, bafilomycin A1 was employed to inhibit the generation of cytoplasmic vacuoles. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, compared to negative control group, bafilomycin A1 alone did not induce significantly LDH release from H9c2 cells and U251 cells, maduramicin and MOMIPP both triggered significant release of LDH (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), however, bafilomycin A1 pretreatment reduced LDH release from maduramicin- or MOMIPP-treated cells. At the same time, PI-positive H9c2 cells induced by maduramicin were decreased by bafilomycin A1 pretreatment, similarly, MOMIPP-induced PI-positive U251 cells were reduced after bafilomycin A1 pre-exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). In addition, the effect of vacuolization inhibition by bafilomycin A1 on DAMPs released from methuotic cells was also evaluated herein. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH, compared to the CRT expression in negative control group, bafilomycin A1 alone did not change CRT expression in H9c2 cells and U251 cells, and maduramicin- and MOMIPP-induced the increase of CRT expression was inhibited by bafilomycin A1 pretreatment. Moreover, immunofluorescence analysis exhibited that maduramicin-triggered enhancement of CRT expression and cytoplasmic distribution in H9c2 cells was markedly inhibited by bafilomycin A1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI), the same findings were observed in MOMIPP-treated U251 cells after bafilomycin A1 pretreatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). Furthermore, compared to negative control group, bafilomycin A1 did not change intracellular ATP levels of H9c2 cells and U251 cells, but significantly attenuated maduramicin- and MOMIPP-induced the decrease of intracellular ATP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Similarly, for extracellular ATP, maduramicin and MOMIPP both significantly increased ATP release from H9c2 cells and U251 cells, respectively, but bafilomycin A1 pretreatment decreased extracellular ATP to baseline level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Based on these above results, excessive cytoplasmic vacuolization induced by maduramicin or MOMIPP mediates final plasma membrane damage in methuotic cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eESCRT-III membrane repair system negatively controls methuosis\u003c/h2\u003e \u003cp\u003eUnder normal condition, cells are able to maintain plasma membrane integrity by activating self-repair system upon suffer from injury. The ESCRT-III repair mechinary has recently been shown as an important membrane repair mechanism for inhibiting necroptosis \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, ferroptosis \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, and pyroptosis \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Herein, the role of ESCRT-III system was investigated in the determinant of methuotic cell death. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, compared to negative control group, the subunits of ESCRT-III complex such as CHMP2B was significantly elevated in plasma membrane of H9c2 cells after maduramicin treatment for 24 h to 72 h, CHMP3 and CHMP5 were significantly elevated after maduramicin treatment for 48 h and 72 h, whereas CHMP4B expression was significantly downregulated at 24 h but not other time points. In U251 cells, compared to the control group, the expression of CHMP2B were significantly decreased after MOMIPP exposed for 48 h but not 12 h, 24 h and 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), the expression of CHMP4B was also significantly reduced by MOMIPP treatment for 48 h-72 h. In contrast, the expression of CHMP3 was significantly increased after MOMIPP treatment for 24 h to 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). However, CHMP5 expression was not changed even MOMIPP exposed for 12 h to 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Furthermore, to understand the role of ESCRT-III system in methuotic cells, siRNA was used to knockdown CHMP3 and CHMP5 in H9c2 cells and CHMP3 in U251 cells, followed by cell viability, LDH release and Hoechst 33342/PI staining assays. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, siRNA-mediated knockdown of CHMP3 and CHMP5 augmented drug-induced cell death of H9c2 cells compared to maduramicin treatment individually. Similarly, compared to MOMIPP group, knockdown of CHMP3 significantly suppressed cell viability of U251 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Moreover, LDH release from H9c2 cells and U251 cells after maduramicin or MOMIPP was significantly elevated when knockdown CHMP3, but knockdown CHMP5 did not affect maduramicin-induced LDH release from H9c2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). In addition, compared to negative control group, maduramicin or MOMIPP alone exposure increased the number of PI-positive cells, knockdown CHMP3 and CHMP5 markedly elevated the number of maduramicin-induced PI-positive H9c2 cells, and knockdown CHMP3 also heightened the number of MOMIPP-triggered PI-positive U251 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). Collectively, ESCRT-III-dependent membrane repair system participates in drug-induced methuosis of H9c2 and U251 cells, and CHMP3 could be exploited as a potential negative regulator of methuosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCytoskeleton together with integral plasma membrane support cellular shape, biological functions and protect cells from extracellular dangers. Cells undergoing death might suffer varying degrees of cytoskeleton damage and plasma membrane rupture. As a well-known cell death model, apoptosis, has been demonstrated that at least three components of cytoskeleton (keratins, polymerized actin and acto-myosin) involve in several steps of apoptotic death \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Recently, a previously uncharacterized cell death form has been coined disulfidptosis, which depends on the vulnerability of actin cytoskeleton to disulfide stress, making this unique cell death form distinct from apoptosis and ferroptosis \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Apart from cytoskeleton, plasma membrane rupture is the final phase of different cell death modalities, including apoptosis, necroptosis, pyroptosis, ferroptosis and other forms \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Although pore-forming proteins such as MLKL and GSDMD are critical to destroy plasma membrane in cells suffering necroptosis and pyroptosis, whereas the plasma membrane damage mode of methuosis remains unclear. Given that aberrant accumulation of macropinososome-derived cytoplasmic vacuoles is the hallmark of methuosis, it is naturally to hypothesize that cytoskeleton and plasma membrane damage might drive cells to methuosis. We herein for the first time reveal that the reversible disruption of cytoskeleton especially microfilament and tubulin, and pore-forming proteins-independent membrane rupture and ESCRT-Ⅲ membrane repair system participate in methuotic cell death.\u003c/p\u003e \u003cp\u003eConcerning that large number of cytoplasmic vacuoles occupy almost area of cytoplasm in methuotic cells, how would the cytoskeleton be altered attracted our considerable interests. In this study, our findings indicate that microfilament protein F-actin distribution and arrangement were disordered in drug-treated H9c2 cells and U251 cells, additionally, actin stress fibers were reduced with the loss of pseudopods. Similar with these findings, recombinant human lactoferrin disrupts F-actin cytoskeleton organization and induces apoptosis in triple-negative breast cancer MDA-MB-231 cells \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. In addition, other microfilament filamin A and filamin B were also suppressed by methuosis-inducing drugs. However, α-tubulin and β-tubulin seemed gather around the nucleus of methuotic cells. These findings suggest cytoskeleton in methuotic cells were disrupted by methuosis-inducing drugs. Studies have shown that knockdown of filamin A promotes cells death \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. It could be concluded that cytoskeleton maintains cellular architecture at the early phase of methuosis, with the prolongation of cytoplasmic vacuoles dilation, cytoskeleton would be further disrupted until cellular final collapse. In other drugs-induced cytotoxicity model, cinnamic acid induces F-actin and tubulin disorganization and apoptotic cell death of human melanoma cells \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, similarly, rotenone exposure results in actin cytoskeleton degradation, followed by programmed necroptosis which contributes to neurodegeneration \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, indicating that disruption of cytoskeleton mediates varied cell death modalities. Furthermore, the underlying mechanisms of cytoskeleton disruption caused by methuosis-inducing drugs are also elucidated in this study. RhoA binds to its substrate ROCK, and activates ROCK phosphorylates myosin light chain phosphatase (MLCP) and inhibits MLCP activity, increasing the level of MLC phosphorylation and promoting stress fiber formation \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. In the present study, we found that the stress fibers were reduced and the expression level of RhoA-ROCK-p-MLC was significantly decreased during cells undergoing methuosis. When RhoA agonist was exploited to activate RhoA, the destruction of stress fibers in methuotic cells could be markedly prevented, suggesting that the RhoA-ROCK signaling pathway involves in drugs-induced methuosis. Similar with maduramicin, another ionophore drug salinomycin has been demonstrated to suppress growth of pancreatic cancer through the inhibition of actin stress fibers due to RhoA downregulation \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. RhoA-mediated cytoskeleton reorganization also contributes to microglia inflammatory reactivity during neuroinflammation\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e, suggesting the critical role of RhoA-related signaling pathways in cell death. Together with our findings, the manipulation of cytoskeleton might function as a potential strategy to reverse or delay drugs-induced methuosis.\u003c/p\u003e \u003cp\u003eMembrane rupture represents the final phase of non-apoptotic cell death forms, there is no doubt that methuotic cells would suffer plasma membrane damage until the end of cellular life. However, the defined membrane rupture form as well as the potential mechanism of methuotic cells have not been characterized. In programmed cell death forms, plasma membrane rupture depends on pore-forming protein MLKL in necroptotic cells but is mediated by non-selective gasdermin D (GSDMD) pores when cells undergoing pyroptosis \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. As a consequence, a variety of damage-associated molecular patterns (DAMPs) release and elicit an immune response. Cellular membrane rupture has been thought to be passive process for a long time, intriguingly, it is recently demonstrated as an actively event in which cell-surface ninjurin-1 (NINJ1) protein aggregates to mediate membrane rupture during toxin-induced death, necroptosis, pyroptosis, ferroptosis and secondary necrosis of apoptotic cells \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. In this study, excessive cytoplasmic vacuoles-derived membrane rupture in methuotic cells is independent of MLKL and GSDMD which forms large pores in the plasma membrane, suggesting alternative membrane damage mechanism remains in methuosis. During this study, we attempted to elucidate the relationship between NINJ1 and methuosis, unfortunately, we were fail to perform native-page to detect the expression of this novel membrane damage protein NINJ1 in methuotic cells, requiring additional efforts to examine that possibility. Although it is not clear the exact plasma membrane damage form of methuosis, we further to explore what DAMPs release from methuotic cells. Intracellular components such as HMGB1, LDH, HSP, CRT and other proteins release through the damaged membrane will alert neighbouring cells injury and elicit immune response \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. For instance, mouse embryonic fibroblasts (MEFs) treated by ML162 (ferroptosis inducer) or methylnitronitrosoguanidine (MNNG, parthanatos inducer) for inducing necrotic cell death, thereby triggering the release of HMGB1 and HSP90 \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. In present study, DAMPs such as LDH, ATP and calreticulin were released from H9c2 cells and U251 cells after methuosis inducers treatment, suggesting that methuosis may act as an inflammatory cell death modality which is distinct from apoptosis. When cytoplasmic vacuoles generation was inhibited by bafilomycin A1, DAMPs released from methuotic cells were largely prevented, concluding that excessive cytoplasmic vacuoles drive membrane damage and DAMPs release during methuosis. However, to date, there are no relevant reports on the detailed DAMPs types released from methuotic cells. Our early study found that pro-inflammatory cytokines TNF-α and IL-8 release from maduramicin-treated myocardial cells \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Unfortunately, the spectrum of DAMPs released from methuotic cells is not clear in this study, proteomics and metabolomics technology could be exploited to reveal large scale of DAMPs from methuosis in further studies.\u003c/p\u003e \u003cp\u003eAlthough plasma membrane injury involved in methuosis is independent of pore-forming proteins, ESCRT-III-dependent membrane repair machinery in programmed cell death forms (necroptosis, pyroptosis and ferroptosis) regulates maduramicin and MOMIPP-induced methuosis \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Given that ESCRT-III acts a critical membrane repair system to allow cells undergoing death switch to survival with integral membrane, we investigated the role of ESCRT-III in methuosis and found the activation of ESCRT-III subunits especially CHMP3 and CHMP5 are potential negative regulators against methuosis. Genetic inhibition of the ESCRT-III machinery highly increased methuosis rates and DAMPs release in H9c2 cells and U251 cells after drugs exposure. Similarly, in immortalized mouse BMDMs (iBMDMs) and HeLa cells, CHMP3 plays a central role in preventing pyroptosis from the activation of GSDMD \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. In addition to CHMP3, other subunits of ESCRT-Ⅲ CHMP4B and CHMP2A are engaged to protect HT-29 cells, L929 cells and RIPK3-2Fv-NIH 3T3 cells from necroptotic death \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Intriguingly, a recent study found that other ESCRT-Ⅲ components CHMP5 and CHMP6 are demonstrated as critical factors which confer human cancer cells (PANC1 and HepG2) to limit lipid peroxidation-derived ferroptosis in vitro and in vivo \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Notably, as a complex, ESCRT-Ⅲ consists of twelve different subunits which involve in multiple biological procedures, including formation of vesicles for the processing of ubiquitin-tagged proteins, membrane repair, nuclear membrane reorganization and viral outgrowth \u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e, eliciting a more complicated scientific questions that what conditions decide the regulation of ESCRT-Ⅲ components in a variety of cell death forms. These results suggest that the precise role of ESCRT-Ⅲ subunits determining the prolongation of cells survival require more experiments to elucidate in future studies. Our findings on the pivotal role of ESCRT-Ⅲ component CHMP3 and CHMP5 to antagonize methuosis may facilitate novel regulation strategy in methuotic cell death.\u003c/p\u003e \u003cp\u003eIn the end, even though our results reveal the role and potential mechanisms of cytoskeleton and plasma membrane damage involved in drugs-induced methuosis in normal and malignant cells, there are still several limitations for this study. First, the exact mechanisms underlying cytoskeleton disruption due to excessive cytoplasmic vacuolization remain unknown in methuosis. Second, pore-forming protein-independent plasma membrane damage mode of methuotic cells is still unclear, requiring more defining morphological characterization by performing fluorescence staining assay. Third, although some of the released DAMPs from drugs-induced methuotic cells are known in present study, a full scale of DAMPs remains to be explored in further studies. Last, the precise regulation mechanism of ESCRT-Ⅲ components during methuosis merits further investigation in vitro and in vivo due to its novelty in cell death field. The answers to these aforementioned scientific questions would greatly accelerate the known of methuosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, our study firstly ascertains the role of cytoskeleton disruption and plasma membrane damage, as well as their underlying mechanisms during chemical drugs-induced methusis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Cytoskeleton such as F-actin and tubulin disruption based on RhoA-ROCK1 in methuotic myocardial cells or glioma cells is reversible. Targeting inhibition of the generation of excessive cytoplasmic vacuoles could impede cytoskeleton damage during methuosis. Furthermore, plasma membrane damage in MLKL and GSDMD-independent manner mediates drugs-induced methuosis, along with the release of several DAMPs, which may elicit immune response in vivo. In addition, ESCRT-Ⅲ components CHMP3 and CHMP5-mediated membrane repair involves in drugs-induced methuosis, suggesting that the regulation of ESCRT-Ⅲ represents a novel strategy to interfere methuotic death in life science.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and cell culture\u003c/h2\u003e \u003cp\u003eThe rat myocardial cell line H9c2 was obtained from the American Type Culture Collection (ATCC, USA). The human glioblastoma cell line U251 was obtained from the National Biomedical Cell-Line Resource, NSTI-BMCR, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cellresource.cn\u003c/span\u003e\u003cspan address=\"http://cellresource.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). H9c2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, L110KJ, BasalMedia), and U251 cells were cultured in Minimum Essential Medium (MEM, L510KJ, BasalMedia), both media supplemented with 10% Fetal Bovine Serum (FBS, FS301, TransGen Biotech), 100 U/mL penicillin and 100 \u0026micro;g/mL streptomycin (SV30010, HyClone). The cells were incubated in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C using an incubator (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhalloidin staining\u003c/h2\u003e \u003cp\u003eCells were seeded in 6-well plates (Corning) at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and incubated for 24 h to allow adherence. To determine the effects of maduramicin or MOMIPP on the F-actin arrangement, H9c2 cells were treated by 0.1% dimethyl sulfoxide (DMSO, D8418, Sigma-Aldrich, negative control group) or 1 \u0026micro;M maduramicin (Mad, obtained from China Institute of Veterinary Drug Control) for 24 h and 48 h, whereas U251 cells were exposed to 0.1% DMSO (negative control group) or 2.5 \u0026micro;M MOMIPP (T33467, TargetMol) for 12 h and 24 h. Similarly, to explore whether drugs-induced alteration of cytoskeleton was reversible, H9c2 cells were exposed to 0.1% DMSO, maduramicin (1 \u0026micro;M, 48 h) and maduramicin (1 \u0026micro;M, 48 h) followed by drug withdraw for 4 h, 12 h, 24 h and 48 h, U251 cells were treated by 0.1% DMSO, MOMIPP (2.5 \u0026micro;M, 24 h) and MOMIPP (2.5 \u0026micro;M, 24 h) followed by drug withdraw for 4 h, 12 h, 24 h and 48 h. In addition, to understand the effect of bafilomycin A1 on the microfilament change, H9c2 cells were pretreated with 1 nM bafilomycin A1 (Baf A1, S1413, Selleck) for 1 h prior to the treatment with 0.1% DMSO or 1 \u0026micro;M maduramicin for 48 h. U251 cells underwent a pretreatment with 100 nM bafilomycin A1 for 1 h, followed by the treatment with 0.1% DMSO (negative control group) or 2.5 \u0026micro;M MOMIPP for additional 24 h. The obtained H9c2 and U251 cells were then fixed with 4% paraformaldehyde (BL539A, Biosharp) in phosphate-buffered saline (PBS) for 15 min, and were washed three times with PBS, and permeabilized with 0.2% Triton X-100 (P0096, Beyotime) in PBS for 5 min at room temperature. After three additional washing with PBS, the obtained cells were incubated with CoraLite 488-Phalloidin dye (PF00001, Proteintech) for 20 min at room temperature. Finally, the cells were counterstained with 4\u0026prime;, 6-diamidino-2-phenylindole (DAPI, C1005, Beyotime) for 5 min and were visualized using an inverted fluorescence microscope (EVOS FL Auto 2, Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence examination\u003c/h2\u003e \u003cp\u003eH9c2 and U251 cells were seeded at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well in 12-well plates (Corning) and were cultured for 24 h. Subsequently, H9c2 cells were treated by 0.1% DMSO (negative control group) or 1 \u0026micro;M maduramicin for 48 h, whereas U251 cells were exposed to 0.1% DMSO (negative control group) or 2.5 \u0026micro;M MOMIPP for 24 h. Cells were fixed with 4% paraformaldehyde at room temperature for 12 min, followed by washing with PBS. Permeabilization was performed using 0.2% Triton X-100 in PBS for 5 min at room temperature, after which the cells were washed again with PBS. Cells were then blocked with 5% bovine serum albumin (BSA, BL736A, Biosharp) for 1 h at room temperature before overnight incubation with primary antibodies against filamin A (sc-17749, 1:200, Santa Cruz), filamin B (TD13572, 1:50, Abmart), α-tubulin (MS00237, 1:200, Abmart), β-tubulin (M2005, 1:100, Abmart) and calreticulin (CRT, DF3139, 1:100, Affinity) at 4\u0026deg;C. On the next day, H9c2 cells and U251 cells were washed three times with PBS containing 0.1% Tween-20 (PBST), and then incubated with a secondary antibody (SA00013-1, SA00013-2, SA00013-3 or SA00013-4, Proteintech) for 1 h at room temperature in the dark. After a final washing with PBST, nuclei of H9c2 cells and U251 cells were counterstained with DAPI (C1005, Beyotime), and photographs were taken using an inverted fluorescence microscope (EVOS FL Auto 2, Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eTo explore the effects of maduramicin and MOMIPP on the expression of target proteins, including filamin A, filamin B, α-actinin, RhoA, ROCK1, p-MLC, β-tubulin, calreticulin (CRT), MLKL, p-MLKL, GSDMD, GSDMD-N, CHMP2B, CHMP3, CHMP4B and CHMP5, H9c2 cells and U251 cells were exposed to maduramicin (1 \u0026micro;M) and MOMIPP (2.5 \u0026micro;M) for 12 h, 24 h, 48 h, and 72 h, respectively. To determine the effect of cytoplasmic vacuolization inhibitor bafilomycin A1 on the expression of target proteins on filamin A, filamin B, and calreticulin, H9c2 cells and U251 cells were treated by 0.1% DMSO (negative control), bafilomycin A1 (1 nM or 100 nM) for 1 h, maduramicin (1 \u0026micro;M) for 48 h or MOMIPP (2.5 \u0026micro;M) for 24 h, and were pretreated by bafilomycin A1 (1 nM or 100 nM) for 1 h followed by maduramicin (1 \u0026micro;M) for 48 h or MOMIPP (2.5 \u0026micro;M) for 24 h, respectively. Subsequently, cells were collected and lysed using a rapid cell lysis buffer to extract total proteins (abs9229, absin) or membrane proteins (A10008, Abmart) according to the manufacturer's protocol. Protein concentrations were measured using a bicinchoninic acid (BCA) protein assay kit (PC0020, Solarbio). Equal aliquots of protein samples were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto nitrocellulose (NC) membranes. The obtained membranes were blocked with skimmed milk for 2 h at room temperature, and were incubated with primary antibodies overnight at 4\u0026deg;C. Primary antibodies filamin A (sc-17749, 1:2000, Santa Cruz), filamin B (TD13572, 1:1500, Abmart), α-actinin1 (sc-17829, 1:2000, Santa Cruz), RhoA (SC-418, 1:1500, Santa Cruz), ROCK1 (sc-17794, 1:1500, Santa Cruz), p-MLC (TA5443, 1:1500, Abmart), β-tubulin (M20005, 1:5000, Abmart), calreticulin (DF3139, 1:1500, Affinity), MLKL (DF-7412, 1:1500, Affinity), p-MLKL (AF-7420, 1:1500, Affinity), GSDMD/GSDMD-N (AF-4012, 1:1500, Affinity), CHMP2B (T510002S, 1:1500, Abmart), CHMP3 (T58143, 1:2000, Abmart), CHMP4B (13683-1-AP, 1:1000, Proteintech), CHMP5 (MG138543, 1:2000, Abmart), Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e-ATPase (sc-48345, 1:2000, Santa Cruz) were used in this section. The next day, the membranes were washed three times with Tris-buffered saline containing Tween-20 (TBST), and were further incubated with secondary antibody (m21003, Abmart) for 1 h at room temperature. Followed three additional washing with TBST, protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit (362-8ES, YEASEN) and were imaged with an automated imaging system (Tanon). Gray values of target proteins were quantified utilizing ImageJ software (1.46 r version by NIH, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLDH release assay\u003c/h2\u003e \u003cp\u003eH9c2 and U251 cells were seeded in 12-well plates at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well and were incubated for 24 h. Subsequently, H9c2 cells were treated with 0.1% DMSO (negative control group) or 1 \u0026micro;M maduramicin, and U251 cells were exposed to 0.1% DMSO (negative control group) or 2.5 \u0026micro;M MOMIPP at intervals of 12 h, 24 h, 48 h, and 72 h. Additionally, to determine the effect of cytoplasmic vacuolization inhibitor bafilomycin A1 on the release of LDH, H9c2 cells were exposed to 0.1% DMSO, 1 nM bafilomycin A1 for 1 h, 1 \u0026micro;M maduramicin for 48 h, 1 nM bafilomycin A1 for 1 h prior to maduramicin (1 \u0026micro;M) for 48 h, similarly, U251 cells were treated by 0.1% DMSO, 100 nM bafilomycin A1 for 1 h, 2.5 \u0026micro;M MOMIPP for 24 h, 100 nM bafilomycin A1 for 1 h prior to MOMIPP (2.5 \u0026micro;M) for 24 h. After that, cell culture supernatants were collected and were centrifuged at 500 \u0026times; g for 5 min to remove pellet debris. Furthermore, lactate dehydrogenase (LDH) release was quantified using an LDH detection kit (A020, Nanjing Jiancheng Bioengineering Institute) according to the manufacture\u0026rsquo;s protocol and previous study \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHoechst 33342/PI staining\u003c/h2\u003e \u003cp\u003eH9c2 and U251 cells were seeded in 12-well plates at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well and were incubated for 24 h. H9c2 cells were treated with 0.1% DMSO (negative control group) or 1 \u0026micro;M maduramicin for 24 h, 48 h, and 72 h, and U251 cells were treated by 0.1% DMSO (negative control group) or 2.5 \u0026micro;M MOMIPP for 24 h, 48 h, and 72 h. In addition, to determine the effect of bafilomycin A1 on the membrane integrity, H9c2 cells were exposed to 0.1% DMSO, 1 nM bafilomycin A1 for 1 h, 1 \u0026micro;M maduramicin for 48 h, 1 nM bafilomycin A1 for 1 h prior to maduramicin (1 \u0026micro;M) for 48 h, simiarly, U251 cells were treated by 0.1% DMSO, 100 nM bafilomycin A1 for 1 h, 2.5 \u0026micro;M MOMIPP for 24 h, 100 nM bafilomycin A1 for 1 h prior to MOMIPP (2.5 \u0026micro;M) for 24 h. Then, Hoechst 33342 solution, propidium iodide (PI) solution, and cell staining buffer were prepared at a 1:1:100 ratio. After that, 500 \u0026micro;L of this mixed Hoechst 33342/PI staining solution was added to each well and was incubated on ice for 20\u0026ndash;30 min. The stained cells were subsequently observed and imaged using an inverted fluorescence microscope (EVOS FL Auto 2, Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular ATP assay\u003c/h2\u003e \u003cp\u003eH9c2 cells and U251 cells were seeded in 6-well plates at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and were incubated for 24 h to allow 60% ~ 70% confluence. To assess the effects of maduramicin and MOMIPP on the level of intracellular ATP, H9c2 cells were treated with 0.1% DMSO (0 h), 1 \u0026micro;M maduramicin for 24 h, 48 h and 72 h, and U251 cells were exposed to 0.1% DMSO (0 h), 2.5 \u0026micro;M MOMIPP for 24 h, 48 h and 72 h. In addition, to determine the effect of bafilomycin A1 on intracellular ATP level, H9c2 cells were treated by 0.1% DMSO, 1 nM bafilomycin A1 for 1 h, 1 \u0026micro;M maduramicin for 48 h, 1 nM bafilomycin A1 for 1 h prior to maduramicin (1 \u0026micro;M) for 48 h, similarly, U251 cells were treated by 0.1% DMSO, 100 nM bafilomycin A1 for 1 h, 2.5 \u0026micro;M MOMIPP for 24 h, 100 nM bafilomycin A1 for 1 h prior to MOMIPP (2.5 \u0026micro;M) for 24 h. After that, intracellular ATP was measured using the commercial ATP assay kit (S0027, Beyotime) according to the manufacturer\u0026rsquo;s instructions. Subsequently, H9c2 and U251 cells were collected and lysed using a rapid cell lysis buffer (abs9229, absin). Protein concentrations were measured using a bicinchoninic acid (BCA) protein assay kit (PC0020, Solarbio). After the addition of an equal volume of rLuciferase/Luciferin reagent, luminescence was measured as readout for ATP levels by using a luminescence system (Snyergy H1, BioTek).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eExtracellular ATP assay\u003c/h2\u003e \u003cp\u003eTo determine the levels of extracellular ATP, H9c2 cells and U251 cells were treated as same as aforementioned drugs exposure procedure in section of 2.7. Then, cell supernatants were collected and were centrifuged at 500 \u0026times; g for 5 min to remove pellet debris. After that, ATP was measured using the Promega's ENLITEN\u0026reg; ATP assay system (FF2000, Promega) according to the manufacturer\u0026rsquo;s instructions by manipulating a luminometer (Snyergy H1, BioTek).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003esiRNA transfection\u003c/h2\u003e \u003cp\u003esiRNAs targeting rat CHMP3 and CHMP5, and siRNAs targeting human CHMP3 were designed and synthesized by Tsingke biotechnology Co.,Ltd., with their sequences provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. H9c2 cells were seeded in 6-well plates at a density of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well and were cultured to reach approximately 50% confluence, at which point siRNA transfection was initiated. Additionally, U251 cells were seeded in 6-well plates at a density of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well and allowed to grow to approximately 50% confluence prior to siRNA transfection. The green fluorescence- labeled siRNAs (50 nM) targeted CHMP3 or CHMP5 were mixed with 200 \u0026micro;L of jetPRIME buffer (100100046, Polyplus), followed by the addition of 4 \u0026micro;L jetPRIME transfection reagent. The mixture was incubated for 10\u0026ndash;15 min to form the transfection mixture, which was then added to the cells. The cell culture plates were gentle shook to ensure homogeneous distribution of the mixture. Transfection efficiency was determined using an inverted fluorescence microscope (EVOS FL Auto 2, Thermo Fisher Scientific) after targeting siRNAs were transfected for 6 h, and siRNAs-transfected cells were further cultured for 24 h to obtain adequate transfection efficiency. H9c2 cells and U251 cells with successfully transfection efficiency were used for further analysis of cellular activity, LDH release and membrane integrity as described.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCCK-8 assay\u003c/h2\u003e \u003cp\u003esiRNAs-transfected H9c2 cells or U251 cells were seeded in 96-well plates at a density of 3 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well and were cultured until 80% confluence. H9c2 cells were then treated with 0.1% DMSO (negative control) and 1 \u0026micro;M maduramicin for 48 h, and U251 cells were treated with 0.1% DMSO (negative control) and 2.5 \u0026micro;M MOMIPP for 24 h. Subsequently, 10 \u0026micro;L of CCK-8 (BS350A, Biosharp) reagent was added to each well and the cell culture plates were incubated for 1\u0026ndash;2 h at 37\u0026deg;C. The optical density (OD) at 450 nm was detected by using a multifunctional microplate reader (Snyergy H1, BioTek), and cell viability for each group was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). GraphPad (version 9.5) was used to analyze these obtained data from at least three independent experiments. Statistical differences were evaluated by performing one-way ANOVA or unpaired Student\u0026rsquo;s t-test. A level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e \u003cp\u003eThe authors declare that no competing financial interests.\u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThis study was supported by the grants from National Natural Science Foundation of China (No:31902326), China Postdoctoral Science Foundation (No: 2018M642271), Jiangsu Province Postdoctoral Research Foundation (No: 2019K166) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).\u003c/p\u003e\u003ch2\u003eAUTHOR CONTRIBUTIONS\u003c/h2\u003e \u003cp\u003e \u003cb\u003eBin Dong\u003c/b\u003e: Investigation, Writing-Original Draft, Validation. \u003cb\u003eJing Xiao\u003c/b\u003e: Methodology, Investigation, Writing-Original Draft. \u003cb\u003eJunqi Wang\u003c/b\u003e: Methodology, Visualization. \u003cb\u003eXinhao Song\u003c/b\u003e: Resources, Writing-Review and Editing. \u003cb\u003eHui Ji\u003c/b\u003e: Resources, Validation, Software. \u003cb\u003eJiurong Peng\u003c/b\u003e: Resources, Methodology. \u003cb\u003eXinru Weng\u003c/b\u003e: Methodology, Validation. \u003cb\u003eDawei Guo\u003c/b\u003e: Writing-Review and Editing. \u003cb\u003eShanxiang Jiang\u003c/b\u003e: Conceptualization, Supervision, Writing-Review and Editing, Funding acquisition. \u003cb\u003eXiuge Gao\u003c/b\u003e: Conceptualization, Supervision, Writing-Review and Editing, Funding acquisition.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eWe thank all our laboratory members for their kindly technical assistance. We acknowledge Chunlei Ji and Yuling Zheng for their their cooperation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOvermeyer JH, Kaul A, Johnson EE, Maltese WA. Active ras triggers death in glioblastoma cells through hyperstimulation of macropinocytosis. Mol Cancer Res 2008, 6(6): 965\u0026ndash;977.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiero EL, Machado-Santelli GM. Cinnamic acid induces apoptotic cell death and cytoskeleton disruption in human melanoma cells. J Exp Clin Cancer Res 2013, 32(1): 31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe T, Shan P, Zhang H. Progress in the discovery and development of small molecule methuosis inducers. 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Curr Opin Cell Biol 2016, 41: 1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Methuosis, Cytoskeleton, Membrane damage, DAMPs, ESCRT-Ⅲ, Maduramicin, MOMIPP, Cell death","lastPublishedDoi":"10.21203/rs.3.rs-5422638/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5422638/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMethuosis represents a novel cell death modality characterized by catastrophic cytoplasmic vacuolization in normal and malignant cells. However, the critical role and the underlying mechanism of cytoskeleton and plasma membrane damage in methuotic cells are largely unknown. Herein, maduramicin-treated myocardial cells (H9c2) and indole chalcone-exposed glioma cells (U251) were used as methuosis model to uncover this secret. We found that cytoskeleton protein F-actin, α-tubulin, β-tubulin and filamin A/B were disrupted in a reversible-dependent manner. In addition, RhoA-ROCK1 signaling pathway mediated cytoskeleton disruption in methuotic cells. Excessive cytoplasmic vacuolization triggered cellular plasma membrane damage and the release of DAMPs, including LDH, ATP and CRT. Furthermore, at the end phase of methuotic cells, plasma membrane was damaged independent of pore-forming protein p-MLKL and GSDMD. Endosomal sorting complex required for transport (ESCRT)-Ⅲ especially its subunit CHMP3 and CHMP5 negatively regulated excessive vacuolization-induced plasma membrane damage in cells undergoing methuosis. In conclusion, for the first time, the critical role and potential mechanism of cytoskeleton and plasma membrane damage in methuotic cells are known, which would facilitate the employment of methuosis in life science and pharmacology.\u003c/p\u003e","manuscriptTitle":"Cytoskeleton disruption and plasma membrane damage determine methuosis of normal and malignant cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-11 07:50:55","doi":"10.21203/rs.3.rs-5422638/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0ee459f9-267e-478c-a0fe-16a9130e5380","owner":[],"postedDate":"December 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":40224911,"name":"Biological sciences/Drug discovery/Toxicology"},{"id":40224912,"name":"Biological sciences/Cell biology/Cell death"}],"tags":[],"updatedAt":"2024-12-11T07:50:57+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-11 07:50:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5422638","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5422638","identity":"rs-5422638","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}