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Mechanosensitive channel of large conductance (MscL) is the most thoroughly studied mechanosensitive channel in prokaryotes. Owing to its small molecular weight, clear mechanical gating mechanism, and nanopore forming ability upon opening, accumulating studies are implemented in regulating cell function by activating mechanosensitive channel of large conductance in mammalian cells. This study aimed to investigate the potentials of mechanosensitive channel of large conductance as a nanomedicine and a mechano-inducer in non-small cell lung cancer (NSCLC) A549 cells from the view of molecular pathways and acoustics. The stable cytoplasmic vacuolization model about NSCLC A549 cells was established via the targeted expression of modified mechanosensitive channel of large conductance channels in different subcellular organelles. Subsequent morphological changes in cellular component and expression levels of cell death markers are analyzed by confocal imaging and western blots. The permeability of mitochondrial inner membrane (MIM) exhibited a vital role in cytoplasmic vacuolization formation. Furthermore, mechanosensitive channel of large conductance channel can be activated by low intensity focused ultrasound (LIFU) in A549 cells, and the suppression of A549 tumors in vivo was achieved by LIFU with sound pressure as low as 0.053 MPa. These findings provide insights into the mechanisms underlying non-apoptotic cell death, and validate the nanochannel-based non-invasive ultrasonic strategy for cancer therapy.
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In order to harvest more light wavelengths to improve the light-assisted electrochemical water splitting capacity, we developed a novel heterostructure of three-dimensional (3D) flower-like CuS architecture with accompanying SnS2 nanoparticles and reduced graphene oxide (rGO) aerogel for outstanding light-assisted electrocatalytic OER performance and good stability. The excellent catalytic kinetics, effective capturing of visible light, and rapid charge transfer of the CuS/SnS2/rGO (CSr) heterostructure were demonstrated. The overpotential (264 mV@10 mA cm-2) under light-assisted conditions is 20% lower than that under light-chopped conditions. SnS2 can harvest more light wavelengths and this boosts its intrinsic activity. However, with the increase of the SnS2 content, the OER activity decreases. The combination of the CS heterostructure and the rGO conductive aerogel achieves rapid charge transfer. Furthermore, the possible mechanism of the light-assisted electrocatalytic OER was also proposed. Overall, this work provides new insights into the simple and scalable fabrication of a highly efficient, low-cost, and stable non-noble-metal-based electrocatalyst.
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Most anticancer therapies trigger apoptosis to eliminate malignant cells. However, the majority of malignant cancer cells are resistant to apoptosis due to genetic mutations or heterogeneity. Here, we report that opening the pore of the bacterial large conductance mechanosensitivity channel (MscL) provides a novel approach of inducing non-apoptotic cell death. The gain-of-function mutant V23A-MscL and chemically responsive mutant G26C-MscL can be functionally expressed in hepatocellular carcinoma HepG2 cells. V23A-MscL spontaneously opens, and G26C-MscL also responds to its chemical activator MTSET. Opening of the MscL channel causes increased intracellular Ca2+ concentration and suppressed cell growth and viability. MTSET-activated G26C channels induce necrosis, while V23A-MscL expression leads to cytoplasmic vacuolization cell death in HepG2 cells and suppresses tumor growth in a mouse model. We propose that MscL may act as a nanovalve through which intracellular homeostasis suffers a disruption and results in malignant tumor cell damage, leading to a new strategy for cancer therapy.
Assuntos
Carcinoma Hepatocelular , Proteínas de Escherichia coli , Neoplasias Hepáticas , Animais , Apoptose , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Células Hep G2 , Humanos , Canais Iônicos , CamundongosRESUMO
The mechanosensitive channel MscS functions as an osmolyte emergency release-valve in the event of a sudden decrease in external environmental osmolarity. MscS has served as a paradigm for studying how channel proteins detect and respond to mechanical stimuli. However, the inter-domain interactions and structural rearrangements that occur in the MscS gating process remain largely unknown. Here, we determined the interactions between the transmembrane domain and cytoplasmic domain of MscS. Using in vivo cellular viability, single-channel electrophysiological recording, and cysteine disulfide trapping, we demonstrated that N117 of the TM3b helix and N167 of the Cyto-helix are critical residues that function at the TM3b-Cyto helix interface. In vivo downshock assays showed that double cysteine substitution at N117 and N167 failed to rescue the osmotic-lysis phenotype of cells in acute osmotic downshock. Single-channel recordings demonstrated that cysteine cross-linking of N117C and N167C led to a non-conductive channel. Consistently, coordination of the histidines of N117H and N167H caused a decrease in channel gating. Moreover, cross-linked N117 and N167 altered the gating of the severe gain-of-function mutant L109S. Our results demonstrate that N117-N167 interactions stabilize the inactivation state by an association of TM3b segments with ß-domains of the cytoplasmic region, providing further insights into the gating mechanism of the MscS channel.
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Externally controlling the excitation of a neuronal subset through ion channels activation can modulate the firing pattern of an entire neural circuit in vivo. As nanovalves in the cell membrane, ion channels can be opened by light (optogenetics) or ultrasonic (sonogenetics) means. A thoroughly analyzed force sensor is the Escherichia coli mechano sensitive channel of large conductance (MscL). Here we expressed MscL in rat hippocampal neurons in a primary culture and showed that it could be activated by low-pressure ultrasound pulses. The gain-of-function mutation, I92L, sensitized MscL's sonic response, triggering action potentials at a peak negative pressure as low as 0.25 MPa. Further, the I92L MscL reliably elicited individual spikes by timed brief pulses, making excitation programmable. Because MscL opens to tension in the lipid bilayer, requiring no other proteins or ligands, it could be developed into a general noninvasive sonogenetic tool to manipulate the activities of neurons or other cells and potential nanodevices.
Assuntos
Membrana Celular/genética , Proteínas de Escherichia coli/química , Canais Iônicos/química , Neurônios/metabolismo , Sequência de Aminoácidos/genética , Animais , Fenômenos Biomecânicos , Membrana Celular/química , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação da Expressão Gênica/genética , Hipocampo/metabolismo , Canais Iônicos/genética , Bicamadas Lipídicas/metabolismo , Neurônios/patologia , Cultura Primária de Células , Ratos , UltrassomRESUMO
How we sense touch is fundamental for many physiological processes. However, the underlying mechanism and molecular identity for touch sensation are largely unknown. Here, we report on defective gentle-touch behavioral responses in brv1 loss-of-function Drosophila larvae. RNAi and Ca2+ imaging confirmed the involvement of Brv1 in sensing touch and demonstrated that Brv1 mediates the mechanotransduction of class III dendritic arborization neurons. Electrophysiological recordings further revealed that the expression of Brv1 protein in HEK293T cells gives rise to stretch-activated cation channels. Purified Brv1 protein reconstituted into liposomes were found to sense stretch stimuli. In addition, co-expression studies suggested that Brv1 amplifies the response of mechanosensitive ion channel NOMPC (no mechanoreceptor potential C) to touch stimuli. Altogether, these findings demonstrate a molecular entity that mediates the gentle-touch response in Drosophila larvae, providing insights into the molecular mechanisms of touch sensation.