Passive diffusion accounts for the majority of intracellular nanovesicle transport.

During membrane trafficking, a vesicle formed at the donor compartment must travel to the acceptor membrane before fusing. For large carriers, it is established that this transport is motor-driven; however, the mode by which small vesicles, which outnumber larger carriers, are transported is poorly characterized. Here, we show that intracellular nanovesicles (INVs), a substantial class of small vesicles, are highly mobile within cells and that this mobility depends almost entirely on passive diffusion (0.1-0.3 µm2 s-1). Using single particle tracking, we describe how other small trafficking vesicles have a similar diffusive mode of transport that contrasts with the motor-dependent movement of larger endolysosomal carriers. We also demonstrate that a subset of INVs is involved in exocytosis and that delivery of cargo to the plasma membrane during exocytosis is decreased when diffusion of INVs is specifically restricted. Our results suggest that passive diffusion is sufficient to explain the majority of small vesicle transport.

Interfacial stresses on droplet interface bilayers using two photon fluorescence lifetime imaging microscopy.

HYPOTHESIS: Response of lipid bilayers to external mechanical stimuli is an active area of research with implications for fundamental and synthetic cell biology. Developing novel tools for systematically imposing mechanical strains and non-invasively mapping out interfacial (membrane) stress distributions on lipid bilayers can accelerate research in this field. EXPERIMENTS: We report a miniature platform to manipulate model cell membranes in the form of droplet interface bilayers (DIBs), and non-invasively measure spatio-temporally resolved interfacial stresses using two photon fluorescence lifetime imaging of an interfacially active molecular flipper (Flipper-TR). We established the effectiveness of the developed framework by investigating interfacial stresses accompanying three key processes associated with DIBs: thin film drainage between lipid monolayer coated droplets, bilayer formation, and bilayer separation. FINDINGS: The measurements revealed fundamental aspects of DIBs including the existence of a radially decaying interfacial stress distribution post bilayer formation, and the simultaneous build up and decay of stress respectively at the bilayer corner and center during bilayer separation. Finally, utilizing interfacial rheology measurements and MD simulations, we also reveal that the tested molecular flipper is sensitive to membrane fluidity that changes with interfacial stress - expanding the scientific understanding of how molecular flippers sense stress.

Crb3 is required to organize the apical domain of multiciliated cells.

Cell shape changes mainly rely on the remodeling of the actin cytoskeleton. Multiciliated cells (MCCs) of the mucociliary epidermis of Xenopus laevis embryos, as they mature, dramatically reshape their apical domain to grow cilia, in coordination with the underlying actin cytoskeleton. Crumbs (Crb) proteins are multifaceted transmembrane apical polarity proteins known to recruit actin linkers and promote apical membrane growth. Here, we identify the homeolog Crb3.L as an important player for the migration of centrioles or basal bodies (collectively centrioles/BBs) and apical domain morphogenesis in MCCs. Crb3.L is present in cytoplasmic vesicles close to the ascending centrioles/BBs, where it partially colocalizes with Rab11a. Crb3.L morpholino-mediated depletion in MCCs caused abnormal migration of centrioles/BBs, a reduction of their apical surface, disorganization of their apical actin meshwork and defective ciliogenesis. Rab11a morpholino-mediated depletion phenocopied Crb3.L loss-of-function in MCCs. Thus, the control of centrioles/BBs migration by Crb3.L might be mediated by Rab11a-dependent apical trafficking. Furthermore, we show that both phospho-activated ERM (pERM; Ezrin-Radixin-Moesin) and Crb3.L are recruited to the growing apical domain of MCCs, where Crb3.L likely anchors pERM, allowing actin-dependent expansion of the apical membrane.

Solar/periodate inhibits ARGs transformation by degradation of DNA without damaging cell membrane.

Antibiotic-resistant bacterial infections are a growing global threat to public health. Chlorine-based water disinfection and some advanced oxidation processes significantly increase the risk of ARGs release and transmission in the aquatic environment. Therefore, it is critical to develop or optimize disinfection methods to reduce the conversion and transmission of ARGs in natural water. This study investigated whether the solar/periodate (PI) system inhibited the natural transmission of ARGs and its mechanism. The results showed that solar/PI systems could effectively inhibit the propagation of ARGs in two simulated natural transformation systems, up to more than 100 times. By characterizing the cellular process of bacteria treated by the solar/PI system, we found that the solar/PI system could directly cause damage to DNA bases and its dual effect with almost no damage to the bacterial cell membrane, which was the main reason why this technology could inhibit natural transformation processes. Specifically, the inhibition effect of solar/PI on bacteria did not result in enhanced membrane permeability under appropriate PI dosage (<200 µM), which greatly reduced the risk of secondary contamination of eARGs released by traditional disinfection. Our findings could help improve existing disinfection strategies to ensure that antibiotic resistance is not spread in the natural water environment.

Application of bioluminescence resonance energy transfer to quantitate cell-surface expression of membrane proteins.

We report a bioluminescence resonance energy transfer (BRET) assay to quantitate the fraction of an engineered membrane protein at the cell surface versus inside the cell. As test cases, we engineered two different G protein-coupled receptors (GPCRs) in which a NanoLuc luciferase (NLuc) and a HaloTag are fused to the extracellular amino-terminal tail of the receptors. We then employed a pulse-chase labeling approach relying on two different fluorescent dyes with distinctive cell permeability properties. The dyes are efficiently excited by luminescence from NLuc, but are spectrally distinct. Measuring BRET from the chemiluminescence of the NLuc to the fluorophores bound to the HaloTag minimizes the limitations of in-cell fluorescence resonance energy transfer (FRET)-based approaches such as photobleaching and autofluorescence. The BRET surface expression assay can quantitatively differentiate between the labeling of receptors at the cell surface and receptors inside of the cell. The assay is shown to be quantitative and robust compared with other approaches to measure cell surface expression of membrane proteins such as enzyme-linked immunosorbent assay or immunoblotting, and significantly increases the throughput because the assay is designed to be carried out in microtiter plate format.

Factors influencing the detergent-free membrane protein isolation using synthetic nanodisc-forming polymers.

The detergent-free isolation of membrane proteins using synthetic polymers is becoming the desired approach for functional and structural studies of membrane proteins. Since the expression levels for many membrane proteins are low and a high yield of functionalized reconstituted membrane proteins is essential for in vitro studies, it is crucial to optimize the experimental conditions for a given polymer to solubilize target membranes/proteins effectively. The factors that affect membrane solubilization and subsequently the isolation of a target membrane protein include polymer concentration, polymer charge, temperature, pH, and concentration of divalent metal ions. Therefore, it is important to have knowledge about the efficacy of different types of polymers in solubilizing cell membranes. In this study, we evaluate the efficacy of inulin-based non-ionic polymers in solubilizing E. coli membranes enriched with rat flavin mononucleotide binding-domain (FBD) of cytochrome-P450-reductase (CPR) and rabbit cytochrome-b5 (Cyt-b5) under various solubilization conditions. Our results show that a 1:1 (w/w) membrane:polymer ratio, low temperature, high pH and sub-millimolar concentration of metal ions favor the solubilization of E. coli membranes enriched with FBD or Cyt-b5. Conversely, the presence of excess divalent metal ions affected the final protein levels in the polymer-solubilized samples. We believe that the results from this study provide knowledge to assess and plan the use of non-ionic polymers in membrane protein studies.

A new MAS-related G protein-coupled receptor X2 cell membrane chromatography analysis model based on HALO-tag technology and its applications.

Cell membrane chromatography (CMC) is an effective method for studying receptors with multiple transmembrane structure such as MAS-related G protein-coupled receptor X2 (MrgX2). CMC relies on the maintenance of the complete biological structure of a membrane receptor; however, it needs to be further improved to obtain a more convenient and stable CMC model. In the present study, the haloalkane dehalogenase protein tag (HALO-tag) technology was used to construct a new MrgX2/CMC model. The fusion receptors of MrgX2 with HALO-tag at the C terminus were expressed in HEK293 cells. The silica gel was modified with a substrate of HALO-tag (chloroalkanes) via one-step acylation for the rapid capture of fusion receptors. The new CMC model (MrgX2-HALO-tag/CMC model) was not only quicker to prepare but also more stable and had a longer lifespan than a previous MrgX2-SNAP-tag/CMC model. In combination with the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) system, the MrgX2-HALO-tag/CMC model was used to screen and identify bioactive components in traditional Chinese medicine. Using this combination, sanggenon C and morusin were identified from Mori Cortex as anti-pseudo-allergic components. The MrgX2-HALO-tag/CMC model alone was also applied to analyze ligand-receptor interaction. The affinity order of four ligands to MrgX2 was as follows: desipramine < imipramine < amitriptyline < clomipramine. This was consistent with the results obtained using the MrgX2-SNAP-tag/CMC model. The MrgX2-HALO-tag/CMC model provides ideas and application prospects for the immobilization of cell membrane that contains receptors with more transmembrane structures.

Cellular excitability and ns-pulsed electric fields: Potential involvement of lipid oxidation in the action potential activation.

Recent studies showed that nanosecond pulsed electric fields (nsPEFs) can activate voltage-gated ion channels (VGICs) and trigger action potentials (APs) in excitable cells. Under physiological conditions, VGICs' activation takes place on time scales of the order 10-100 µs. These time scales are considerably longer than the applied pulse duration, thus activation of VGICs by nsPEFs remains puzzling and there is no clear consensus on the mechanisms involved. Here we propose that changes in local electrical properties of the cell membrane due to lipid oxidation might be implicated in AP activation. We first use MD simulations of model lipid bilayers with increasing concentration of primary and secondary lipid oxidation products and demonstrate that oxidation not only increases the bilayer conductance, but also the bilayer capacitance. Equipped with MD-based characterization of electrical properties of oxidized bilayers, we then resort to AP modelling at the cell level with Hodgkin-Huxley-type models. We confirm that a local change in membrane properties, particularly the increase in membrane conductance, due to formation of oxidized membrane lesions can be high enough to trigger an AP, even when no external stimulus is applied. However, excessive accumulation of oxidized lesions (or other conductive defects) can lead to altered cell excitability.

Nanosecond Pulsed Electric Fields (nsPEFs) Modulate Electron Transport in the Plasma Membrane and the Mitochondria.

Nanosecond pulsed electric fields (nsPEFs) are a pulsed power technology known for ablating tumors, but they also modulate diverse biological mechanisms. Here we show that nsPEFs regulate trans-plasma membrane electron transport (tPMET) rates in the plasma membrane redox system (PMRS) shown as a reduction of the cell-impermeable, WST-8 tetrazolium dye. At lower charging conditions, nsPEFs enhance, and at higher charging conditions inhibit tPMET in H9c2 non-cancerous cardiac myoblasts and 4T1-luc breast cancer cells. This biphasic nsPEF-induced modulation of tPMET is typical of a hormetic stimulus that is beneficial and stress-adaptive at lower levels and damaging at higher levels. NsPEFs also attenuated mitochondrial electron transport system (ETS) activity (O2 consumption) at Complex I when coupled and uncoupled to oxidative phosphorylation. NsPEFs generated more reactive oxygen species (ROS) in mitochondria (mROS) than in the cytosol (cROS) in non-cancer H9c2 heart cells but more cROS than mROS in 4T1-luc cancer cells. Under lower charging conditions, nsPEFs support glycolysis while under higher charging conditions, nsPEFs inhibit electron transport in the PMRS and the mitochondrial ETS producing ROS, ultimately causing cell death. The impact of nsPEF on ETS presents a new paradigm for considering nsPEF modulation of redox functions, including redox homeostasis and metabolism.

Plant P4-ATPase lipid flippases: How are they regulated?

P4 ATPases are active membrane transporters that translocate lipids towards the cytosolic side of the biological membranes in eukaryotic cells. Due to their essential cellular functions, P4 ATPase activity is expected to be tightly controlled, but fundamental aspects of the regulation of plant P4 ATPases remain unstudied. In this mini-review, our knowledge of the regulatory mechanisms of yeast and mammalian P4 ATPases will be summarized, and sequence comparison and structural modelling will be used as a basis to discuss the putative regulation of the corresponding plant lipid transporters.

Relevance of the Entry by Fusion at the Cytoplasmic Membrane vs. Fusion After Endocytosis in the HIV and SARS-Cov-2 Infections.

HIV-1 and SARS-Cov-2 fuse at the cell surface or at endosomal compartments for entry into target cells; entry at the cell surface associates to productive infection, whereas endocytosis of low pH-independent viruses may lead to virus inactivation, slow replication, or alternatively, to productive infection. Endocytosis and fusion at the cell surface are conditioned by cell type-specific restriction factors and the presence of enzymes required for activation of the viral fusogen. Whereas fusion with the plasma membrane is considered the main pathway to productive infection of low pH-independent entry viruses, endocytosis is also productive and may be the main route of the highly efficient cell-to-cell dissemination of viruses. Alternative receptors, membrane cofactors, and the presence of enzymes processing the fusion protein at the cell membrane, determine the balance between fusion and endocytosis in specific target cells. Characterization of the mode of entry in particular cell culture conditions is desirable to better assess the effect of neutralizing and blocking agents and their mechanism of action. Whatever the pathway of virus internalization, production of the viral proteins into the cells can lead to the expression of the viral fusion protein on the cell surface; if this protein is able to induce membrane fusion at physiological pH, it promotes the fusion of the infected cell with surrounding uninfected cells, leading to the formation of syncytia or heterokaryons. Importantly, particular membrane proteins and lipids act as cofactors to support fusion. Virus-induced cell-cell fusion leads to efficient virus replication into fused cells, cell death, inflammation, and severe disease.

Coordination of RAB-8 and RAB-11 during unconventional protein secretion.

Multiple physiology-pertinent transmembrane proteins reach the cell surface via the Golgi-bypassing unconventional protein secretion (UcPS) pathway. By employing C. elegans-polarized intestine epithelia, we recently have revealed that the small GTPase RAB-8/Rab8 serves as an important player in the process. Nonetheless, its function and the relevant UcPS itinerary remain poorly understood. Here, we show that deregulated RAB-8 activity resulted in impaired apical UcPS, which increased sensitivity to infection and environmental stress. We also identified the SNARE VTI-1/Vti1a/b as a new RAB-8-interacting factor involved in the apical UcPS. Besides, RAB-11/Rab11 was capable of recruiting RABI-8/Rabin8 to reduce the guanine nucleotide exchange activity of SMGL-1/GEF toward RAB-8, indicating the necessity of a finely tuned RAB-8/RAB-11 network. Populations of RAB-8- and RAB-11-positive endosomal structures containing the apical UcPS cargo moved toward the apical side. In the absence of RAB-11 or its effectors, the cargo was retained in RAB-8- and RAB-11-positive endosomes, respectively, suggesting that these endosomes are utilized as intermediate carriers for the UcPS.

Super-resolution imaging of folate receptor alpha on cell membranes using peptide-based probes.

Folate receptor alpha (FRα) is a vital membrane protein which have great association with cancers and involved in various biological processes including folate transport and cell signaling. However, the distribution and organization pattern of FRα on cell membranes remains unclear. Previous studies relied on antibodies to recognize the proteins. However, multivalent crosslinking and large size of antibodies confuse the direct observation to some extent. Fortunately, the emergence of peptide, which are small-sized and monovalent, has supplied us an unprecedented choice. Here, we applied fluorophore-conjugated peptide probe to recognize the FRα and study the distribution pattern of FRα on cell membrane using dSTORM super-resolution imaging technique. FRα were found to organized as clusters on cell surface with different sizes. And they have a higher expression level and formed larger clusters on various cancer cells than normal cells, which hinted that its specific distribution could be utilized for cancer diagnosis. Furthermore, we revealed that the lipid raft and cortical actin as restrictive factors for the FRα clustering, suggesting a potential assembly mechanism insight into FRα clustering on cell membrane. Collectively, our work clarified the morphology distribution and clustered organization of FRα with peptide probes at the nanometer scale, which paves the way for further revealing the relationship between the spatial organization and functions of membranal proteins.

Interplay between the plasma membrane and cell-cell adhesion maintains epithelial identity for correct polarised cell divisions.

Polarised epithelial cell divisions represent a fundamental mechanism for tissue maintenance and morphogenesis. Morphological and mechanical changes in the plasma membrane influence the organisation and crosstalk of microtubules and actin at the cell cortex, thereby regulating the mitotic spindle machinery and chromosome segregation. Yet, the precise mechanisms linking plasma membrane remodelling to cell polarity and cortical cytoskeleton dynamics to ensure accurate execution of mitosis in mammalian epithelial cells remain poorly understood. Here, we manipulated the density of mammary epithelial cells in culture, which led to several mitotic defects. Perturbation of cell-cell adhesion formation impairs the dynamics of the plasma membrane, affecting the shape and size of mitotic cells and resulting in defects in mitotic progression and the generation of daughter cells with aberrant architecture. In these conditions, F- actin-astral microtubule crosstalk is impaired, leading to mitotic spindle misassembly and misorientation, which in turn contributes to chromosome mis-segregation. Mechanistically, we identify S100 Ca2+-binding protein A11 (S100A11) as a key membrane-associated regulator that forms a complex with E-cadherin (CDH1) and the leucine-glycine-asparagine repeat protein LGN (also known as GPSM2) to coordinate plasma membrane remodelling with E-cadherin-mediated cell adhesion and LGN-dependent mitotic spindle machinery. Thus, plasma membrane-mediated maintenance of mammalian epithelial cell identity is crucial for correct execution of polarised cell divisions, genome maintenance and safeguarding tissue integrity.

Regulation of the apico-basolateral trafficking polarity of the homologous copper-ATPases ATP7A and ATP7B.

The homologous P-type copper-ATPases (Cu-ATPases) ATP7A and ATP7B are the key regulators of copper homeostasis in mammalian cells. In polarized epithelia, upon copper treatment, ATP7A and ATP7B traffic from the trans-Golgi network (TGN) to basolateral and apical membranes, respectively. We characterized the sorting pathways of Cu-ATPases between TGN and the plasma membrane and identified the machinery involved. ATP7A and ATP7B reside on distinct domains of TGN in limiting copper conditions, and in high copper, ATP7A traffics to basolateral membrane, whereas ATP7B traverses common recycling, apical sorting and apical recycling endosomes en route to apical membrane. Mass spectrometry identified regulatory partners of ATP7A and ATP7B that include the adaptor protein-1 complex. Upon knocking out pan-AP-1, sorting of both Cu-ATPases is disrupted. ATP7A loses its trafficking polarity and localizes on both apical and basolateral surfaces in high copper. By contrast, ATP7B loses TGN retention but retained its trafficking polarity to the apical domain, which became copper independent. Using isoform-specific knockouts, we found that the AP-1A complex provides directionality and TGN retention for both Cu-ATPases, whereas the AP-1B complex governs copper-independent trafficking of ATP7B solely. Trafficking phenotypes of Wilson disease-causing ATP7B mutants that disrupts putative ATP7B-AP1 interaction further substantiates the role of AP-1 in apical sorting of ATP7B.

Quantification of Trophoblast Syncytialization by Fluorescent Membrane Labeling.

Trophoblast fusion or syncytialization is a fundamental yet poorly understood process during placental development. Primary cultured cytotrophoblasts and human choriocarcinoma cell lines are commonly used to study trophoblast fusion mechanisms in vitro. Quantification of trophoblast fusion index is a key for the in vitro studies. In this chapter, we describe a new method to quantify fusion index, which is based on fluorescent labeling of the plasma membrane using Di-8-ANEPPS, a membrane potential dye. This method directly works on live cells, thereby is simple, economic, and reliable.

Assaying the Effect of Peptide Treatment on H+-Pumping Activity in Plasma Membranes from Arabidopsis Seedlings.

Extracellular acidification or alkalization is a common response to many plant-signaling peptides and microbial elicitors. This may be a result of peptide-mediated regulation of plasma membrane-localized ion transporters, such as the plasma membrane H+-ATPase. Early responses to some signaling peptides can therefore be analyzed by assaying H+-pumping across the plasma membrane.We describe a set-up suited for the purification of plasma membranes by aqueous two-phase partitioning from a small sample of Arabidopsis seedlings. Seedlings are grown in a liquid culture, suited for the analysis of in vivo peptide treatment. Additionally, we describe how to measure the H+-pumping activity of the plasma membrane H+-ATPase using the fluorescent probe ACMA.

Microscale Thermophoresis (MST) to Study Rapid Alkalinization Factor (RALF)-Receptor Interactions.

Microscale thermophoresis (MST) is a simple but powerful tool to study the in vitro interaction among biomolecules, and to quantify binding affinities. MST curves describe the change in the fluorescence level of a fluorescent target as a result of an IR-laser-induced temperature change. The degree and nature of the change in fluorescence signal depends on the size, charge, and solvation shell of the molecules, properties that change in function of the binding of a ligand to the fluorescent target.We used MST to describe the interaction between components of a regulatory module involved in plant cell wall integrity control. This module comprises the secreted peptide Rapid Alkalinization Factor 23 (RALF23) and its receptor complex consisting of the GPI-anchored receptor Lorelei-Like Glycoprotein 1 (LLG1) and a receptor kinase of the CrRLK1L family, FERONIA. Here we show how MST can also be used to study three-partner interactions.

CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane.

CFTR is a membrane protein that functions as an ion channel. Mutations that disrupt its biosynthesis, trafficking or function cause cystic fibrosis (CF). Here, we present a novel in vitro model system prepared using CRISPR/Cas9 genome editing with endogenously expressed WT-CFTR tagged with a HiBiT peptide. To enable the detection of CFTR in the plasma membrane of live cells, we inserted the HiBiT tag in the fourth extracellular loop of WT-CFTR. The 11-amino acid HiBiT tag binds with high affinity to a large inactive subunit (LgBiT), generating a reporter luciferase with bright luminescence. Nine homozygous clones with the HiBiT knock-in were identified from the 182 screened clones; two were genetically and functionally validated. In summary, this work describes the preparation and validation of a novel reporter cell line with the potential to be used as an ultimate building block for developing unique cellular CF models by CRISPR-mediated insertion of CF-causing mutations.

Localization of the Remnant of a Cell Wall Sorting Signal and Its Interaction with a Sensor Kinase.

Sortases are highly conserved enzymes with endopeptidase and transpeptidase activities in Gram-positive bacteria. Sortase A cleaves within an LPXTG-motif and covalently crosslinks cell wall proteins to become anchored to the peptidoglycan of the cell wall. We showed that a peptide cleaved by sortase A from the C-terminus (C-pep) of the LPXTG-adhesin SspA intercalates in the cell membrane. Nested in the membrane, this C-pep docks with the intramembrane sensor histidine kinase, SraS, to activate the response regulator, SraR. SraR signals that the C-pep has been cleaved as an indicator of the fidelity of sortase A processing. SraSR also signals that key LPXTG-proteins in concert with lipoteichoic acid engage the mucin, MUC5B, which elicits a different transcriptional response than the binding of other salivary constituents. To visualize the C-pep intercalating in the cell membrane in vivo, we used Structured Illumination Microscopy (SIM). And to show that the C-pep complexes with SraS, we used bimolecular fluorescence experiments. The C-pep and SraS were each expressed with one or the other half of yellow fluorescence protein (YFP). Reconstitution of the complete YFP signal indicated that the C-pep and SraS interacted at molecular distances within the cell membrane in vivo. Using these imaging protocols, we learned that the C-pep functions as a signaling molecule within the cell membrane of the streptococcal cell.