Search Results (24)

The efficiency of virus internalization into target cells is a major determinant of infectivity. SARS-CoV-2 internalization occurs via S-protein-mediated cell binding followed either by direct fusion with the plasma membrane or endocytosis and subsequent fusion with the endosomal membrane. Despite the crucial role of virus internalization, the precise kinetics of the processes involved remains elusive. We developed a pipeline, which combines live-cell microscopy and advanced image analysis, for measuring the rates of multiple internalization-associated molecular events of single SARS-CoV-2-virus-like particles (VLPs), including endosome ingression and pH change. Our live-cell imaging experiments demonstrate that only a few minutes after binding to the plasma membrane, VLPs ingress into RAP5-negative endosomes via dynamin-dependent scission. Less than two minutes later, VLP speed increases in parallel with a pH drop below 5, yet these two events are not interrelated. By co-imaging fluorescently labeled nucleocapsid proteins, we show that nucleocapsid release occurs with similar kinetics to VLP acidification. Neither Omicron mutations nor abrogation of the S protein polybasic cleavage site affected the rate of VLP internalization, indicating that they do not confer any significant advantages or disadvantages during this process. Finally, we observe that VLP internalization occurs two to three times faster in VeroE6 than in A549 cells, which may contribute to the greater susceptibility of the former cell line to SARS-CoV-2 infection. Taken together, our precise measurements of the kinetics of VLP internalization-associated processes shed light on their contribution to the effectiveness of SARS-CoV-2 propagation in cells. Full article

Mechanically robust anion-exchange membranes (AEMs) with high conductivity and long-term alkali resistance are needed for water electrolysis application. In this work, aryl-ether free polyaromatics containing isatin moieties were prepared via super acid-catalyzed copolymerization, followed by functionalization with alkaline stable cyclic quaternary ammonium (QA) cationic groups, to afford high performance AEMs for application in water electrolysis. The incorporation of side functional cationic groups (pyrrolidinium and piperidinium) onto a polymer backbone via a flexible alkyl spacer aimed at conductivity and alkaline stability improvement. The effect of cation structure on the properties of prepared AEMs was thoroughly studied. Pyrrolidinium- and piperidinium-based AEMs showed similar electrolyte uptakes and no obvious phase separation, as revealed by SAXS and further supported by AFM and TEM data. In addition, these AEMs displayed high conductivity values (81. 5 and 120 mS cm⁻¹ for pyrrolidinium- and piperidinium-based AEM, respectively, at 80 °C) and excellent alkaline stability after 1 month aging in 2M KOH at 80 °C. Especially, a pyrrolidinium-based AEM membrane preserved 87% of its initial conductivity value, while at the same time retaining its flexibility and mechanical robustness after storage in alkaline media (2M KOH) for 1 month at 80 °C. Based on ¹H NMR data, the conductivity loss observed after the aging test is mainly related to the piperidinium degradation that took place, probably via ring-opening Hofmann elimination, alkyl spacer scission and nucleophilic substitution reactions as well. The synthesized AEMs were also tested in an alkaline water electrolysis cell. Piperidinium-based AEM showed superior performance compared to its pyrrolidinium analogue, owing to its higher conductivity as revealed by EIS data, further confirming the ex situ conductivity measurements. Full article

The mechanical effects of membrane compositional inhomogeneities are analyzed in a process analogous to neck formation in cellular membranes. We cast on the Canham–Helfrich model of fluid membranes with both the spontaneous curvature and the surface tension being non-homogeneous functions along the cell membrane. The inhomogeneous distribution of necking forces is determined by the equilibrium mechanical equations and the boundary conditions as considered in the axisymmetric setting compatible with the necking process. To establish the role played by mechanical inhomogeneity, we focus on the catenoid, a surface of zero mean curvature. Analytic solutions are shown to exist for the spontaneous curvature and the constrictive forces in terms of the border radii. Our theoretical analysis shows that the inhomogeneous distribution of spontaneous curvature in a mosaic-like neck constrictional forces potentially contributes to the membrane scission under minimized work in living cells. Full article

Schiff bases and similar molecules forming metal complexes may cause redox effects, which may also be influenced by light. Anthraquinones such as doxorubicin and idarubicin are widely used antitumor agents, which can generate reactive oxygen species (ROS), stimulated by both the presence of iron and copper ions and also by light. The generated ROS can cause DNA scission, cell membrane oxidation, and many other toxic effects. The redox activity of the quinone-quinoline chelator 2-phenyl-4-(butylamino)naphtho [2,3-h]quinoline-7,12-dione (Q1) was investigated in the presence of iron, copper, and zinc. The influence of light in these interactions was also examined. The chemically induced dynamic nuclear polarization (CIDNP), nuclear magnetic resonance (NMR), and electron paramagnetic resonance (EPR) methods were used to elucidate the molecular changes and ROS generation effects of the Q1 metal interactions. A model electron transfer reaction system between 1,4-dihydropyridine and Q1 was utilized to demonstrate that the chelate complexes of Q1 with both Fe(III) and Cu(II) ions were more redox active than Q1 itself. Similarly, CIDNP and NMR data showed that the concentration dependence of the free radicals yield is much higher in the presence of Fe(III) and Cu(II) ions, in comparison to Zn(II), and also that it increased in the presence of light. These findings underline the role of transition metal ions and Q1 in cyclic redox chain reactions and increase the prospect of the development of copper- and iron-based chelating agents, including Q1 and its derivatives, for anticancer therapy. Furthermore, these findings also signify the effect of light on enhancing ROS formation by Q1 and the prospect of utilizing such information for designing target specific anticancer drugs for photodynamic therapy. Full article

Pattern recognition receptor (PRR) trafficking to the plasma membrane and endocytosis plays a crucial role in pattern triggered immunity (PTI). Dynamin-related proteins (DRPs) participate in endocytosis and recycling. In Arabidopsis, DRP1 and DRP2 are involved in plasma membrane scission during endocytosis. They are required for the PRR FLS2 endocytosis induction and PTI activation after elicitation with flg22, the MAMP recognized by FLS2. In tomato, SlDRP2A regulates the PRR LeEIX2 endocytosis and PTI activation in response to EIX, the MAMP recognized by LeEIX2. However, it is unknown if other DRPs participate in these processes. Taking advantage of bioinformatics tools, we selected SlDRP2B among the eight DRP2 tomato orthologues to study its functionality in trafficking and plant immunity. Through transient expression of SlDRP1B and its dominant-negative mutant on Nicotiana benthamiana and Nicotiana tabacum, we analyzed SlDRP1B function. We observed that SlDRP1B is physically associated with the LeEIX2 and modifies LeEIX2 trafficking, increasing its presence in endosomes. An enhancement of EIX-elicitated defense responses accompanies the role of SlDRP1B on LeEIX endocytosis. In addition, SlDRP1B overexpression enhanced flg22-elicited defense response. With these results, we conclude that SlDRP1B regulates PRR trafficking and, therefore, plant immunity, similarly to the SlDRP2A role. Full article

The quantification of low abundant membrane-binding proteins such as transcriptional factors and chaperones has proven difficult, even with the most sophisticated analytical technologies. Here, we exploit and optimise the non-invasive Fluorescence Correlation Spectroscopy (FCS) for the quantitation of low abundance proteins, and as proof of principle, we choose two interacting proteins involved in the fission of mitochondria in yeast, Fis1p and Mdv1p. In Saccharomyces cerevisiae, the recruitment of Fis1p and Mdv1p to mitochondria is essential for the scission of the organelles and the retention of functional mitochondrial structures in the cell. We use FCS in single GFP-labelled live yeast cells to quantify the protein abundance in homozygote and heterozygote cells and to investigate the impact of the environments on protein copy number, bound/unbound protein state and mobility kinetics. Both proteins were observed to localise predominantly at mitochondrial structures, with the Mdv1p bound state increasing significantly in a strictly respiratory environment. Moreover, a compensatory mechanism that controls Fis1p abundance upon deletion of one allele was observed in Fis1p but not in Mdv1p, suggesting differential regulation of Fis1p and Mdv1p protein expression. Full article

Membrane bioreactors have been widely used in textile wastewater treatment. Intensive chemical cleaning is indispensable in the MBR for textile wastewater treatment due to the severe membrane fouling implied. This work investigated the aging of three different membranes, polyvinylidene fluoride (PVDF), polyether sulfone (PES), and polytetrafluoroethylene (PTFE), in the MBRs for textile wastewater treatment. Pilot-scale MBRs were operated and the used membrane was characterized. Batch chemical soaking tests were conducted to elucidate the aging properties of the membranes. The results indicated that the PVDF membrane was most liable to the chemical cleaning, and the PES and PTFE membranes were rather stable. The surface hydrophobicity of the PVDF increased in the acid aging test, and the pore size and pure water flux decreased due to the elevated hydrophobic effect; alkaline oxide aging destructed the structure of the PVDF membrane, enlarged pore size, and increased pure water flux. Chemical cleaning only altered the interfacial properties (hydrophobicity and surface zeta potential) of the PES and PTFE membranes. The fluoro-substitution and the dehydrofluorination of the PVDF, chain scission of the PES molecules, and dehydrofluorination of the PTFE were observed in aging. A chemically stable and anti-aging membrane would be of great importance in the MBR for textile wastewater treatment due to the intensive chemical cleaning applied. Full article

The ESCRT machinery is an evolutionarily conserved membrane remodeling complex that is used by the cell to perform reverse membrane scission in essential processes like protein degradation, cell division, and release of enveloped retroviruses. ESCRT-III, together with the AAA ATPase VPS4, harbors the main remodeling and scission function of the ESCRT machinery, whereas early-acting ESCRTs mainly contribute to protein sorting and ESCRT-III recruitment through association with upstream targeting factors. Here, we review recent advances in our understanding of the molecular mechanisms that underlie membrane constriction and scission by ESCRT-III and describe the involvement of this machinery in the sealing and repairing of damaged cellular membranes, a key function to preserve cellular viability and organellar function. Full article

CHMP2B is a protein that coordinates membrane scission events as a core component of the ESCRT machinery. Mutations in CHMP2B are an uncommon cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two neurodegenerative diseases with clinical, genetic, and pathological overlap. Different mutations have now been identified across the ALS-FTD spectrum. Disruption of the neuromuscular junction is an early pathogenic event in ALS. Currently, the links between neuromuscular junction functionality and ALS-associated genes, such as CHMP2B, remain poorly understood. We have previously shown that CHMP2B transgenic mice expressing the CHMP2B^intron5 mutant specifically in neurons develop a progressive motor phenotype reminiscent of ALS. In this study, we used complementary approaches (behavior, histology, electroneuromyography, and biochemistry) to determine the extent to which neuron-specific expression of CHMP2B^intron5 could impact the skeletal muscle characteristics. We show that neuronal expression of the CHMP2B^intron5 mutant is sufficient to trigger progressive gait impairment associated with structural and functional changes in the neuromuscular junction. Indeed, CHMP2B^intron5 alters the pre-synaptic terminal organization and the synaptic transmission that ultimately lead to a switch of fast-twitch glycolytic muscle fibers to more oxidative slow-twitch muscle fibers. Taken together these data indicate that neuronal expression of CHMP2B^intron5 is sufficient to induce a synaptopathy with molecular and functional changes in the motor unit reminiscent of those found in ALS patients. Full article

Although once perceived as inert structures that merely serve for lipid storage, lipid droplets (LDs) have proven to be the dynamic organelles that hold many cellular functions. The LDs’ basic structure of a hydrophobic core consisting of neutral lipids and enclosed in a phospholipid monolayer allows for quick lipid accessibility for intracellular energy and membrane production. Whereas formed at the peripheral and perinuclear endoplasmic reticulum, LDs are degraded either in the cytosol by lipolysis or in the vacuoles/lysosomes by autophagy. Autophagy is a regulated breakdown of dysfunctional, damaged, or surplus cellular components. The selective autophagy of LDs is called lipophagy. Here, we review LDs and their degradation by lipophagy in yeast, which proceeds via the micrometer-scale raft-like lipid domains in the vacuolar membrane. These vacuolar microdomains form during nutrient deprivation and facilitate internalization of LDs via the vacuolar membrane invagination and scission. The resultant intra-vacuolar autophagic bodies with LDs inside are broken down by vacuolar lipases and proteases. This type of lipophagy is called microlipophagy as it resembles microautophagy, the type of autophagy when substrates are sequestered right at the surface of a lytic compartment. Yeast microlipophagy via the raft-like vacuolar microdomains is a great model system to study the role of lipid domains in microautophagic pathways. Full article

The proper assembly and dissemination of progeny virions is a fundamental step in virus replication. As a whole, viruses have evolved a myriad of strategies to exploit cellular compartments and mechanisms to ensure a successful round of infection. For enveloped viruses such as retroviruses and herpesviruses, acquisition and incorporation of cellular membrane is an essential process during the formation of infectious viral particles. To do this, these viruses have evolved to hijack the host Endosomal Sorting Complexes Required for Transport (ESCRT-I, -II, and -III) to coordinate the sculpting of cellular membrane at virus assembly and dissemination sites, in seemingly different, yet fundamentally similar ways. For instance, at the plasma membrane, ESCRT-I recruitment is essential for HIV-1 assembly and budding, while it is dispensable for the release of HSV-1. Further, HSV-1 was shown to recruit ESCRT-III for nuclear particle assembly and egress, a process not used by retroviruses during replication. Although the cooption of ESCRTs occurs in two separate subcellular compartments and at two distinct steps for these viral lifecycles, the role fulfilled by ESCRTs at these sites appears to be conserved. This review discusses recent findings that shed some light on the potential parallels between retroviral budding and nuclear egress and proposes a model where HSV-1 nuclear egress may occur through an ESCRT-dependent mechanism. Full article

The molecular mechanism affecting translocation of newly synthesized herpesvirus nucleocapsids from the nucleus into the cytoplasm is still not fully understood. The viral nuclear egress complex (NEC) mediates budding at and scission from the inner nuclear membrane, but the NEC is not sufficient for efficient fusion of the primary virion envelope with the outer nuclear membrane. Since no other viral protein was found to be essential for this process, it was suggested that a cellular machinery is recruited by viral proteins. However, knowledge on fusion mechanisms involving the nuclear membranes is rare. Recently, vesicle-associated membrane protein-associated protein B (VAPB) was shown to play a role in nuclear egress of herpes simplex virus 1 (HSV-1). To test this for the related alphaherpesvirus pseudorabies virus (PrV), we mutated genes encoding VAPB and VAPA by CRISPR/Cas9-based genome editing in our standard rabbit kidney cells (RK13), either individually or in combination. Single as well as double knockout cells were tested for virus propagation and for defects in nuclear egress. However, no deficiency in virus replication nor any effect on nuclear egress was obvious suggesting that VAPB and VAPA do not play a significant role in this process during PrV infection in RK13 cells. Full article

Peroxisomes proliferate by sequential processes comprising elongation, constriction, and scission of peroxisomal membrane. It is known that the constriction step is mediated by a GTPase named dynamin-like protein 1 (DLP1) upon efficient loading of GTP. However, mechanism of fuelling GTP to DLP1 remains unknown in mammals. We earlier show that nucleoside diphosphate (NDP) kinase-like protein, termed dynamin-based ring motive-force organizer 1 (DYNAMO1), generates GTP for DLP1 in a red alga, Cyanidioschyzon merolae. In the present study, we identified that nucleoside diphosphate kinase 3 (NME3), a mammalian homologue of DYNAMO1, localizes to peroxisomes. Elongated peroxisomes were observed in cells with suppressed expression of NME3 and fibroblasts from a patient lacking NME3 due to the homozygous mutation at the initiation codon of NME3. Peroxisomes proliferated by elevation of NME3 upon silencing the expression of ATPase family AAA domain containing 1, ATAD1. In the wild-type cells expressing catalytically-inactive NME3, peroxisomes were elongated. These results suggest that NME3 plays an important role in peroxisome division in a manner dependent on its NDP kinase activity. Moreover, the impairment of peroxisome division reduces the level of ether-linked glycerophospholipids, ethanolamine plasmalogens, implying the physiological importance of regulation of peroxisome morphology. Full article

Mitochondrial morphology, distribution and function are maintained by the opposing forces of mitochondrial fission and fusion, the perturbation of which gives rise to several neurodegenerative disorders. The large guanosine triphosphate (GTP)ase dynamin-related protein 1 (Drp1) is a critical regulator of mitochondrial fission by mediating membrane scission, often at points of mitochondrial constriction at endoplasmic reticulum (ER)-mitochondrial contacts. Hereditary spastic paraplegia (HSP) subtype SPG61 is a rare neurodegenerative disorder caused by mutations in the ER-shaping protein Arl6IP1. We have previously reported defects in both the ER and mitochondrial networks in a Drosophila model of SPG61. In this study, we report that knockdown of Arl6IP1 lowers Drp1 protein levels, resulting in reduced ER–mitochondrial contacts and impaired mitochondrial load at the distal ends of long motor neurons. Increasing mitochondrial fission, by overexpression of wild-type Drp1 but not a dominant negative Drp1, increases ER–mitochondrial contacts, restores mitochondrial load within axons and partially rescues locomotor deficits. Arl6IP1 knockdown Drosophila also demonstrate impaired autophagic flux and an accumulation of ubiquitinated proteins, which occur independent of Drp1-mediated mitochondrial fission defects. Together, these findings provide evidence that impaired mitochondrial fission contributes to neurodegeneration in this in vivo model of HSP. Full article

The exceptional reactivity of the azide group makes organic azides a highly versatile family of compounds in chemistry and the material sciences. One of the most prominent reactions employing organic azides is the regioselective copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition with alkynes yielding 1,2,3-triazoles. Other named reactions include the Staudinger reduction, the aza-Wittig reaction, and the Curtius rearrangement. The popularity of organic azides in material sciences is mostly based on their propensity to release nitrogen by thermal activation or photolysis. On the one hand, this scission reaction is accompanied with a considerable output of energy, making them interesting as highly energetic materials. On the other hand, it produces highly reactive nitrenes that show extraordinary efficiency in polymer crosslinking, a process used to alter the physical properties of polymers and to boost efficiencies of polymer-based devices such as membrane fuel cells, organic solar cells (OSCs), light-emitting diodes (LEDs), and organic field-effect transistors (OFETs). Thermosets are also suitable application areas. In most cases, organic azides with multiple azide functions are employed which can either be small molecules or oligo- and polymers. This review focuses on nitrene-based applications of multivalent organic azides in the material and life sciences. Full article