Search

Photodynamic therapy (PDT) takes advantage of reactive oxygen species (ROS) to trigger the apoptosis for cancer therapy. Given that cell apoptosis is a form of programmed cell death involved with multiple suborganelles and cancer cells are more sensitive to ROS than normal cells, early confirmation of the apoptosis induced by ROS would effectively avoid overtreatment. Herein, we highlight an aggregation-induced emission (AIE)-based theranostic agent ( TPA3 ) to in situ dynamically track mitophagy prior to late apoptosis. TPA3 showed high specificity to autophagy vacuoles (AVs), of which appearance is the signature event of mitophagy during early apoptosis and delivered photocytotoxicity to cancer cells and skin cancer tumors in nude mice under irradiation of white light. Furthermore, in situ monitoring of the dynamical mitophagy process involved with mitochondria, AVs, and lysosomes was performed for the first time under confocal microscopy, providing a real-time self-monitoring system for assessing the curative effect prior to late apoptosis. This fluorescence imaging guided PDT witness great advances for applying in the clinical application.

Light-initiated fusion between vesicles has attracted much attention in the research community. In particular, fusion between photoresponsive and non-photoresponsive vesicles has been of much interest in the development of systems for the delivery of therapeutic agents to cells. We have performed fusion between giant vesicles (GVs) and photoresponsive smaller vesicles containing malachite green (MG) derivative, which undergoes ionization to afford a positive charge on the molecule by irradiation. The fusion proceeds as the concentration of GV lipid increases toward equimolarity with the lipid of the smaller vesicle. It is also dependent on the molar percentage of photoionized MG in the lipid of the smaller vesicle. On the other hand, the fusion is hardly affected by the anionic component of the GV. The photoinduced fusion was characterized by two methods, involving the mixing of lipid membranes and of aqueous contents. Fluorescence microscopy revealed that irradiation triggered the fusion of a single GV with the smaller vesicles containing MG., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Remote control over fusion of single cells and vesicles has a great potential in biological and chemical research allowing both transfer of genetic material between cells and transfer of molecular content between vesicles. Membrane fusion is a critical process in biology that facilitates molecular transport and mixing of cellular cytoplasms with potential formation of hybrid cells. Cells precisely regulate internal membrane fusions with the aid of specialized fusion complexes that physically provide the energy necessary for mediating fusion. Physical factors like membrane curvature, tension and temperature, affect biological membrane fusion by lowering the associated energy barrier. This has inspired the development of physical approaches to harness the fusion process at a single cell level by using remotely controlled electromagnetic fields to trigger membrane fusion. Here, we critically review various approaches, based on lasers or electric pulses, to control fusion between individual cells or between individual lipid vesicles and discuss their potential and limitations for present and future applications within biochemistry, biology and soft matter.

The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin-related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin-D-induced depolymerization or phalloidin-induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild-type (WT) Arabidopsis plants. Light-induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening., (© 2012 Blackwell Publishing Ltd.)

Glucose oxidase (GOD) was encapsulated in vesicles containing a photoionizable Malachite Green leuconitrile derivative (MGL). Subsequent UV irradiation of MGL afforded the fusion of GOD- and glucose-encapsulating vesicles and thus decreased the concentration of glucose in the vesicles. The time dependence of the vesicle fusion was studied using fluorescent probe molecules. This phototriggered fusion could be instrumental in the development of a system for the production of nanometer-sized bioreactors., (Copyright © 2011 Elsevier B.V. All rights reserved.)

To characterize the sites of synaptic vesicle fusion in photoreceptors, we evaluated the three-dimensional structure of rod spherules from mice exposed to steady bright light or dark-adapted for periods ranging from 3 to 180 minutes using conical electron tomography. Conical tilt series from mice retinas were reconstructed using the weighted back projection algorithm, refined by projection matching and analyzed using semiautomatic density segmentation. In the light, rod spherules contained ∼470 vesicles that were hemi-fused and ∼187 vesicles that were fully fused (omega figures) with the plasma membrane. Active zones, defined by the presence of fully fused vesicles, extended along the entire area of contact between the rod spherule and the horizontal cell ending, and included the base of the ribbon, the slope of the synaptic ridge and ribbon-free regions apposed to horizontal cell axonal endings. There were transient changes of the rod spherules during dark adaptation. At early periods in the dark (3-15 minutes), there was a) an increase in the number of fully fused synaptic vesicles, b) a decrease in rod spherule volume, and c) an increase in the surface area of the contact between the rod spherule and horizontal cell endings. These changes partially compensate for the increase in the rod spherule plasma membrane following vesicle fusion. After 30 minutes of dark-adaptation, the rod spherules returned to dimensions similar to those measured in the light. These findings show that vesicle fusion occurs at both ribbon-associated and ribbon-free regions, and that transient changes in rod spherules and horizontal cell endings occur shortly after dark onset.

The authors present the use of electric-field constriction created by a microfabricated structure to realise high-yield electrofusion of biological cells. The method uses an orifice on an electrically insulating wall (orifice plate) whose diameter is as small as that of the cells. Owing to the field constriction created by the orifice, we can induce the controlled magnitude of membrane voltage selectively around the contact point, regardless of the cell size. The field constriction also ensures 1:1 fusion even when more than two cells are forming a chain at the orifice. A device for electrofusion has been made with a standard SU-8 lithography and PDMS molding, and real-time observation of the electrofusion process is made. Experiments using plant protoplasts or mammalian cells show that the process is highly reproducible, and the yield higher than 90% is achieved.

This paper reports a microfabricated device with high aspect-ratio electrodes and low power consumption for the electrofusion of liposomes and cells. The applications may range from gene transfection or cell tracking to biophysical studies of membrane proteins. The device consists of 250 microm thick silicon electrodes bonded to a glass substrate and covered by a PDMS-coated glass slide. Liposomes were first aligned by AC voltage at 300 kHz and then fused with short DC pulses. The fusion yield can reach 75% and is globally better for liposome diameters larger than 10 microm. The encapsulation of microbeads inside liposomes has also been demonstrated and opens up the route towards fusion-based delivery of artificial microstructures into cells.

We performed a detailed kinetic analysis of influenza virus fusion with the endosomal and plasma membranes of Madin Darby canine kidney (MDCK) cells and provided a comparison of the kinetic parameters obtained for both cases at 20 degrees C and 37 degrees C. Using our mass action kinetic model, we determined that the fusion rate constant, f, for influenza virus with the endosomal membrane was 0.02 s(-1) at 37 degrees C and 0.0035 s(-1) at 20 degrees C. The analysis of the fusion kinetics of influenza virus with the plasma membrane yielded that the fusion rate constants were close to those deduced with the endosomal membrane. The systematic kinetic analysis performed in this study provides for the first time a biophysical support for studies on influenza virus-cell fusion where the acidic endosomal internal environment is simulated artificially by lowering the pH of the medium.

The potential biomedical utility of the photoinduced destabilization of liposomes depends in part on the use of green to near infrared light with its inherent therapeutic advantages. The polymerization of bilayers can be sensitized to green light by associating selected amphiphilic cyanine dyes, i.e. the cationic 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine (DiI), or the corresponding anionic disulfonated DiI (DiI-DS), with the lipid bilayer. The DiI sensitization of the polymerization of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine/1,2-bis[10-(2', 4'-hexadienoyloxy)-decanoyl]-sn-glycero-3-phosphocholine liposomes caused liposome destabilization with release of encapsulated aqueous markers. In separate experiments, similar photosensitive liposomes were endocytosed by cultured HeLa cells. Exposure of the cells and liposomes to 550 nm light caused a net movement of the liposome-encapsulated 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) from low pH compartment(s) to higher pH compartment(s). This suggests that photolysis of DiI-labelled liposomes results in delivery of the contents of the endocytosed liposomes to the cytoplasm. The release of HPTS into the cytoplasm appears to require the photoactivated fusion of the labelled liposomes with the endosomal membrane. These studies aid in the design of visible light sensitive liposomes for the delivery of liposome-encapsulated reagents to the cytoplasm.

The surge of Ca(2+) that triggers vesicle fusion is shaped by the distribution of Ca(2+) channels and the physical relationship between those channels and the exocytotic apparatus. Although channels and the release apparatus are thought to be tightly associated at fast synapses, the arrangement at neuroendocrine cells is less clear. The distribution of Ca(2+) influx near release sites is difficult to determine because of spatial and temporal limitations on Ca(2+) imaging techniques. We now present spatially resolved images of Ca(2+) influx into rat neuroendocrine terminals on a millisecond time scale. Images of voltage-dependent Ca(2+) influx into neurohypophysial terminals were captured after excitation of Ca(2+)-sensitive dyes with pulses of laser light lasting a fraction of a microsecond. Submembranous Ca(2+) increases were detected during the first millisecond of an evoked Ca(2+) tail current. Steep gradients of Ca(2+) were evident, with concentrations near the membrane reaching above 1 microM during a 30 msec depolarization. Ca(2+) influx appeared evenly distributed, even when diffusion was restricted with an exogenous Ca(2+) chelator. During longer depolarizations, mean and peak Ca(2+) concentrations reached an asymptote in parallel, suggesting that Ca(2+) binding proteins near the membrane rapidly buffer Ca(2+) and do not become saturated during prolonged influx. These data support the hypothesis that exocytosis is activated in these terminals by the summation of influx through multiple, randomly spaced Ca(2+) channels.

We have investigated the effects of membrane lipid composition on biological membrane fusion triggered by low pH and mediated by the baculovirus envelope glycoprotein gp64. Lysolipids, either added exogenously or produced in situ by phospholipase A2 treatment of cell membranes, reversibly inhibited syncytium formation. Lysolipids also decreased the baculovirus infection rate. In contrast, oleic and arachidonic acids and monoolein promoted cell-cell fusion. Membrane lipid composition affected pH-independent processes which followed the low-pH-induced change in fusion protein conformation. Inhibition and promotion of membrane fusion by a number of lipids could not be explained by mere binding or incorporation into membranes, but rather was correlated with the effective molecular shape of exogenous lipids. Our data are consistent with the hypothesis that membrane fusion proceeds through highly bent membrane intermediates (stalks) having a net negative curvature. Consequently, inverted cone-shaped lysolipids inhibit and cone-shaped cis-unsaturated fatty acids promote stalk formation and, ultimately, membrane fusion.

The photopolymerization of two-component large unilamellar liposomes (LUV) composed of 3:1 dioleoylphosphatidylethanolamine (DOPE) and either 1,2-bis[10-(2'-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphatidylc holine (bis-SorbPC) or 1-palmitoyl-2-[10-(2'-hexadienoyloxy)decanoyl]-sn- glycero-3-phosphatidylcholine (mono-SorbPC) facilitated liposome fusion. Fusion was characterized by fluorescent assays for lipid mixing, aqueous contents mixing, and aqueous contents leakage. The rate and extent of the liposome fusion was dependent on the extent of photopolymerization, temperature, and the fusion initiation conditions, including the pH and the presence of Mg2+ ions. Examination of the temperature dependence of fusion for unpolymerized and polymerized liposomes showed that an enhancement of the rate of fusion occurred in the temperature range delta TI, which previous NMR studies have identified as the initial appearance of precursors to the formation of the inverted cubic phase [Barry, J. A., et al. (1992) Biochemistry 31, 10114]. The phase behavior and fusion characteristics of the DOPE/bis-SorbPC (3:1) membranes provide unequivocal evidence that liposome fusion is mediated via intermediates associated with the lamellar to QII phase transition rather than the HII phase. Photopolymerization of SorbPC-containing liposomes forms poly-SorbPC, which enhances the lateral separation of the liposome components. The formation of enriched domains of polymorphic lipids, e.g., DOPE, causes isothermal induction of fusion by lowering the critical fusion temperature of the membranes.

The fusion kinetics of cells expressing the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein with CD4 target cells was continuously monitored by image-enhanced Nomarski differential interference contrast optics. The analysis of the videotape recordings showed that (i) cells made contact relatively rapidly (within minutes), in many cases by using microspikes to "touch" and adhere to adjoining cells; (ii) the adhered cells fused after a relatively long waiting period, which varied from 15 min to hours; (iii) the morphological changes after membrane fusion, which led to disappearance of the interface separating the two cells, were rapid (less than 1 min); and (iv) the process of syncytium formation involved subsequent fusion with other cells and not simultaneous fusion of many cells. To measure the kinetics of early stages of cell fusion, we used the recently developed very stable membrane-soluble dye, PKH26, which redistributes between labeled and unlabeled membranes after fusion but does not exchange spontaneously between membranes for prolonged periods. We found that photoactivation of this dye by illumination with green light inhibits fusion of cell membranes as indicated by the lack of dye transfer from the labeled HIV-1 envelope-expressing cells to unlabeled CD4 cells. The inhibitory effect was localized in space and time, which allowed us to develop a new assay for measuring the kinetics of membrane fusion by illuminating the cell mixture at different times after coculture. This assay has also been used to monitor the fusion kinetics of HIV-1 and recombinant vaccinia virus. The photoactivation of nonexchangeable membrane-soluble fluorescent dyes may be useful for development of new assays for measuring the kinetics of membrane fusion and could also be important in designing new antiviral approaches.

We have recently demonstrated using dielectic relaxation studies in the radiowave frequency range that sinusoidal 50 Hz magnetic fields (with intensities ranging from 1 to 10 mT) induce a nonlinear change in both membrane conductivity and permittivity of primary chick embryo myoblasts in vitro. It was the aim of the present study to determine if a DC-induced static magnetic field is capable of generating similar variations in the membrane conductivity and/or the membrane permittivity of chick embryo myoblasts. The results indicate that when the myogenic cells are exposed to a static magnetic field of either 1, 3 or 5 mT (values comparable with the previous extremely low frequency study), no changes in the membrane electrical parameters can be observed with respect to controls. Differences in the characteristics of static and extremely low frequency fields as well as the possible mechanisms underlying the contrasting results with these two types of magnetic fields are discussed.

Rose bengal inactivated influenza virus upon exposure to light. Infectivity and fusion were inactivated with the same dose dependence, supporting the suggestion that the virucidal activity of photodynamic agents against enveloped viruses may be generally due to inactivation of their fusion protein(s). Concentrations required for inactivation were found to depend upon the ratio of rose bengal to virus, rather than on the nominal aqueous concentration. Fusion-competent virosomes were inactivated similarly to intact virus particles. The HA2 portion of the influenza fusion protein HA underwent two different, apparently mutually exclusive modifications upon illumination with rose bengal: cross-linking, and conversion to a form that moved slightly more slowly on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Inactivation of viral fusion was inhibited by oxygen removal or addition of azide or beta-carotene, and was enhanced by D2O, consistent with partial involvement of singlet oxygen. The possibility of a second mechanism of viral photoinactivation, by direct interaction between the viral fusion protein and the photoactivated dye, is also discussed.

Primary chick embryo myoblasts can be a useful tool for studying the developmental events which accompany myoblast differentiation, particularly myoblast membrane fusion. To determine whether the electrical properties and/or fusion in these systems are affected by 50 Hz magnetic fields, chick embryo myoblast cultures were exposed to B-field intensities ranging from 1 to 10 mT. The electrical parameters of the myoblasts, i.e. membrane conductivity, membrane permittivity and the conductivity of the cell interior (cytosol) were determined by the analysis of conductivity dispersion data in the radio frequency range (10 kHz-100 MHz). Preliminary results indicate that the time of fusion (60 h) is not affected by these fields, but that the absolute values of the two membrane electrical parameters are affected. In particular, a B-field intensity-dependent decrease was observed. The maximum effect resulted after a 1 h exposure to a magnetic flux density of about 5 mT. The conductivity of the cytosol remained unchanged. These data seem to indicate that exposure to 50 Hz magnetic fields affects both static and dynamic membrane properties in primary chick embryo myoblasts.

Liposomes have been prepared from dipalmitoylphosphatidylcholine containing small amounts of a synthetic photochromic phospholipid, 'Bis-Azo PC'. In the dark, these are stable at room temperature, and contents do not significantly leak over weeks. Photoisomerisation results in immediate release of trapped marker, and in liposome fusion to form larger structures. Fusion has been detected using a fluorescence polarisation assay, and confirmed by electron microscopy. In mixtures, fusion occurs between 'photochromic' liposomes and those of pure lipid. Bis-Azo PC contains two photochromic acyl chains; analogues bearing a single photochromic chain appear to have little effect on bilayer permeability after isomerisation. Photo-induced leakage and liposome fusion suggest possible applications for localised drug delivery as an adjunct to phototherapy. The ability to non-invasively trigger fusion processes should be useful in fundamental studies of membrane interactions. We believe this to be the first report of photo-induced fusion to date.