HeLa Cell Membranes Research Articles

Towards Selective Light‐Activated RuII‐Based Prodrug Candidates

AbstractPhotoactivated chemotherapy (PACT) relies on the use of a drug and light to cause cell death. Unlike photodynamic therapy (PDT), its mechanism of action is independent of the presence of oxygen in tissues. This approach is therefore also effective for tumours characterized by hypoxic conditions. Herein we present the use of three novel RuII–polypyridyl complexes derivatized with a light‐sensitive cage and targeting peptides as selective PACT agents. Light irradiation allowed the selective release of the cytotoxic compounds from the molecular cages in living cells. Further selectivity was conferred on the system by a peptide carrier (i.e., bombesin), which targets receptors overexpressed on the membranes of HeLa cells. As expected, this peptide was found to provide an increased uptake of the systems in HeLa cells compared with normal MRC‐5 cells. As a result, for two of the compounds presented in this study, no cyto‐ or phototoxicity was observed in MRC‐5 cells, whereas in HeLa cells, upon light irradiation, IC50 values of 42.2 and 60.0 μM were obtained, which correspond to a two‐fold increase in toxicity.

Site of fluorescent label modifies interaction of melittin with live cells and model membranes

The mechanism of membrane disruption by melittin (MLT) of giant unilamellar vesicles (GUVs) and live cells was studied using fluorescence microscopy and two fluorescent synthetic analogues of MLT. The N-terminus of one of these was acylated with thiopropionic acid to enable labeling with maleimido-AlexaFluor 430 to study the interaction of MLT with live cells. It was compared with a second analogue labeled at P14C. The results indicated that the fluorescent peptides adhered to the membrane bilayer of phosphatidylcholine GUVs and inserted into the plasma membrane of HeLa cells. Fluorescence and light microscopy revealed changes in cell morphology after exposure to MLT peptides and showed bleb formation in the plasma membrane of HeLa cells. However, the membrane disruptive effect was dependent upon the location of the fluorescent label on the peptide and was greater when MLT was labeled at the N-terminus. Proline at position 14 appeared to be important for antimicrobial activity, hemolysis and cytotoxicity, but not essential for cell membrane disruption.

Discreet and distinct clustering of five model membrane proteins revealed by single molecule localization microscopy.

Compartmentalization is a functionally important property of the plasma membrane, yet the underlying principles that organize membrane proteins into distinct domains are not well understood. Using single molecule localization microscopy, we assessed the clustering of five model membrane proteins in the plasma membrane of HeLa cells. All five proteins formed discrete and distinct nano-scaled clusters. The extent of clustering of the five proteins, independent of their membrane anchors, increased significantly when the fluorescent protein mEOS2 was employed, suggesting that protein-protein interactions are a key driver for clustering. Further, actin depolymerization or reduction of membrane order had a greater, and in some instances opposing effects on the clustering of membrane proteins fused to mEOS2 compared to PS-CFP2-fusion proteins. The data propose that protein interactions can override the lateral organization imposed by membrane anchors to provide an exquisite regulation of the mosaic-like compartmentalization of the plasma membrane.

A luminescent poly(amidoamine)-iridium complex as a new singlet-oxygen sensitizer for photodynamic therapy.

A polymer complex (1P) was synthesized by binding bis(cyclometalated) Ir(ppy)2(+) fragments (ppy = 2-phenylpyridyl) to phenanthroline (phen) pendants of a poly(amidoamine) copolymer (PhenISA, in which the phen pendants involved ∼6% of the repeating units). The corresponding molecular complex [Ir(ppy)2(bap)](+) (1M, bap = 4-(butyl-4-amino)-1,10-phenanthroline) was also prepared for comparison. In water solution 1P gives nanoaggregates with a hydrodynamic diameter of 30 nm in which the lipophilic metal centers are presumed to be segregated within polymer tasks to reduce their interaction with water. Such confinement, combined with the dilution of triplet emitters along the polymer chains, led to 1P having a photoluminescence quantum yield greater than that of 1M (0.061 vs 0.034, respectively, in an aerated water solution) with a longer lifetime of the (3)MLCT excited states and a blue-shifted emission (595 nm vs 604 nm, respectively). NMR data supported segregation of the metal centers. Photoreaction of O2 with 1,5-dihydroxynaphthalene showed that 1P is able to sensitize (1)O2 generation but with half the quantum yield of 1M. Cellular uptake experiments showed that both 1M and 1P are efficient cell staining agents endowed with two-photon excitation (TPE) imaging capability. TPE microscopy at 840 nm indicated that both complexes penetrate the cellular membrane of HeLa cells, localizing in the perinuclear region. Cellular photodynamic therapy tests showed that both 1M and 1P are able to induce cell apoptosis upon exposure to Xe lamp irradiation. The fraction of apoptotic cells for 1M was higher than that for 1P (74 and 38%, respectively) 6 h after being irradiated for 5 min, but cells incubated with 1P showed much lower levels of necrosis as well as lower toxicity in the absence of irradiation. More generally, the results indicate that cell damage induced by 1M was avoided by binding the iridium sensitizers to the poly(amidoamine).

Size-dependent and real-time effect of SiO2 nanoparticles on a single living HeLa Cell's membrane permeability.

In this study, we provide the quantitative data on the membrane permeability of a HeLa cell in the presence of different sizes of SiO2 nanoparticles (NPs, 50, 100 and 200 nm). SiO2 NPs have low cytotoxicity, but 50 and 100 nm SiO2 NPs can increase the cell membrane permeability (a reversible effect) by 12.5% (0.02 mg mL-1, 4 min) and 9% (0.02 mg mL-1, 6 min), respectively. However, the 200 nm SiO2 NPs cannot affect the cell membrane permeability.

Branched dimerization of Tat peptide improves permeability to HeLa and hippocampal neuronal cells.

A dimeric branched peptide TATp-D designed as an analogue of the HIV-Tat protein transduction domain (TATp), a prototypical cell penetrating peptide (CPP), demonstrates significantly enhanced cell uptake at 0.25 to 2.5 μM. Live cell confocal laser scanning microscopy revealed that multivalency dramatically improved the permeation potency of TATp-D to HeLa and primary hippocampal neuronal cells. The observed enhanced ability of TATp-D to translocate through the membrane is highlighted by a non-linear dependence on concentration, exhibiting the greatest uptake at sub-micromolar concentrations as compared to TATp. Multimerization via bis-Fmoc Lysine offered a synthetically straightforward method to investigate the effects of multivalent CPPs while offering orthogonal handles for cargo attachment, increasing the utility of CPPs at significantly lower concentrations.

Two hits are better than one: synergistic anticancer activity of α-helical peptides and doxorubicin/epirubicin.

This study explored combinational anticancer therapy using α-helical peptides HPRP-A1/HPRP-A2 with the chemical drugs doxorubicin (DOX) and epirubicin (EPI). The in vitro activity of these drugs against different cancer cell lines was synergistically increased, as was their activity in a HeLa xenograft model in BALB/c nude mice. We delineated the mechanism of this synergy by studying the apoptosis pathway and morphologic changes in the HeLa cell membrane. The mechanism of the HPRP-A1/DOX combination was found to involve enhanced apoptosis, which seemed to be caspase-dependent and involved both the extrinsic and intrinsic parts of the caspase cascade in HeLa cells. Combined application of HPRP-A1 and DOX at low concentrations was significantly more effective than either drug alone against HeLa tumors in the mouse xenograft model. This type of combination therapy appears to have great clinical potential.

The connexin 30.3 of zebrafish homologue of human connexin 26 may play similar role in the inner ear

The intercellular gap junction channels formed by connexins (CXs) are important for recycling potassium ions in the inner ear. CXs are encoded by a family of the CX gene, such as GJB2, and the mechanism leading to mutant connexin-associated diseases, including hearing loss, remains to be elucidated. In this study, using bioinformatics, we found that two zebrafish cx genes, cx27.5 and cx30.3, are likely homologous to human and mouse GJB2. During embryogenesis, zebrafish cx27.5 was rarely expressed at 1.5–3 h post-fertilization (hpf), but a relatively high level of cx27.5 expression was detected from 6 to 96 hpf. However, zebrafish cx30.3 transcripts were hardly detected until 9 hpf. The temporal experiment was conducted in whole larvae. Both cx27.5 and cx30.3 transcripts were revealed significantly in the inner ear by reverse transcription polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization (WISH). In the HeLa cell model, we found that zebrafish Cx27.5 was distributed intracellularly in the cytoplasm, whereas Cx30.3 was localized in the plasma membrane of HeLa cells stably expressing Cx proteins. The expression pattern of zebrafish Cx30.3 in HeLa cells was more similar to that of cells expressing human CX26 than Cx27.5. In addition, we found that Cx30.3 was localized in the cell membrane of hair cells within the inner ear by immunohistochemistry (IHC), suggesting that zebrafish cx30.3 might play an essential role in the development of the inner ear, in the same manner as human GJB2. We then performed morpholino knockdown studies in zebrafish embryos to elucidate the physiological functions of Cx30.3. The zebrafish cx30.3 morphants exhibited wild-type-like and heart edema phenotypes with smaller inner ears at 72 hpf. Based on these results, we suggest that the zebrafish Cx30.3 and mammalian CX26 may play alike roles in the inner ear. Thus, zebrafish can potentially serve as a model for studying hearing loss disorders that result from human CX26 mutations.

Investigating the role of F-actin in human immunodeficiency virus assembly by live-cell microscopy.

Human immunodeficiency virus type 1 (HIV-1) particles assemble at the plasma membrane, which is lined by a dense network of filamentous actin (F-actin). Large amounts of actin have been detected in HIV-1 virions, proposed to be incorporated by interactions with the nucleocapsid domain of the viral polyprotein Gag. Previous studies addressing the role of F-actin in HIV-1 particle formation using F-actin-interfering drugs did not yield consistent results. Filamentous structures pointing toward nascent HIV-1 budding sites, detected by cryo-electron tomography and atomic force microscopy, prompted us to revisit the role of F-actin in HIV-1 assembly by live-cell microscopy. HeLa cells coexpressing HIV-1 carrying fluorescently labeled Gag and a labeled F-actin-binding peptide were imaged by live-cell total internal reflection fluorescence microscopy (TIR-FM). Computational analysis of image series did not reveal characteristic patterns of F-actin in the vicinity of viral budding sites. Furthermore, no transient recruitment of F-actin during bud formation was detected by monitoring fluorescence intensity changes at nascent HIV-1 assembly sites. The chosen approach allowed us to measure the effect of F-actin-interfering drugs on the assembly of individual virions in parallel with monitoring changes in the F-actin network of the respective cell. Treatment of cells with latrunculin did not affect the efficiency and dynamics of Gag assembly under conditions resulting in the disruption of F-actin filaments. Normal assembly rates were also observed upon transient stabilization of F-actin by short-term treatment with jasplakinolide. Taken together, these findings indicate that actin filament dynamics are dispensable for HIV-1 Gag assembly at the plasma membrane of HeLa cells. Importance: HIV-1 particles assemble at the plasma membrane of virus-producing cells. This membrane is lined by a dense network of actin filaments that might either present a physical obstacle to the formation of virus particles or generate force promoting the assembly process. Drug-mediated interference with the actin cytoskeleton showed different results for the formation of retroviral particles in different studies, likely due to general effects on the cell upon prolonged drug treatment. Here, we characterized the effect of actin-interfering compounds on the HIV-1 assembly process by direct observation of virus formation in live cells, which allowed us to measure assembly rate constants directly upon drug addition. Virus assembly proceeded with normal rates when actin filaments were either disrupted or stabilized. Taken together with the absence of characteristic actin filament patterns at viral budding sites in our analyses, this indicates that the actin network is dispensable for HIV-1 assembly.

Morphological Change of Cell Membrane by Interaction with Domain‐Swapped Cytochrome c Oligomers

Monomeric cyt c has been reported to bind to the mitochondrial membrane by electrostatic and hydrophobic interactions with anionic phospholipids. We have previously shown that domain-swapped oligomeric cyt c retains the secondary structure of the monomer, and its surface possesses a larger area and more charges compared to the monomer. However, the effect of oligomerization of cyt c on cells has yet to be revealed. Herein, we investigated the interaction of oligomeric cyt c with anionic phospholipid-containing vesicles and the outer membrane of HeLa cells. Oligomeric cyt c interacted more strongly than monomeric cyt c with anionic phospholipid-containing vesicles and the outer membrane of HeLa cells. Oligomeric cyt c induced lateral phase separation of lipids in LUVs and GUVs, thereby leading to membrane disruption, whereas monomeric cyt c did not. Morphological changes in HeLa cells resulted from interaction with oligomeric cyt c, but little from interaction with the monomer. These results show that domain-swapped oligomeric proteins might exhibit properties different to those of monomer in cell systems.

Separation of the Inner and Outer Mitochondrial Membrane in HeLa Cells

Separation of the Inner and Outer Mitochondrial Membrane in HeLa Cells

Quantitative and real-time effects of carbon quantum dots on single living HeLa cell membrane permeability.

The interaction between carbon quantum dots (CQDs) and a single living cell was explored in real time. Here, we provide the quantitative data on the permeability of the HeLa cell membrane in the presence of CQDs with different surface functional groups (CQDs terminated with -OH/-COOH (CQD-OH), -PEG (CQD-PEG), and -NH2 (CQD-NH2)). Although these CQDs have very low toxicity towards HeLa cells, they still increase the cell membrane permeability by 8%, 13%, and 19% for CQD-PEG, CQD-OH, and CQD-NH2, respectively, and this kind of permeability was irreversible. These observations are valuable for promoting the bio-applications of carbon nanostructures in living systems.

Development of a FRET Biosensor with High Specificity for Akt

The serine/threonine kinase Akt plays a critical role in cell proliferation, survival, and tumorigenesis. As a central kinase in the phosphatidylinositol 3-kinase pathway, its activation mechanism at the plasma membrane has been well characterized. However, the subcellular Akt activity in living cells is still largely unknown. Fluorescence resonance energy transfer (FRET)-based biosensors have emerged as indispensable tools to visualize the subcellular activities of signaling molecules. In this study, we developed a highly specific FRET biosensor for Akt based on the Eevee backbone, called Eevee-iAkt. Using inhibitors targeting kinases upstream and downstream of Akt, we showed that Eevee-iAkt specifically monitors Akt activity in living cells. To visualize Akt activity at different subcellular compartments, we targeted Eevee-iAkt to raft and non-raft regions of the plasma membrane, mitochondria, and nucleus in HeLa and Cos7 cells. Interestingly, we revealed substantial differences in Akt activation between HeLa and Cos7 cells upon epidermal growth factor (EGF) stimulation: Akt was transiently activated in HeLa cells with comparable levels at the plasma membrane, cytosol, and mitochondria. In contrast, sustained and spatially localized Akt activation was observed in EGF-stimulated Cos7 cells. We found high Akt activity at the plasma membrane, low activity in the cytosol, and no detectable activity at the mitochondria and nucleus in Cos7 cells. The Eevee-iAkt biosensor was shown to be a valuable tool to study the functional relationship between subcellular Akt activation and its anti-apoptotic role in living cells.

PH luminescence switching, dihydrogen phosphate sensing, and cellular uptake of a heterobimetallic ruthenium(ii)–rhenium(i) complex

A new heterobimetallic ruthenium(II)-rhenium(I) complex of [Ru(bpy)2(HL)Re(CO)3Cl](ClO4)2·6H2O (RuHLRe) {bpy = 2,2'-bipyridine and HL = 2-(4-(2,6-di(pyridin-2-yl)pyridin-4-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} was synthesised and characterised by elemental analysis, proton nuclear magnetic resonance spectroscopy, and mass spectrometry. The ground- and excited-state acid-base properties of RuHLRe were studied using UV-Vis absorption spectrophotometric and spectrofluorimetric titrations in a 100 : 1 (v/v) Britton-Robinson buffer-CH3CN solution combined with luminescence lifetime measurements. The complex exhibited two-step separate protonation-deprotonation processes in both the ground and excited states. The complex acted as pH-induced "off-on-off" luminescence switches (I(on)/I(off) = 31.0 and 14.6), with one of the switching actions being driven by pH variations over the physiological pH range (5.3-8.0). Importantly, cellular imaging and cytotoxicity experiments demonstrated that RuHLRe rapidly and selectively illuminated the membrane of HeLa cells over fixed cells and exhibited reduced cytotoxicity at the imaging concentration compared to the Re(I)-free parent Ru(II) complex. In addition, RuHLRe acted as an efficient "turn on" emission sensor for H2PO4(-) and "turn off" emission sensor for F(-) and OAc(-).

Factors influencing the cell adhesion and invasion capacity of Mycoplasma gallisepticum

BackgroundThe cell invasiveness of Mycoplasma gallisepticum, the causative agent of respiratory disease in chickens and infectious sinusitis in turkeys, may be a substantial factor in the well-known chronicity of these diseases and in the systemic spread of infection. To date, not much is known about the host factors and mechanisms involved in promotion or obstruction of M. gallisepticum adherence and/or cell invasion.In the current study, the influence of extracellular matrix (ECM) proteins such as fibronectin, collagen type IV and heparin, as well as plasminogen/plasmin, on the adhesion and cell invasion levels of M. gallisepticum to chicken erythrocytes and HeLa cells was investigated in vitro. Two strains, Rhigh and Rlow, which differ in their adhesion and invasion capacity, were analyzed by applying a modified gentamicin invasion assay. Binding of selected ECM molecules to M. gallisepticum was proven by Western blot analysis.ResultsCollagen type IV, fibronectin, and plasminogen exerted positive effects on adhesion and cell invasion of M. gallisepticum, with varying degrees, depending on the strain used. Especially strain Rhigh, with its highly reduced cell adhesion and invasion capabilities seemed to profit from the addition of plasminogen. Western and dot blot analyses showed that Rhigh as well as Rlow are able to adsorb horse fibronectin and plasminogen present in the growth medium. Depletion of HeLa cell membranes from cholesterol resulted in increased adhesion, but decreased cell invasion.ConclusionECM molecules seem to play a supportive role in the adhesion/cell invasion process of M. gallisepticum. Cholesterol depletion known to affect lipid rafts on the host cell surface had contrary effects on cell adherence and cell invasion of M. gallisepticum.

Resiliency of the plasma membrane and actin cortex to large‐scale deformation

The tight coupling between the plasma membrane and actin cortex allows cells to rapidly change shape in response to mechanical cues and during physiological processes. Mechanical properties of the membrane are critical for organizing the actin cortex, which ultimately governs the conversion of mechanical information into signaling. The cortex has been shown to rapidly remodel on timescales of seconds to minutes, facilitating localized deformations and bundling dynamics that arise during the exertion of mechanical forces and cellular deformations. Here, we directly visualized and quantified the time-dependent deformation and recovery of the membrane and actin cortex of HeLa cells in response to externally applied loads both on- and off-nucleus using simultaneous confocal and atomic force microscopy. The local creep-like deformation of the membrane and actin cortex depends on both load magnitude and duration and does not appear to depend on cell confluency. The membrane and actin cortex rapidly recover their initial shape after prolonged loading (up to 10 min) with large forces (up to 20 nN) and high aspect ratio deformations. Cytoplasmic regions surrounding the nucleus are shown to be more resistant to long-term creep than nuclear regions. These dynamics are highly regulated by actomyosin contractility and an intact actin cytoskeleton. Results suggest that in response to local deformations, the nucleus does not appear to provide significant resistance or play a major role in cell shape recovery. The membrane and actin cortex clearly possess remarkable mechanical stability, critical for the transduction of mechanical deformation into long term biochemical signals and cellular remodeling.

Photoluminescent ZnO Quantum Dots Coated by Polymethacrylamide and Their Application in Cell Imaging

ZnO@polymer core-shell nanoparticles with stable luminescence in aqueous solutions and tunable emission were synthesized by sol-gel method,studied in cytotoxicity tests towards HeLa cells and measured by laser confocal scanning microscope.The shell of such luminescent probes is a copolymer connected the ZnO core through coordination bonds,which contains an external hydrophilic layer of polymethacrylamide and an internal hydrophobic layer of polymethacrylate.Such design protected the ZnO cores from decomposition by aqueous solutions on one hand,and assured the highly dispersity and long-term stability in water on the other hand.Cytotoxicity measurements proved this material is biocompatible and suitable for biological labels.The results of the laser confocal imaging showed these nanoparticles could penetrate the membranes of HeLa cells and exhibited stable fluorescence in the cytoplasm.

Fluorescent Modular Boron Systems Based on NNN- and ONO-Tridentate Ligands: Self-Assembly and Cell Imaging

We have synthesized a series of new fluorescent boron systems 1a-c and 2a-d based on nitrogen (NNN) or nitrogen and oxygen (ONO)-containing tridentate ligands. These novel dyes are characterized by high thermal and chemical stability. They show large Stokes shifts (mostly above 3200 cm(-1)) and quantum yields in solution and in the solid state up to 40%. The easy, modular synthesis facilitates the convenient variation of the axial substituent on the central boron atom, allowing the functionalization of this dye for biochemical use. Introducing a long alkyl chain with a phenyl spacer at this axial position enables the self-assembly of the boron compound 2d to form a fluorescent vesicle, which is able to encapsulate small molecules such as sulforhodamine. Additionally, boron compound 2d was found to serve as a dye for cell imaging since it has the capability of binding to the nuclear membranes of HeLa cells. With phospholipids such as DOPC, giant unilamelar vesicles (GUV) are formed. These results demonstrate the wide applicability of this new boron system in supramolecular and medicinal chemistry.

Dual modes of antitumor action of an amphiphilic peptide A9K

Following our recent report of attractive antibacterial properties of a designed amphiphilic peptide, A9K, we have investigated its antitumor activities by examining the modes of its action against different mammalian cell types. The peptide strongly inhibited the growth of cancerous HeLa cells and human promyelocytic leukemia HL60 cells whilst remaining benign to the host cells, including Cos 7 cells, mouse fibroblast NIH3T3 cells and human red blood cells. Images from SEM and fluorescence microscopy showed that A9K penetrated HeLa cell membranes and disrupted membrane structures, a feature broadly similar to that observed from its bactericidal actions. Further interactions of A9K with inner cellular membranes caused mitochondrial dysfunction associated with the F-actin reorganization and the decreased transcription of bcl-2 and c-myc genes, resulting in HeLa cell apoptosis in a mitochondria-induced apoptosis pathway. Thus A9K has high selectivity against cancerous cells and kills them by dual modes of action: membrane disruption and cell apoptosis. In addition, the peptide does not induce non-specific immunological effects and is not degraded by proteases. These features are crucial for developing their applications in future research.

Resistance of cervical adenocarcinoma cells (HeLa) to venom from the scorpion Centruroides limpidus limpidus.

BackgroundThe venom of Centruroides limpidus limpidus (Cll) is a mixture of pharmacologically active principles. The most important of these are toxic proteins that interact both selectively and specifically with different cellular targets such as ion channels. Recently, anticancer properties of the venom from other scorpion species have been described. Studies in vitro have shown that scorpion venom induces cell death, inhibits proliferation and triggers the apoptotic pathway in different cancer cell lines. Herein, after treating human cervical adenocarcinoma (HeLa) cells with Cll crude venom, their cytotoxic activity and apoptosis induction were assessed.ResultsCll crude venom induced cell death in normal macrophages in a dose-dependent manner. However, through viability assays, HeLa cells showed high survival rates after exposure to Cll venom. Also, Cll venom did not induce apoptosis after performing ethidium bromide/acridine orange assays, nor was there any evidence of chromatin condensation or DNA fragmentation.ConclusionsCrude Cll venom exposure was not detrimental to HeLa cell cultures. This may be partially attributable to the absence of specific HeLa cell membrane targets for molecules present in the venom of Centruroides limpidus limpidus. Although these results might discourage additional studies exploring the potential of Cll venom to treat human papilloma cervical cancer, further research is required to explore positive effects of crude Cll venom on other cancer cell lines.