Plant actin controls membrane permeability

The biological effects of electric pulses with low rise time, high field strength, and durations in the nanosecond range (nsPEFs) have attracted considerable biotechnological and medical interest. However, the cellular mechanisms causing membrane permeabilization by nanosecond pulsed electric fields are still far from being understood. We investigated the role of actin filaments for membrane permeability in plant cells using cell lines where different degrees of actin bundling had been introduced by genetic engineering. We demonstrate that stabilization of actin increases the stability of the plasma membrane against electric permeabilization recorded by penetration of Trypan Blue into the cytoplasm. By use of a cell line expressing the actin bundling WLIM domain under control of an inducible promotor we can activate membrane stabilization by the glucocorticoid analog dexamethasone. By total internal reflection fluorescence microscopy we can visualize a subset of the cytoskeleton that is directly adjacent to the plasma membrane. We conclude that this submembrane cytoskeleton stabilizes the plasma membrane against permeabilization through electric pulses.     

Nanosecond pulsed electric fields act as a novel cellular stress that induces translational suppression accompanied by eIF2α phosphorylation and 4E-BP1 dephosphorylation

Recent advances in electrical engineering enable the generation of ultrashort electric fields, namely nanosecond pulsed electric fields (nsPEFs). Contrary to conventional electric fields used for DNA electroporation, nsPEFs can directly reach intracellular components without membrane destruction. Although nsPEFs are now recognized as a unique tool in life sciences, the molecular mechanism of nsPEF action remains largely unclear. Here, we present evidence that nsPEFs act as a novel cellular stress. Exposure of HeLa S3 cells to nsPEFs quickly induced phosphorylation of eIF2α, activation of its upstream stress-responsive kinases, PERK and GCN2, and translational suppression. Experiments using PERK- and GCN2-knockout cells demonstrated dual contribution of PERK and GCN2 to nsPEF-induced eIF2α phosphorylation. Moreover, nsPEF exposure yielded the elevated GADD34 expression, which is known to downregulate the phosphorylated eIF2α. In addition, nsPEF exposure caused a rapid decrease in 4E-BP1 phosphorylation irrespective of the PERK/GCN2 status, suggesting participation of both eIF2α and 4E-BP1 in nsPEF-induced translational suppression. RT-PCR analysis of stress-inducible genes demonstrated that cellular responses to nsPEFs are distinct from those induced by previously known forms of cellular stress. These results provide new mechanistic insights into nsPEF action and implicate the therapeutic potential of nsPEFs for stress response-associated diseases.     

Enhanced Killing Effect of Nanosecond Pulse Electric Fields on PANC1 and Jurkat Cell Lines in the Presence of Tween 80

We investigated the effects of nanosecond pulse electric fields (nsPEFs) on Jurkat and PANC1 cells, which are human carcinoma cell lines, in the presence of Tween 80 (T80) at a concentration of 0.18?% and demonstarted an enhanced killing effect. We used two biological assays to determine cell viability after exposing cells to nsPEFs in the presence of T80 and observed a significant increase in the killing effect of nsPEFs. We did not see a toxic effect of T80 when cells were exposed to surfactant alone. However, we saw a synergistic effect when cells exposed to T80 were combined with the nsPEFs. Increasing the time of exposure for up to 8?h in T80 led to a significant decrease in cell viability when nsPEFs were applied to cells compared to control cells. We also observed cell type–specific swelling in the presence of T80. We suggest that T80 acts as an adjuvant in facilitating the effects of nsPEFs on the cell membrane; however, the limitations of the viability assays were addressed. We conclude that T80 may increase the fragility of the cell membrane, which makes it more susceptible to nsPEF-mediated killing.     

Nanosecond pulsed electric fields (nsPEF) induce direct electric field effects and biological effects on human colon carcinoma cells

Nanosecond pulsed electric fields (nsPEFs) are ultrashort pulses with high electric field intensity (kV/cm) and high power (megawatts), but low energy density (mJ/cc). To determine roles for p53 in response to nsPEFs, HCT116 cells (p53+/+ and p53-/-) were exposed to nsPEF and analyzed for membrane integrity, phosphatidylserine externalization, caspase activation, and cell survival. Decreasing plasma membrane effects were observed in both HCT116p53+/+ and p53-/- cells with decreasing pulse durations and/or decreasing electric fields. However, addition of ethidium homodimer-1 and Annexin-V-FITC post-pulse demonstrated greater fluorescence in p53-/- versus p53+/+ cells, suggesting a postpulse p53-dependent biological effect at the plasma membrane. Caspase activity was significantly higher than nonpulsed cells only in the p53-/- cells. HCT116 cells exhibited greater survival in response to nsPEFs than HL-60 and Jurkat cells, but survival was more evident for HCT116p53+/+ cells than for HCT116p53-/- cells. These results indicate that nsPEF effects on HCT116 cells include (1) apparent direct electric field effects, (2) biological effects that are p53-dependent and p53-independent, (3) actions on mechanisms that originate at the plasma membranes and at intracellular structures, and (4) an apparent p53 protective effect. NsPEF applications provide a means to explore intracellular structures and functions that can reveal mechanisms in health and disease.     

Ultrashort electric pulse induced changes in cellular dielectric properties

The interaction of nanosecond duration pulsed electric fields (nsPEFs) with biological cells, and the models describing this behavior, depend critically on the electrical properties of the cells being pulsed. Here, we used time domain dielectric spectroscopy to measure the dielectric properties of Jurkat cells, a malignant human T-cell line, before and after exposure to five 10ns, 150kV/cm electrical pulses. The cytoplasm and nucleoplasm conductivities decreased dramatically following pulsing, corresponding to previously observed rises in cell suspension conductivity. This suggests that electropermeabilization occurred, resulting in ion transport from the cell's interior to the exterior. A delayed decrease in cell membrane conductivity after the nsPEFs possibly suggests long-term ion channel damage or use dependence due to repeated membrane charging and discharging. This data could be used in models describing the phenomena at work.     

Eukaryotic localization, activation and ubiquitinylation of a bacterial type III secreted toxin

Type III secretion is a widespread method whereby Gram-negative bacteria introduce toxins into eukaryotic cells. These toxins mimic or subvert a normal cellular process by interacting with a specific target, although how toxins reach their site of action is unclear. We set out to investigate the intracellular localization of a type III toxin of Pseudomonas aeruginosa called ExoU, which has phospholipase activity and requires a eukaryotic factor for activity. We found that ExoU is localized to the plasma membrane and undergoes modification within the cell by addition of two ubiquitin molecules at lysine-178. A region of five amino acids at position 679-683 near the C-terminus of the ExoU protein controls both membrane localization and ubiquitinylation. Site-directed mutagenesis identified a tryptophan at position 681 as crucial for these effects. We found that the same region at position 679-683 was also required for cell toxicity produced by ExoU as well as in vitro phospholipase activity. Localization of the phospholipase ExoU to the plasma membrane is thus required for activation and allows efficient utilization of adjacent substrate phospholipids.     

Amino Acid Residues in the ω-Minus Region Participate in Cellular Localization of Yeast Glycosylphosphatidylinositol-Attached Proteins

The final destination of glycosylphosphatidylinositol (GPI)-attached proteins in Saccharomyces cerevisiae is the plasma membrane or the cell wall. Two kinds of signals have been proposed for their cellular localization: (i) the specific amino acid residues V, I, or L at the site 4 or 5 amino acids upstream of the GPI attachment site (the omega site) and Y or N at the site 2 amino acids upstream of the omega site for cell wall localization and (ii) dibasic residues in the region upstream of the omega site (the omega-minus region) for plasma membrane localization. The relationships between these amino acid residues and efficiencies of cell wall incorporation were examined by constructing fusion reporter proteins from open reading frames encoding putative GPI-attached proteins. The levels of incorporation were high in the constructs containing the specific amino acid residues and quite low in those containing two basic amino acid residues in the omega-minus region. With constructs that contained neither specific residues nor two basic residues, levels of incorporation were moderate. These correlations clearly suggest that GPI-attached proteins have two different signals which act positively or negatively in cell wall incorporation for their cellular localization.     

Cyclical regulation of the exocyst and cell polarity determinants for polarized cell growth

Polarized exocytosis is important for morphogenesis and cell growth. The exocyst is a multiprotein complex implicated in tethering secretory vesicles at specific sites of the plasma membrane for exocytosis. In the budding yeast, the exocyst is localized to sites of bud emergence or the tips of small daughter cells, where it mediates secretion and cell surface expansion. To understand how exocytosis is spatially controlled, we systematically analyzed the localization of Sec15p, a member of the exocyst complex and downstream effector of the rab protein Sec4p, in various mutants. We found that the polarized localization of Sec15p relies on functional upstream membrane traffic, activated rab protein Sec4p, and its guanine exchange factor Sec2p. The initial targeting of both Sec4p and Sec15p to the bud tip depends on polarized actin cable. However, different recycling mechanisms for rab and Sec15p may account for the different kinetics of polarization for these two proteins. We also found that Sec3p and Sec15p, though both members of the exocyst complex, rely on distinctive targeting mechanisms for their localization. The assembly of the exocyst may integrate various cellular signals to ensure that exocytosis is tightly controlled. Key regulators of cell polarity such as Cdc42p are important for the recruitment of the exocyst to the budding site. Conversely, we found that the proper localization of these cell polarity regulators themselves also requires a functional exocytosis pathway. We further report that Bem1p, a protein essential for the recruitment of signaling molecules for the establishment of cell polarity, interacts with the exocyst complex. We propose that a cyclical regulatory network contributes to the establishment and maintenance of polarized cell growth in yeast.     

Polyglutamine expansion disturbs the endoplasmic reticulum formation,leading to caspase-7 activation through Bax

The endoplasmic reticulum (ER) plays a pivotal role in cellular functions such as the ER stress response. However, the effect of the ER membrane on caspase activation remains unclear. This study reveals that polyglutamine oligomers augmented at ER induce insertion of Bax into the ER membrane, thereby activating caspase-7. In line with the role of ER in cell death induced by polyglutamine expansion, the ER membrane was found to be disrupted and dilated in the brain of a murine model of Huntington’s disease. We can conclude that polyglutamine expansion may drive caspase-7 activation by disrupting the ER membrane.     

Nanosecond pulsed electric fields have differential effects on cells in the S-phase

Nanosecond pulsed electric fields (nsPEFs) are a type of nonthermal, nonionizing radiation that exhibit intense electric fields with high power, but low energy. NsPEFs extend conventional electroporation (EP) to affect intracellular structures and functions and depending on the intensity, can induce lethal and nonlethal cell signaling. In this study, HCT116 human colon carcinoma cells were synchronized to the S-phase or remained unsynchronized, exposed to electric fields of 60 kV/cm with either 60-ns or 300-ns durations, and analyzed for apoptosis and proliferative markers. Several nsPEF structural and functional targets were identified. Unlike unsynchronized cells, S-phase cells under limiting conditions exhibited greater membrane integrity and caspase activation and maintained cytoskeletal structure. Regardless of synchronization, cells exposed to nsPEFs under these conditions primarily survived, but exhibited some turnover and delayed proliferation in cell populations, as well as reversible increases in phosphatidylserine externalization, membrane integrity, and nuclei size. These results show that nsPEFs can act as a nonligand agonist to modulate plasma membrane (PM) and intracellular structures and functions, as well as differentially affect cells in the S-phase, but without effect on cell survival. Furthermore, nsPEF effects on the nucleus and cytoskeleton may provide synergistic therapeutic actions with other agents, such as ionizing radiation or chemotherapeutics that affect these same structures.     

Yersinia pseudotuberculosis spatially controls activation and misregulation of host cell Rac1

Yersinia pseudotuberculosis binds host cells and modulates the mammalian Rac1 guanosine triphosphatase (GTPase) at two levels. Activation of Rac1 results from integrin receptor engagement, while misregulation is promoted by translocation of YopE and YopT proteins into target cells. Little is known regarding how these various factors interplay to control Rac1 dynamics. To investigate these competing processes, the localization of Rac1 activation was imaged microscopically using fluorescence resonance energy transfer. In the absence of translocated effectors, bacteria induced activation of the GTPase at the site of bacterial binding. In contrast, the entire cellular pool of Rac1 was inactivated shortly after translocation of YopE RhoGAP. Inactivation required membrane localization of Rac1. The translocated protease YopT had very different effects on Rac1. This protein, which removes the membrane localization site of Rac1, did not inactivate Rac1, but promoted entry of cleaved activated Rac1 molecules into the host cell nucleus, allowing Rac1 to localize with nuclear guanosine nucleotide exchange factors. As was true for YopE, membrane-associated Rac1 was the target for YopT, indicating that the two translocated effectors may compete for the same pool of target protein. Consistent with the observation that YopE inactivation requires membrane localization of Rac1, the presence of YopT in the cell interfered with the action of the YopE RhoGAP. As a result, interaction of target cells with a strain that produces both YopT and YopE resulted in two spatially distinct pools of Rac1: an inactive cytoplasmic pool and an activated nuclear pool. These studies demonstrate that competition between bacterial virulence factors for access to host substrates is controlled by the spatial arrangement of a target protein. In turn, the combined effects of translocated bacterial proteins are to generate pools of a single signaling molecule with distinct localization and activation states in a single cell.     

Comparative Study of Nanosecond Electric Fields In Vitro and In Vivo on Hepatocellular Carcinoma Indicate Macrophage Infiltration Contribute to Tumor Ablation In Vivo

Background and Aim

Recurrence and metastasis are associated with poor prognosis in hepatocellular carcinoma even in the patients who have undergone radical resection. Therefore, effective treatment is urgently needed for improvement of patients'' survival. Previously, we reported that nanosecond pulse electric fields (nsPEFs) can ablate melanoma by induction of apoptosis and inhibition of angiogenesis. This study aims to investigate the in vivo ablation strategy by comparing the dose effect of nanosecond electric fields in vitro and in vivo on hepatocellular carcinoma.

Materials and Methods

Four hepatocellular carcinoma cell lines HepG2, SMMC7721, Hep1-6, and HCCLM3 were pulsed to test the anti-proliferation and anti-migration ability of 100 ns nsPEFs in vitro. The animal model of human subdermal xenograft HCCLM3 cells into BALB/c nude mouse was used to test the anti-tumor growth and macrophage infiltration in vivo.

Results

In vitro assays showed anti-tumor effect of nsPEFs is dose-dependant. But the in vivo study showed the strategy of low dose and multiple treatments is superior to high dose single treatment. The macrophages infiltration significantly increased in the tumors which were treated by multiple low dose nsPEFs.

Conclusion

The low dose multiple nsPEFs application is more efficient than high dose single treatment in inhibiting the tumor volume in vivo, which is quite different from the dose-effect relationship in vitro. Beside the electric field strength, the macrophage involvement must be considered to account for effect variability and toxicology in vivo.     

Plasma membrane charging of Jurkat cells by nanosecond pulsed electric fields

The initial effect of nanosecond pulsed electric fields (nsPEFs) on cells is a change of charge distributions along membranes. This first response is observed as a sudden shift in the plasma transmembrane potential that is faster than can be attributed to any physiological event. These immediate, yet transient, effects are only measurable if the diagnostic is faster than the exposure, i.e., on a nanosecond time scale. In this study, we monitored changes in the plasma transmembrane potential of Jurkat cells exposed to nsPEFs of 60 ns and amplitudes from 5 to 90 kV/cm with a temporal resolution of 5 ns by means of the fast voltage-sensitive dye Annine-6. The measurements suggest the contribution of both dipole effects and asymmetric conduction currents across opposite sides of the cell to the charging. With the application of higher field strengths the membrane charges until a threshold voltage value of 1.4–1.6 V is attained at the anodic pole. This indicates when the ion exchange rates exceed charging currents, thus providing strong evidence for pore formation. Prior to reaching this threshold, the time for the charging of the membrane by conductive currents is qualitatively in agreement with accepted models of membrane charging, which predict longer charging times for lower field strengths. The comparison of the data with previous studies suggests that the sub-physiological induced ionic imbalances may trigger other intracellular signaling events leading to dramatic outcomes, such as apoptosis.     

Regulation of intracellular calcium concentration by nanosecond pulsed electric fields

Changes in [Ca²⁺]_i response of individual Jurkat cells to nanosecond pulsed electric fields (nsPEFs) of 60 ns and field strengths of 25, 50, and 100 kV/cm were investigated. The magnitude of the nsPEF-induced rise in [Ca²⁺]_i was dependent on the electric field strength. With 25 and 50 kV/cm, the [Ca²⁺]_i response was due to the release of Ca²⁺ from intracellular stores and occurred in less than 18 ms. With 100 kV/cm, the increase in [Ca²⁺]_i was due to both internal release and to influx across the plasma membrane. Spontaneous changes in [Ca²⁺]_i exhibited a more gradual increase over several seconds. The initial, pulse-induced [Ca²⁺]_i response initiates at the poles of the cell with respect to electrode placement and co-localizes with the endoplasmic reticulum. The results suggest that nsPEFs target both the plasma membrane and subcellular membranes and that one of the mechanisms for Ca²⁺ release may be due to nanopore formation in the endoplasmic reticulum.     

P-glycoprotein subcellular localization and cell morphotype in MDR1 gene-transfected human osteosarcoma cells.

Efflux of chemotherapy agents by P-glycoprotein at the plasma membrane is thought to be a major cause of cancer multidrug-resistance (MDR). However, the mechanism underlying the cellular accumulation and distribution of cytotoxic drugs is still poorly defined. We have recently found that P-glycoprotein is expressed also in the nucleus of MDR cell lines selected in doxorubicin (DXR), suggesting the possible involvement of this protein in the direct extrusion of the drug from the nucleus of resistant cells. In this study, we analyzed the subcellular localization of P-glycoprotein, in a series of U-2 OS osteosarcoma cell clones transfected with MDR1 gene in order to verify whether the nucleus is a constant site for the localization and functional activity of P-glycoprotein, and in which way some aspects of cell morphology related to MDR depend on the subcellular P-glycoprotein localization rather than on the exposure to the selective drug. Our results indicate that to achieve a subcellular drug distribution prevailing in the cytoplasm but not in the nucleus, a significant increase in the expression of P-glycoprotein at the different cellular compartments, including the plasma membrane, the cytoplasm, and the nucleus, is needed, although the in vitro drug resistance appears to be mainly dependent on the expression of P-glycoprotein at the cell surface. With regard to the morphological characteristics of MDR cells involving the cell surface and the chromatin arrangement, the influence of DXR appears to be prevalent, although P-glycoprotein overexpression cannot be excluded.     

Memo-RhoA-mDia1 signaling controls microtubules, the actin network, and adhesion site formation in migrating cells

Actin assembly at the cell front drives membrane protrusion and initiates the cell migration cycle. Microtubules (MTs) extend within forward protrusions to sustain cell polarity and promote adhesion site turnover. Memo is an effector of the ErbB2 receptor tyrosine kinase involved in breast carcinoma cell migration. However, its mechanism of action remained unknown. We report in this study that Memo controls ErbB2-regulated MT dynamics by altering the transition frequency between MT growth and shortening phases. Moreover, although Memo-depleted cells can assemble the Rac1-dependent actin meshwork and form lamellipodia, they show defective localization of lamellipodial markers such as α-actinin-1 and a reduced number of short-lived adhesion sites underlying the advancing edge of migrating cells. Finally, we demonstrate that Memo is required for the localization of the RhoA guanosine triphosphatase and its effector mDia1 to the plasma membrane and that Memo–RhoA–mDia1 signaling coordinates the organization of the lamellipodial actin network, adhesion site formation, and MT outgrowth within the cell leading edge to sustain cell motility.     

DNA electrophoretic migration patterns change after exposure of Jurkat cells to a single intense nanosecond electric pulse

Intense nanosecond pulsed electric fields (nsPEFs) interact with cellular membranes and intracellular structures. Investigating how cells respond to nanosecond pulses is essential for a) development of biomedical applications of nsPEFs, including cancer therapy, and b) better understanding of the mechanisms underlying such bioelectrical effects. In this work, we explored relatively mild exposure conditions to provide insight into weak, reversible effects, laying a foundation for a better understanding of the interaction mechanisms and kinetics underlying nsPEF bio-effects. In particular, we report changes in the nucleus of Jurkat cells (human lymphoblastoid T cells) exposed to single pulses of 60 ns duration and 1.0, 1.5 and 2.5 MV/m amplitudes, which do not affect cell growth and viability. A dose-dependent reduction in alkaline comet-assayed DNA migration is observed immediately after nsPEF exposure, accompanied by permeabilization of the plasma membrane (YO-PRO-1 uptake). Comet assay profiles return to normal within 60 minutes after pulse delivery at the highest pulse amplitude tested, indicating that our exposure protocol affects the nucleus, modifying DNA electrophoretic migration patterns.     

Intracellular localization of RORalpha is isoform and cell line-dependent

The retinoid-related orphan receptor alpha (RORalpha) belongs to the nuclear receptor superfamily and comprises four isoforms generated by different promotor usage and alternative splicing. To better understand its function, the subcellular distribution of RORalpha was investigated. We could show that subcellular distribution of RORalpha is cell line and isoform-dependent. Isoform specific differences were mediated by the A/B domains which with the exception of RORalpha1 contain a signal that mediates cytoplasmic localization. The lack of this signal in RORalpha1 results in a complete nuclear localization and prevents cell membrane association observed for RORalpha2, 3, and 4. The region responsible for membrane association was identified as the C-terminal alpha-helix 12. Furthermore, the hinge region/ligand binding domain mediates nuclear localization. Our results show that isoform specific activity of RORalpha is not only regulated by different expression and DNA binding affinities but also by different subcellular distribution. Different access to the nucleus reveals an important mechanism regulating the activity of this constitutively active nuclear receptor.     

Proteins in different Synechocystis compartments have distinguishing N-terminal features: a combined proteomics and multivariate sequence analysis

Cyanobacteria have a cell envelope consisting of a plasma membrane, a periplasmic space with a peptidoglycan layer, and an outer membrane. A third, separate membrane system, the intracellular thylakoid membranes, is the site for both photosynthesis and respiration. All membranes and luminal spaces have unique protein compositions, which impose an intriguing mechanism for protein sorting of extracytoplasmic proteins due to single sets of translocation protein genes. It is shown here by multivariate sequence analyses of many experimentally identified proteins in Synechocystis, that proteins routed for the different extracytosolic compartments have correspondingly different physicochemical properties in their signal peptide and mature N-terminal segments. The full-length mature sequences contain less significant information. From these multivariate, N-terminal property-profile models for proteins with single experimental localization, proteins with ambiguous localization could, to a large extent, be predicted to a defined compartment. The sequence properties involve amino acids varying especially in volume and polarizability and at certain positions in the sequence segments, in a manner typical for the various compartment classes. Potential means of the cell to recognize the property features are discussed, involving the translocation channels and two Type I signal peptidases with different cellular localization, and charge features at their membrane interfaces.