1Department of Internal Medicine and Biomedical Engineering, University of Iowa, Iowa City 52242; 2Department of Mechanical, Aerospace, and Biomedical Engineering, University of Tennessee, Knoxville, Tennessee 37996; and 3Department of Electrical and Computer Engineering, University of Iowa, Iowa City, Iowa 52242
Submitted 26 November 2003 ; accepted in final form 3 June 2004
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ABSTRACT |
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adhesion; motility; computational modeling; focal contact; quantitation
The cell cytoskeleton is critical to the viral life cycle. Several viruses enter, replicate, and are released from infected cells by exploiting and remodeling the host cells cytoskeleton (5, 27, 45). Viruses exploit microtubules, microfilaments, and motor proteins to facilitate viral transport to the nucleus or perinuclear region for replication. Viral assembly and release during the lytic phase for several viruses such as herpes simplex, human immunodeficiency virus, adenovirus, and vaccinia virus are dependent on antegrade movement along microtubules and microfilaments (5, 31). Adenoviral proteinases are activated only after adenovirus first binds to the carboxy-terminal region on the actin cytoskeleton (8). Finally, adenovirus disassembles the actin cytoskeleton, which impairs cytoskeletal membrane stability (27). Agents that target the host cells actin cytoskeleton may interfere with virus-mediated cell injury through structural cytoskeletal and nonstructural mechanisms. Thus approaches that target the actin cytoskeleton represent a potential therapeutic strategy in treating viral infections.
Pharmacological remodeling of the actin cytoskeleton alters the virulence of several viruses. Cytochalasin inhibits viral infections under in vitro conditions (15, 26, 32, 48). However, cytochalasin causes significant cell toxicity, which prevents its clinical use as an antiviral agent. The actin cytoskeleton regulates a diverse series of housekeeping functions such as mitosis, cytokinesis, contraction, cell motility, and barrier function (6, 11, 13, 14, 17, 18, 21, 22, 24, 25, 3638, 40, 42, 43, 49). Thus antiviral therapies that target the actin cytoskeleton have to balance efficacy against perturbing housekeeping cellular functions.
Motor proteins for regulating actin-myosin contraction in smooth muscle and nonmuscle cells are dependent on phosphorylation of the regulatory myosin light chain (MLC). Phosphorylation of MLC in smooth muscle and nonmuscle cells is regulated by calcium-calmodulin activation of myosin light chain kinase (MLCK), which increases the actin-myosin ATPase activity (13, 12, 39). In addition, the GTPase Rho also regulates phosphorylation of MLC by activating Rho-kinase, which, in turn, phosphorylates MLC (4). GTPase Rho also phosphorylates the myosin-binding subunit of MLC phosphatase, which inhibits MLC dephosphorylation (29). A decrease in steady-state dephosphorylation leads to unopposed MLC phosphorylation, which increases myosin ATPase activity. MLC is phosphorylated by other kinases (28, 44), which adds to the redundancy of signaling pathways that activate myosin motors. Because there is a redundancy of upstream signaling pathways that activate myosin motors, pharmacological inhibitors that interfere with these pathways may represent ineffective strategies that interfere with virus-mediated cell injury and can pose unrealized dose-dependent toxic side effects.
The actin-binding protein caldesmon (CaD) also regulates actin-myosin contraction. Caldesmon exists in two forms: h-CaD, a high-molecular-weight form (120,000150,000 kDa) that is predominately expressed in smooth muscle, and l-CaD, a low-molecular-weight form (70,00080,000 kDa) that is predominately expressed in nonmuscle cells (34, 35). l-CaD is structurally similar to h-CaD except that it lacks a central repeating region. l-CaD is distributed along stress fibers in nonmuscle cells and is colocalized with tropomyosin (50). Unphosphorylated l-CaD constitutively inhibits the myosin ATPase activity (30, 47), and phosphorylation of l-CaD under in vitro conditions increases myosin ATPase activity through dysinhibition of l-CaD. However, it is not well documented that l-CaD inhibits myosin ATPase in intact cells. Because myosin ATPase activity is mechanically coupled to cell adhesion sites (37, 40) and can stabilize the actin cytoskeleton, l-CaD overexpression may represent a potential strategy that could preserve actin stability and cellular integrity in response to viral pathogens. Furthermore, gene delivery of l-CaD represents a potential targeted therapy to prevent viruses from exploiting the actin cytoskeleton and myosin motors without interfering with generalized upstream signal transduction pathways that could affect a diversity of other important cellular functions.
This study evaluated whether gene delivery of wild-type human l-CaD inhibits any adenovirus-mediated cell membrane properties and whether it achieves this benefit with less cell toxicity than cytochalasin. These studies were performed using a DF-1 cell line because it is a spontaneously immortalized but nontransformed avian fibroblast line that is null for l-CaD. The use of DF-1 cells as a knockout cell line permits accurate quantification of transfection efficiency. Heterologous human wild-type l-CaD was transiently expressed and localized with avian microfilaments in DF-1 cells by using a replication-deficient adenovirus expression system, which allowed the dual study of adenoviral infection and l-CaD incorporation in these cells. A number of quantitative and dynamic biophysical assays were performed and integrated with molecular biology approaches to evaluate whether gene delivery of wild-type human l-CaD could inhibit any cytopathic effect of adenovirus in cultured fibroblast function with less cell toxicity than cytochalasin. A novel numerical model based on the experimental transcellular impedance across a cell-covered electrode was used to evaluate the impact of l-CaD on several cytoskeletal membrane properties. Also, a series of interdisciplinary molecular and bioengineering studies were performed to evaluate the mechanism of this protection.
This study demonstrates that gene delivery of heterologous human wild-type l-CaD affords protection of cell membrane integrity against an adenoviral infection even at high multiplicity-of-infection (MOI) dosages. A unique aspect of this report is that low transfection efficiency of l-CaD gene delivery proved effective in stabilizing cell membrane integrity in the face of a competing adenovirus infection through a mechanism independent of actin assembly and myosin ATPase activity. In addition, gene delivery of l-CaD attenuated these adenovirus-mediated effects with less cell toxicity than cytochalasin.
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MATERIALS AND METHODS |
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Cell lysis and total RNA extraction. Approximately 4 x 106 cultured HUVEC were lysed with Sigma TRI Reagent lysis solution (Sigma-Aldrich, St. Louis, MO), and total RNA was extracted according to the manufacturers instructions. Lysate was incubated at room temperature with reagent chloroform, incubated at room temperature for 15 min, and then centrifuged at 12,000 g for 15 min at 4°C. The aqueous phase was removed and mixed with isopropyl alcohol, incubated at room temperature for 10 min, and then centrifuged at 12,000 g for 10 min at 4°C. The RNA pellet was washed with 75% ethanol and then centrifuged at 7,500 g for 5 min at 4°C. The RNA pellet was then air dried at room temperature and resuspended in diethyl pyrocarbonate-treated water and quantitated using a spectrophotometer with settings based on the ratio of absorbance at 260 and 280 nm (A260/A280) and by formaldehyde-agarose gel electrophoresis.
Reverse transcription and polymerase chain reaction. HUVEC total RNA was amplified in a one-step reverse transcriptase-polymerase chain reaction by using the Access RT-PCR system according to the manufacturers instructions (Promega, Madison, WI). Specific primers corresponding to the full-length HUVEC l-CaD were designed with BamHI restriction sites; the sense primer sequence was 5'-GGATCCATGGATGATTTTGAGCGTCG-3', and the antisense primer was 5'-GGATCCAACCTTAGTGGGGGAAGTGA-3'. Avian fibroblast total RNA was amplified using the same technique; the sense primer sequence was 5'-ATGATCAGCAGATCATACTGCAGG-3', and the antisense primer was 5'-CTACGGTTCTTTCTCAAATTGTCT-3'.
HUVEC l-CaD subcloning and adenovirus vector construction.
The RT-PCR product was ligated overnight at 14°C by using the pGEM-T-Easy vector according to the manufacturers instructions (Promega). The PCR product was ligated with T4 DNA ligase. Competent DH5 Escherichia coli bacteria were transformed by standard heat shock methods and plated on LB-agar plates containing 100 µg/µl ampicillin. Positive transformants were selected and grown in 5 ml of liquid culture containing ampicillin. Bacterial culture was subjected to standard alkaline-lysis miniprep DNA isolations. Plasmid and insert orientation was examined by BamHI restriction digestion and confirmed by Sanger dideoxy DNA sequencing. Positive transformants were scaled up in 1 liter of LB medium containing ampicillin, and plasmid DNA isolation was then performed using the Qiagen MaxiPrep kit (Qiagen, Hilden, Germany).
pGEM-T-CaD was digested using BamHI and separated on a 1% TAE (Tris-acetate-EDTA)-agarose gel. The l-CaD band was isolated using the Bio-Rad Quantum Prep gel slice kit (Bio-Rad Laboratories, Hercules, CA). The DNA insert was then ligated to pacAd5CMV, an adenovirus shuttle vector (University of Iowa Gene Vector Core Facility) using the BamHI restriction site. Adenovirus virions were produced from HEK-293 cells, purified, and titered at the University of Iowa Gene Vector Core Facility. The vector was under control of a cytomegalovirus (CMV) promoter.
Transfection protocol. Cultured DF-1 cells were transiently transfected with recombinant, replication-deficient adenovirions expressing wild-type l-CaD (Ad-l-CaD) by using a calcium phosphate (CaPi) coprecipitation procedure described by Fasbender et al. (16). Briefly, recombinant adenovirus particles [with plaque-forming units according to the final MOI] were incubated with Eagles minimal essential medium at a pH of 8.0, containing 1.8 mM Ca2+ and 0.86 mM inorganic phosphate. An aliquot of 2 M CaCl2 was also added to this solution to increase the Ca2+ concentration. Culture medium was aspirated, and the coprecipitate was exposed to cells for 30 min at room temperature. Cells were then immediately washed with fresh medium and then washed daily for 2 days. DF-1 cells were grown in DMEM in 10% fetal bovine serum at 39°C. Cultured cells were transfected at doses between MOI 10 and 500. After 48 h, cells were detached for use in different experiments.
For evaluation of transfection efficiency, cultured cells were fixed with 3.7% paraformaldehyde, permeabilized with 0.1% Triton X-100, and sequentially exposed to a mouse IgG anti-l-CaD primary antibody (5 µg/ml dilution; Transduction Laboratories), an Oregon green-conjugated goat anti-mouse secondary antibody (10 µg/ml), and Texas red-phalloidin (1 unit/slide). Cells were viewed with a Zeiss Axiovert 135 TV microscope equipped with epifluorescence using rhodamine or fluorescein excitation filters and a x40/0.75 Plan Neofluar objective. Images were captured with a 10-bit digital camera (ORCA; Hamamatsu), and images were computer digitized and processed with custom algorithms written in LabVIEW and IMAQ Vision software (National Instruments, Austin, TX). Groups of 100 cells, which were identified by Texas red staining of microfilaments, were evaluated for the presence of Oregon green-labeled l-CaD-decorated microfilaments. The transfection efficiency was defined as the fraction of l-CaD-decorated microfilaments out of the total number of actin-stained cells.
Images for colocalization of l-CaD with microfilaments were captured using a x100 Plan Neofluar objective. Images containing Oregon green-labeled l-CaD filaments were assigned a green pseudocolor using Adobe Photoshop, whereas images containing Texas red-labeled actin filaments were assigned a magenta pseudocolor. Images of green and magenta filters were registered, and colocalization of l-CaD to the actin cytoskeleton was determined by the resulting formation of white or lighter magenta-colored filaments.
In vitro wound closure assay to measure cell motility. DF-1 cell motility was measured by wounding the cell monolayer and measuring wound closure by monitoring the distance traversed over time. Cells were transfected with Ad-l-CaD or Ad-empty (empty vector) and allowed to grow to 100% confluence (2 days posttransfection). A wound was made in the monolayer with the use of a sterile pipette tip with an outer diameter of 300 µm. The same area was captured sequentially at time intervals of 0, 1, 3, and 6 h under phase-contrast microscopy by using a diamond-etched mark as a reference. The migrated distance was assessed at three locations that were separated by equal distance. Cell motility was determined as the average velocity for the three measurements for each time point. Replicate experiments were performed, and cell motility was averaged for each time period and for each condition. Unpaired Students t-test and analysis of variance were performed to compare intrasubject and intersubject differences.
Transcellular impedance measurements.
Quantitative transcellular impedance measurements were acquired in real time by using a previously reported technique of inoculating cultured cells grown on the surface of microelectrodes coated with 100 µg/ml fibronectin (38, 40, 41). A 1-V, 4,000-Hz alternating current signal was supplied through a 1-M resistor to approximate a constant current source. Voltage and phase data were measured with an SR830 lock-in amplifier (Stanford Research Systems, Sunnyvale, CA) and later stored and processed with a personal computer. The in-phase voltage, proportional to the resistance, and the out-of-phase voltage, proportional to the capacitive reactance, were measured. For most situations, resistance is reported because it is most sensitive to cytoskeletal membrane properties. The resistance of the naked electrode was subtracted from the resistance of a cell-covered electrode to control for variance in biosensor properties. A uniform number of viable cells (
650,000 cells/sample), as determined by the amount of cells that excluded Trypan blue, were inoculated on the electrode to control for the effect of cell viability. Transcellular resistance was expressed as either the average total transcellular resistance as a function of time or the fractional change at 15 h normalized to the cell resistance of wild-type DF-1 cells treated with mock CaPi precipitation.
Breakdown of cytoskeletal membrane properties using numerical modeling of experimental transcellular impedance.
A numerical analysis was used to calculate specific cell-cell and cell-substrate adhesion and membrane capacitance based on the measured transcellular impedance. The procedure is described in more detail elsewhere for endothelial cells (37, 41), fibroblasts (19), and epithelial cells (33). The total impedance across a cell-covered electrode is composed of the impedance created between the ventral surface of the cell and the electrode (related to ; due to cell-matrix adhesion), the impedance created between cells (indicated as Rb; due to cell-cell adhesion), the transcellular impedance created from transcellular current conduction (Zm), and the impedance of a naked electrode (Zn). For these calculations, Zm is inversely related to membrane capacitance (Cm), which is dependent on membrane convolution, which, in turn, is dependent on the cortical cytoskeleton. The data were expressed as a ratio of the real or imaginary measurement (Zc) of a cell-covered electrode to that of a naked electrode as a function of current frequency between 25 and 60,000 Hz. A calculated real and imaginary value (Zs) was generated from the solutions of
, Rb, and Cm obtained from a multiresponse Levenberg-Marquardt nonlinear optimization model of the real, imaginary, real and imaginary (in complex form), and real and imaginary (in magnitude form) data. An error evaluation was calculated using a
2 analysis of the squared sum of the calculated and the experimental residuals as a function of current frequency. Solutions of
, Rb, and Cm were obtained by selecting the frequency subset (5,00060,000 Hz) and Levenberg-Marquardt approach that best approximated the experimental data determined by curve fitting and
2 analysis.
Quantitation of focal contact mass. DF-1 cells were plated at 70% confluence on glass coverslips coated with 100 µg/ml fibronectin. The next day, cells were transfected with Ad-l-CaD or Ad-empty at a dose of MOI 200. Two days posttransfection, cells were fixed in PBS containing 3.7% formaldehyde for 10 min. Cells were then permeabilized with 0.1% Triton X-100 and sequentially exposed to a mouse IgG anti-vinculin primary antibody (1:75 dilution) and an Oregon green-conjugated goat anti-mouse secondary antibody (10 µg/ml). Cells were viewed with a Zeiss Axiovert 135 TV microscope equipped with epifluorescence using fluorescein excitation filters and a x100 Plan Neofluar objective.
Images were captured with a 10-bit digital camera (ORCA; Hamamatsu) and were computer digitized and processed with custom algorithms written with the LabVIEW and IMAQ Vision software language for the Macintosh computer (National Instruments). Immunolabeled vinculin focal contacts were isolated by filtering the original images, thresholding the filtered images, and closing (dilation followed by an erosion) the thresholded images to help join plaque outlines by connecting broken lines and filling in unwanted holes. The resulting images were used as masks over the original images to quantitate total focal contact area and total pixel intensity of vinculin-labeled plaques. With the use of a nonlinear high-pass filter, the focal contact outlines and total cell area outlines were identified on the processed images and overlaid with the original images for comparison and display in monolayers transfected with the Ad-l-CaD or Ad-empty construct. Focal contact mass was expressed using several criteria: mean pixel area/plaque, total focal contact area (pixels)/total cell area (pixels), total plaque intensity/total cell intensity, total number of plaques/total cell area (pixels), and [total focal contact area (pixels) x total focal contact intensity]/[total cell area (pixels) x total cell intensity].
Measurement of prestress force. Prestress was determined by the measured loss of constitutive isometric tension in response to cytochalasin D. Isometric tension was measured in cultured DF-1 cells inoculated on the surface of polymerized type 1 collagen membranes as described by Bodmer et al. (7). Isometric tension was simultaneously monitored in two separate isometric vectors to account for the presence of anisotropic tension. Constitutive tension was measured in cell-collagen lattices under unloaded conditions. To determine the optimal length-tension relationship at the unloaded state, the collagen gel was stretched with a micromanipulator that was attached to the transducer until a 10-mg increase in force was observed. The gel was progressively unloaded until no further decline in preload was observed. The length (l0) of the gel was chosen as the no-load state. After a steady-state baseline was achieved, a sham response composed of the carrier buffer for cytochalasin D was used to assess for nonspecific mechanical effects. Cells were subsequently exposed to 3 µM cytochalasin D, and the abolished constitutive tension was quantitated in real time. The constitutive tension was defined as the measured loss in tension upon the addition of cytochalasin D.
Measurement of actin assembly. Actin assembly was quantitated by the fraction of insoluble actin in a Triton X-100-based buffer as previously described by Carson et al. (9) with the following modifications. The soluble actin fraction was precipitated by adding ice-cold TCA to a final concentration of 10%, incubated on ice for 30 min, and spun for 15 min at 14,000 rpm at 4°C. The pellet was then washed three times in ice-cold acetone and resuspended in 2x SDS sample buffer to a volume equal to the final Triton-insoluble fraction volume defined below. The cytoskeleton remaining on the dish was solubilized with 2% SDS, 0.5% 2-mercaptoethanol, and 100 mM NaCl, sheered through a 26-gauge needle, and centrifuged at 100,000 g for 15 min. The supernatant from this centrifugation was combined with the pellet saved from the Triton-soluble fraction, and then an equal volume of 2x SDS sample buffer was added; this fraction was defined as the Triton-insoluble fraction. Equivalent volume fractions of the soluble and insoluble fractions were subjected to electrophoresis on an 815% SDS polyacrylamide gradient gel, along with known concentrations of purified nonmuscle actin (Cytoskeleton, Denver, CO). The gel was stained with Coomassie blue and dried, and actin content was quantitated by laser densitometry. The concentration of actin in each cell fraction was quantitated from an in vitro standard calibration curve of the measured radiance volume of known amounts of purified nonmuscle actin separated on the same gel.
Cell viability assay. Cultured cells were transfected with Ad-l-CaD or the controlled virus by using a CaPi coprecipitation technique. Cells were washed free of virus and incubated for an additional 48 h in serum-containing medium. Transfected cells were exposed to Trypan blue dye, and the fraction of viable cells was quantitated. Viability of cells transfected with Ad-l-CaD and controlled virus was compared with the cell viability measured in monolayers exposed to cytochalasin for the same corresponding period.
Statistical analysis.
Data are reported as means ± SE. Comparisons between groups were made using an unpaired Students t-test. Comparisons between more than two groups were made using analysis of variance (ANOVA). Individual group comparisons were done using Tukeys honest significant difference test for post hoc comparison of means. Differences were considered significant at the P 0.05 level.
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RESULTS |
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Transfection and expression of human l-CaD in DF-1 cells using an adenovirus expression system. HUVEC cDNA of l-CaD amplified by RT-PCR procedures was subcloned into an adenovirus shuttle vector. Recombinant deficient subtype 5 adenovirus (Ad-l-CaD) was prepared from HEK-293 cells with l-CaD transcription regulated by a CMV promoter. Cultured DF-1 cells were transfected with Ad-l-CaD by using a CaPi coprecipitation protocol as described in MATERIALS AND METHODS. Figure 2 shows that transfection of cultured DF-1 cells with Ad-l-CaD caused a dose-dependent increase in protein expression of human l-CaD between MOI 10 and 1,000 on the basis of Western blot analysis.
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Figure 7C shows the effects of cytochalasin D on , Rb, and Cm in wild-type DF-1 cells exposed to cytochalasin D. Cytochalasin D mediated a decrease in
and Rb in cultured DF-1 cells, indicating that cytochalasin D decreased cell-cell and cell-matrix adhesion. A statistically significant increase in Cm was also observed in response to cytochalasin D, indicating that cytochalasin D decreased the experimental transcellular resistance by also increasing membrane capacitance. Thus disassembly of the actin cytoskeleton decreased transcellular resistance by lowering cell-cell and cell-matrix adhesion and by increasing membrane capacitance, which was consistent with the pattern observed in cells exposed to only adenovirus.
A statistically significant lower Cm was observed in cells expressing l-CaD than in cells transfected with the controlled virus, based on ANOVA performed on a Tukeys multiple comparison (Fig. 7A). There was no statistical difference between Cm in cells expressing l-CaD and in wild-type CaPi-treated cells, indicating that expression of l-CaD rescued the effects of adenovirus on membrane capacitance. There was, however, no significant difference in the mean value of between monolayers transfected with Ad-l-CaD and either mock-transfected or controlled virus-treated monolayers. These data suggest that expression of l-CaD mediated a modest rescue of adenovirus-induced reduction in cell-matrix adhesion. The measured mean Rb values between cells transfected with Ad-l-CaD and controlled virus were not statistically different, whereas there was a statistically significant difference in the mean Rb value between cells expressing either l-CaD or controlled virus and wild-type CaPi-treated cells. These data suggest that expression of l-CaD did not rescue adenovirus-mediated reduction in cell-cell adhesion. Taken together, these data indicate that l-CaD expression quantitatively attenuated adenovirus-mediated loss in transcellular resistance predominately by decreasing membrane capacitance, with modest effects on cell-matrix adhesion. Cells exposed to controlled adenovirus experienced an increase in cell death compared with wild-type cells (Fig. 7D). Expression of wild-type l-CaD mediated a modest additional increase in cell death compared with cells exposed to controlled adenovirus, although this did not achieve statistical significance. In contrast, exposure of cells to cytochalasin D for the same time period decreased cell viability in a statistically significant fashion compared with cells exposed to Ad-l-CaD or controlled virus.
Because the numerical model predicted modest rescuing in in cells transfected with the Ad-l-CaD construct, we further tested whether changes in cell-matrix adhesion could be explained, in part, by a change in focal contact mass. Monolayers treated with Ad-empty or Ad-l-CaD were immunolabeled for vinculin, and images were processed using custom-written algorithms. Individual and total focal contact mass measurements were expressed according to several criteria as described in MATERIALS AND METHODS. Focal contact mass measurements were then compared between cells treated with Ad-empty and Ad-l-CaD. Figure 8 depicts a processed image sequence of Ad-l-CaD-treated cells immunolabeled for vinculin. Figure 8A shows the original image, which was then filtered to identify edge-detected focal contact plaques (Fig. 8B). Next, the filtered image was thresholded to create a binary image, and then plaques were closed by a dilation and erosion process (Fig. 8C). Figure 8D shows the outlines of focal contact plaques (green) and cell boundaries (magenta) generated by the image-processing algorithms overlaid on the original image from Fig. 8A. The areas where focal contact and cell boundary overlap are outlined in white.
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DISCUSSION |
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Cytochalasin inhibits viral infections under in vitro conditions. However, cytochalasin induces significant drug toxicity, which precludes its use as a clinical antiviral agent. Thus gene delivery of whole or partial cytoskeleton proteins or the delivery of small molecules that antagonize or mimic specific cytoskeleton properties represents an ideal strategy to inhibit the virus-mediated cell injury if three requirements can be satisfied. First, gene delivery should be effective against high virus loads. Second, the antiviral effect of gene therapy should be potent at low transfection efficiency, which is a logistical consideration when contemplating gene delivery under in vivo conditions. Last, gene delivery of cytoskeletal targets should not unduly interfere with cell housekeeping functions. In this study, we demonstrated that gene delivery of human wild-type l-CaD inhibits structural cytoskeletal membrane dysfunction induced by adenovirus at low transfection efficiency with much less cell toxicity than cytochalasin.
An adenovirus delivery system expressing human wild-type l-CaD in DF-1 cells was used to precisely quantitate transfection efficiency, evaluate the impact and mechanism by which gene delivery of l-CaD attenuated adenovirus-mediated cell injury, and evaluate whether gene delivery had a significant impact on housekeeping functions like cell viability and wound repair. Human cDNA of l-CaD was cloned from cultured HUVEC and subcloned into an adenovirus expression system to achieve sufficient transfection efficiency. The efficacy and safety of gene delivery of l-CaD in attenuating the cytopathic effect of adenovirus was assessed using a number of quantitative and direct biophysical, computational, biochemical, and microscopic approaches. The level of transfection efficiency that was necessary to detect changes in cytoskeletal membrane properties was precisely evaluated, and it was determined whether myosin ATPase activity and actin assembly regulated such changes. Constitutive myosin ATPase activity was quantitatively and dynamically measured by recording the loss in isometric tension in response to cytochalasin D. Levels of actin assembly were measured by quantitating the amount of soluble and insoluble actin in cell lysates. Cytoskeletal membrane properties were also measured by quantitating changes in transcellular resistance, a technique that has been reported in cultured endothelial cells (38, 40), cultured fibroblasts (19, 20), and cultured epithelial cells (33). The cytoskeletal membrane regions that human l-CaD affects were specifically localized using a numerical model that breaks down the experimental transcellular resistance into independent parameters of cell-cell adhesion, cell-matrix adhesion, and membrane capacitance that was previously reported for endothelial cells (37, 41) and was originally developed for cultured fibroblasts (19).
We confirmed that DF-1 cells are null for l-CaD protein and mRNA expression on the basis of Western blot and RT-PCR procedures. The absence of l-CaD protein expression was attributed to gene knockout mRNA expression. CaPi coprecipitation with Ad-l-CaD mediated a dose-dependent increase in transfection efficiency and protein expression. More importantly, transfection with Ad-l-CaD mediated a colocalization of human wild-type l-CaD with avian microfilaments. These data demonstrate that this cell model system fulfills the criteria to precisely characterize the mechanical properties of human l-CaD under in situ conditions in the presence of adenovirus.
Adenovirus decreased transcellular resistance in confluent cultured DF-1 cells in a dose-dependent fashion. The decline in transcellular resistance indicates that adenovirus mediates a dose-dependent decline in cell-cell adhesion, cell-matrix adhesion, or transmembrane impedance (41). A decrease in transmembrane impedance is inversely proportional to an increase in membrane capacitance, which, in turn, is proportional to the amount of membrane convolution (41). Membrane convolution, in turn, is dependent on cortical cytoskeletal membrane properties. To resolve the spatial effects of adenovirus on cytoskeletal membrane rheology, a mathematical model previously reported and validated in endothelial cells (37, 41) and originally reported in fibroblasts (19) and epithelial cells (33) was used.
Based on the model, adenovirus decreased transcellular resistance by decreasing cell-cell and cell-matrix adhesion and by increasing membrane capacitance. The notion that these membrane properties are dependent on the actin cytoskeleton is supported by the association between actin disassembly and changes in , Rb, and Cm upon exposure to adenovirus. In addition, the dependence of the actin cytoskeleton on these parameters is illustrated by the similar decline in
and Rb and increase in Cm in response to cytochalasin D.
Expression of l-CaD attenuated the adenovirus-mediated loss in transcellular resistance. Using the same numerical modeling approach, we found that expression of l-CaD had the most impact on transcellular resistance by rescuing adenovirus-mediated increase in membrane capacitance. The effect of l-CaD on membrane capacitance suggests that l-CaD stabilized the cortical actin cytoskeleton, which, in turn, stabilized membrane convolution. Expression of l-CaD had limited effects on rescuing adenovirus-mediated reduction in cell-cell adhesion. Expression of l-CaD had an intermediate rescuing effect on adenovirus-mediated reduction in cell-matrix adhesion.
The loss in transcellular resistance seen in transfected cells was controlled for effects on cell viability. These effects were specifically controlled for by inoculating a confluent density of cells based on a consistent number of cells determined by Trypan blue exclusion. The resistance of the naked electrode was also subtracted and expressed as a function of the culture monolayer only, which eliminated the variability introduced by differences in electrode fabrication. The resistance at the 15-h time point was also compared, which reflected a steady-state time point at which cells were well attached and spread over the electrode. Last, the transcellular resistance was normalized to the resistance level of wild-type CaPi-treated cells to control for variability in cell culture behavior by culture passaging. The failure to observe a statistical difference in the measured values of Rb and between controlled cells and cells expressing l-CaD could be due to data variance. Data variance is due to biological and nonbiological factors; the latter include issues such as electrical noise, temporal changes in the electrical properties of the sensor, and model bias. However, these nonbiological factors are generally influenced more at low-frequency currents and can be limited by selecting a higher frequency current bandwidth. Numerical stability of the solution parameters provided by the model is critical, and confidence levels, error parameters, and methods to assess model stability are important factors in evaluating the strength of any numerical modeling approach. These criteria were achieved by developing a multiresponse Levenberg-Maquardt nonlinear optimization model of the real, imaginary, real and imaginary (in complex form), and real and imaginary (in magnitude form) data and by calculating a normalized
2 analysis for the solution parameters. These error parameters and the different Levenberg-Marquardt approaches provide greater intuition regarding the stability of the model solutions, and they provide a mechanism to evaluate whether data were corrupted. Future developments must include numerical and computational algorithms to make further corrections for effects of electrical noise, model bias, and temporal changes in sensor properties.
Changes in membrane capacitance were detected in cultured monolayers upon coprecipitation of Ad-l-CaD at transfection efficiency as low as 2025%. The lower requirement for transfection efficiency is encouraging because it suggests that low transfection efficiency may be sufficient to attenuate an adenovirus infection under in vivo conditions.
Because there was a modest rescue in adenovirus-mediated reduction in total cell-matrix adhesion in cells transfected with the Ad-l-CaD construct, we also evaluated whether some of these functional changes based on the numerical model could be associated with changes in focal contact mass. Whereas there was no increase in the average plaque size, normalized total number of plaques, and total focal contact area, there was an increase in the normalized total focal contact intensity of immunolabeled vinculin plaques in cells transfected with Ad-l-CaD. These results suggest that expression of l-CaD increased the total mass of focal contacts independently of increasing plaque area or number.
These observations are inconsistent with those of Helfman et al. (23), who reported a decrease in focal contacts in mammalian fibroblasts overexpressing l-CaD. There are several explanations to account for these differences. There may be differences in behavior between mammalian and avian fibroblasts. Yet, there were significant methodological differences in the manner in which focal contact was measured. As reported by Helfman et al., focal adhesion was indirectly defined by the distribution of cells that met a focal contact size criterion, whereas in our report, focal contact mass was directly quantitated and included size, number, and intensity determinants and was normalized as a function of cell area or total cellular intensity.
It is important to note that focal contacts represent only 1316% of the total cell-covered area, leaving a large portion of cell-covered area excluded from the calculation. The determinant reflects the average separation distance between substrate and cell and includes an integration of both focal and nonfocal contact adhesion (41). Taken together, these results suggest that inclusion of nonfocal contacts may be necessary to completely evaluate cytoskeletal cell-substrate interactions to understand the role of focal contact and nonfocal contact adhesion on the
determinant. These data suggest that l-CaD expression in DF-1 cells has a direct antagonistic effect on adenovirus-mediated loss in transcellular resistance by modulating membrane capacitance, possibly by stabilizing the actin cytoskeleton and adhesion plaques.
An important aspect of the present study is that gene delivery of l-CaD did not adversely affect static and dynamic housekeeping cellular functions, as did cytochalasin D. Expression of wild-type l-CaD had no significant impact on wound closure in response to mechanical wounding. There was no observed significant difference in cell velocity upon wound closure between cells expressing l-CaD and controlled cells. In contrast, cytochalasin D completely prevented wound repair. Gene delivery of l-CaD also caused less cell toxicity, based on Trypan blue dye exclusion, than that observed in response to cytochalasin during the same exposure period. Surgucheva et al. (46) reported inconsistent changes in cell motility in cultured fibroblasts that overexpressed smooth muscle caldesmon. Our study suggests that gene delivery of l-CaD protects cell membrane integrity in the face of an adenoviral infection with minimal impact on cell toxicity and housekeeping functions such as wound repair and cell adhesion.
The observed change in membrane capacitance in cells expressing l-CaD was not associated with a reduction in myosin ATPase activity. Cytochalasin D abolished constitutive isometric tension to the same degree in both cells expressing l-CaD and controlled cells. Furthermore, the effect of l-CaD to modulate adenovirus-mediated change in membrane capacitance was not attributed to effects on actin assembly. Expression of l-CaD did not significantly change the fraction of insoluble actin compared with cells transfected with the control virus, which indicates that l-CaD did not affect actin assembly. In contrast, a statistically significant difference in F-actin content between wild-type CaPi-treated cells and cells exposed to the controlled virus was observed. Taken together, these data suggest that human wild-type l-CaD stabilizes the actin cytoskeleton in DF-1 cells independently of its effects on actin assembly or myosin ATPase activity.
This report presents new and unexpected antiviral properties of l-CaD that were not predicted from data previously reported in studies performed under in vitro and in situ conditions. Previous studies have attempted to evaluate the role of l-CaD in intact cells by overexpressing l-CaD and measuring its impact on cell mechanics. Helfman et al. (23) reported that overexpression of wild-type l-CaD decreased myosin ATPase activity and inhibited focal contact formation in contrast to the present study, which showed a total increase in focal contact mass in cells transfected with Ad-l-CaD. Helfman et al. assessed myosin ATPase and cell-matrix adhesion in a qualitative manner and did not comprehensively measure the physical properties of cytoskeletal membrane structures or myosin ATPase activity. More importantly, focal contact measurements represent only a fraction of the entire cytoskeletal membrane properties of a cell. Also, it is difficult in overexpression cell models to resolve the function of heterologous l-CaD from that of endogenous l-CaD.
There are several potential explanations to account for the observed differences between the findings of Helfman et al. (23) and those of the present study. First, Helfman et al. used a silicone-wrinkling assay to measure myosin ATPase activity, which was reported by demonstrating selected images of wrinkled substrate generated by a few cells. This assay is limited by its qualitative nature and its potential for sampling error from intrasubject variability and bias when reported microscopically. In contrast, in our study, we used a quantitative physical approach that was not prone to the same level of statistical bias, because tension was averaged over a population of cultured cells. The report of Helfman et al. also defined cell adhesion only according to morphological criteria and not according to functional determinants of cell adhesion. Their report ignored the physical properties of close contacts, cell-cell adhesion sites, and membrane properties affected by the cytoskeleton. Adhesion is a physical property that should be defined by strain, adhesivity, or force measurements. Although Helfman et al. reported less mature focal contact formation in cells overexpressing l-CaD, cells were still more resistant to trypsin-mediated cell rounding. The authors argued that the greater resistance to cell rounding in response to trypsin was attributed to a reduction in constitutive myosin ATPase activity. Our data are inconsistent with this notion and demonstrate novel properties of l-CaD on cell mechanics that have not been previously documented.
Our report represents the first comprehensive and detailed analysis that precisely evaluates the rheological physical properties of l-CaD in an intact cell. These data demonstrate that expression of wild-type l-CaD attenuates adenovirus-mediated cell injury through additional mechanisms other than modulating myosin ATPase activity or actin assembly. The precise mechanism by which l-CaD modulates actin-dependent mechanical forces to stabilize membrane capacitance remains to be elucidated.
In summary, this study takes a direct and integrated molecular and bioengineering approach, which demonstrates that gene delivery of human wild-type l-CaD attenuates the effect of adenovirus on cytoskeletal membrane integrity. Heterologous expression of human l-CaD attenuated the adenovirus-mediated loss in transcellular resistance predominately by stabilizing membrane capacitance, which suggests a stabilization of the cortical actin cytoskeleton and focal contacts. This effect on membrane capacitance was detected at low transfection efficiency and was mediated independently of its effects on constitutive myosin ATPase activity or actin assembly. We acknowledge that this study represents the first step toward recognizing the potential of gene delivery of l-CaD as an antiviral therapy against adenoviral infections. Further investigation is needed to assess whether gene delivery of l-CaD through a replicate-deficient viral delivery system can attenuate cell injury from a challenge of wild-type adenovirus. Additional studies remain to be done to evaluate whether gene delivery of l-CaD can attenuate adenovirus in permissive cells and in mammalian cells. Also, further investigation is required to determine whether gene delivery of l-CaD antagonizes adenovirus through nonstructural mechanisms such as inhibition of viral replication and viral protein synthesis. In addition, further investigation is required to determine whether gene delivery of l-CaD antagonizes RNA and DNA viruses in a generalized fashion.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* K. Haxhinasto and A. Kamath contributed equally to this work.
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