Topographical and physicochemical modification of material surface to enable patterning of living cells.

Precise control of the architecture of multiple cells in culture and in vivo via precise engineering of the material surface properties is described as cell patterning. Substrate patterning by control of the surface physicochemical and topographic features enables selective localization and phenotypic and genotypic control of living cells. In culture, control over spatial and temporal dynamics of cells and heterotypic interactions draws inspiration from in vivo embryogenesis and haptotaxis. Patterned arrays of single or multiple cell types in culture serve as model systems for exploration of cell-cell and cell-matrix interactions. More recently, the patterned arrays and assemblies of tissues have found practical applications in the fields of Biosensors and cell-based assays for Drug Discovery. Although the field of cell patterning has its origins early in this century, an improved understanding of cell-substrate interactions and the use of microfabrication techniques borrowed from the microelectronics industry have enabled significant recent progress. This review presents the important early discoveries and emphasizes results of recent state-of-the-art cell patterning methods. The review concludes by illustrating the growing impact of cell patterning in the areas of bioelectronic devices and cell-based assays for drug discovery.

Real-time molecular and cellular analysis: the new frontier of drug discovery.

The pharmaceutical industry is currently facing the challenge of maintaining increased efficiency and productivity while contending with a deluge of genomic and high-throughput screening data. To ease the bottlenecks at target validation and lead optimization, the industry must look to the living cell, the ultimate target of all drugs, as a source of new biological knowledge. This new 'cell-centric' perspective must integrate reagents that report on the state of molecular processes within the cell, automated detection and analysis of these processes, and cellular knowledge, building into a single platform.

Fluorescent-protein biosensors: new tools for drug discovery.

Recent improvements in target discovery and high-throughput screening have increased the pressure at key points along the drug-discovery pipeline. High-content screening was developed to ease the bottlenecks formed at the target-validation and lead-optimization points, and a new generation of reagents that report on specific molecular processes in living cells (fluorescent-protein biosensors) have been important in its development. Creative designs of fluorescent-protein biosensors have emerged and been used to measure the molecular dynamics of macromolecules, metabolites and ions. Recent applications of fluorescent-protein biosensors to biological problems have provided a foundation for their use in biotechnology.

Immunofluorescence signal amplification by the enzyme-catalyzed deposition of a fluorescent reporter substrate (CARD).

Progress has been made in improving the immunohistochemical detection of antigens for imaging and flow cytometry. We report the synthesis of a novel fluorescent horseradish peroxidase substrate, Cy3.29-tyramide, and its application in an enzyme-based signal amplification system, catalyzed reporter deposition (CARD). The catalyzed deposition of Cy3.29-tyramide was used to detect cell surface markers such as CD8 and CD25 on tonsil tissue and human lymphocytes. We compared the fluorescence CARD method to standard indirect immunofluorescence detection methods and found that an amplification of up to 15-fold was possible with CARD. The detection of the intracellular protein myosin II in fibroblastic cells and rabbit serum proteins blotted onto nitrocellulose was also improved. Thus, fluorescent CARD is a simple modification that can be made to standard immunofluorescence staining protocols to enhance significantly the detection of antigens.

Dissecting the individuality of cancer cells: the morphological and molecular dynamics of single human glioma cells.

A glioma produces some of the most heterogeneously growing, angiogenic, and invasive primary brain tumor cells known. To dissect cellular individuality, and therefore tumor heterogeneity, multiple morphological and molecular processes in single living human glioma cells were measured using multimode light microscopy. Feature extraction of time-lapse image series of spreading, locomoting, and interacting cells either in the presence or absence of physiological modulators was performed by defining five parameters that described cell shape, movement, and cell-cell contacts. Concurrent visualization of all five parameters with a scatterplot matrix revealed temporal as well as time-independent relationships between the parameters that were sufficient to define the individuality of normal and transformed glial cells. Because the actin-cytoskeleton plays a role in regulating the cellular processes described above, the dynamics of a fluorescent analog of non-muscle actin within motile glioma cells were measured in addition to the morphological parameters. The actin-cytoskeleton within the thin sweeping lamellipodia of a glioma exhibited a paucity of large stress fibers, a rich collection of microvillar structures containing actin, and dynamics that were distinct from those of normal motile cells. This approach can therefore potentially be used to dissect the molecular origins of transformation using a small number of representative tumor cells.

Morphologic, immunologic, biochemical, and cytogenetic characteristics of the human glioblastoma-derived cell line, SNB-19.

Human glioma-derived cell cultures and lines have proven to be of significant value in the study of the basic properties that contribute to the highly malignant, invasive and angiogenic phenotype of glioblastoma multiforme tumors. It is frequently difficult to establish lines that retain glial tumor properties in long term culture. The SNB-19 cell line has maintained and exhibited properties of transformation, differentiation, autocrine growth response, and tumorigenesis while remaining in culture for over 13 yr and undergoing over 200 passages. This human line has been utilized in a wide range of studies related to the basic properties of human glioblastoma multiforme. In this report, we summarize the immunologic, biochemical, and cytogenetic properties of this versatile cell line and its utility for additional mechanistic investigation into the pathophysiology of the progression of human malignant gliomas.

Measurement and manipulation of cytoskeletal dynamics in living cells.

A new ear in cell biology is at hand with the development of tools for imaging molecular functions in living cells and tissues. Specific chemical and molecular events can now be measured and manipulated in cells in order to explore the mechanisms of cell functions. In particular, cytoskeletal processes are being dissected temporally and spatially in single cells from lower eukaryotes, plants, and animals using light-based reagents and electronic light microscopy.

Fluorescent protein biosensors: measurement of molecular dynamics in living cells.

A new generation of reagents that report on specific molecular events in living cells, called fluorescent protein biosensors, has evolved from in vitro fluorescence spectroscopy and fluorescent analogue cytochemistry. Creative designs of fluorescent protein biosensors to measure the molecular dynamics of macromolecules, metabolites, and ions in single cells emerge from the integrative use of contemporary synthetic organic chemistry, biochemistry, and molecular biology. Future advances in fluorescent probe design, computer-driven optical instrumentation, and software will allow us to engineer endogenous cellular components that localize and function as reporters of their activities, thus moving molecular measurement beyond the single cell to living tissues and the whole organism.

Fluorescent actin analogs with a high affinity for profilin in vitro exhibit an enhanced gradient of assembly in living cells.

Constitutive centripetal transport of the actin-based cytoskeleton has been detected in cells spreading on a substrate, locomoting fibroblasts and keratocytes, and non-locomoting serum-deprived fibroblasts. These results suggest a gradient of actin assembly, highest in the cortex at the cytoplasm-membrane interface and lowest in the non-cortical perinuclear cytoplasm. We predicted that such a gradient would be maintained in part by phosphoinositide-regulated actin binding proteins because the intracellular free Ca2+ and pH are low and spatially constant in serum-deprived cells. The cytoplasm-membrane interface presents one surface where the assembly of actin is differentially regulated relative to the non-cortical cytoplasm. Several models, based on in vitro biochemistry, propose that phosphoinositide-regulated actin binding proteins are involved in local actin assembly. To test these models in living cells using imaging techniques, we prepared a new fluorescent analog of actin that bound profilin, a protein that interacts with phosphoinositides and actin-monomers in a mutually exclusive manner, with an order of magnitude greater affinity (Kd = 3.6 microM) than cys-374-labeled actin (Kd > 30 microM), yet retained the ability to inhibit DNase I. Hence, we were able to directly compare the distribution and activity of a biochemical mutant of actin with an analog possessing closer to wild-type activity. Three-dimensional fluorescence microscopy of the fluorescent analog of actin with a high affinity for profilin revealed that it incorporated into cortical cytoplasmic fibers and was also distributed diffusely in the non-cortical cytoplasm consistent with a bias of actin assembly near the surface of the cell. Fluorescence ratio imaging revealed that serum-deprived and migrating fibroblasts concentrated the new actin analog into fibers up to four-fold in the periphery and leading edge of these cells, respectively, relative to a soluble fluorescent dextran volume marker, consistent with the formation of a gradient of actin filament density relative to cell volume. Comparison of these gradients in the same living cell using analogs of actin with high and low affinities for profilin demonstrated that increased profilin binding enhanced the gradient. Profilin and related proteins may therefore function in part to bias the assembly of actin at the membrane-cytoplasm interface.

Relative distribution of actin, myosin I, and myosin II during the wound healing response of fibroblasts.

Myosin I is present in Swiss 3T3 fibroblasts and its localization reflects a possible involvement in the extension and/or retraction of protrusions at the leading edge of locomoting cells and the transport of vesicles, but not in the contraction of stress fibers or transverse fibers. An affinity-purified polyclonal antibody to brush border myosin I colocalizes with a polypeptide of 120 kD in fibroblast extracts. Within initial protrusions of polarized, migrating fibroblasts, myosin I exhibits a punctate distribution, whereas actin is diffuse and myosin II is absent. Myosin I also exists in linear arrays parallel to the direction of migration in filopodia and microspikes, established protrusions, and within the leading lamellae of migrating cells. Myosin II and actin colocalize along transverse fibers in the lamellae of migrating cells, while myosin I displays no definitive organization along these fibers. During contractions of actin-based fibers, myosin II is concentrated in the center of the cell, while the distribution of myosin I does not change. Thus, myosin I is found at the correct location and time to be involved in the extension and/or retraction of protrusions and the transport of vesicles. Myosin II-based contractions in more posterior cellular regions could generate forces to separate cells, maintain a polarized cell shape, maintain the direction of locomotion, maximize the rate of locomotion, and/or aid in the delivery of cytoskeletal/contractile subunits to the leading edge.

Myosin II phosphorylation and the dynamics of stress fibers in serum-deprived and stimulated fibroblasts.

The actin-based cytomatrix generates stress fibers containing a host of proteins including actin and myosin II and whose dynamics are easily observable in living cells. We developed a dual-radioisotope-based assay of myosin II phosphorylation and applied it to serum-deprived fibroblasts treated with agents that modified the dynamic distribution of stress fibers and/or altered the phosphorylation state of myosin II. Serum-stimulation induced an immediate and sustained increase in the level of myosin II heavy chain (MHC) and 20-kDa light chain (LC20) phosphorylation over the same time course that it caused stress fiber contraction. Cytochalasin D, shown to cause stress fiber fragmentation and contraction, had little effect on myosin II phosphorylation. Okadaic acid, a protein phosphatase inhibitor, induced a delayed but massive cell shortening preceded by a large increase in MHC and LC20 phosphorylation. Staurosporine, a kinase inhibitor known to effect dissolution but not contraction of stress fibers, immediately caused an increase in MHC and LC20 phosphorylation followed within minutes by the dephosphorylation of LC20 to a level below that of untreated cells. We therefore propose that the contractility of the actin-based cytomatrix is regulated by both modulating the activity of molecular motors such as myosin II and by altering the gel structure in such a manner as to either resist or yield to the tension applied by the motors.

Chromatography of proteins on columns of polyvinylpolypyrrolidone using adsorbed textile dyes as affinity ligands.

A simple and inexpensive chromatography system for proteins is introduced. When the amino derivatives of chlorotriazine dyes or other azo dyes were added to an aqueous slurry of the crosslinked polymer polyvinylpolypyrrolidone they were adsorbed, thus forming an immobilized dye chromatographic matrix. The association of the textile dyes with polyvinylpolypyrrolidone did not prevent them from acting as affinity ligands for proteins. Parameters such as ionic strength, dye concentration, and column size modulated the affinity effect exerted by the immobilized dyes. Lysozyme present in an egg white protein mixture bound to a column onto which the amino derivative of Procion Brown H-A was adsorbed and was eluted with a linear gradient of KCl. The resulting purification of the enzyme was 37-fold with 80% of the original activity being recovered. Free dye eluting with the lysozyme was removed on a column of polyvinylpolypyrrolidone equilibrated with 0.5 M KCl. After chromatography, the dye column was regenerated with 0.5 M NaOH and recharged with dye. The system presented here allows one to initially screen large numbers of potentially useful protein ligands to optimize a protein separation, followed by scaleup to a system size determined by the user.

Aqueous two-phase protein partitioning using textile dyes as affinity ligands.

A simple and inexpensive aqueous two-phase system for the affinity partitioning of proteins is introduced. An aqueous solution consisting of maltodextrin (M100; molecular mass, 1800) and polyvinylpyrrolidone (PVP360; molecular mass, 360,000) formed two phases at 4 degrees C when the concentration of the polymers was 22.5% (w/w) and 4.0% (w/w), respectively. When the amino derivatives of chlorotriazine textile dyes or other azo textile dyes were added to the two-phase system they partitioned asymmetrically, favoring the upper, less dense, PVP360-rich phase. The association of the textile dyes with PVP360 did not prevent them from acting as affinity ligands for proteins. Three of the dyes screened increased the partition coefficient of purified lysozyme nearly 50-fold over a control containing no dye. Parameters such as pH, ionic strength, and dye concentration modulated the affinity-partitioning effect of the system. The partition coefficient of lysozyme in an egg white protein mixture increased severalfold as the total protein content of the system approached 4% (w/w), indicating that protein concentration is also important in determining the partitioning characteristics of this two-phase system. Proteins were efficiently freed of PVP360 and textile dye by recovery in a high-salt solution when another two-phase system was formed upon the addition of a solution of concentrated potassium phosphate to the isolated upper phase of a PVP360/M100/textile dye two-phase system. The affinity-partitioning system presented here allows one to screen large numbers of potentially useful protein ligands to optimize protein separation, followed by direct scaleup to a system size determined by the user.

Formation, transport, contraction, and disassembly of stress fibers in fibroblasts.

Swiss mouse 3T3 fibroblasts grown on a solid substrate in the presence of 10% serum exhibit cell movement, organelle transport, and cytokinesis. When the serum concentration in the culture medium is decreased to 0.2% for 48 h the serum-deprived cells virtually stop locomoting, spread, decreased organelle transport, and exhibit extensive arrays of stress fibers that are visible with video-enhanced differential interference contrast microscopy and that also incorporate fluorescent analogs of actin and conventional myosin (myosin II). The stress fibers form in a constitutive manner at the cytoplasm-membrane interface, transport toward the nucleus, and then disappear. The rate of transport of these fibers is quite heterogeneous with average rates in the range of 10-20 microns/h. When serum-deprived cells are stimulated with mitogens such as 10% serum or 10 nM thrombin, many of the stress fibers immediately begin to shorten, suggesting a contraction. The rate of shortening is approximately two orders of magnitude slower than that of unloaded smooth muscle cells. The fiber shortening is often accompanied by retraction of the edges of the cell and continues for at least the 1st hour post-stimulation.

Properties of purified actin depolymerizing factor from chick brain.

Actin depolymerizing factor (ADF) from 19-day embryonic chick brains has been purified to greater than 98% homogeneity with a yield of 7.2 mg/100 g of brain. Quantitative immunoblotting with a monospecific antibody to ADF indicated that ADF comprises 0.3% of the total brain protein, resulting in an actual purification yield of about 20%. Brain ADF migrates as a single polypeptide of 19,000 kDa on SDS-containing polyacrylamide gels. The molecular weight of the native protein determined from sedimentation equilibrium in buffers containing from 50 to 200 mM KCl is 20,000. The secondary structure of ADF calculated from the circular dichroic spectrum consists of about 22% alpha-helix, 24% beta-sheet, and 18% beta-turn. ADF contains a blocked N-terminus, a single tryptophan residue located about one-third of the way from one end of the protein, and six cysteine residues (all in reduced form in the native protein). All six cysteine residues could be chemically modified with eosinylmaleimide under nondenaturing conditions; however, ADF activity was lost when more than one cysteine residue was modified. ADF microheterogeneity has been observed upon nonequilibrium pH gradient electrophoresis in polyacrylamide gels containing 9 M urea, the major isoform having a pI of congruent to 7.9-8.0. ADF can interact with either monomeric or filamentous actin to give a complex which can be isolated by gel filtration chromatography. Both major and minor isoforms of the ADF are found in the complex. Assembly-competent actin and active ADF can both be recovered from the complex by chromatography on ATP-saturated DEAE-cellulose.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular acidification inhibits the proliferative response in BALB/c-3T3 cells.

One of the earliest events to occur upon the addition of serum to quiescent cells is an increase in the intracellular pH (pHin). The relationship between this pH change and proliferation is not known. In the present study, we investigate the consequences of acidifying the cytosol using the weak acid, 5', 5"-dimethyl oxazolidine 2,4-dione (DMO). At a concentration of 50 mM, DMO inhibits the serum-induced increases in pHin, DNA synthesis, and cell number. This concentration of DMO is shown not to inhibit the steady-state rate of mitochondrial respiration and not to inhibit DNA synthesis in a pH-independent fashion. The effects of DMO treatments are also shown to be reversible, indicating that this compound is not cytotoxic. These observations indicate that DMO inhibits cell proliferation by lowering intracellular pH. One important event that must occur prior to the initiation of DNA synthesis is an elevated rate of protein synthesis. The rate of protein synthesis in situ is extremely pH sensitive. Addition of 50 mM DMO to serum-stimulated cultures reduces the rate of leucine incorporation to unstimulated levels. These observations suggest that cytoplasmic acidification may inhibit proliferation through its effects on protein synthesis.

Effect of serum on the intracellular pH of BALB/c-3T3 cells: serum deprivation causes changes in sensitivity of cells to serum.

One of the earliest responses of quiescent mammalian cells to the addition of serum is an increase in intracellular pH (pHin). This pHin change is generally believed to be due to an increased activity of Na+/H+ exchange. A number of investigators have observed steady-state differences in pHin between cells in the presence and absence of serum. However, no one has examined differences in pHin regulation that may exist between cells chronically exposed to, or deprived of serum. In this study, we investigated the effects of serum deprivation to identify those components of pHin regulation that were associated with quiescence. To do this, we examined pHin in cells growing chronically in 10% serum as well as in cells that were either acutely (1.5-2 hr) or chronically (48 hr) deprived of serum. Intracellular pH was monitored using the fluorescence of intracellularly loaded pyranine dye. Our results indicate that the resting pHin values of chronically or acutely serum-deprived cells were not significantly different from each other yet, in both cases, were lower than those observed in cells exposed to 10% serum. Furthermore, we observed significant increases in pHin of both acutely or chronically serum-deprived cells in response to the addition of serum at various concentrations, in the presence of 24 mM bicarbonate. Chronically serum-deprived cells had slightly smaller responses and were more sensitive to lower concentrations of serum than were acutely deprived cells. Therefore, our data suggest that long-term serum deprivation affects the magnitude and sensitivity of pHin to serum stimulation and causes the loss of some form of pHin regulatory mechanism(s).

Determination of intracellular pH of BALB/c-3T3 cells using the fluorescence of pyranine.

In terms of accuracy and sensitivity, intracellularly trapped, pH-dependent fluorescent probes are appropriate to accurately measure intracellular pH. These probes are commonly introduced into living cells in esterified form, wherein the free acid is produced through enzymatic hydrolysis. The fluorescence characteristics of the ester and the free acid can differ markedly and spectral uncertainty can occur. We describe here the measurement of intracellular pH using 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine) that has been scrape-loaded into BALB/c-3T3 mouse cells. The excitation spectrum of pyranine is pH sensitive, with an isosbestic point at 415 nm and peaks at 405 and 465 nm which decrease and increase with pH, respectively. The 465/405 ratio can be used to monitor the pH, while the fluorescence at 415 nm indicates the total dye-dependent signal remaining. The scrape-loaded dye persists in cells for periods up to 6 h. We have calibrated this dye in situ using nigericin/high K+, and have found that the pKa of the dye in situ is 7.82, as compared to 7.68 in vitro. We have observed that the cells can slowly equilibrate their intracellular pH to near control levels when presented with either an acute alkaline or acid load.