Automated measurement of cell motility and proliferation

Background

Time-lapse microscopic imaging provides a powerful approach for following changes in cell phenotype over time. Visible responses of whole cells can yield insight into functional changes that underlie physiological processes in health and disease. For example, features of cell motility accompany molecular changes that are central to the immune response, to carcinogenesis and metastasis, to wound healing and tissue regeneration, and to the myriad developmental processes that generate an organism. Previously reported image processing methods for motility analysis required custom viewing devices and manual interactions that may introduce bias, that slow throughput, and that constrain the scope of experiments in terms of the number of treatment variables, time period of observation, replication and statistical options. Here we describe a fully automated system in which images are acquired 24/7 from 384 well plates and are automatically processed to yield high-content motility and morphological data.

Results

We have applied this technology to study the effects of different extracellular matrix compounds on human osteoblast-like cell lines to explore functional changes that may underlie processes involved in bone formation and maintenance. We show dose-response and kinetic data for induction of increased motility by laminin and collagen type I without significant effects on growth rate. Differential motility response was evident within 4 hours of plating cells; long-term responses differed depending upon cell type and surface coating. Average velocities were increased approximately 0.1 um/min by ten-fold increases in laminin coating concentration in some cases. Comparison with manual tracking demonstrated the accuracy of the automated method and highlighted the comparative imprecision of human tracking for analysis of cell motility data. Quality statistics are reported that associate with stage noise, interference by non-cell objects, and uncertainty in the outlining and positioning of cells by automated image analysis. Exponential growth, as monitored by total cell area, did not linearly correlate with absolute cell number, but proved valuable for selection of reliable tracking data and for disclosing between-experiment variations in cell growth.

Conclusion

These results demonstrate the applicability of a system that uses fully automated image acquisition and analysis to study cell motility and growth. Cellular motility response is determined in an unbiased and comparatively high throughput manner. Abundant ancillary data provide opportunities for uniform filtering according to criteria that select for biological relevance and for providing insight into features of system performance. Data quality measures have been developed that can serve as a basis for the design and quality control of experiments that are facilitated by automation and the 384 well plate format. This system is applicable to large-scale studies such as drug screening and research into effects of complex combinations of factors and matrices on cell phenotype.     

Using the ecology model to describe the impact of asthma on patterns of health care

Background

The recent increase in childhood asthma has been a puzzling one. Recent views focus on the role of infection in the education of the immune system of young children. However, this so called hygiene hypothesis fails to answer some important questions about the current trends in asthma or to account for environmental influences that bear little relation to infection.

Discussion

The multi-factorial nature of asthma, reflecting the different ways we tend to interact with our environment, mandates that we look at the asthma epidemic from a broader perspective. Seemingly modern affluent lifestyles are placing us increasingly in static, artificial, microenvironments very different from the conditions prevailed for most part of our evolution and shaped our organisms. Changes that occurred during the second half of the 20th century in industrialized nations with the spread of central heating/conditioning, building insulation, hygiene, TV/PC/games, manufactured food, indoor entertainment, cars, medical care, and sedentary lifestyles all seem to be depriving our children from the essential inputs needed to develop normal airway function (resistance). Asthma according to this view is a manifestation of our respiratory maladaptation to modern lifestyles, or in other words to our increasingly artificial habitats. The basis of the artificial habitat notion may lie in reduced exposure of innate immunity to a variety of environmental stimuli, infectious and non-infectious, leading to reduced formulation of regulatory cells/cytokines as well as inscribed regulatory pathways. This could contribute to a faulty checking mechanism of non-functional Th2 (and likely Th1) responses, resulting in asthma and other immuno-dysregulation disorders.

Summary

In this piece I discuss the artificial habitat concept, its correspondence with epidemiological data of asthma and allergy, and provide possible immunological underpinning for it from an evolutionary perspective of health and disease.     

E3LSI research: an essential element of biodefense

The threat of bioterrorism has prompted the U.S. to undertake a vast biodefense initiative, including funding biodefense-related scientific research at unprecedented levels. Unfortunately, the many ethical, economic, environmental, legal, and social implications (E(3)LSI) of biodefense research and activities are not yet receiving the attention they warrant. Previously, in laudable demonstrations of foresight and responsibility, the federal government has funded research into the E(3)LSI of other recent scientific endeavors--namely, the Human Genome Project and the nanotechnology research program--through directed appropriations from their respective research budgets. This article advocates and proposes a model for a portion of biodefense funding to be similarly set aside for an E(3)LSI research program to complement biodefense research, to ensure that bioterror preparedness does not give rise to harmful or otherwise undesirable unintended consequences.     

Involvement of poly(ADP-ribose) polymerase activity in regulating Chk1-dependent apoptotic cell death

The activity of poly(ADP-ribose) polymerase (PARP) is highly stimulated following DNA damage resulting in formation of DNA nicks and strand breaks. This leads to modification of numerous proteins, including itself, using NAD(+) as substrate and to exhaustion of intracellular ATP. A highly cytotoxic concentration of the DNA methylating agent methyl methanesulfonate (MMS) results in cellular ATP depletion and cell death primarily by necrosis in both wild-type and DNA polymerase beta null mouse fibroblasts. The loss of ATP can be prevented by the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN), and now cells die by an energy-dependent apoptotic pathway. We find that inhibition of PARP activity transforms a sub-lethal exposure to MMS into a highly cytotoxic event. Under this condition, ATP is not depleted and cell death is by apoptosis. The caspase inhibitor, Z-VAD, shifts the mechanism of cell death to necrosis indicating a caspase-dependent component of the apoptotic cell death. Co-exposure to the Chk1 inhibitor UCN-01 also produces a decrease in apoptotic cell death, but now there is an increase in viable cells and an enhancement in long-term survival. Taken together, our results suggest that inhibition of PARP activity, induced as a result of low dose MMS exposure, signals via a Chk1-dependent pathway for cell death by apoptosis.     

Heparin II domain of fibronectin uses alpha4beta1 integrin to control focal adhesion and stress fiber formation, independent of syndecan-4

Co-signaling events between integrins and cell surface proteoglycans play a critical role in the organization of the cytoskeleton and adhesion forces of cells. These processes, which appear to be responsible for maintaining intraocular pressure in the human eye, involve a novel cooperative co-signaling pathway between alpha5beta1 and alpha4beta1 integrins and are independent of heparan sulfate proteoglycans. Human trabecular meshwork cells isolated from the eye were plated on type III 7-10 repeats of fibronectin (alpha5beta1 ligand) in the absence or presence of the heparin (Hep) II domain of fibronectin. In the absence of the Hep II domain, cells had a bipolar morphology with few focal adhesions and stress fibers. The addition of the Hep II domain increased cell spreading and the numbers of focal adhesions and stress fibers. Cell spreading and stress fiber formation were not mediated by heparan sulfate proteoglycans because treatment with chlorate, heparinase, or soluble heparin did not prevent Hep II domain-mediated cell spreading. Cell spreading and stress fiber formation were mediated by alpha4beta1 integrin because soluble anti-alpha4 integrin antibodies inhibited Hep II domain-mediated cell spreading and soluble vascular cell adhesion molecule-1 (alpha4beta1 ligand)-induced cell spreading. This is the first demonstration of the Hep II domain mediating cell spreading and stress fiber formation through alpha4beta1 integrin. This novel pathway demonstrates a cooperative, rather than antagonistic, role between alpha5beta1 and alpha4beta1 integrins and suggests that interactions between the Hep II domain and alpha4beta1 integrin could modulate the strength of cytoskeleton-mediated processes in the trabecular meshwork of the human eye.     

Plant gamma-glutamyl hydrolases and folate polyglutamates: characterization, compartmentation, and co-occurrence in vacuoles

gamma-Glutamyl hydrolase (GGH, EC 3.4.19.9) catalyzes removal of the polyglutamyl tail from folyl and p-aminobenzoyl polyglutamates. Plants typically have one or a few GGH genes; Arabidopsis has three, tandemly arranged on chromosome 1, which encode proteins with predicted secretory pathway signal peptides. Two representative Arabidopsis GGH proteins, AtGGH1 and AtGGH2 (the At1g78660 and At1g78680 gene products, respectively) were expressed in truncated form in Escherichia coli and purified. Both enzymes were active as dimers, had low K(m) values (0.5-2 microm) for folyl and p-aminobenzoyl pentaglutamates, and acted as endopeptidases. However, despite 80% sequence identity, they differed in that AtGGH1 cleaved pentaglutamates, mainly to di- and triglutamates, whereas AtGGH2 yielded mainly monoglutamates. Analysis of subcellular fractions of pea leaves and red beet roots established that GGH activity is confined to the vacuole and that this activity, if not so sequestered, would deglutamylate all cellular folylpolyglutamates within minutes. Purified pea leaf vacuoles contained an average of 20% of the total cellular folate compared with approximately 50 and approximately 10%, respectively, in mitochondria and chloroplasts. The main vacuolar folate was 5-methyltetrahydrofolate, of which 51% was polyglutamylated. In contrast, the principal mitochondrial and chloroplastic forms were 5-formyl- and 5,10-methenyltetrahydrofolate polyglutamates, respectively. In beet roots, 16-60% of the folate was vacuolar and was again mainly 5-methyltetrahydrofolate, of which 76% was polyglutamylated. These data point to a hitherto unsuspected role for vacuoles in folate storage. Furthermore, the paradoxical co-occurrence of GGH and folylpolyglutamates in vacuoles implies that the polyglutamates are somehow protected from GGH attack.     

Nonaqueous synthesis of a selectively modified, highly anionic sulfopropyl ether derivative of cyclomaltoheptaose (beta-cyclodextrin) in the presence of 18-crown-6

A highly anionic cyclomaltooligosaccharide (cyclodextrin, CD) derivative containing sulfopropyl functional groups on the primary face of the CD was synthesized. Heptakis(2,3-di-O-methyl)cyclomaltoheptaose [heptakis(2,3-di-O-methyl)-beta-cyclodextrin] was reacted with 1,3-propane sultone and potassium hydride (KH) in anhydrous tetrahydrofuran in the presence of 18-crown-6 to yield highly substituted potassium heptakis(2,3-di-O-methyl-6-O-sulfopropyl)cyclomaltoheptaose [heptakis(KSPDM)-beta-CD] with an average degree of substitution (DSCE) of 6.9 as determined by inverse detection capillary electrophoresis (CE). The principal species in the product is the fully substituted heptakis(KSPDM)-beta-CD. Complete NMR assignments of the hydrogen and carbon atoms are made using a combination of gCOSY and gHSQC. In the absence of 18-crown-6, the reaction generates a mixture of multiply charged derivatives with average DSCE of 4.1. The possible roles of the crown ether in the reaction are discussed. The ROESY NMR spectrum of the inclusion complex that forms between heptakis(KSPDM)-beta-CD and 2-naphthoic acid in D2O reveals that 2-naphthoic acid inserts with the carboxyl group toward the derivatized primary rim of the cyclodextrin.     

Preferential phosphorylation of R-domain Serine 768 dampens activation of CFTR channels by PKA

CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (P(o)) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted P(o)-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The observed reduced sensitivity to activation by [PKA] imparted by Ser 768 might serve to ensure activation of WT CFTR by strong stimuli while dampening responses to weak signals.     

PD98059 and U0126 activate AMP-activated protein kinase by increasing the cellular AMP:ATP ratio and not via inhibition of the MAP kinase pathway

The MAP kinase pathway inhibitor U0126 caused phosphorylation and activation of AMP-activated protein kinase (AMPK) and increased phosphorylation of its downstream target acetyl-CoA carboxylase, in HEK293 cells. This effect only occurred in cells expressing the upstream kinase, LKB1. Of two other widely used MAP kinase pathway inhibitors not closely related in structure to U0126, PD98059 also activated AMPK but PD184352 did not. U0126 and PD98059, but not PD184352, also increased the cellular ADP:ATP and AMP:ATP ratios, accounting for their ability to activate AMPK. These results suggest the need for caution in interpreting experiments conducted using U0126 and PD98059.