Identification of kinases and regulatory proteins required for cell migration using a transfected cell-microarray system.

BACKGROUND: Cell migration plays a major role in a variety of normal biological processes, and a detailed understanding of the associated mechanisms should lead to advances in the medical sciences in areas such as cancer therapy. Previously, we developed a simple chip, based on transfected-cell microarray (TCM) technology, for the identification of genes related to cell migration. In the present study, we used the TCM chip for high-throughput screening (HTS) of a kinome siRNA library to identify genes involved in the motility of highly invasive NBT-L2b cells. RESULTS: We performed HTS using TCM coupled with a programmed image tracer to capture time-lapse fluorescence images of siRNA-transfected cells and calculated speeds of cell movement. This first screening allowed us to identify 52 genes. After quantitative PCR (qPCR) and a second screening by a conventional transfection method, we confirmed that 32 of these genes were associated with the migration of NBT-L2b cells. We investigated the subcellular localization of proteins and levels of expression of these 32 genes, and then we used our results and databases of protein-protein interactions (PPIs) to construct a hypothetic but comprehensive signal network for cell migration. CONCLUSIONS: The genes that we identified belonged to several functional categories, and our pathway analysis suggested that some of the encoded proteins functioned as the hubs of networks required for cell migration. Our signal pathways suggest that epidermal growth factor receptor (EGFR) is an upstream regulator in the network, while Src and GRB2 seem to represent nodes for control of respective the downstream proteins that are required to coordinate the many cellular events that are involved in migration. Our microarray appears to be a useful tool for the analysis of protein networks and signal pathways related to cancer metastasis.

Development of a microfabricated device for low-voltage electropermeabilization of adherent cells.

A microfabricated device for low-voltage electropermeabilization of adherent cells has been developed. The device, which is fabricated on a cover glass using mainly transparent materials, allows optical observation of cells through it. This device consists of an array of 6-µm circular electrodes connected via a transparent and conductive layer made of ITO (indium tin oxide) to give pulsed voltages for electropermeabilization. The surface of the electrode is coated with a thin Au film to reduce impedance. The insulation layer contains holes aligned on the electrodes to constrict the electric field so that the applied voltage drops mainly at the hole and is effectively used for electropermeabilization. Adherent cells for electropermeabilization are cultured on the device and those in the vicinity of the holes are influenced by the relatively strong electric field induced by the holes in the insulation layer. We demonstrated the culture of adherent cells on the device and their optical observation by phase-contrast microscopy. We further demonstrated the delivery of YO-PRO-1 into various cells including HeLa, NIH3T3, and smooth muscle cells, by applying pulsed voltages of 6.0 V.

Motility behavior of rpoS-deficient Escherichia coli analyzed by individual cell tracking.

Motility is one of the most extensively studied cellular events conducted by bacteria, including Escherichia coli. A motility agar plate assay showed that deletion of the rpoS gene enhanced the apparent motility of the E. coli BW25113 strain, which inherently had negligible motility compared to wild-type E. coli strains, such as MG1655, with no effect on cell growth. This enhancement of motility was accompanied by drastic up-regulation of genes involved in the formation and rotation of flagella. Furthermore, an individual cell motility assay showed that the population of ΔrpoS cells had bimodal motility character, and that a minority of this population exhibited a much higher motility rate. These results support a view that a minority population contributes to increasing in apparent motility of the whole population of ΔrpoS cells.

Evaluation method for gene transfection by using the period of onset of gene expression and cell division.

Using single-cells time-lapse analysis, we investigated the mechanism of gene expression using nine transfection reagents. Although onset of gene expression occurred after cell division by all reagents, 91.6% periods, which depended on onset and cell division, had statistical significance. Evaluation of those periods is useful for elucidating mechanism of transfection.

Transfection microarrays for high-throughput phenotypic screening of genes involved in cell migration.

Cell migration is important in several biological phenomena, such as cancer metastasis. Therefore, the identification of genes involved in cell migration might facilitate the discovery of antimetastatic drugs. However, screening of genes by the current methods can be complicated by factors related to cell stimulation, for example, abolition of contact inhibition and the release inflammatory cytokines from wounded cells during examinations of wound healing in vitro. To overcome these problems and identify genes involved in cell migration, in this chapter we describe the use of transfection microarrays for high-throughput phenotypic screening.

Onset timing of transient gene expression depends on cell division.

By using the time lapse of both phase-contrast and fluorescent images, we examined the morphology of cells and the dynamics of gene expression (EGFP). We applied k-means clustering to the time course data of fluorescent intensity of EGFP and successfully found four subpopulations. Discriminating the appropriate clusters and investigating the details of them, we found that almost all cells express the transfected gene after cell division and also found there is a strong correlation between onset timing and cell division. This result suggested that it is possible to normalize the dynamics of gene by arranging the onset timing of gene expression or by arranging the cell division.

A system for analyzing the event timing profile of single cells by using a model of neurite maturation in PC12D cells.

Nerve growth factor (NGF)-induced neurite maturation in PC12D cells involves neuritogenesis and neurite outgrowth. Actions of compounds affecting the neurite maturation are sometimes invisible behind the individually variable events in nature even in the clonal population. In this study, we designed a time-lapse imaging system to determine the timing of each event in individual PC12D cells. To examine the system, we analyzed the effect of staurosporine on the neurite maturation in PC12D cells. By using the system, we obtained four event timing data sets (stimulation by NGF with and without staurosporine at the concentrations of 0.01, 0.1, and 1 microM). A permutation test of these data sets revealed that staurosporine caused an early induction of neurite outgrowth during neurite maturation in PC12D cells. These results suggest that the time-lapse imaging system to determine the timing of each event in individual cells can provide a novel insight into features of a cell mass by single-cell analysis and is expected to be utilized for profiling of compounds that can serve as candidate drugs.

On-chip screening method for cell migration genes based on a transfection microarray.

Cell migration plays a major role in a variety of biological processes and a detailed understanding of associated mechanisms should lead to advances in the medical sciences, for example, in drug discovery for cancer therapy. However, the traditional methods used for analysis of cell migration cannot easily be scaled up for high-throughput screening. In this study, we have attempted to develop a novel simple method for high-throughput phenotypic screening for the identification of genes that are required for cell migration. As the appropriate cell line for the method, we found NBT-L2b cells that would be suitable for screening of migration-related genes in our method without influence by other cellular processes. Moreover, the idea for printing both the labeled fibronectin, for identification of the starting region of a cell, and the green fluorescent protein (GFP) expression vector, for identification of cells that had been transfected with siRNA and of the end point of migration, brings a rapid and efficient high-throughput screening procedure. Our new method will lead to an enhanced understanding of cell migration.

Modified fuzzy gap statistic for estimating preferable number of clusters in fuzzy k-means clustering.

In clustering methods, the estimation of the optimal number of clusters is significant for subsequent analysis. Without detailed biological information on the genes involved, the evaluation of the number of clusters becomes difficult, and we have to rely on an internal measure that is based on the distribution of the data of the clustering result. The Gap statistic has been proposed as a superior method for estimating the number of clusters in crisp clustering. In this study, we proposed a modified Fuzzy Gap statistic (MFGS) and applied it to fuzzy k-means clustering. For estimating the number of clusters, fuzzy k-means clustering with the MFGS was applied to two artificial data sets with noise and to two experimentally observed gene expression data sets. For the artificial data sets, compared with other internal measures, the MFGS showed a higher performance in terms of robustness against noise for estimating the optimal number of clusters. Moreover, it could be used to estimate the optimal number of clusters in experimental data sets. It was confirmed that the proposed MFGS is a useful method for estimating the number of clusters for microarray data sets.

Novel technique for preprocessing high dimensional time-course data from DNA microarray: mathematical model-based clustering.

MOTIVATION: Classifying genes into clusters depending on their expression profiles is one of the most important analysis techniques for microarray data. Because temporal gene expression profiles are indicative of the dynamic functional properties of genes, the application of clustering analysis to time-course data allows the more precise division of genes into functional classes. Conventional clustering methods treat the sampling data at each time point as data obtained under different experimental conditions without considering the continuity of time-course data between time periods t and t+1. Here, we propose a method designated mathematical model-based clustering (MMBC). RESULTS: The proposed method, designated MMBC, was applied to artificial data and time-course data obtained using Saccharomyces cerevisiae. Our method is able to divide data into clusters more accurately and coherently than conventional clustering methods. Furthermore, MMBC is more tolerant to noise than conventional clustering methods. AVAILABILITY: Software is available upon request. CONTACT: taizo@brs.kyushu-u.ac.jp.

A preprocessing method for inferring genetic interaction from gene expression data using Boolean algorithm.

Unknown genetic regulation mechanisms are expected to be discovered by information technology using large amount of biological data especially for gene expression data. In this study, we propose a novel inferring method for genetic interactions that combines our original preprocessing method and the Boolean algorithm. First, the performance of our method was evaluated using artificial data. The results showed that our method was able to infer genetic interactions with high specificity (specificity=0.629). Then, using our method, the genetic interaction was inferred from the experimental time course data collected using microarray on 69 genes of cell cycle for Saccharomyces cerevisiae. Our method estimated about 80% of all genetic interactions in Kyoto Encyclopedia Genes and Genomes (KEGG) for these genes. Furthermore, our method was able to infer several other genetic interactions that are not included in KEGG but whose existence is supported by other biological reports.