BackgroundFilopodia are small cellular projections that help cells to move through and sense their environment. Filopodia play crucial roles in processes such as development and wound-healing. Also, increases in filopodia number or size are characteristic of many invasive cancers and are correlated with increased rates of metastasis in mouse experiments. Thus, one possible route to developing anti-metastatic therapies is to target factors that influence the filopodia system. Filopodia can be detected by eye using confocal fluorescence microscopy, and they can be manually annotated in images to quantify filopodia parameters. Although this approach is accurate, it is slow, tedious and not entirely objective. Manual detection is a significant barrier to the discovery and quantification of new factors that influence the filopodia system.ResultsHere, we present FiloDetect, an automated tool for detecting, counting and measuring the length of filopodia in fluorescence microscopy images. The method first segments the cell from the background, using a modified triangle threshold method, and then extracts the filopodia using a series of morphological operations. We verified the accuracy of FiloDetect on Rat2 and B16F1 cell images from three different labs, showing that per-cell filopodia counts and length estimates are highly correlated with the manual annotations. We then used FiloDetect to assess the role of a lipid kinase on filopodia production in breast cancer cells. Experimental results show that PI4KIII β expression leads to an increase in filopodia number and length, suggesting that PI4KIII β is involved in driving filopodia production.ConclusionFiloDetect provides accurate and objective quantification of filopodia in microscopy images, and will enable large scale comparative studies to assess the effects of different genetic and chemical perturbations on filopodia production in different cell types, including cancer cell lines.

Emerging evidence now implicates phosphatidylinositol 4-kinases (PI4K), enzymes that generate PI(4)P from phosphatidylinositol (PtdIns), in cancer. In this study, we investigate the role of

Eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a transforming gene product that is highly expressed in human tumors of the ovary, lung, and breast. eEF1A2 also stimulates actin remodeling, and the expression of this factor is sufficient to induce the formation of filopodia, long cellular processes composed of bundles of parallel actin filaments. Here, we find that eEF1A2 stimulates formation of filopodia by increasing the cellular abundance of cytosolic and plasma membrane-bound phosphatidylinositol-4,5 bisphosphate [PI(4,5)P 2 ]. We have previously reported that the eEF1A2 protein binds and activates phosphatidylinositol-4 kinase III beta (PI4KIII␤), and we find that production of eEF1A2-dependent PI(4,5)P 2 and generation of filopodia require PI4KIII␤. Furthermore, PI4KIII␤ is itself capable of activating both the production of PI(4,5)P 2 and the creation of filopodia. We propose a model for extrusion of filopodia in which eEF1A2 activates PI4KIII␤, and activated PI4KIII␤ stimulates production of PI(4,5)P 2 and filopodia by increasing PI4P abundance. Our work suggests an important role for both eEF1A2 and PI4KIII␤ in the control of PI(4,5)P 2 signaling and actin remodeling.Filopodia are fingerlike projections from the plasma membrane that are the first cellular structures to reach new spaces during cell migration. Filopodia are composed of bundled actin filaments and actin-associated proteins (9, 13). Transmembrane receptors within filopodia respond to extracellular cues and guide directional movement toward chemoattractants (26). In addition, filopodia contain abundant adhesion molecules that regulate cellular attachment to growth substrates and cell-cell interactions (37). As such, filopodia regulate several key physiological processes, including cell migration, wound healing, and development. For example, filopodia are essential for neurogenesis in mice and for cell-cell adhesion during Drosophila melanogaster embryogenesis (9, 13).We have previously described a role for eukaryotic elongation factor 1 alpha 2 (eEF1A2) in the initiation and maintenance of filopodia (1). In several types of mammalian cells, eEF1A2 expression is sufficient to stimulate formation of filopodia (1). eEF1A2 is one of two members of the eEF1A family of proteins, eEF1A1 and eEF1A2. During the elongation phase of protein synthesis, GTP-bound eEF1A proteins interact with amino-acylated tRNA and recruit them to the ribosome (18). While eEF1A1 and eEF1A2 are believed to have equivalent roles in protein translation, their tissue-specific expression patterns are each markedly different. eEF1A1 is expressed ubiquitously, whereas eEF1A2 is detectably expressed only in normal tissues of mammalian heart, brain, and skeletal muscle (24a, 24b, 28). Homozygous deletion of eEF1A2 occurs in the wasted mouse (7). These mice develop normally but suffer from neuromuscular abnormalities and immunodeficiency and die at approximately 1 month of age (35,36). Importantly, eEF1A2 is likely to be a human oncogene, it is highly expressed, and its gene is...

BackgroundFilopodia are actin-based cellular projections that have a critical role in initiating and sustaining directional migration in vertebrate cells. Filopodia are highly dynamic structures that show a rich diversity in appearance and behavior. While there are several mathematical models of filopodia initiation and growth, testing the capacity of these theoretical models in predicting empirical behavior has been hampered by a surprising shortage of quantitative data related to filopodia. Neither is it clear how quantitatively robust the cellular filopodial network is and how perturbations alter it.ResultsWe have measured the length and interfilopodial separation distances of several thousand filopodia in the rodent cell line Rat2 and measured these parameters in response to genetic, chemical and physical perturbation. Our work shows that length and separation distance have a lognormal pattern distribution over their entire detection range (0.4 μm to 50 μm).ConclusionsWe find that the lognormal distribution of length and separation is robust and highly resistant to perturbation. We also find that length and separation are independent variables. Most importantly, our empirical data is not entirely in agreement with predictions made based on existing theoretical models and that filopodial size and separation are an order of magnitude larger than what existing models suggest.

<p>Supplementary Figure 3</p>