Lilian Soon

Phospholipase C and cofilin are required for carcinoma cell directionality in response to EGF stimulation

The epidermal growth factor (EGF)–induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.

N-WASP and cortactin are involved in invadopodium-dependent chemotaxis to EGF in breast tumor cells

Metastatic mammary carcinoma cells, which have previously been observed to form mature, matrix degrading invadopodia on a thick ECM matrix, are able to form invadopodia with similar characteristics on glass without previously applied matrix. They form in response to epidermal growth factor (EGF), and contain the usual invadopodium core proteins N-WASP, Arp2/3, cortactin, cofilin, and F-actin. The study of invadopodia on glass allows for higher resolution analysis including the use of total internal reflection microscopy and analysis of their relationship to other cell motility events, in particular, lamellipodium extension and chemotaxis toward an EGF gradient. Invadopodium formation on glass requires N-WASP and cortactin but not microtubules. In a gradient of EGF more invadopodia form on the side of the cells facing the source of EGF. In addition, depletion of N-WASP or cortactin, which blocks invadopodium fromation, inhibits chemotaxis of cells towards EGF. This appears to be a localized defect in chemotaxis since depletion of N-WASP or cortactin via siRNA had no effect on lamellipodium protrusion or barbed end generation at the lamellipodiums leading edge. Since chemotaxis to EGF by breast tumor cells is involved in metastasis, inhibiting N-WASP activity in breast tumor cells might prevent metastasis of tumor cells while not affecting chemotaxis-dependent innate immunity which depends on WASp function in macrophages.

Correlative fluorescence and transmission electron microscopy: an elegant tool to study the actin cytoskeleton of whole-mount (breast) cancer cells

Elucidating the structure and dynamics of lamellipodia and filopodia in response to different stimuli is a topic of continuing interest in cancer cells as these structures may be attractive targets for therapeutic purposes. Interestingly, a close functional relationship between these actin‐rich protrusions and specialized membrane domains has been recently demonstrated. The aim of this study was therefore to investigate the fine organization of these actin‐rich structures and examine how they structurally may relate to detergent‐resistant membrane (DRM) domains in the MTLn3 EGF/serum starvation model. For this reason, we designed a straightforward and alternative method to study cytoskeleton arrays and their associated structures by means of correlative fluorescence (/laser)‐ and electron microscopy (CFEM).

Actin-Binding Drugs: An Elegant Tool to Dissect Subcellular Processes in Endothelial and Cancer Cells

Until a decade or so ago there were only a few agents available that interfered with cellular activities by binding to actin. In fact, most of our initial knowledge concerning the involvement of actin in basic cellular processes was based on the extensive use of the mold metabolites cytochalasins. However, the actin-binding activities and cellular effects of cytochalasins are complex and difficult to interpret (Cooper 1987; Sampath and Pollard 1991), so that the functions and dynamics of the actin cytoskeleton in various organisms remained elusive. Another widely used class of actin-binding drugs, the mushroom-derived phallotoxins, stabilizes actin filaments and promotes actin polymerization (Cooper 1987; Sampath and Pollard 1991), but they do not enter most cell types and are predominantly used as fluorescent derivatives to visualize actin filaments in fixed cells. With the growing awareness that the actin cytoskeleton is involved in practically all aspects of cell behavior, as well as in cancer and other human diseases, it has become increasingly important to identify new agents with well-defined actin-binding properties that preferentially affect certain aspects of actin filament organization and dynamics in a reversible manner (Table 1). Such agents are not only necessary as research tools to better understand how the different actin structures are assembled, organized, and function in cells, but may also be potentially useful as therapeutic agents in the treatment of diseases (Spector et al. 1999; Yeung and Paterson 2002; Fenteany and Zhu 2003; Giganti and Friederich 2003). So far, however, there is no actin-binding drug in the clinic, and there are only limited attempts to explore the pharmaceutical usefulness of antiactin drugs for the treatment of diseases (Table 2). Inhibition of cell growth and motility, control of viral budding, eye outflow, transendothelial transport, and neointimal hyperplasia are a few examples in which actin-binding drugs were found to have some effects. Of these, the topical application of microfilament-disrupting drugs (mainly the family of latrunculins) to the eye seems to reduce intraocular pressure in such an effective way that this might 3 Actin-Binding Drugs: An Elegant Tool to Dissect Subcellular Processes in Endothelial and Cancer Cells