RNA interference (RNAi) has become a powerful technique for reverse genetics and drug discovery and, in both of these areas, large-scale high-throughput RNAi screens are commonly performed. The statistical techniques used to analyze these screens are frequently borrowed directly from smallmolecule screening; however small-molecule and RNAi data characteristics differ in meaningful ways. We examine the similarities and differences between RNAi and small-molecule screens, highlighting particular characteristics of RNAi screen data that must be addressed during analysis. Additionally, we provide guidance on selection of analysis techniques in the context of a sample workflow.

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Crawling T cell locomotion in which activated lymphocyte function-associated antigen 1 (LFA-1) integrins participate is associated with translocation of the protein kinase C-beta (PKC-beta) isoenzyme to the microtubule cytoskeleton. In normal T cells and T lymphoma cell lines, this type of motility is accompanied by PKC-beta-sensitive cytoskeletal rearrangements and the formation of trailing cell extensions, which are supported by microtubules. Expression of PKC-beta(I) and enhanced green fluorescent protein (EGFP) in nonmotile PKC-beta-deficient T cells restored their locomotory behavior in response to a triggering stimulus delivered via LFA-1 and correlated directly with the degree of cell polarization. We have also shown that PKC-beta(I) is a component of the tubulin-enriched LFA-1-cytoskeletal complex assembled upon LFA-1 cross-linking. These observations may have physiological equivalents at advanced (post-integrin activation) stages of lymphocyte extravasation.

Chemokines such as SDF-1α play a crucial role in orchestrating T lymphocyte polarity and migration via polymerization and reorganization of the F-actin cytoskeleton, but the role of actin-associated proteins in this process is not well characterized. In this study, we have investigated a role for L-plastin, a leukocyte-specific F-actin–bundling protein, in SDF-1α–stimulated human T lymphocyte polarization and migration. We found that L-plastin colocalized with F-actin at the leading edge of SDF-1α–stimulated T lymphocytes and was also phosphorylated at Ser5, a site that when phosphorylated regulates the ability of L-plastin to bundle F-actin. L-plastin phosphorylation was sensitive to pharmacological inhibitors of protein kinase C (PKC), and several PKC isoforms colocalized with L-plastin at the leading edge of SDF-1α–stimulated lymphocytes. However, PKC ζ, an established regulator of cell polarity, was the only isoform that regulated L-plastin phosphorylation. Knockdown of L-plastin expression with small interfering RNAs demonstrated that this protein regulated the localization of F-actin at the leading edge of chemokine-stimulated cells and was also required for polarization, lamellipodia formation, and chemotaxis. Knockdown of L-plastin expression also impaired the Rac1 activation cycle and Akt phosphorylation in response to SDF-1α stimulation. Furthermore, L-plastin also regulated SDF-1α–mediated lymphocyte migration on the integrin ligand ICAM-1 by influencing velocity and persistence, but in a manner that was independent of LFA-1 integrin activation or adhesion. This study, therefore, demonstrates an important role for L-plastin and the signaling pathways that regulate its phosphorylation in response to chemokines and adds L-plastin to a growing list of proteins implicated in T lymphocyte polarity and migration.

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T-cell migration is a complex highly coordinated process that involves cell adhesion to the high endothelial venules or to the extracellular matrix by surface receptor/ligand interactions, cytoskeletal rearrangements, and phosphorylation-dependent signaling cascades. The mechanism(s) that regulates T-cell migration is of considerable relevance for understanding the pathogenesis of various diseases, such as chronic inflammatory diseases and cancer metastasis. This study was designed to identify potential involvement of STAT3, a latent transcription factor, in mediating integrin-induced T-cell migration. Using our previously characterized in vitro model for lymphocyte migration, we demonstrate that STAT3 is activated and translocated to the nucleus during the process of active motility of Hut78 T-lymphoma cells triggered via LFA-1. Blocking STAT3 signaling by multiple approaches inhibited LFA-1-induced T-cell locomotion via destabilization of microtubules and post-translational modification of tubulin. Here, we show that STAT3 physically interacts with stathmin to regulate microtubule dynamics in migrating T-cells. These observations strongly indicate that STAT3 is critically important for T-cell migration and associated signaling events.Efficient operation of the adaptive immune system requires migration of T-lymphocytes from the vascular compartment across tissue barriers and through the extracellular matrix. This process involves a series of integrin ligand-receptor interactions (1) that initially retards lymphocyte flow and ultimately leads to arrest and diapedesis across the endothelium (2, 3). T-cells utilize the integrin, lymphocyte function-associated antigen-1 (LFA-1), 3 when migrating in response to chemoattractants across the vasculature into lymph nodes or inflamed tissues (1, 4, 5). By engagement with ligands from the intercellular adhesion molecule group (ICAMs), in particular ICAM-1, LFA-1 also provides a strong adhesive force to promote and stabilize T-cell and antigen-presenting cell conjugate formation. We have demonstrated that LFA-1 transduces a variety of transmembrane signals in crawling T-cells involving protein kinase C activation and cytoskeletal rearrangement (4, 6 -9). However, the exact sequence of downstream integrin-mediated signaling events resulting in cytoskeletal rearrangements and cell locomotion is not fully understood.T-cell migration involves cross-talk between integrins and the cytoskeleton, coordinated changes in the cytoskeleton, and the controlled formation and dispersal of adhesion sites (10). Motile lymphocytes develop trailing extensions, which contain cytoskeletal and signaling elements (11). Microtubules (MTs) are essential components of the cytoskeleton and are important for many aspects of mammalian cell responses, including cell division, growth, migration, and signaling (12)(13)(14). Whereas the actin cytoskeleton provides the driving force at the cell front, the MT network assumes a regulatory function in coordinating rear retraction (15). MT retraction into the ...

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L-plastin, a gene that codes for an actin-bundling protein, is upregulated in the metastatic colon cancer cell line SW620, when compared to its premetastatic counterpart SW480. The aim of our study was to characterise the effect of L-plastin overexpression on SW480 cells in the context of the acquisition of a metastatic phenotype. SW480 cell lines overexpressing L-plastin were established (SW480-LPL). Analysis of these cell lines revealed significantly higher rates of proliferation and invasion than the control cell line (SW480-Ctrl). In addition, the expression of E-cadherin was lost from SW480-LPL cells. Treatment of SW480-LPL cells with cytochalasin B, an inhibitor of endocytosis, attenuated the loss of Ecadherin expression in these cells. The association of L-plastin overexpression with an increased rate of proliferation and invasion, and loss of E-cadherin expression in the SW480 colon cancer cell line indicates that L-plastin plays an important mechanistic role in colorectal cancer metastasis (supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/ suppmat/index.html). ' 2005 Wiley-Liss, Inc.Key words: colon cancer; metastasis; L-plastin; E-cadherin Colorectal cancer (CRC) is a leading cause of morbidity and mortality and accounted for 203,700 deaths in Europe in 2004. 1 A genetic model for colorectal tumourigenesis has previously been described, but the molecular mechanisms of metastasis are not well elucidated. Decrease in cell-cell adhesion, loss of contact inhibition, increase in proliferation and dedifferentiation are all observed in the progression of tumours to the invasive phenotype. In recent years, research into metastasis has focussed increasingly on the actin cytoskeleton and actin-associated proteins.L-plastin is an actin-bundling protein usually expressed only in cells of haemopoietic origin. However, the L-plastin gene is frequently activated in human cancers. Sixty-eight percent of carcinomas and 53 percent of other nonhaemopoietic cancers exhibit constitutive activation of the L-plastin gene, and L-plastin synthesis has been detected in a primary culture of ovarian carcinomas. 2,3 L-plastin is phosphorylated on Serine 5 in response to the activation of the integrin a(M) b(2) on polymorphonuclear neutrophils. 4 Additionally, the yeast homologue of L-plastin, Sac6, is involved in yeast fluid phase endocytosis, and L-plastin restores normal function in yeast with a null mutation for Sac6. 5 The colon carcinoma cell lines SW480 and SW620 represent a good model for the study of CRC progression in that they are derived from a single patient. SW480 was established from a primary colon adenocarcinoma and SW620 was established, 1 year later, from a lymph node metastasis. 6 More recently, the cell lines have been validated as showing appropriate phenotypic differences following long-term culture in vitro. 7 As such, these cell lines represent an interesting model for studying molecular events in the la...