Despite their importance in cell biology, the mechanisms that maintain the nucleus in its proper position in the cell are not well understood. This is primarily the result of an incomplete knowledge of the proteins in the outer nuclear membrane (ONM) that are able to associate with the different cytoskeletal systems. Two related ONM proteins, nuclear envelope spectrin repeat (nesprin)–1 and –2, are known to make direct connections with the actin cytoskeleton through their NH₂-terminal actin-binding domain (ABD). We have now isolated a third member of the nesprin family that lacks an ABD and instead binds to the plakin family member plectin, which can associate with the intermediate filament (IF) system. Overexpression of nesprin-3 results in a dramatic recruitment of plectin to the nuclear perimeter, which is where these two molecules are colocalized with both keratin-6 and -14. Importantly, plectin binds to the integrin α6β4 at the cell surface and to nesprin-3 at the ONM in keratinocytes, suggesting that there is a continuous connection between the nucleus and the extracellular matrix through the IF cytoskeleton.

Background: The estrogen metabolite 2-methoxyestradiol (2ME2) and a number of synthesised derivatives have been shown to bind to microtubules thereby arresting cancer cells in mitosis which leads to apoptosis. In interphase cells, microtubules play an important role in the delivery of proteins to subcellular locations including the focal adhesions. In fact, focal adhesion dynamics and cell migration are in part regulated by microtubules. We hypothesised that novel 2ME2 derivatives can alter cell migration by influencing microtubule dynamics in interphase cells. In this report we describe 2ME2 derivatives that display anti-migratory capabilities in a metastatic breast cancer cell line through their effects on the microtubule network resulting in altered focal adhesion signalling and RhoA activity. Methods: Cell migration was assayed using wound healing assays. To eliminate mitosis blockage and cell rounding as a confounding factor cell migration was also assessed in interphase blocked cells. Fluorescence confocal microscopy was used to visualise microtubule dynamics and actin cytoskeleton organisation while western blot analysis was performed to analyse focal adhesion signalling and RhoA activation. Results: 2ME2 derivatives, ESE-one and ESE-15-one, inhibited cell migration in cycling cells as expected but equally diminished migration in cells blocked in interphase. While no significant effects were observed on the actin cytoskeleton, focal adhesion kinase activity was increased while RhoA GTPase activity was inhibited after exposure to either compound. Microtubule stability was increased as evidenced by the increased length and number of detyrosinated microtubules while at the same time clear disorganisation of the normal radial microtubule organisation was observed including multiple foci. Conclusions: ESE-15-one and ESE-one are potent migration inhibitors of metastatic breast cancer cells. This ability is coupled to alterations in focal adhesion signalling but more importantly is associated with severe disorganisation of microtubule dynamics and polarity. Therefore, these compounds may offer potential as anti-metastatic therapies.

It has long been known that phosphoinositides are present in cellular membranes, but only in the past four decades has our understanding of their importance for proper cell function advanced significantly. Key to determining the biological roles of phosphoinositides is understanding the enzymes involved in their metabolism. Although many such enzymes have now been identified, there is still much to learn about their cellular functions. Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) are a group of kinases that catalyse the production of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2]. As well as being a substrate for the enzymes phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI3K), PtdIns(4,5)P2 acts as a second messenger in its own right, influencing a variety of cellular processes. In this Commentary, we review how PIP5Ks are modulated to achieve regulated PtdIns(4,5)P2 production, and discuss the role of these proteins in different cellular processes.

Introduction of the integrin β1- but not the β3-subunit in GE11 cells induces an epithelial-to-mesenchymal-transition (EMT)-like phenomenon that is characterized by the loss of cell-cell contacts, cell scattering, increased cell migration and RhoA activity, and fibronectin fibrillogenesis. Because galactose-binding lectins (galectins) have been implicated in these phenomena, we investigated whether galectins are involved in the β1-induced phenotype. We examined 9 galectins and, intriguingly, found that the expression of galectin-3 (Gal-3) is specifically induced by β1 but not by β3. Using β1-β3 chimeric integrins, we show that the induction of Gal-3 expression requires the hypervariable region in the extracellular domain of β1, but not its cytoplasmic tail. Furthermore, Gal-3 expression does not depend on RhoA signaling, serum factors, or any of the major signal transduction pathways involving protein kinase C (PKC), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase-1/-2 (ERK-1/2), phosphatidylinositol-3-OH kinase (PI3-K), or Src kinases. Instead, Gal-3 expression is controlled in an epigenetic manner. Whereas DNA methylation of the Lgals3 promoter maintains Gal-3 silencing in GE11 cells, expression of β1 causes its demethylation, leading to transcriptional activation of the Lgals3 gene. In turn, Gal-3 expression enhances β1 integrin-mediated cell adhesion to fibronectin (FN) and laminin (LN), as well as cell migration. Gal-3 also promotes β1-mediated cell adhesion to LN and Collagen-1 (Col)-1 in cells that endogenously express Gal-3 and β1 integrins. In conclusion, we identify a functional feedback-loop between β1 integrins and Gal-3 that involves the epigenetic induction of Gal-3 expression during integrin-induced EMT and cell scattering.

In N1E-115 cells, neurite retraction induced by neurite remodelling factors such as lysophosphatidic acid, sphingosine 1-phosphate and semaphorin 3A require the activity of phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks). PIP5Ks synthesise the phosphoinositide lipid second messenger phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P₂], and overexpression of active PIP5K is sufficient to induce neurite retraction in both N1E-115 cells and cerebellar granule neurones. However, how PIP5Ks are regulated or how they induce neurite retraction is not well defined. Here, we show that neurite retraction induced by PIP5Kβ is dependent on its interaction with the low molecular weight G protein Rac. We identified the interaction site between PIP5Kβ and Rac1 and generated a point mutant of PIP5Kβ that no longer interacts with endogenous Rac. Using this mutant, we show that Rac controls the plasma membrane localisation of PIP5Kβ and thereby the localised synthesis of PtdIns(4,5)P₂ required to induce neurite retraction. Mutation of this residue in other PIP5K isoforms also attenuates their ability to induce neurite retraction and to localise at the membrane. To clarify how increased levels of PtdIns(4,5)P₂ induce neurite retraction, we show that mutants of vinculin that are unable to interact with PtdIns(4,5)P₂, attenuate PIP5K- and LPA-induced neurite retraction. Our findings support a role for PtdIns(4,5)P₂ synthesis in the regulation of vinculin localisation at focal complexes and ultimately in the regulation of neurite dynamics.

Increased activity of the proto-oncogene c-Src and elevated levels of integrin α_vβ₃ are found in melanomas and multiple carcinomas. Regulation of c-Src involves “priming” through disruption of intramolecular interactions followed by “activation” through phosphorylation in the kinase domain. Interactions with overexpressed receptor tyrosine kinases or mutations in the SRC gene can induce priming of c-Src in cancer. Here, we show that α_vβ₃ promotes activation of primed c-Src, causing enhanced phosphorylation of established Src substrates, survival, proliferation, and tumor growth. The β₃ cytoplasmic tail is required and sufficient for integrin-mediated stimulation of all these events through a mechanism that is independent of β₃ tyrosine phosphorylation. Instead, experiments using Src variants containing the v-Src Src homology 3 (SH3) domain and using mutant β₃ subunits indicate that a functional interaction of the β₃ cytoplasmic tail with the c-Src SH3 domain is required. These findings delineate a novel integrin-controlled oncogenic signaling cascade and suggest that the interaction of α_vβ₃ with c-Src may represent a novel target for therapeutic intervention. [Cancer Res 2007;67(6):2693–700]