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The sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), can induce or inhibit cellular migration. The intermediate filament protein vimentin is an inducer of migration and a marker for epithelial–mesenchymal transition. Given that keratin intermediate filaments are regulated by SPC, with consequences for cell motility, we wanted to determine whether vimentin is also regulated by sphingolipid signalling and whether it is a determinant for sphingolipid-mediated functions. In cancer cells where S1P and SPC inhibited migration, we observed that S1P and SPC induced phosphorylation of vimentin on S71, leading to a corresponding reorganization of vimentin filaments. These effects were sphingolipid-signalling-dependent, because inhibition of either the S1P2 receptor (also known as S1PR2) or its downstream effector Rho-associated kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) nullified the sphingolipid-induced effects on vimentin organization and S71 phosphorylation. Furthermore, the anti-migratory effect of S1P and SPC could be prevented by expressing S71-phosphorylation-deficient vimentin. In addition, we demonstrated, by using wild-type and vimentin-knockout mouse embryonic fibroblasts, that the sphingolipid-mediated inhibition of migration is dependent on vimentin. These results imply that this newly discovered sphingolipid–vimentin signalling axis exerts brake-and-throttle functions in the regulation of cell migration.

Implementation of the swine umbilical vein endothelial cells (SUVECs) model in vitro can be instrumental in determining the biology of endothelial cells. We have generated an immortalized endothelial cell line, G-1410, using Simian virus 40 T-antigen (SV40 T-ag) primarily to overcome the short life span before the onset of senescence and high variability among enzymatically isolated cells of primary cultures. Fast proliferating cells were selected from cultures and, after a fifth passage, examined for the presence of the SV40 T-ag by PCR and immunocytochemistry. Phase contrast and transmission electron microscopy revealed that G-1410 cells did not differ morphologically from SUVECs. The G-1410 cells exhibited positive staining for vascular endothelial (VE)-cadherin and von Willebrand factor (vWF), and formed capillary-like tube structures on Matrigel. Despite the strong oncogenic signal provided by SV40 T-ag, these transformed G-1410 cells have remained karyotypically normal and non-tumorigenic. G-1410 cells also responded to stimulation with VEGF, FGF-2, and newborn calf serum. Moreover, G-1410 cells showed elevated expression of VEGF120, VEGF164 (VEGF-A), and FGF-2 at both mRNA and protein levels. In conclusion, based on the cytological and functional evaluation of the newly obtained immortalized cell line, it can be concluded that G-1410 cells provide a useful tool for studying the effects of VEGF and FGF systems, and other signal transduction pathways related to angiogenesis. Mol. Reprod. Dev. 78:597–610, 2011. © 2011 Wiley-Liss, Inc.

Intermediate filament (IF) proteins comprise a large family with more than 70 members. Initially, IFs were assumed to provide only structural reinforcement for the cell. However, IFs are now known to be dynamic structures that are involved in a wide range of cellular processes during all stages of life, from development to ageing, and during homeostasis and stress. This Commentary discusses some lesser-known functional and regulatory aspects of IFs. We specifically address the emerging roles of nestin in myogenesis and cancer cell migration, and examine exciting evidence on the regulation of nestin and lamin A by the notch signalling pathway, which could have repercussions for our understanding of the roles of IF proteins in development and ageing. In addition, we discuss the modulation of the post-translational modifications of neuronally expressed IFs and their protein–protein interactions, as well as IF glycosylation, which not only has a role in stress and ageing, but might also regulate IFs during development. Although many of these recent findings are still preliminary, they nevertheless open new doors to explore the functionality of the IF family of proteins.

The heat shock response is a primordial defense mechanism against cell stress and protein misfolding. It proceeds with the minimum number of mechanisms that any regulatory network must include, a stress-induced activation and a feedback regulation, and can thus be regarded as the archetype for a cellular regulatory process. We propose here a simple mechanistic model for the eukaryotic heat shock response, including its mathematical validation. Based on numerical predictions of the model and on its sensitivity analysis, we minimize the model by identifying the reactions with marginal contribution to the heat shock response. As the heat shock response is a very basic and conserved regulatory network, our analysis of the network provides a useful foundation for modeling strategies of more complex cellular processes.

Intermediate filaments are dynamically regulated by their post-translational modifications. Initially these modifications were found to regulate filament dynamics and organization. In the last few years, their roles have extended significantly to facilitating, for example, the recruitment and sequestration of signaling molecules that regulate a wide range of cellular functions. While phosphorylation has been established as the principal post-translational modification regulating intermediate filament function, other modifications with co-operative roles are emerging, adding a further dimensions to intermediate filament-mediated signaling.

Nestin, an intermediate filament protein and marker of undifferentiated cells, is expressed in several cancers. Nestin is important for neuronal survival and is a regulator of myogenesis but its function in malignancy is ambiguous. We show that nestin downregulation leads to a redistribution of phosphorylated focal adhesion kinase (pFAK, also known as PTK2) to focal adhesions and alterations in focal adhesion turnover. Nestin downregulation also leads to an increase in the protein levels of integrin α5β1 at the cell membrane, activation of integrin β1 and an increase in integrin clustering. These effects have striking consequences for cell invasion, as nestin downregulation leads to a significant increase in pFAK- and integrin-dependent matrix degradation and cell invasion. Our results indicate that nestin regulates the localisation and functions of FAK and integrin. Because nestin has been shown to be prevalent in a number of specific cancers, our observations have broad ramifications for the roles of nestin in malignant transformation.

Nestin, an intermediate filament protein and marker of undifferentiated cells, is expressed in several cancers. Nestin is important for neuronal survival and is a regulator of myogenesis but its function in malignancy is ambiguous. We show that nestin downregulation leads to a redistribution of phosphorylated focal adhesion kinase (pFAK, also known as PTK2) to focal adhesions and alterations in focal adhesion turnover. Nestin downregulation also leads to an increase in the protein levels of integrin α5β1 at the cell membrane, activation of integrin β1 and an increase in integrin clustering. These effects have striking consequences for cell invasion, as nestin downregulation leads to a significant increase in pFAK- and integrin-dependent matrix degradation and cell invasion. Our results indicate that nestin regulates the localisation and functions of FAK and integrin. Because nestin has been shown to be prevalent in a number of specific cancers, our observations have broad ramifications for the roles of nestin in malignant transformation.

Implementation of the swine umbilical vein endothelial cells (SUVECs) model in vitro can be instrumental in determining the biology of endothelial cells. We have generated an immortalized endothelial cell line, G-1410, using Simian virus 40 T-antigen (SV40 T-ag) primarily to overcome the short life span before the onset of senescence and high variability among enzymatically isolated cells of primary cultures. Fast proliferating cells were selected from cultures and, after a fifth passage, examined for the presence of the SV40 T-ag by PCR and immunocytochemistry. Phase contrast and transmission electron microscopy revealed that G-1410 cells did not differ morphologically from SUVECs. The G-1410 cells exhibited positive staining for vascular endothelial (VE)-cadherin and von Willebrand factor (vWF), and formed capillary-like tube structures on Matrigel. Despite the strong oncogenic signal provided by SV40 T-ag, these transformed G-1410 cells have remained karyotypically normal and non-tumorigenic. G-1410 cells also responded to stimulation with VEGF, FGF-2, and newborn calf serum. Moreover, G-1410 cells showed elevated expression of VEGF120, VEGF164 (VEGF-A), and FGF-2 at both mRNA and protein levels. In conclusion, based on the cytological and functional evaluation of the newly obtained immortalized cell line, it can be concluded that G-1410 cells provide a useful tool for studying the effects of VEGF and FGF systems, and other signal transduction pathways related to angiogenesis.

We present in this paper a novel molecular model for the gene regulatory network responsible for the eukaryotic heat shock response. Our model includes the temperature-induced protein misfolding, the chaperone activity of the heat shock proteins, and the backregulation of their gene transcription. We then build a mathematical model for it, based on ordinary differential equations. Finally, we discuss the parameter fit and the implications of the sensitivity analysis for our model.