The 'invisible hand': regulation of RHO GTPases by RHOGDIs

The 'invisible hand' is a term originally coined by Adam Smith in the Theory of Moral Sentiments to describe the forces of self-interest, competition, and supply and demand that regulate the resources in society. This metaphor continues to be used by economists to describe the self-regulating nature of a market economy. The same metaphor can be used to describe the RHO-specific guanine nucleotide dissociation inhibitor (RHOGDI) family, which operates in the background, as an invisible hand, using similar forces to regulate the RHO GTPase cycle

RhoG regulates endothelial apical cup assembly downstream from ICAM1 engagement and is involved in leukocyte trans-endothelial migration

During trans-endothelial migration (TEM), leukocytes use adhesion receptors such as intercellular adhesion molecule-1 (ICAM1) to adhere to the endothelium. In response to this interaction, the endothelium throws up dynamic membrane protrusions, forming a cup that partially surrounds the adherent leukocyte. Little is known about the signaling pathways that regulate cup formation. In this study, we show that RhoG is activated downstream from ICAM1 engagement. This activation requires the intracellular domain of ICAM1. ICAM1 colocalizes with RhoG and binds to the RhoG-specific SH3-containing guanine-nucleotide exchange factor (SGEF). The SH3 domain of SGEF mediates this interaction. Depletion of endothelial RhoG by small interfering RNA does not affect leukocyte adhesion but decreases cup formation and inhibits leukocyte TEM. Silencing SGEF also results in a substantial reduction in RhoG activity, cup formation, and TEM. Together, these results identify a new signaling pathway involving RhoG and its exchange factor SGEF downstream from ICAM1 that is critical for leukocyte TEM

Regulation of Rho GTPase crosstalk, degradation and activity by RhoGDI1

At steady state, most Rho GTPases are bound in the cytosol to Rho Guanine nucleotide Dissociation Inhibitors (RhoGDI) 1. RhoGDIs have generally been considered to passively hold Rho proteins in an inactive state within the cytoplasm. Here we describe an evolutionarily conserved mechanism by which RhoGDI1 controls the homeostasis of Rho proteins in eukaryotic cells. We found that depletion of RhoGDI1 promotes misfolding and degradation of the cytosolic geranylgeranylated pool of Rho GTPases while unexpectedly activating the remaining membrane-bound fraction. Since RhoGDI1 levels are limiting, and Rho proteins compete for binding to RhoGDI1, overexpression of an exogenous Rho GTPase displaces endogenous Rho proteins bound to RhoGDI1, inducing their degradation and inactivation. These results raise important questions about the conclusions drawn from studies that manipulate Rho protein levels. In many cases the response observed may arise not simply from the overexpression per se, but from additional effects on the levels and activity of other Rho GTPases due to competition for binding to RhoGDI1, and may require a re-evaluation of previously published studies that rely exclusively on these techniques

Knockout of Vdac1 activates hypoxia-inducible factor through reactive oxygen species generation and induces tumor growth by promoting metabolic reprogramming and inflammation

BACKGROUND: Mitochondria are more than just the powerhouse of cells; they dictate if a cell dies or survives. Mitochondria are dynamic organelles that constantly undergo fusion and fission in response to environmental conditions. We showed previously that mitochondria of cells in a low oxygen environment (hypoxia) hyperfuse to form enlarged or highly interconnected networks with enhanced metabolic efficacy and resistance to apoptosis. Modifications to the appearance and metabolic capacity of mitochondria have been reported in cancer. However, the precise mechanisms regulating mitochondrial dynamics and metabolism in cancer are unknown. Since hypoxia plays a role in the generation of these abnormal mitochondria, we questioned if it modulates mitochondrial function. The mitochondrial outer-membrane voltage-dependent anion channel 1 (VDAC1) is at center stage in regulating metabolism and apoptosis. We demonstrated previously that VDAC1 was post-translationally C-terminal cleaved not only in various hypoxic cancer cells but also in tumor tissues of patients with lung adenocarcinomas. Cells with enlarged mitochondria and cleaved VDAC1 were also more resistant to chemotherapy-stimulated cell death than normoxic cancer cells. RESULTS: Transcriptome analysis of mouse embryonic fibroblasts (MEF) knocked out for Vdac1 highlighted alterations in not only cancer and inflammatory pathways but also in the activation of the hypoxia-inducible factor-1 (HIF-1) signaling pathway in normoxia. HIF-1α was stable in normoxia due to accumulation of reactive oxygen species (ROS), which decreased respiration and glycolysis and maintained basal apoptosis. However, in hypoxia, activation of extracellular signal-regulated kinase (ERK) in combination with maintenance of respiration and increased glycolysis counterbalanced the deleterious effects of enhanced ROS, thereby allowing Vdac1 (-/-) MEF to proliferate better than wild-type MEF in hypoxia. Allografts of RAS-transformed Vdac1 (-/-) MEF exhibited stabilization of both HIF-1α and HIF-2α, blood vessel destabilization, and a strong inflammatory response. Moreover, expression of Cdkn2a, a HIF-1-target and tumor suppressor gene, was markedly decreased. Consequently, RAS-transformed Vdac1 (-/-) MEF tumors grew faster than wild-type MEF tumors. CONCLUSIONS: Metabolic reprogramming in cancer cells may be regulated by VDAC1 through vascular destabilization and inflammation. These findings provide new perspectives into the understanding of VDAC1 in the function of mitochondria not only in cancer but also in inflammatory diseases

Young children's understanding of disabilities: the influence of development, context and cognition

Throughout Europe, educational support for children with disabilities has moved towards a model of inclusive education. Such policy changes mean that for all children there will be an increased likelihood of working with and encountering children with differing disabilities and difficulties. Previous research had indicated that children had poorly differentiated views of developmental differences. The present study investigated children?s representations of different disabilities. Seventy-nine 8-9 and 10-11 year old Greek children from an urban school and a rural school completed an attitudes toward school inclusion rating scale and a semi-structured interview. Responses to the attitude scale provided generally positive views of educational inclusion. However, children were less positive about activities that might directly reflect upon themselves. Children?s responses in the interviews indicated that they were developing rich representations of differences and diversities. Children had the greatest understanding of sensory and physical disabilities, followed by learning disabilities. There was limited knowledge of dyslexia and hyperactivity and no child was familiar with the term autism. Both groups of children identified a range of developmental difficulties, with older children being more aware of specific learning disabilities, their origin and impact. Results are discussed in terms of children?s developing knowledge systems and the implications for educational practices

The Small GTPase RhoA Localizes to the Nucleus and Is Activated by Net1 and DNA Damage Signals

Rho GTPases control many cellular processes, including cell survival, gene expression and migration. Rho proteins reside mainly in the cytosol and are targeted to the plasma membrane (PM) upon specific activation by guanine nucleotide exchange factors (GEFs). Accordingly, most GEFs are also cytosolic or associated with the PM. However, Net1, a RhoA-specific GEF predominantly localizes to the cell nucleus at steady-state. Nuclear localization for Net1 has been seen as a mechanism for sequestering the GEF away from RhoA, effectively rendering the protein inactive. However, considering the prominence of nuclear Net1 and the fact that a biological stimulus that promotes Net1 translocation out the nucleus to the cytosol has yet to be discovered, we hypothesized that Net1 might have a previously unidentified function in the nucleus of cells.Using an affinity precipitation method to pulldown the active form of Rho GEFs from different cellular fractions, we show here that nuclear Net1 does in fact exist in an active form, contrary to previous expectations. We further demonstrate that a fraction of RhoA resides in the nucleus, and can also be found in a GTP-bound active form and that Net1 plays a role in the activation of nuclear RhoA. In addition, we show that ionizing radiation (IR) specifically promotes the activation of the nuclear pool of RhoA in a Net1-dependent manner, while the cytoplasmic activity remains unchanged. Surprisingly, irradiating isolated nuclei alone also increases nuclear RhoA activity via Net1, suggesting that all the signals required for IR-induced nuclear RhoA signaling are contained within the nucleus.These results demonstrate the existence of a functional Net1/RhoA signaling pathway within the nucleus of the cell and implicate them in the DNA damage response

Cyclic uniaxial cell stretching in tissue culture using a LEGO®-based mechanical stretcher and a polydimethylsiloxane stretchable vessel

Summary: Mechanical signals are essential for the regulation of many biological processes. Therefore, it has become paramount to account for these mechanical parameters when exploring biological processes. Here, we describe a protocol to apply cyclic uniaxial stretch on cells in culture using a LEGO®-based mechanical stretcher and a flexible custom-made polydimethylsiloxane culture vessel as well as validated downstream applications. While this system offers an out-of-the-box limited type of simulation, it provides a reliable and low-cost opportunity to perform cell stretching.For complete details on the use and execution of this protocol, please refer to Boulter et al. (2020)

Integrin-linked kinase and its partners: a modular platform regulating cell-matrix adhesion dynamics and cytoskeletal organization.

International audienceIntegrin-linked kinase (ILK) represents a key component of integrin signaling complexes that functions in concert with multiple binding partners to transmit cues from the extracellular matrix environment to the actin cytoskeleton. Both gain- and loss-of-function approaches to study ILK have confirmed the essential role of this protein in regulating cell-matrix adhesion dynamics and cytoskeletal organization