RhoU forms homo-oligomers to regulate cellular responses.

RhoU is an atypical member of the Rho family of small G-proteins, which has N- and C-terminal extensions compared to the classic Rho GTPases RhoA, Rac1 and Cdc42, and associates with membranes through C-terminal palmitoylation rather than prenylation. RhoU mRNA expression is upregulated in prostate cancer and is considered a marker for disease progression. Here, we show that RhoU overexpression in prostate cancer cells increases cell migration and invasion. To identify RhoU targets that contribute to its function, we found that RhoU homodimerizes in cells. We map the region involved in this interaction to the C-terminal extension and show that C-terminal palmitoylation is required for self-association. Expression of the isolated C-terminal extension reduces RhoU-induced activation of p21-activated kinases (PAKs), which are known downstream targets for RhoU, and induces cell morphological changes consistent with inhibiting RhoU function. Our results show for the first time that the activity of a Rho family member is stimulated by self-association, and this is important for its activity.

Intermediate filaments: Integration of cell mechanical properties during migration.

Cell migration is a vital and dynamic process required for the development of multicellular organisms and for immune system responses, tissue renewal and wound healing in adults. It also contributes to a variety of human diseases such as cancers, autoimmune diseases, chronic inflammation and fibrosis. The cytoskeleton, which includes actin microfilaments, microtubules, and intermediate filaments (IFs), is responsible for the maintenance of animal cell shape and structural integrity. Each cytoskeletal network contributes its unique properties to dynamic cell behaviour, such as cell polarization, membrane protrusion, cell adhesion and contraction. Hence, cell migration requires the dynamic orchestration of all cytoskeleton components. Among these, IFs have emerged as a molecular scaffold with unique mechanical features and a key player in the cell resilience to mechanical stresses during migration through complex 3D environment. Moreover, accumulating evidence illustrates the participation of IFs in signalling cascades and cytoskeletal crosstalk. Teaming up with actin and microtubules, IFs contribute to the active generation of forces required for cell adhesion and mesenchymal migration and invasion. Here we summarize and discuss how IFs integrate mechanical properties and signalling functions to control cell migration in a wide spectrum of physiological and pathological situations.

The ESCRT machinery counteracts Nesprin-2G-mediated mechanical forces during nuclear envelope repair.

Transient nuclear envelope ruptures during interphase (NERDI) occur due to cytoskeletal compressive forces at sites of weakened lamina, and delayed NERDI repair results in genomic instability. Nuclear envelope (NE) sealing is completed by endosomal sorting complex required for transport (ESCRT) machinery. A key unanswered question is how local compressive forces are counteracted to allow efficient membrane resealing. Here, we identify the ESCRT-associated protein BROX as a crucial factor required to accelerate repair of the NE. Critically, BROX binds Nesprin-2G, a component of the linker of nucleoskeleton and cytoskeleton complex (LINC). This interaction promotes Nesprin-2G ubiquitination and facilitates the relaxation of mechanical stress imposed by compressive actin fibers at the rupture site. Thus, BROX rebalances excessive cytoskeletal forces in cells experiencing NE instability to promote effective NERDI repair. Our results demonstrate that BROX coordinates mechanoregulation with membrane remodeling to ensure the maintenance of nuclear-cytoplasmic compartmentalization and genomic stability.

Intersection of TKS5 and FGD1/CDC42 signaling cascades directs the formation of invadopodia.

Tumor cells exposed to a physiological matrix of type I collagen fibers form elongated collagenolytic invadopodia, which differ from dotty-like invadopodia forming on the gelatin substratum model. The related scaffold proteins, TKS5 and TKS4, are key components of the mechanism of invadopodia assembly. The molecular events through which TKS proteins direct collagenolytic invadopodia formation are poorly defined. Using coimmunoprecipitation experiments, identification of bound proteins by mass spectrometry, and in vitro pull-down experiments, we found an interaction between TKS5 and FGD1, a guanine nucleotide exchange factor for the Rho-GTPase CDC42, which is known for its role in the assembly of invadopodial actin core structure. A novel cell polarity network is uncovered comprising TKS5, FGD1, and CDC42, directing invadopodia formation and the polarization of MT1-MMP recycling compartments, required for invadopodia activity and invasion in a 3D collagen matrix. Additionally, our data unveil distinct signaling pathways involved in collagenolytic invadopodia formation downstream of TKS4 or TKS5 in breast cancer cells.

Rnd3 interacts with TAO kinases and contributes to mitotic cell rounding and spindle positioning.

Rnd3 is an atypical Rho family protein that is constitutively GTP bound, and acts on membranes to induce loss of actin stress fibers and cell rounding. Phosphorylation of Rnd3 promotes 14-3-3 binding and its relocation to the cytosol. Here, we show that Rnd3 binds to the thousand-and-one amino acid kinases TAOK1 and TAOK2 in vitro and in cells. TAOK1 and TAOK2 can phosphorylate serine residues 210, 218 and 240 near the C-terminus of Rnd3, and induce Rnd3 translocation from the plasma membrane to the cytosol. TAOKs are activated catalytically during mitosis and Rnd3 phosphorylation on serine 210 increases in dividing cells. Rnd3 depletion by RNAi inhibits mitotic cell rounding and spindle centralization, and delays breakdown of the intercellular bridge between two daughter cells. Our results show that TAOKs bind, phosphorylate and relocate Rnd3 to the cytosol and that Rnd3 contributes to mitotic cell rounding, spindle positioning and cytokinesis. Rnd3 can therefore participate in the regulation of early and late mitosis and may also act downstream of TAOKs to affect the cytoskeleton.

Nucleus-Invadopodia Duo During Cancer Invasion.

Matrix proteolysis mediated by MT1-MMP facilitates the invasive migration of tumor cells in dense tissues, which otherwise get trapped in the matrix because of limited nuclear deformability. A digest-on-demand response has been identified, which requires nucleus-microtubule linkage through the LINC complex and triggers MT1-MMP surface-exposure to facilitate nucleus movement.

The mechanical stability of proteins regulates their translocation rate into the cell nucleus.

The translocation of mechanosensitive transcription factors (TFs) across the nuclear envelope is a crucial step in cellular mechanotransduction. Yet the molecular mechanisms by which external mechanical cues control the nuclear shuttling dynamics of TFs through the nuclear pore complex (NPC) to activate gene expression are poorly understood. Here, we show that the nuclear import rate of myocardin-related transcription factor A (MRTFA) - a protein that regulates cytoskeletal dynamics via the activation of the TF serum response factor (SRF) - inversely correlates with the protein's nanomechanical stability and does not relate to its thermodynamic stability. Tagging MRTFA with mechanically resistant proteins results in the downregulation of SRF-mediated myosin light-chain 9 (MYL9) gene expression and subsequent slowing down of cell migration. We conclude that the mechanical unfolding of proteins regulates their nuclear translocation rate through the NPC, and highlight the role of the NPC as a selective mechanosensor able to discriminate forces as low as ~10 pN. The modulation of the mechanical stability of TFs may represent a new strategy for the control of gene expression.

Calcium-RasGRP2-Rap1 signaling mediates CD38-induced migration of chronic lymphocytic leukemia cells.

CD38 is a transmembrane exoenzyme that is associated with poor prognosis in chronic lymphocytic leukemia (CLL). High CD38 levels in CLL cells are linked to increased cell migration, but the molecular basis is unknown. CD38 produces nicotinic acid adenine dinucleotide phosphate and adenosine 5'-diphosphate-ribose, both of which can act to increase intracellular Ca2+ levels. Here we show that CD38 expression increases basal intracellular Ca2+ levels and stimulates CLL cell migration both with and without chemokine stimulation. We find that CD38 acts via intracellular Ca2+ to increase the activity of the Ras family GTPase Rap1, which is in turn regulated by the Ca2+-sensitive Rap1 guanine-nucleotide exchange factor RasGRP2. Both Rap1 and RasGRP2 are required for CLL cell migration, and RasGRP2 is polarized in primary CLL cells with high CD38 levels. These results indicate that CD38 promotes RasGRP2/Rap1-mediated CLL cell adhesion and migration by increasing intracellular Ca2+ levels.

LINC complex-Lis1 interplay controls MT1-MMP matrix digest-on-demand response for confined tumor cell migration.

Cancer cells' ability to migrate through constricting pores in the tissue matrix is limited by nuclear stiffness. MT1-MMP contributes to metastasis by widening matrix pores, facilitating confined migration. Here, we show that modulation of matrix pore size or of lamin A expression known to modulate nuclear stiffness directly impinges on levels of MT1-MMP-mediated pericellular collagenolysis by cancer cells. A component of this adaptive response is the centrosome-centered distribution of MT1-MMP intracellular storage compartments ahead of the nucleus. We further show that this response, including invadopodia formation in association with confining matrix fibrils, requires an intact connection between the nucleus and the centrosome via the linker of nucleoskeleton and cytoskeleton (LINC) complex protein nesprin-2 and dynein adaptor Lis1. Our results uncover a digest-on-demand strategy for nuclear translocation through constricted spaces whereby confined migration triggers polarization of MT1-MMP storage compartments and matrix proteolysis in front of the nucleus depending on nucleus-microtubule linkage.

[Hyponatremia and its etiology in pediatric patients admitted to the emergency room]. / Hiponatremia y su etiología en pacientes pediátricos ingresados a sala de urgencias.

BACKGROUND: The importance of hyponatremia is underestimated despite it is a frequent alteration in a hospital environment. Usually no cause is investigated and it is treated as an isolated data. The aim was to determine the prevalence and etiology of hyponatremia in pediatric patients. METHODS: Cross-sectional study of 72 patients from 1-month to 15-years of age admitted to the emergency room, from May to September, 2015, with lower serum determination of Na < 135 mEq/L at admission. For statistical analysis, frequencies and percentages, as well as medians and ranges were used. RESULTS: 72 patients (3%) out of 2400 admissions to the emergency room were detected. 42 were male (58%), 28 infant (30.5%), and 46 eutrophic (68.5%). The diagnosis at admission was sepsis in 13 (18%), mild hyponatremia in 41 (56.9%), real hyponatremia in 71 (98.6%) and dilutional in 1 (1.4%). The median serum sodium was 130 mEq/L (range 112-134), and the median serum osmolality 266 mOsm/L (range 236-288). The most usual management was the increase of sodium in solutions in 21 cases (29%), and the use of diuretics (n = 21, 29%). The median of sodium input per m2 (BSA) per day was 45 mEq (range 0-158). CONCLUSIONS: Mild and real hyponatremia were the most frequent diagnoses. The infection at admission was the more common etiology. The increase in sodium input in solution was the most common management. The use of intravenous solutions 2:1 (isotonic) did not produce hypernatremia and avoided administration of hypotonic solutions.

Introducción: la importancia de la hiponatremia es subestimada a pesar de ser una alteración frecuente en el medio hospitalario. Habitualmente no se investiga su causa y se trata como dato aislado. Se buscó determinar la prevalencia de hiponatremia y su etiología en pacientes en urgencias pediátricas. Métodos: estudio transversal en 72 pacientes de un mes a 15 años ingresados de mayo a septiembre de 2015 en urgencias con determinación sérica de Na < 135 mEq/L a su ingreso. Para la estadística se emplearon frecuencias y porcentajes, medianas y rangos. Resultados: se detectaron 72 pacientes de 2400 ingresos a urgencias (3%). Fueron varones 42 (58%), 28 lactantes (30.5%) y 46 eutróficos (68.5%). El diagnóstico de ingreso fue de sepsis en 13 (18%), hiponatremia leve en 41 (56.9%), real (hipoosomolar) en 71 (98.6%) y dilucional en 1 (1.4%). La mediana del sodio sérico fue de 130 mEq/L (rango 112-134) y la de la osmolaridad sérica de 266 mOsm/L (rango 236-288). El manejo más frecuente fue el incremento del aporte de sodio en soluciones en 21 casos (29.1%) y el uso de diuréticos en 21 (29%). La mediana del aporte de sodio por m2 SCT al día fue de 45 mEq (rango 0-158). Conclusiones: la hiponatremia leve y la hipoosomolar fueron las más frecuentes. El diagnóstico de origen infeccioso fue la causa más común. El incremento en el aporte de sodio en soluciones fue el manejo más habitual. El uso de soluciones intravenosas 2:1 (isotónicas) no produjo hipernatremia y evitó la administración de soluciones hipotónicas.

Intervención educativa: alimentación para unos dientes y encías sanos en el adulto mayor / Educational intervention: food for healthy teeth and gingiva in the elderly

Se realizó una intervención educativa en un círculo de abuelos del área Norte del municipio Guantánamo en el período septiembre de 2015 a septiembre de 2016, con el objetivo de evaluar el nivel de conocimientos sobre alimentación que poseían los ancianos. A través de una encuesta se determinó el nivel de información que poseían sobre alimentación saludable para el cuidado de los dientes. El universo estuvo constituido por 40 ancianos. Las variables estudiadas fueron: edad, sexo, nivel de escolaridad, conocimientos sobre alimentación saludable. La percepción de los ancianos con respecto a la intervención educativa fue muy favorable, pues todos reconocieron haber sobrepasado sus expectativas, lo cual se manifestó en los cambios beneficiosos que para su salud le propició la intervención. Las actividades en la práctica propiciaron los conocimientos para elevar la calidad de vida de los ancianos, optimizando la labor preventiva y una alimentación adecuada(AU)

An educational intervention was performed in a home of grandparents North area of the municipality Guantanamo from September 2015 to September 2016 in order to assess the level of knowledge of food possessed by the elderly. Through a survey was determined the level of information they had about healthy tooth and healthy feeding. The universe consisted of 40 elderly. The variables were studied: age, sex, level of education, knowledge about healthy eating. The perception of the elderly regarding the educational intervention that was very favorable, all of them were having exceeded their expectations, which was manifested in the beneficial health changes that will lead to the intervention. Activities put into practice knowledges to improve the quality of life of the elderly, optimizing preventive work and adequate food

ARF6-JIP3/4 regulate endosomal tubules for MT1-MMP exocytosis in cancer invasion.

Invasion of cancer cells into collagen-rich extracellular matrix requires membrane-tethered membrane type 1-matrix metalloproteinase (MT1-MMP) as the key protease for collagen breakdown. Understanding how MT1-MMP is delivered to the surface of tumor cells is essential for cancer cell biology. In this study, we identify ARF6 together with c-Jun NH2-terminal kinase-interacting protein 3 and 4 (JIP3 and JIP4) effectors as critical regulators of this process. Silencing ARF6 or JIP3/JIP4 in breast tumor cells results in MT1-MMP endosome mispositioning and reduces MT1-MMP exocytosis and tumor cell invasion. JIPs are recruited by Wiskott-Aldrich syndrome protein and scar homologue (WASH) on MT1-MMP endosomes on which they recruit dynein-dynactin and kinesin-1. The interaction of plasma membrane ARF6 with endosomal JIPs coordinates dynactin-dynein and kinesin-1 activity in a tug-of-war mechanism, leading to MT1-MMP endosome tubulation and exocytosis. In addition, we find that ARF6, MT1-MMP, and kinesin-1 are up-regulated in high-grade triple-negative breast cancers. These data identify a critical ARF6-JIP-MT1-MMP-dynein-dynactin-kinesin-1 axis promoting an invasive phenotype of breast cancer cells.

Self-recognition of the endothelium enables regulatory T-cell trafficking and defines the kinetics of immune regulation.

Localization of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells to lymphoid and non-lymphoid tissue is instrumental for the effective control of immune responses. Compared with conventional T cells, Treg cells constitute a minute fraction of the T-cell repertoire. Despite this numeric disadvantage, Tregs efficiently migrate to sites of immune responses reaching an optimal number for the regulation of T effector (Teff) cells. The array and levels of adhesion and chemokine receptor expression by Tregs do not explain their powerful migratory capacity. Here we show that recognition of self-antigens expressed by endothelial cells in target tissue is instrumental for efficient Treg recruitment in vivo. This event relies upon IFN-γ-mediated induction of MHC-class-II molecule expression by the endothelium and requires optimal PI3K p110δ activation by the T-cell receptor. We also show that, once in the tissue, Tregs inhibit Teff recruitment, further enabling a Teff:Treg ratio optimal for regulation.

Roles of Rho GTPases in leucocyte and leukaemia cell transendothelial migration.

Leucocytes migrate into and out of blood vessels at multiple points during their development and maturation, and during immune surveillance. In response to tissue damage and infection, they are rapidly recruited through the endothelium lining blood vessels into the tissues. Leukaemia cells also move in and out of the bloodstream during leukaemia progression. Rho GTPases are intracellular signalling proteins that regulate cytoskeletal dynamics and are key coordinators of cell migration. Here, we describe how different members of the Rho GTPase family act in leucocytes and leukaemia cells to regulate steps of transendothelial migration. We discuss how inhibitors of Rho signalling could be used to reduce leucocyte or leukaemia cell entry into tissues.

FMNL1 promotes proliferation and migration of leukemia cells.

The human FMNL1 is expressed predominantly in hematopoietic cells and has been described previously as overexpressed in hematopoietic malignancies. However, it is not known whether FMNL1 contributes to leukemogenesis. Here, we investigate the FMNL1 function using two different human leukemia models: Namalwa and K562 cell lines. FMNL1 depletion reduced cell proliferation and colony formation in both leukemic cell types, as well as a decrease in the tumor growth of FMNL1-depleted Namalwa cell xenografts. In addition, there was a decrease in migration and in TEM in FMNL1-depleted Namalwa cells. FMNL1 endogenously associates with Rac1, and FMNL1 silencing resulted in an increased Rac1 activity. The reduced migration observed in FMNL1-depleted cells was restored by inhibiting Rac activity. Our results indicate that FMNL1 stimulates leukemia cell proliferation as well as migration. This suggests that FMNL1 contributes to leukemogenesis and could act in part through Rac1 regulation.

Statins inhibit T-acute lymphoblastic leukemia cell adhesion and migration through Rap1b.

Statins are known to inhibit signaling of Ras superfamily GTPases and reduce T cell adhesion to ICAM-1. Here, we address the hypothesis that statins affect T cell adhesion and migration by modulating the function of specific GTPases. Statins inhibit the synthesis of mevalonic acid, which is required for farnesyl and geranylgeranyl isoprenoid synthesis. Ras superfamily GTPases are post-translationally isoprenylated to facilitate their anchorage to membranes, where they function to stimulate signal transduction processes. We demonstrate that 1 µM statin inhibits the adhesion, migration, and chemotaxis of the T-ALL cell line CCRF-CEM and TEM of CCRF-CEM and PEER T-ALL cells, but higher statin concentrations are needed to inhibit adhesion of primary T cells. Similar effects are observed following treatment with GGTI-298 or RNA interference-mediated knockdown of Rap1b but not Rap1a, Rac1, Rac2, RhoA, or Cdc42. Statins also alter Rap1 activity and Rap1b localization. Rap1 levels are higher in primary T cells than T-ALL cells, which could explain their reduced sensitivity to statins. These results demonstrate for the first time that the closely related Rap1a and Rap1b isoforms have different functions and suggest that statins or Rap1b depletion could be used to reduce tissue invasion in T-ALL.