Dasatinib induces endothelial dysfunction leading to impaired recovery from ischaemia.

Chronic myeloid leukaemia (CML) management is complicated by treatment-emergent vascular adverse events seen with tyrosine kinase inhibitors (TKIs) such as nilotinib, dasatinib and ponatinib. Pleural effusion and pulmonary arterial hypertension (PAH) have been associated with dasatinib treatment. Endothelial dysfunction and impaired angiogenesis are hallmarks of PAH. In this study, we explored, at cellular and whole animal levels, the connection between dasatinib exposure and disruption of endothelial barrier integrity and function, leading to impaired angiogenesis. Understanding the mechanisms whereby dasatinib initiates PAH will provide opportunities for intervention and prevention of such adverse effects, and for future development of safer TKIs, thereby improving CML management.

Allosteric activation of vinculin by talin.

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single-molecule magnetic tweezers experiments, Molecular Dynamics simulations, actin-bundling assays, and adhesion assembly experiments in live cells, we here describe a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding, which reinforces the binding to talin at a rate of 0.03 s-1. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at forces up to 10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The 'allosteric vinculin mutant' is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.

Multisite assessment of reproducibility in high-content cell migration imaging data.

High-content image-based cell phenotyping provides fundamental insights into a broad variety of life science disciplines. Striving for accurate conclusions and meaningful impact demands high reproducibility standards, with particular relevance for high-quality open-access data sharing and meta-analysis. However, the sources and degree of biological and technical variability, and thus the reproducibility and usefulness of meta-analysis of results from live-cell microscopy, have not been systematically investigated. Here, using high-content data describing features of cell migration and morphology, we determine the sources of variability across different scales, including between laboratories, persons, experiments, technical repeats, cells, and time points. Significant technical variability occurred between laboratories and, to lesser extent, between persons, providing low value to direct meta-analysis on the data from different laboratories. However, batch effect removal markedly improved the possibility to combine image-based datasets of perturbation experiments. Thus, reproducible quantitative high-content cell image analysis of perturbation effects and meta-analysis depend on standardized procedures combined with batch correction.

The Actin Network Interfacing Diverse Integrin-Mediated Adhesions.

The interface between the cellular actin network and diverse forms of integrin-mediated cell adhesions displays a unique capacity to serve as accurate chemical and mechanical sensors of the cell's microenvironment. Focal adhesion-like structures of diverse cell types, podosomes in osteoclasts, and invadopodia of invading cancer cells display distinct morphologies and apparent functions. Yet, all three share a similar composition and mode of coupling between a protrusive structure (the lamellipodium, the core actin bundle of the podosome, and the invadopodia protrusion, respectively), and a nearby adhesion site. Cytoskeletal or external forces, applied to the adhesion sites, trigger a cascade of unfolding and activation of key adhesome components (e.g., talin, vinculin, integrin), which in turn, trigger the assembly of adhesion sites and generation of adhesion-mediated signals that affect cell behavior and fate. The structural and molecular mechanisms underlying the dynamic crosstalk between the actin cytoskeleton and the adhesome network are discussed.

A new function for the serine protease HtrA2 in controlling radiation-induced senescence in cancer cells.

Radiation therapy can induce cellular senescence in cancer cells, leading to short-term tumor growth arrest but increased long-term recurrence. To better understand the molecular mechanisms involved, we developed a model of radiation-induced senescence in cultured cancer cells. The irradiated cells exhibited a typical senescent phenotype, including upregulation of p53 and its main target, p21, followed by a sustained reduction in cellular proliferation, changes in cell size and cytoskeleton organization, and senescence-associated beta-galactosidase activity. Mass spectrometry-based proteomic profiling of the senescent cells indicated downregulation of proteins involved in cell cycle progression and DNA repair, and upregulation of proteins associated with malignancy. A functional siRNA screen using a cell death-related library identified mitochondrial serine protease HtrA2 as being necessary for sustained growth arrest of the senescent cells. In search of direct HtrA2 substrates following radiation, we determined that HtrA2 cleaves the intermediate filament protein vimentin, affecting its cytoplasmic organization. Ectopic expression of active cytosolic HtrA2 resulted in similar changes to vimentin filament assembly. Thus, HtrA2 is involved in the cytoskeletal reorganization that accompanies radiation-induced senescence and the continuous maintenance of proliferation arrest.

Sorting Nexin 10 as a Key Regulator of Membrane Trafficking in Bone-Resorbing Osteoclasts: Lessons Learned From Osteopetrosis.

Bone homeostasis is a complex, multi-step process, which is based primarily on a tightly orchestrated interplay between bone formation and bone resorption that is executed by osteoblasts and osteoclasts (OCLs), respectively. The essential physiological balance between these cells is maintained and controlled at multiple levels, ranging from regulated gene expression to endocrine signals, yet the underlying cellular and molecular mechanisms are still poorly understood. One approach for deciphering the mechanisms that regulate bone homeostasis is the characterization of relevant pathological states in which this balance is disturbed. In this article we describe one such "error of nature," namely the development of acute recessive osteopetrosis (ARO) in humans that is caused by mutations in sorting nexin 10 (SNX10) that affect OCL functioning. We hypothesize here that, by virtue of its specific roles in vesicular trafficking, SNX10 serves as a key selective regulator of the composition of diverse membrane compartments in OCLs, thereby affecting critical processes in the sequence of events that link the plasma membrane with formation of the ruffled border and with extracellular acidification. As a result, SNX10 determines multiple features of these cells either directly or, as in regulation of cell-cell fusion, indirectly. This hypothesis is further supported by the similarities between the cellular defects observed in OCLs form various models of ARO, induced by mutations in SNX10 and in other genes, which suggest that mutations in the known ARO-associated genes act by disrupting the same plasma membrane-to-ruffled border axis, albeit to different degrees. In this article, we describe the population genetics and spread of the original arginine-to-glutamine mutation at position 51 (R51Q) in SNX10 in the Palestinian community. We further review recent studies, conducted in animal and cellular model systems, that highlight the essential roles of SNX10 in critical membrane functions in OCLs, and discuss possible future research directions that are needed for challenging or substantiating our hypothesis.

PD-L1 recruits phospholipase C and enhances tumorigenicity of lung tumors harboring mutant forms of EGFR.

Cancer immunotherapy focuses on inhibitors of checkpoint proteins, such as programmed death ligand 1 (PD-L1). Unlike RAS-mutated lung cancers, EGFR mutant tumors have a generally low response to immunotherapy. Because treatment outcomes vary by EGFR allele, intrinsic and microenvironmental factors may be involved. Among all non-immunological signaling pathways surveyed in patients' datasets, EGFR signaling is best associated with high PD-L1. Correspondingly, active EGFRs stabilize PD-L1 transcripts and depletion of PD-L1 severely inhibits EGFR-driven tumorigenicity and metastasis in mice. The underlying mechanisms involve the recruitment of phospholipase C-γ1 (PLC-γ1) to a cytoplasmic motif of PD-L1, which enhances PLC-γ1 activation by EGFR. Once stimulated, PLC-γ1 activates calcium flux, Rho GTPases, and protein kinase C, collectively promoting an aggressive phenotype. Anti-PD-L1 antibodies can inhibit these intrinsic functions of PD-L1. Our results portray PD-L1 as a molecular amplifier of EGFR signaling and improve the understanding of the resistance of EGFR+ tumors to immunotherapy.

An SNX10-dependent mechanism downregulates fusion between mature osteoclasts.

Homozygosity for the R51Q mutation in sorting nexin 10 (SNX10) inactivates osteoclasts (OCLs) and induces autosomal recessive osteopetrosis in humans and in mice. We show here that the fusion of wild-type murine monocytes to form OCLs is highly regulated, and that its extent is limited by blocking fusion between mature OCLs. In contrast, monocytes from homozygous R51Q SNX10 mice fuse uncontrollably, forming giant dysfunctional OCLs that can become 10- to 100-fold larger than their wild-type counterparts. Furthermore, mutant OCLs display reduced endocytotic activity, suggesting that their deregulated fusion is due to alterations in membrane homeostasis caused by loss of SNX10 function. This is supported by the finding that the R51Q SNX10 protein is unstable and exhibits altered lipid-binding properties, and is consistent with a key role for SNX10 in vesicular trafficking. We propose that OCL size and functionality are regulated by a cell-autonomous SNX10-dependent mechanism that downregulates fusion between mature OCLs. The R51Q mutation abolishes this regulatory activity, leading to excessive fusion, loss of bone resorption capacity and, consequently, to an osteopetrotic phenotype in vivo. This article has an associated First Person interview with the joint first authors of the paper.

Massive osteopetrosis caused by non-functional osteoclasts in R51Q SNX10 mutant mice.

The R51Q mutation in sorting nexin 10 (SNX10) was shown to cause a lethal genetic disease in humans, namely autosomal recessive osteopetrosis (ARO). We describe here the first R51Q SNX10 knock-in mouse model and show that mice homozygous for this mutation exhibit massive, early-onset, and widespread osteopetrosis. The mutant mice exhibit multiple additional characteristics of the corresponding human disease, including stunted growth, failure to thrive, missing or impacted teeth, occasional osteomyelitis, and a significantly-reduced lifespan. Osteopetrosis in this model is the result of osteoclast inactivity that, in turn, is caused by absence of ruffled borders in the mutant osteoclasts and by their inability to secrete protons. These results confirm that the R51Q mutation in SNX10 is a causative factor in ARO and provide a model system for studying this rare disease.

The involvement of mutant Rac1 in the formation of invadopodia in cultured melanoma cells.

In this article, we discuss the complex involvement of a Rho-family GTPase, Rac1, in cell migration and in invadopodia-mediated matrix degradation. We discuss the involvement of invadopodia in invasive cell migration, and their capacity to promote cancer metastasis. Considering the regulation of invadopodia formation, we describe studies that demonstrate the role of Rac1 in the metastatic process, and the suggestion that this effect is attributable to the capacity of Rac1 to promote invadopodia formation. This notion is demonstrated here by showing that knockdown of Rac1 in melanoma cells expressing a wild-type form of this GTPase, reduces invadopodia-dependent matrix degradation. Interestingly, we also show that excessive activity of Rac1, displayed by the P29S, hyperactive, "fast cycling" mutant of Rac1, which is present in 5-10% of melanoma tumors, inhibits invadopodia function. Moreover, knockdown of this hyperactive mutant enhanced matrix degradation, indicating that excessive Rac1 activity by this mutant can negatively regulate invadopodia formation and function.

Recurrent inactivating RASA2 mutations in melanoma.

Analysis of 501 melanoma exomes identified RASA2, encoding a RasGAP, as a tumor-suppressor gene mutated in 5% of melanomas. Recurrent loss-of-function mutations in RASA2 were found to increase RAS activation, melanoma cell growth and migration. RASA2 expression was lost in ≥30% of human melanomas and was associated with reduced patient survival. These findings identify RASA2 inactivation as a melanoma driver and highlight the importance of RasGAPs in cancer.

The integrin adhesome: from genes and proteins to human disease.

The adhesive interactions of cells with their environment through the integrin family of transmembrane receptors have key roles in regulating multiple aspects of cellular physiology, including cell proliferation, viability, differentiation and migration. Consequently, failure to establish functional cell adhesions, and thus the assembly of associated cytoplasmic scaffolding and signalling networks, can have severe pathological effects. The roles of specific constituents of integrin-mediated adhesions, which are collectively known as the 'integrin adhesome', in diverse pathological states are becoming clear. Indeed, the prominence of mutations in specific adhesome molecules in various human diseases is now appreciated, and experimental as well as in silico approaches provide insights into the molecular mechanisms underlying these pathological conditions.

Involvement of actin polymerization in podosome dynamics.

Podosomes, which are formed by different monocyte derivatives, are small adhesion structures whose coordinated dynamics and cytoskeletal reorganization drive their motile and invasive features. Using live-cell microscopy, we explored the temporal molecular steps of the de novo assembly and disassembly of podosomes in cultured osteoclasts. We demonstrate here that the earliest visible step in podosome assembly is the local accumulation of the plaque protein paxillin, along with cortactin, which stabilizes actin networks, followed by robust polymerization of actin filaments and their association with α-actinin. Only then is a local increase in integrin ß3 levels apparent in the podosome ring domain. Thus, local actin polymerization in cortactin- and paxillin-rich locations nucleates podosome assembly before the local accumulation of ß3 integrin. We further show that actin polymerization is also important for the recruitment and maintenance of plaque proteins in the mature podosome ring domain. Our model implies that core bundle dynamics play a central role in regulating podosome stability.

Analysis of the signaling pathways regulating Src-dependent remodeling of the actin cytoskeleton.

Cell adhesion to the extracellular matrix is mediated by adhesion receptors, mainly integrins, which upon interaction with the extracellular matrix, bind to the actin cytoskeleton via their cytoplasmic domains. This association is mediated by a variety of scaffold and signaling proteins, which control the mechanical and signaling activities of the adhesion site. Upon transformation of fibroblasts with active forms of Src (e.g., v-Src), focal adhesions are disrupted, and transformed into dot-like contacts known as podosomes, and consisting of a central actin core surrounded by an adhesion ring. To clarify the mechanism underlying Src-dependent modulation of the adhesive phenotype, and its influence on podosome organization, we screened for the effect of siRNA-mediated knockdown of tyrosine kinases, MAP kinases and phosphatases on the reorganization of the adhesion-cytoskeleton complex, induced by a constitutively active Src mutant (SrcY527F). In this screen, we discovered several genes that are involved in Src-induced remodeling of the actin cytoskeleton. We further showed that knockdown of Src in osteoclasts abolishes the formation of the podosome-based rings and impairs cell spreading, without inducing stress fiber development. Our work points to several genes that are involved in this process, and sheds new light on the molecular plasticity of integrin adhesions.

Multiparametric analysis of focal adhesion formation by RNAi-mediated gene knockdown.

Cell adhesion to the extracellular matrix is mediated by elaborate networks of multiprotein complexes consisting of adhesion receptors, cytoskeletal components, signaling molecules, and diverse adaptor proteins. To explore how specific molecular pathways function in the assembly of focal adhesions (FAs), we performed a high-throughput, high-resolution, microscopy-based screen. We used small interfering RNAs (siRNAs) to target human kinases, phosphatases, and migration- and adhesion-related genes. Multiparametric image analysis of control and of siRNA-treated cells revealed major correlations between distinct morphological FA features. Clustering analysis identified different gene families whose perturbation induced similar effects, some of which uncoupled the interfeature correlations. Based on these findings, we propose a model for the molecular hierarchy of FA formation, and tested its validity by dynamic analysis of FA formation and turnover. This study provides a comprehensive information resource on the molecular regulation of multiple cell adhesion features, and sheds light on signaling mechanisms regulating the formation of integrin adhesions.

Promoción de la donación de sangre, estudio de las motivaciones con la técnica de grupos focales / Promotion of blood donation, study of motivations in focal groups technique

Dos aspectos podrían resumir las preocupaciones más importantes de todo profesional abocado a la tarea de gestionar un Banco de Sangre: la suficiente provisión, y la seguridad de los hemocomponentes a transfundir. Sin duda, mucho se avanzó en conseguir hoy un producto muy seguro, con bajísimos porcentajes de riesgo en cuanto a la transmisión de enfermedades, pero sigue siendo materia pendiente conseguir una adecuada cantidad de donantes que permitan abastecer la demanda transfusional. Si bien es cierto que bastaría que un 3 al 5 por ciento de la población concurriera como donante de sangre voluntario y habitual para cubrir las necesidades, todavía es bajísimo el porcentaje conseguido, y, por lo tanto muy poco lo que hemos hecho para revertir esta situación. Agreguemos que no basta con llegar a la cifra requerida de donantes sino además, que estos sean habituales, ya que son considerados más seguros, permiten una mejor planificación de los stocks, y terminan con la práctica aberrante de pedir donantes en los momentos de mayor angustia y necesidad, y a veces hasta condicionando prácticas como la cirugía. Últimos datos referidos a Argentina, recopilados por la OPS en 2004 muestran que el porcentaje de donantes voluntarios regulares (o habituales) es de 7 por ciento. El restante 93 por ciento son donantes de reposición y no existen donantes pagos. Siempre resulta difícil para un banco de sangre destinar recursos para la promoción de la donación de sangre, dado que no los tiene, y generarlos a partir del precio de los hemocomponentes no es aceptado por el mercado. Como otras cuestiones vinculadas a la salud, no debe ser el mercado quien establezca las pautas y es necesario un grado mayor de involucramiento del estado para resolver estas cuestiones... (TRUNCADO)

A qualitative research was carried out on focus groups with Gesell dome (one-way mirror). Four groups of twelve people each were studied, divided into two categories, those who have previously donated and those who have not. Characteristics such as age, sex and residence place were taken into account. The evaluation for each group took approximately 1 :30 hr. General objectives of the market research. We asked questions in order to make the members of the group express themselves to achieve the following objectives: To detect donor's motivations. To find out negative values and barriers against blood donation. To detect free association concerning institutions that work as Blood Bank. To detect the degree of knowledge about Donor's Organizations and their positive or negative assumptions. To test some potential advertising concepts.

Development and application of automatic high-resolution light microscopy for cell-based screens.

Large-scale microscopy-based screens offer compelling advantages for assessing the effects of genetic and pharmacological modulations on a wide variety of cellular features. However, development of such assays is often confronted by an apparent conflict between the need for high throughput, which usually provides limited information on a large number of samples, and a high-content approach, providing detailed information on each sample. This chapter describes a novel high-resolution screening (HRS) platform that is able to acquire large sets of data at a high rate and light microscope resolution using specific "reporter cells," cultured in multiwell plates. To harvest extensive morphological and molecular information in these automated screens, we have constructed a general analysis pipeline that is capable of assigning scores to multiparameter-based comparisons between treated cells and controls. This chapter demonstrates the structure of this system and its application for several research projects, including screening of chemical compound libraries for their effect on cell adhesion, discovery of novel cytoskeletal genes, discovery of cell migration-related genes, and a siRNA screen for perturbation of cell adhesion.