The Interplay between Cytoskeleton and Calcium Dynamics

Cell motility is a complex cellular event that involves reorganization of cytoskeleton. This reorganization encompasses the transient polarization of the cell to facilitate the plasma membrane ruffling, a rearrangement of cortical actin cytoskeleton required for the development of cellular protrusions. It is known that extracellular Ca2+ influx is essential for cell migration and for the positive-feedback cycle that maintains leading-edge structures and ruffling activity. The aim of this review is to summarize our knowledge regarding the Ca2+-dependent signaling pathways, Ca2+ transporters and sensors involved in cell migration. Also, we show here reported evidences that support for a crosstalk between Ca2+ transport and the reorganization of the cytoskeleton required for cell migration. In this regard, we will analyze the role of store-operated Ca2+ entry (SOCE) as a modulator of cytoskeleton and cell migration, but also the modulation of this Ca2+ entry pathway by microtubules and the actin cytoskeleton. As a main conclusion, this review will show that data reported in the last years support a role for SOCE in shaping cytoskeleton, but at the same time, SOCE is strongly dependent on cytoskeletal proteins, in an interesting interplay between cytoskeleton and Ca2+ dynamics

Regulation of Calcium Signaling by STIM1 and ORAI1

STIM1 and ORAI1 proteins are regulators of intracellular Ca2+ mobilization. This Ca2+ mobilization is essential to shape Ca2+ signaling in eukaryotic cells. STIM1 is a transmembrane protein located at the endoplasmic reticulum, where it acts as an intraluminal Ca2+ sensor. The transient drop of intraluminal Ca2+ concentration triggers STIM1 activation, which relocates to plasma membrane-endoplasmic reticulum junctions to bind and activate ORAI1, a plasma membrane Ca2+ channel. Thus, the Ca2+ influx pathway mediated by STIM1/ORAI1 is termed store-operated Ca2+ entry (SOCE). STIM and ORAI proteins are also involved in non-SOCE Ca2+ influx pathways, as we discuss here. In this chapter, we review the current knowledge regarding the role of SOCE, STIM1, and ORAI1 in cell signaling, with special focus on the modulation of the activity of kinases, phosphatases, and transcription factors that are strongly influenced by the extracellular Ca2+ influx mediated by these regulators

STIM1 deficiency is linked to Alzheimer’s disease and triggers cell death in SH-SY5Y cells by upregulation of L-type voltage-operated Ca2+ entry

La STIM1 es una proteína del retículo endoplásmico con un papel en la movilización y señalización del Ca2+. Como sensor de los niveles intraluminales de Ca2+, STIM1 modula los canales de Ca2+ de la membrana plasmática para regular la entrada de Ca2+. En las células de neuroblastoma SH-SY5Y y en los fibroblastos cutáneos familiares de pacientes con la enfermedad de Alzheimer, STIM1 se divide en el dominio transmembrana por la presenilina-1-asociada a γ-secretase, lo que lleva a una desregulación de la homeostasis del Ca2+. En este informe, investigamos los niveles de expresión de STIM1 en los tejidos cerebrales (giro frontal medio) de pacientes con enfermedad de Alzheimer confirmada patológicamente, y observamos que el nivel de expresión de la proteína STIM1 disminuyó con la progresión de la neurodegeneración. Para estudiar el papel de STIM1 en la neurodegeneración, se diseñó una estrategia para eliminar la expresión del gen STIM1 en la línea de células de neuroblastoma SH-SY5Y mediante la edición del genoma mediado por CRISPR/Cas9, como un modelo in vitro para examinar el fenotipo de las células neuronales deficientes de STIM1. Se demostró que, si bien la STIM1 no es necesaria para la diferenciación de las células SH-SY5Y, es absolutamente esencial para la supervivencia de las células en la diferenciación. Las células STIM1-KO diferenciadas mostraron una disminución significativa de la actividad del complejo I de la cadena respiratoria mitocondrial, la despolarización de la membrana interna de la mitocondria, la reducción de la concentración de Ca2+ libre en la mitocondria y mayores niveles de senescencia en comparación con las células de tipo salvaje. En paralelo, las células STIM1-KO mostraron una entrada de Ca2+ potenciada en respuesta a la despolarización, que era sensible a la nifedipina, apuntando a los canales de Ca2+ operados por voltaje de tipo L como mediadores de la entrada de Ca2+ aumentada. El derribo estable de las transcripciones de CACNA1C restauró la función mitocondrial, aumentó los niveles mitocondriales de Ca2+ y redujo la senescencia a los niveles basales, demostrando el papel esencial de la regulación de la entrada de Ca2+ operada por voltaje a través de los canales Cav1.2 en la muerte celular deficiente de STIM1 SHSY5Y.STIM1 is an endoplasmic reticulum protein with a role in Ca2+ mobilization and signaling. As a sensor of intraluminal Ca2+ levels, STIM1 modulates plasma membrane Ca2+ channels to regulate Ca2+ entry. In neuroblastoma SH-SY5Y cells and in familial Alzheimer’s disease patient skin fibroblasts, STIM1 is cleaved at the transmembrane domain by the presenilin-1-associated γ-secretase, leading to dysregulation of Ca2+ homeostasis. In this report, we investigated expression levels of STIM1 in brain tissues (medium frontal gyrus) of pathologically confirmed Alzheimer’s disease patients, and observed that STIM1 protein expression level decreased with the progression of neurodegeneration. To study the role of STIM1 in neurodegeneration, a strategy was designed to knock-out the expression of STIM1 gene in the SH-SY5Y neuroblastoma cell line by CRISPR/Cas9-mediated genome editing, as an in vitro model to examine the phenotype of STIM1-deficient neuronal cells. It was proved that, while STIM1 is not required for the differentiation of SH-SY5Y cells, it is absolutely essential for cell survival in differentiating cells. Differentiated STIM1-KO cells showed a significant decrease of mitochondrial respiratory chain complex I activity, mitochondrial inner membrane depolarization, reduced mitochondrial free Ca2+ concentration, and higher levels of senescence as compared with wild-type cells. In parallel, STIM1-KO cells showed a potentiated Ca2+ entry in response to depolarization, which was sensitive to nifedipine, pointing to L-type voltage-operated Ca2+ channels as mediators of the upregulated Ca2+ entry. The stable knocking-down of CACNA1C transcripts restored mitocondrial function, increased mitochondrial Ca2+ levels, and dropped senescence to basal levels, emonstrating the essential role of the upregulation of voltage-operated Ca2+ entry through Cav1.2 channels in STIM1-deficient SHSY5Y cell death.• Ministerio de Educación, Cultura y Deporte. Beca FPU13/03430 • The Company of Biologists. Ayuda JCSTF-170507 • Ministerio de Economía, y Competitividad. Proyectos BFU2014-52401-P y BFU2017-82716, para Francisco Javier Martín Romero • Ministerio de Economía, y Competitividad. Proyectos BFU2014-53641-P y BFU2017-85723-P, para Ana María Mata Durán y Carlos Gutiérrez Merino • Junta de Extremadura. Ayudas GRU15077 e IB16088, para Francisco Javier Martín Romero • Junta de Extremadura. Ayuda GRU15139, para Ana María Mata DuránpeerReviewe

Connections between postparotid terminal branches of the facial nerve: An immunohistochemistry study

It has been assumed that connections between the postparotid terminal branches of the facial nerve are purely motor. However, the nature of their fibers remains unexplored. The aim of this study is to determine whether these connections comprise motor fibers exclusively. In total 17 connections between terminal facial nerve branches were obtained from 13 different facial nerves. Choline acetyltransferase antibody (ChAT) was used to stain the fibers in the connections and determine whether or not all of them were motor. All connections contained ChAT positive and negative fibers. The average number of fibers overall was 287 (84–587) and the average proportion of positive fibers was 63% (37.7%–91.5%). In 29% of the nerves, >75% of the fibers were ChAT+ (strongly positive); in 52.94%, 50%–75% were ChAT+ (intermediately positive); and in 17.65%, <50% were ChAT+ (weakly positive). Fibers traveling inside the postparotid terminal cranial nerve VII branch connections are not exclusively motor

CA 15-3 prognostic biomarker in SARS-CoV-2 pneumonia.

The severity of lung involvement is the main prognostic factor in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Carbohydrate antigen 15-3 (CA 15-3), a marker of lung damage and fibrosis, could help predict the prognosis of SARS-CoV-2 pneumonia. This was a retrospective and observational study. CA 15-3 was analyzed in the blood samples of patients consecutively admitted for SARS-CoV-2 pneumonia and whose blood samples were available in the biobank. Other prognostic markers were also measured (interleukin 6 [IL6], C-reactive protein [CRP], D-dimer, troponin T, and NT-ProBNP). The occurrence of in-hospital complications was registered, including death, the need for medical intensive care, and oxygen therapy at discharge. In this study, 539 patients were recruited (54.9% men, mean age: 59.6 ± 16.4 years). At admission, the mean concentrations of CA 15-3 was 20.5 ± 15.8 U/mL, and the concentration was correlated with male sex, older age, and other severity markers of coronavirus disease of 2019 (COVID-19) (IL6, CRP, D-dimer, troponine T, and NT-ProBNP). CA 15-3 levels were higher in patients who died (n = 56, 10.4%) (35.33 ± 30.45 vs. 18.8 ± 12.11, p < 0.001), who required intensive medical support (n = 78, 14.4%; 31.17 ± 27.83 vs. 18.68 ± 11.83; p < 0.001), and who were discharged with supplemental oxygen (n = 64, 13.3%; 22.65 ± 14.41 vs. 18.2 ± 11.7; p = 0.011). Elevated CA 15-3 levels (above 34.5 U/mL) were a strong predictor of a complicated in-hospital course, in terms of a higher risk of death (adjusted odds ratio [OR] 3.74, 95% confidence interval [CI]: 1.22-11.9, p = 0.022) and need for intensive care (adjusted OR 4.56, 95% CI: 1.37-15.8) after adjusting for all other risk factors. The degree of lung damage and fibrosis evaluated in terms of CA 15-3 concentrations may allow early identification of the increased risk of complications in patients with SARS-CoV-2 pneumonia.S

Vertebrate populations' trends across the Iberian Peninsula

Resumen del trabajo presentado a: 2nd Meeting of the Iberian Ecological Society (SIBECOL); XXI conference of the Iberian Association of Limnology (AIL) and 21st National Congress of Ecology of the Portuguese Ecological Society (SPECO), celebrados en Aveiro (Portugal) del 3 al 8 de julio de 2022.Understanding and identifying the impacts of multiple stressors on natural populations has become a key objective for post-2020 biodiversity conservation. Of particular concern are the impacts of stressors at the local scale, which are not necessarily reflected in current global conservation assessments. As a hotspot of biodiversity, the Iberian Peninsula is an area of special interest for conservation, but it is currently facing myriad environmental problems. Yet, a synthesis of the impacts of multiple stressors on the Iberian faunal populations is glaringly missing. To contribute to this challenge, we join our efforts in an Early Career Researchers project funded by SIBECOL, which aims to synthesise current knowledge on population trends of Iberian marine, terrestrial and freshwater vertebrates, and, eventually, to identify the most common stressors for Iberian fauna populations, and their differences between major taxonomic groups and ecosystems. Here, we will present the results of the first phase of this project, whose objective was to describe the trends of Iberian vertebrate populations and identify knowledge gaps related to taxonomic groups and ecosystems. To that aim, we conducted an extensive literature review, with more than 5,000 scientific manuscripts and grey literature screened. From this initial review, and after applying data quality controls, we obtained > 1,000 population time series of Iberian vertebrates across marine, terrestrial, and freshwater ecosystems. Once we compiled this database, we analysed the population trends using a two-step modelling process: (i) first, we applied state-space models to derive the population trend of each population time series; and (ii) second, we used multilevel Bayesian models to determine the factors influencing these population trends. Overall, we found that Iberian vertebrate populations display a disparity in trends, with both declines and increases, with fishes (both marine and freshwater) being the taxa showing clear signs of decline. Additionally, we identified important gaps in monitoring data across the Iberian Peninsula, with amphibians and reptiles severely underrepresented in our dataset, despite being among the most threatened taxonomic groups. These gaps may limit our understanding of the impacts of stressors at local scales and eventually bias current conservation assessments. Overall, our approach will bridge the significant gap in our knowledge between the global and local scale conservation status of wild Iberian populations and help focus future conservation policies.Peer reviewe

Regulación de la homeostasis de Ca²⁺ y viabilidad celular por STIM1 en la línea de neuroblastoma SH-SY5Y

Tesis doctoral con la Mención de "Doctor Internacional"En esta Tesis Doctoral se ha descrito el papel de STIM1, uno de los principales mediadores de la entrada extracelular de Ca²⁺ (SOCE), en la regulación de la homeostasis de Ca²⁺ y viabilidad celular de la línea de neuroblastoma SH-SY5Y. Para realizar este estudio se ha generado una línea STIM1 knock-out y, por tanto, defectiva en SOCE, mediante el sistema de edición genómica CRISPR/Cas9. Nuestros resultados demuestran que STIM1 no es esencial para la diferenciación de células SH-SY5Y. Sin embargo, se observa una pérdida de viabilidad celular durante la diferenciación de las células deficientes en STIM1. En este sentido, nuestros resultados indican que las células STIM1-KO muestran una mayor expresión y entrada de Ca²⁺ a través del canal Caᵥ1.2, lo cual se traduce en una alteración de la homeostasis de Ca²⁺ intracelular desencadenando una pérdida de la funcionalidad mitocondrial y un incremento de la muerte celular. El silenciamiento génico del canal Caᵥ1.2 logró normalizar la viabilidad y funcionalidad mitocondrial a niveles basales, confirmando que la sobreactivación de Caᵥ1.2 es la principal causa de la muerte celular observada en células STIM1-KO. Por otro lado, nuestros resultados indican que STIM1 es clave en la transferencia de Ca²⁺ entre el retículo endoplasmático y la mitocondria, en un control mediado por el receptor de IP₃ tipo 3. Todo ello permite concluir que STIM1 es clave en el mantenimiento de la homeostasis de Ca²⁺ intracelular y que las células STIM1-KO constituyen un modelo in vitro útil para comprender el papel de STIM1 y la señalización por Ca²⁺ intracelular en la neurodegeneración.The role of STIM1, one of the main mediators of the extracelular entry of Ca²⁺ (SOCE), in the regulation of Ca²⁺ homeostasis and cell viability of the SH-SY5Y neuroblastoma line, has been described in this Doctoral Thesis. To carry out this study, a STIM1 knock-out and therefore a SOCE-defective cell line has been generated using the CRISPR/Cas9 genomic editing system. Our results show that STIM1 is not essential for SH-SY5Y cell differentiation. However, a loss of cell viability is observed during differentiation of STIM1- deficient cells. In this regard, our results indicate that STIM1-KO cells show a greater expression and Ca²⁺ entry through the Caᵥ1.2 channel, which leads to an alteration of the intracellular Ca²⁺ homeostasis, triggering a loss of mitochondrial functionality and an increase in cell death. Gene silencing of the Caᵥ1.2 channel normalised mitochondrial viability and functionality, confirming that the upregulation of Caᵥ1.2 is the main cause of cell death observed in STIM1-KO cells. On the other hand, our results indicate that STIM1 is a key regulator in the transfer of Ca²⁺ between the endoplasmic reticulum and the mitochondria, a role mediated by the IP3 type 3 receptor. All this allows us to conclude that STIM1 shows a key role in the maintenance of intracellular Ca²⁺ homeostasis and that STIM1-KO cells constitute a useful in vitro model to understand the role of STIM1 and intracellular Ca²⁺ signalling in neurodegeneration.• Ministerio de Economía y Competitividad: Proyecto BFU2014-52401-P • Ministerio de Ciencia, Innovación y Universidades: Proyecto BFU2017-82716-P • Junta de Extremadura-Fondo Social Europeo: Proyectos IB16088 y GR15077 • Ministerio de Educación, Cultura y Deporte: Beca predoctoral de Formación del Profesorado Universitario FPU13/03430 • The Company of Biologists: Beca de estancia en el extranjero (Travelling Fellowship, JCSTF-170507)

Regulación de la homeostasis de Ca²⁺ y viabilidad celular por STIM1 en la línea de neuroblastoma SH-SY5Y

Tesis doctoral con la Mención de "Doctor Internacional"En esta Tesis Doctoral se ha descrito el papel de STIM1, uno de los principales mediadores de la entrada extracelular de Ca²⁺ (SOCE), en la regulación de la homeostasis de Ca²⁺ y viabilidad celular de la línea de neuroblastoma SH-SY5Y. Para realizar este estudio se ha generado una línea STIM1 knock-out y, por tanto, defectiva en SOCE, mediante el sistema de edición genómica CRISPR/Cas9. Nuestros resultados demuestran que STIM1 no es esencial para la diferenciación de células SH-SY5Y. Sin embargo, se observa una pérdida de viabilidad celular durante la diferenciación de las células deficientes en STIM1. En este sentido, nuestros resultados indican que las células STIM1-KO muestran una mayor expresión y entrada de Ca²⁺ a través del canal Caᵥ1.2, lo cual se traduce en una alteración de la homeostasis de Ca²⁺ intracelular desencadenando una pérdida de la funcionalidad mitocondrial y un incremento de la muerte celular. El silenciamiento génico del canal Caᵥ1.2 logró normalizar la viabilidad y funcionalidad mitocondrial a niveles basales, confirmando que la sobreactivación de Caᵥ1.2 es la principal causa de la muerte celular observada en células STIM1-KO. Por otro lado, nuestros resultados indican que STIM1 es clave en la transferencia de Ca²⁺ entre el retículo endoplasmático y la mitocondria, en un control mediado por el receptor de IP₃ tipo 3. Todo ello permite concluir que STIM1 es clave en el mantenimiento de la homeostasis de Ca²⁺ intracelular y que las células STIM1-KO constituyen un modelo in vitro útil para comprender el papel de STIM1 y la señalización por Ca²⁺ intracelular en la neurodegeneración.The role of STIM1, one of the main mediators of the extracelular entry of Ca²⁺ (SOCE), in the regulation of Ca²⁺ homeostasis and cell viability of the SH-SY5Y neuroblastoma line, has been described in this Doctoral Thesis. To carry out this study, a STIM1 knock-out and therefore a SOCE-defective cell line has been generated using the CRISPR/Cas9 genomic editing system. Our results show that STIM1 is not essential for SH-SY5Y cell differentiation. However, a loss of cell viability is observed during differentiation of STIM1- deficient cells. In this regard, our results indicate that STIM1-KO cells show a greater expression and Ca²⁺ entry through the Caᵥ1.2 channel, which leads to an alteration of the intracellular Ca²⁺ homeostasis, triggering a loss of mitochondrial functionality and an increase in cell death. Gene silencing of the Caᵥ1.2 channel normalised mitochondrial viability and functionality, confirming that the upregulation of Caᵥ1.2 is the main cause of cell death observed in STIM1-KO cells. On the other hand, our results indicate that STIM1 is a key regulator in the transfer of Ca²⁺ between the endoplasmic reticulum and the mitochondria, a role mediated by the IP3 type 3 receptor. All this allows us to conclude that STIM1 shows a key role in the maintenance of intracellular Ca²⁺ homeostasis and that STIM1-KO cells constitute a useful in vitro model to understand the role of STIM1 and intracellular Ca²⁺ signalling in neurodegeneration.• Ministerio de Economía y Competitividad: Proyecto BFU2014-52401-P • Ministerio de Ciencia, Innovación y Universidades: Proyecto BFU2017-82716-P • Junta de Extremadura-Fondo Social Europeo: Proyectos IB16088 y GR15077 • Ministerio de Educación, Cultura y Deporte: Beca predoctoral de Formación del Profesorado Universitario FPU13/03430 • The Company of Biologists: Beca de estancia en el extranjero (Travelling Fellowship, JCSTF-170507)

Store-operated calcium entry is dispensable for the activation of ERK1/2 pathway in prostate cancer cells

STIM1, el sensor de Ca2 + del retículo endoplásmico que modula la actividad de los canales de Ca2 + de la membrana plasmática, se fosforila en los sitios objetivo de ERK1 / 2 durante el agotamiento del almacenamiento de Ca2 + desencadenado por thapsigargin o factor de crecimiento epidérmico (EGF). Esta fosforilación dependiente de ERK1 / 2 regula la localización y disociación de STIM1 de los microtúbulos, y se sabe que mejora la unión a ORAI1, un canal de entrada de Ca2 + (SOCE) operado en el almacén, lo que lleva a la activación de esta vía de afluencia de Ca2 +. Sin embargo, quedaron algunas pruebas de un papel para SOCE en la activación de ERK1 / 2, y aquí evaluamos la contribución de SOCE a la activación de ERK1 / 2 generando una línea celular deficiente en STIM1 mediante la edición del genoma CRISPR / Cas9 del locus STIM1 En células de cáncer de próstata PC3. La modificación genómica consistió en una inserción de pares de 16 bases en el exón 5 de ambos alelos, anulando así la síntesis de STIM1. Las células STIM1-KO mostraron una disminución sorprendente en el flujo de Ca2 + en respuesta a thapsigargin o EGF, un resultado que demuestra que SOCE media la entrada de Ca2 + en las células PC3 durante la estimulación con EGF. Además, se encontraron niveles idénticos de ERK1 / 2 total en células STIM1-KO y en la línea celular parental, y la activación de ERK1 / 2 se activó completamente en células KO, tanto en presencia como en ausencia de Ca2 + extracelular, un resultado que apoya que STIM1 y SOCE no son necesarios para la activación de ERK1 / 2. Esta activación fue sensible a la inhibición de la Src quinasa, pero no a la inhibición de CAMKII ni a la PKC, un resultado que establece a STIM1 y SOCE como objetivos descendentes del eje Src-Raf-MEK-ERK, en lugar de reguladores ascendentes.STIM1, the endoplasmic reticulum Ca2+ sensor that modulates the activity of plasma membrane Ca2+ channels, becomes phosphorylated at ERK1/2 target sites during Ca2+ store depletion triggered by thapsigargin or epidermal growth factor (EGF). This ERK1/2-dependent phosphorylation regulates STIM1 localization and dissociation from microtubules, and it is known that enhances the binding to ORAI1, a store-operated Ca2+ entry (SOCE) channel, leading to the activation of this Ca2+ influx pathway. However, there remained some evidence of a role for SOCE in the activation of ERK1/2, and here we assessed the contribution of SOCE to ERK1/2 activation by generating a STIM1-deficient cell line by CRISPR/Cas9 genome editing of the STIM1 locus in prostate cancer PC3 cells. The genomic modification consisted of a 16 base -pair insertion in exon 5 of both alleles, therefore abrogating STIM1 synthesis. STIM1-KO cells did show a striking decrease in Ca2+ influx in response to thapsigargin or EGF, a result that demonstrates that SOCE mediates Ca2+ entry in PC3 cells during stimulation with EGF. Moreover, identical levels of total ERK1/2 were found in STIM1-KO cells and the parental cell line, and ERK1/2 activation was fully activated in KO cells, both in the presence and in the absence of extracellular Ca2+, a result that supports that STIM1 and SOCE are not required for ERK1/2 activation. This activation was sensitive to Src kinase inhibition, but not to CAMKII nor PKC inhibition, a result that sets STIM1 and SOCE as downstream targets of the axis Src-Raf-MEK-ERK, rather than upstream regulators.• Ministerio de Economía y Competitividad y Fondo Social Europeo. Beca BFU2014-52401-p (I+D+i), para Francisco Javier Martín Romero • Ministerio de Economía y Competitividad. Beca BES2012-052061, para Aida María López Guerrero • Ministerio de Educación, Cultura y Deporte. Beca FPU12/03430, para Carlos Pascual CaropeerReviewe