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

Regulation of activity and localization of the WNK1 protein kinase by hyperosmotic stress

Mutations within the WNK1 (with-no-K[Lys] kinase-1) gene cause Gordon's hypertension syndrome. Little is known about how WNK1 is regulated. We demonstrate that WNK1 is rapidly activated and phosphorylated at multiple residues after exposure of cells to hyperosmotic conditions and that activation is mediated by the phosphorylation of its T-loop Ser382 residue, possibly triggered by a transautophosphorylation reaction. Activation of WNK1 coincides with the phosphorylation and activation of two WNK1 substrates, namely, the protein kinases STE20/SPS1-related proline alanine–rich kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Small interfering RNA depletion of WNK1 impairs SPAK/OSR1 activity and phosphorylation of residues targeted by WNK1. Hyperosmotic stress induces rapid redistribution of WNK1 from the cytosol to vesicular structures that may comprise trans-Golgi network (TGN)/recycling endosomes, as they display rapid movement, colocalize with clathrin, adaptor protein complex 1 (AP-1), and TGN46, but not the AP-2 plasma membrane–coated pit marker nor the endosomal markers EEA1, Hrs, and LAMP1. Mutational analysis suggests that the WNK1 C-terminal noncatalytic domain mediates vesicle localization. Our observations shed light on the mechanism by which WNK1 is regulated by hyperosmotic stress

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

Regulation of cell survival by resveratrol involves inhibition of NFκB-regulated gene expression in prostate cancer cells

BACKGROUND. Polyphenols have been proposed as antitumoral agents. We have shown that resveratrol (RES) induced cell cycle arrest and promoted apoptosis in prostate cancer cells by inhibition of the PI3K pathway. The RES effects on NFκB activity in LNCaP cells (inducible NFκB), and PC-3 cells (constitutive NFκB) are reported. METHODS. Cells were treated with 1-150 μM of RES during 36 hr. NFκB subcellular localization was analyzed by western blot and immunofluorescence. IκBα was evaluated by immunoprecipitation followed by Western blot. Specific DNA binding of NFκB was determined by EMSA assays and NFκB-mediated transcriptional activity by transient transfection with a luciferase gene reporter system. RESULTS. RES induced a dose-dependent cytoplasmic retention of NFκB mediated by IκBα in PC-3 cells but not in LNCaP. RES-induced inhibition ofNFκB specific binding toDNAwas more significant in PC-3 cells. NFκB-mediated transcriptional activity induced by EGF and TNFα were inhibited by RES