RESUMEN
The role of zinc ion in cytotoxicity following ischemic stroke, prolonged status epilepticus, and traumatic brain injury remains controversial, but likely is the result of mitochondrial dysfunction. We describe an excitation ratiometric fluorescence biosensor based on human carbonic anhydrase II variants expressed in the mitochondrial matrix, permitting free zinc levels to be quantitatively imaged therein. We observed an average mitochondrial matrix free zinc concentration of 0.2 pM in the PC12 rat pheochromacytoma cell culture line. Cytoplasmic and mitochondrial free zinc levels were imaged in a cellular oxygen glucose deprivation (OGD) model of ischemia/reperfusion. We observed a significant increase in mitochondrial zinc 1 h following 3 h OGD, at a time point when cytosolic zinc levels were depressed. Following the increase, mitochondrial zinc levels returned to physiological levels, while cytosolic zinc increased gradually over a 24 h time period in viable cells. The increase in intramitochondrial zinc observed during reoxygenation after OGD may contribute to bioenergetic dysfunction and cell death that occurs with both in vitro and in vivo models of reperfusion.
Asunto(s)
Mitocondrias/metabolismo , Mitocondrias/patología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Zinc/metabolismo , Animales , Anhidrasa Carbónica II/genética , Anhidrasa Carbónica II/metabolismo , Muerte Celular/genética , Hipoxia de la Célula/genética , Glucosa/metabolismo , Humanos , Microscopía Fluorescente , Mitocondrias/genética , Células PC12 , Ratas , Daño por Reperfusión/genéticaRESUMEN
Viruses modulate the actin cytoskeleton at almost every step of their cellular journey from entry to egress. Cellular sensing of these cytoskeletal changes may function in the recognition of viral infection. Here we show that focal adhesion kinase (FAK), a focal adhesion localized tyrosine kinase that transmits signals between the extracellular matrix and the cytoplasm, serves as a RIG-I-like receptor antiviral signaling component by directing mitochondrial antiviral signaling adaptor (MAVS) activation. Cells deficient in FAK are highly susceptible to RNA virus infection and attenuated in antiviral signaling. We show that FAK interacts with MAVS at the mitochondrial membrane in a virus infection-dependent manner and potentiates MAVS-mediated signaling via a kinase-independent mechanism. A cysteine protease encoded by enteroviruses cleaves FAK to suppress its role in innate immune signaling. These findings suggest that FAK serves as a link between cytoskeletal perturbations that occur during virus infection and activation of innate immune signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Virus ARN/inmunología , Transducción de Señal , Línea Celular , Humanos , Membranas Mitocondriales/metabolismo , Mapeo de Interacción de Proteínas , Ensayo de Placa ViralRESUMEN
Coxsackievirus B (CVB), a member of the enterovirus family, targets the polarized epithelial cells lining the intestinal tract early in infection. Although the polarized epithelium functions as a protective barrier, this barrier is likely exploited by CVB to promote viral entry and subsequent egress. Here we show that, in contrast to nonpolarized cells, CVB-infected polarized intestinal Caco-2 cells undergo nonapoptotic necrotic cell death triggered by inositol 1,4,5-trisphosphate receptor-dependent calcium release. We further show that CVB-induced cellular necrosis depends on the Ca(2+)-activated protease calpain-2 and that this protease is involved in CVB-induced disruption of the junctional complex and rearrangements of the actin cytoskeleton. Our study illustrates the cell signaling pathways hijacked by CVB, and perhaps other viral pathogens, to promote their replication and spread in polarized cell types.
Asunto(s)
Señalización del Calcio , Calpaína/metabolismo , Enterovirus Humano B/fisiología , Infecciones por Enterovirus/virología , Mucosa Intestinal/virología , Necrosis/virología , Liberación del Virus , Células CACO-2 , Proteínas de la Cápside/metabolismo , Caspasa 3/metabolismo , Polaridad Celular , Citoesqueleto/metabolismo , Retículo Endoplásmico/metabolismo , Pruebas de Enzimas , Células HeLa , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , Microscopía Fluorescente , Necrosis/metabolismo , Uniones Estrechas/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
Group B coxsackieviruses (CVB) are associated with viral-induced heart disease and are among the leading causes of aseptic meningitis worldwide. Here we show that CVB entry into polarized brain microvasculature and aortic endothelial cells triggers a depletion of intracellular calcium stores initiated through viral attachment to the apical attachment factor decay-accelerating factor. Calcium release was dependent upon a signaling cascade that required the activity of the Src family of tyrosine kinases, phospholipase C, and the inositol 1,4,5-trisphosphate receptor isoform 3. CVB-mediated calcium release was required for the activation of calpain-2, a calcium-dependent cysteine protease, which controlled the vesicular trafficking of internalized CVB particles. These data point to a specific role for calcium signaling in CVB entry into polarized endothelial monolayers and highlight the unique signaling mechanisms used by these viruses to cross endothelial barriers.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Polaridad Celular , Células Endoteliales/virología , Enterovirus/fisiología , Internalización del Virus , Calpaína/metabolismo , Calpaína/fisiología , Polaridad Celular/fisiología , Células Cultivadas , Infecciones por Coxsackievirus/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Endotelio Vascular/virología , Epitelio/metabolismo , Epitelio/fisiología , Epitelio/virología , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/fisiología , Espacio Intracelular/metabolismo , Fosfolipasa C gamma/metabolismo , Fosfolipasa C gamma/fisiologíaRESUMEN
The zinc(II) ion has recently been implicated in a number of novel functions and pathologies in loci as diverse as the brain, retina, small intestine, prostate, heart, pancreas, and immune system. Zinc ions are a required nutrient but elevated concentrations are known to kill cells in vitro. Paradoxical observations regarding zinc's effects have appeared frequently in the literature, and often their physiological relevance is unclear. We found that for PC-12, HeLa and HT-29 cell lines as well as primary cultures of cardiac myocytes and neurons in vitro in differing media, approximately 5 nmol/L free zinc (pZn = 8.3, where pZn is defined as--log(10) [free Zn(2+)]) produced apparently healthy cells, but 20-fold higher or (in one case) lower concentrations were usually harmful as judged by multiple criteria. These results indicate that (1) the free zinc ion levels of media should be controlled with a metal ion buffer; (2) adding zinc or strong zinc ligands to an insufficiently buffered medium may lead to unpredictably low or high free zinc levels that are often harmful to cells; and (3) it is generally desirable to measure free zinc ion levels due to the presence of contaminating zinc in many biochemicals and unknown buffering capacity of many media.
Asunto(s)
Zinc/toxicidad , Animales , Línea Celular , Células Cultivadas , Medios de Cultivo/química , Células Epiteliales/efectos de los fármacos , Humanos , Iones/toxicidad , Ratones , Células Musculares/efectos de los fármacos , Neuronas/efectos de los fármacos , RatasRESUMEN
Zinc plays both physiological and pathological roles in biology, making it of increasing interest. To date, intracellular free zinc has been measured in cell types supplemented with or enriched in zinc, such as hippocampal neurons. Here we quantitatively image intracellular exchangeable zinc in an ordinary resting cell culture line (PC-12), using an excitation ratiometric fluorescent biosensor based on carbonic anhydrase (CA). Human CA II has a K d of 4 pM for zinc and suffers no interference from millimolar calcium or magnesium ions. The CA-based biosensor was readily introduced into the cell by a novel approach: fusing a transactivator of transcription (TAT)-derived cell penetrating peptide to the CA molecule and adding it to the cells. Our results indicate that the resting concentration is approximately 5-10 pM in cytoplasm and nucleus. Interestingly, the tetrakis(2-pyridylmethyl)ethylenediamine (TPEN)-Zn complex and TPEN are both apoptogenic for this cell line.