RESUMEN
Activation of G protein-coupled receptors upon chemoattractant stimulation induces activation of multiple signaling pathways. To fully understand how these signaling pathway coordinates to achieve directional migration of neutrophils, it is essential to determine the dynamics of the spatiotemporal activation profile of signaling components at the level of single living cells. Here, we describe a detailed methodology for monitoring and quantitatively analyzing the spatiotemporal dynamics of 1,4,5-inositol trisphosphate (IP3) in neutrophil-like HL60 cells in response to various chemoattractant fields by applying Förster resonance energy transfer (FRET) fluorescence microscopy.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Inositol 1,4,5-Trifosfato , Microscopía Confocal , Microscopía Fluorescente , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Células HL-60 , Microscopía Fluorescente/métodos , Microscopía Confocal/métodos , Inositol 1,4,5-Trifosfato/metabolismo , Transducción de Señal , Neutrófilos/metabolismoRESUMEN
This study focuses on a subset of medical students who participated in an anatomy dissection program and undertook an additional self-directed learning (SDL) project investigating incidental findings of cadaveric pathology. The value of SDL activity is explored as a means of enhancing medical student education, particularly its student perceived value in preparing and developing them as future medical educators. It was assessed whether the project advanced student interest in medical education by analyzing their motivations for participation. The results of the study highlight the potential of SDL as an experiential learning opportunity for medical students and the role of anatomic pathology in connecting multiple domains of medical education.
RESUMEN
The current dogma is that chemoattractants G protein-coupled receptors activate ß phospholipase C while receptor tyrosine kinases activate γ phospholipase C. Here, we show that chemoattractant/G protein-coupled receptor-mediated membrane recruitment of γ2 phospholipase C constitutes G protein-coupled receptor-mediated phospholipase C signaling and is essential for neutrophil polarization and migration during chemotaxis. In response to a chemoattractant stimulation, cells lacking γ2 phospholipase C (plcg2kd) displayed altered dynamics of diacylglycerol production and calcium response, increased Ras/PI3K/Akt activation, elevated GSK3 phosphorylation and cofilin activation, impaired dynamics of actin polymerization, and, consequently, defects in cell polarization and migration during chemotaxis. The study reveals a molecular mechanism of membrane targeting of γ2 phospholipase C and the signaling pathways by which γ2 phospholipase C plays an essential role in neutrophil chemotaxis.