5-fluorocytosine sale Previous research has suggested that d

Previous research has suggested that differences in spatiotemporal regulation of intracellular signaling pathways can confer specificity to cellular responses (Marshall, 1995). Conventional approaches based on gain- or loss-of-function genetic manipulations or small-molecule inhibitors, however, lack the necessary resolution to modulate specific changes in space and time to test this hypothesis. A better understanding of signaling mechanisms therefore calls for new tools that can precisely control intracellular signaling in both space and time. Recently, several optogenetic tools have emerged, and they could potentially transform conventional ways of studying intracellular signaling (Kennedy et al., 2010, Levskaya et al., 2009, Wu et al., 2009, Yazawa et al., 2009). Optogenetics relies on light-induced protein interactions to control the activation state of engineered signaling components in cells. Kim and colleagues use blue light-induced cryptochrome oligomerization to trigger the activation of an engineered FGFR (optoFGFR1) and subsequent signaling pathways (Kim et al., 2014). Light-controlled activation of this pathway opens the door for experiments that rely on spatial and temporal regulation aimed at dissecting the roles of specific 5-fluorocytosine sale types (Figure 1). To make an FGFR that can be activated by blue light (optoFGFR1), the authors engineered a chimeric receptor by inserting the cytoplasmic regions of FGFR1 between the N-terminal photolyase homology domain of cryptochrome (CRY2PHR) and a membrane-targeting myristoylation peptide. CRY2PHR has been shown to undergo blue light-mediated oligomerization (Bugaj et al., 2013, Wend et al., 2013). Therefore, when optoFGFR1 is exposed to blue light, CRY2PHR oligomerizes and brings the catalytic domains of FGFR into proximity, mimicking ligand-induced FGFR dimerization and subsequent activation. Using live cell imaging, a FRET based sensor, and other standard approaches for analyzing signaling pathways, the authors demonstrated that blue light can indeed induce phosphorylation of optoFGFR1 and activate downstream ERK, AKT, and PLCγ signaling cascades. By controlling the temporal patterns of excitation light, the authors characterized ERK signaling in response to modulated light frequency and duration. They found that high-frequency light stimulation (10 min interval) leads to sustained ERK activation, whereas low-frequency light stimulation (30 min and 60 min) gives pulsatile patterns of ERK activation. This result is consistent with a previous study showing that the Ras/ERK signaling module functions as a low-pass filter in transmitting extracellular growth factor signals (Toettcher et al., 2013). Several previous reports have used the PhyB-PIF or CRY2-CIB1 binding pairs to demonstrate optogenetic control of individual signaling cascades downstream of growth factor stimulation including the ERK (Toettcher et al., 2013, Zhang et al., 2014) and the AKT (Idevall-Hagren et al., 2012) pathways. This work adds another node of regulation at the level of membrane receptor. Overall, these light-based regulation studies promise greater insights into understanding the spatial and temporal dimensions of intracellular signal transduction.
Introduction Carcinogenesis follows the stepwise progression of normal benign cells to cancer cells that have acquired abilities in independent growth, to evade anti-apoptotic signals, to promote angiogenesis, and to invade and metastasise to distant organs [1]. This progression can be characterised by multiple gene mutations, often happening in a specific temporal fashion. Such mutations may include activating mutations, gene amplifications and overexpression of oncogenes, or inactivating mutations and epigenetic silencing of tumour suppressors. However, simple association of the mutations is not sufficient in providing the mechanism underlying cancer progression. The mutations may be ‘passenger’ mutations, and may not always be the critical ‘driver’ events that provide the malignant cells survival advantage and the ability for clonal expansion.