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Intracellular levels of the RNA-binding protein and pluripotency factor, Lin28a, are tightly controlled to govern cellular and organismal growth. Lin28a is extensively regulated at the posttranscriptional level, and can undergo mitogen-activated protein kinase (MAPK)-mediated elevation from low basal levels in differentiated cells by phosphorylation-dependent stabilizing interaction with the RNA-silencing factor HIV TAR RNA-binding protein (TRBP). However, molecular and spatiotemporal details of this critical control mechanism remained unknown. In this work, we dissect the interacting regions of Lin28a and TRBP proteins and develop biosensors to visualize this interaction. We identify truncated domains of Lin28a and of TRBP that are sufficient to support coassociation and mutual elevation of protein levels, and a requirement for MAPK-dependent phosphorylation of TRBP at putative Erk-target serine 152, as well as Lin28a serine 200 phosphorylation, in mediating the increase of Lin28a protein by TRBP. The phosphorylation-dependent association of Lin28a and TRBP truncated constructs is leveraged to develop fluorescence resonance energy transfer (FRET)-based sensors for dynamic monitoring of Lin28a and TRBP interaction. We demonstrate the response of bimolecular and unimolecular FRET sensors to growth factor stimulation in living cells, with coimaging of Erk activation to achieve further understanding of the role of MAPK signaling in Lin28a regulation.
Pubmed ID: 29540527 RIS Download
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Software tool for automated microscope acquisition, device control, and image analysis. Used for integrating dissimilar fluorescent microscope hardware and peripherals into a single custom workstation, while providing all the tools needed to perform analysis of acquired images. Offers user friendly application modules for analysis such as cell signaling, cell counting, and protein expression.
View all literature mentionsSoftware designed for single or multi-wavelength intracellular ion measurements. It provides simultaneous display of raw data, ratio image, graphs of intensities, ratios, ion concentrations, and a non-ratiometric image such as a brightfield or phase-contrast image. Two different ratiometric indicators can be imaged and measured simultaneously to provide greater insight to ion exchange and intracellular function regardless of dye loading concentrations, conditions, or emission intensities.
View all literature mentionsCell line HEK293T is a Transformed cell line with a species of origin Homo sapiens (Human)
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