Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal-regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors.

Growth cone localization of MKK7 mRNA switches the classic function of MKK7 protein in transcriptional control to regulation of microtubules necessary for neuronal outgrowth.

Embryonic stem cells (ESCs) are pluripotent cells able to grow indefinitely in culture and to differentiate into all cell types of embryos upon specific stimuli. Molecular mechanisms controlling the unique characteristics of ESCs are still largely unknown. We identified Dies1 (Differentiation of ESCs 1), an unpublished gene, that encodes a type I membrane protein. ESCs stably transfected with Dies1 small hairpin RNAs failed to properly differentiate toward neural and cardiac cell fate upon appropriate stimuli and continued to express markers of undifferentiated cells, such as the membrane-associated alkaline phosphatase, and transcription factors, like Oct3/4 and Nanog, when grown under conditions promoting differentiation. Our results demonstrated that Dies1 is required for BMP4/Smad1 signaling cascade; in undifferentiated ESCs Dies1 knockdown did not affect the expression of leukemia inhibitory factor downstream targets, whereas it resulted in a strong decrease of BMP4 signaling, as demonstrated by the decrease of Id1, -2, and -3 mRNAs, the decreased activity of Id1 gene promoter, and the reduced phospho-Smad1 levels. Dies1 knockdown had no effect in murine ESCs when the expression of the BMP4 receptor Alk3 was suppressed. The phenotype induced by Dies1 suppression in ESCs is due to the indirect activation of the Nodal/Activin pathway, which is a consequence of the BMP4 pathway inhibition and is sufficient to support the mESC undifferentiated state in the absence of leukemia inhibitory factor.

NeuriteTracker is a computer vision approach used to analyze neuronal morphodynamics and to examine spatiotemporal Rho GTPase signaling networks regulating neurite outgrowth.

Growth cones are complex, motile structures at the tip of an outgrowing neurite. They often exhibit a high density of filopodia (thin actin bundles), which complicates the unbiased quantification of their morphologies by software. Contemporary image processing methods require extensive tuning of segmentation parameters, require significant manual curation, and are often not sufficiently adaptable to capture morphology changes associated with switches in regulatory signals. To overcome these limitations, we developed Growth Cone Analyzer (GCA). GCA is designed to quantify growth cone morphodynamics from time-lapse sequences imaged both in vitro and in vivo, but is sufficiently generic that it may be applied to nonneuronal cellular structures. We demonstrate the adaptability of GCA through the analysis of growth cone morphological variation and its relation to motility in both an unperturbed system and in the context of modified Rho GTPase signaling. We find that perturbations inducing similar changes in neurite length exhibit underappreciated phenotypic nuance at the scale of the growth cone.