Arabidopsis thaliana cryptochrome 2 (AtCRY2), a light-sensitive photosensory protein, was previously adapted for use controling protein-protein interactions through light-dependent binding to a partner protein, CIB1. While the existing CRY2/CIB dimerization system has been used extensively for optogenetic applications, some limitations exist. Here, we set out to optimize function of the CRY2/CIB system, to identify versions of CRY2/CIB that are smaller, show reduced dark interaction, and maintain longer or shorter signaling states in response to a pulse of light. We describe minimal functional CRY2 and CIB1 domains maintaining light-dependent interaction and new signaling mutations affecting AtCRY2 photocycle kinetics. The latter work implicates a α13-α14 turn motif within plant CRYs where perturbations alter signaling state lifetime. Using a long-lived L348F photocycle mutant, we engineered a second generation photoactivatable Cre recombinase, PA-Cre2.0, that shows five-fold improved dynamic range allowing robust recombination following exposure to a single, brief pulse of light.

Transition metals are essential enzyme cofactors that are required for a wide range of cellular processes. Paradoxically, whereas metal ions are essential for numerous cellular processes, they are also toxic. Therefore cells must tightly regulate metal accumulation, transport, distribution, and export. Improved tools to interrogate metal ion availability and spatial distribution within living cells would greatly advance our understanding of cellular metal homeostasis. In this work, we present genetically encoded sensors for Zn 2؉ based on the principle of fluorescence resonance energy transfer. We also develop methodology to calibrate the probes within the cellular environment. To identify both sources of and sinks for Zn 2؉ , these sensors are genetically targeted to specific locations within the cell, including cytosol, plasma membrane, and mitochondria. Localized probes reveal that mitochondria contain an elevated pool of Zn 2؉ under resting conditions that can be released into the cytosol upon glutamate stimulation of hippocampal neurons. We also observed that Zn 2؉ is taken up into mitochondria following glutamate/Zn 2؉ treatment and that there is heterogeneity in both the magnitude and kinetics of the response. Our results suggest that mitochondria serve as a source of and a sink for Zn 2؉ signals under different cellular conditions.Although mammalian cells are known to concentrate transition metals, it is now well established that under resting conditions, "free" (e.g. unbound) metals are maintained at extremely low levels. Estimates of the total Zn 2ϩ concentration in mammalian cells typically range from 100 to 500 M (1); yet free Zn 2ϩ concentrations are tightly buffered by proteins such as metallothionein to maintain cytosolic Zn 2ϩ concentrations in the picomolar to nanomolar range (2-5). However, there is emerging evidence that this static picture is dramatically altered by different cellular conditions, such as redox perturbations caused by oxidative stress (6, 7) and cellular signals such as nitric oxide (8 as mitochondrial function (7, 9, 10). Elucidation of the sources and dynamics of these Zn 2ϩ signals would greatly advance our understanding of the interplay between metal regulation and cellular function.There has been a huge effort in the past few years to develop sensitive and selective fluorescent probes to monitor Zn 2ϩ in biological systems. The majority of this work has focused on the generation of small molecule fluorescent indicators (reviewed by Que et al. (11)). Yet there are also examples of sensors based partially on Zn 2ϩ -binding proteins, such as carbonic anhydrase (12) and metallothionein (13), and peptide scaffolds (14). Although many of these sensors have begun to provide insight into Zn 2ϩ concentrations within cells, one limitation is that it is challenging to explicitly target them to subdomains within the cell. Localized probes are necessary to generate a complete picture of cellular Zn 2ϩ homeostasis in mammalian cells. For this reason, sensors that are genetically encode...

SUMMARY Melanoma and other cancers harbor oncogenic mutations in the protein kinase B-Raf, which leads to constitutive activation and dysregulation of MAP kinase signaling. In order to elucidate molecular determinants responsible for B-Raf control of cancer phenotypes, we present a method for phosphoprotein profiling, using negative ionization mass spectrometry to detect phosphopeptides based on their fragment ion signature caused by release of PO3−. The method provides an alternative strategy for phosphoproteomics, circumventing affinity enrichment of phosphopeptides and isotopic labeling of samples. Ninety phosphorylation events were regulated by oncogenic B-Raf signaling, based on their responses to treating melanoma cells with MKK1/2 inhibitor. Regulated phosphoproteins included known signaling effectors and cytoskeletal regulators. We investigated MINERVA/FAM129B, a target belonging to a protein family with unknown category and function, and established the importance of this protein and its MAP kinase-dependent phosphorylation in controlling melanoma cell invasion into 3-dimensional collagen matrix.

Fluorescent sensors are powerful tools for visualizing and quantifying molecules and ions in living cells. A variety of small molecule and genetically encoded sensors have been developed for studying intracellular Zn2+ homeostasis and signaling, but no direct comparisons exist making it challenging for researchers to identify the appropriate sensor for a given application. Here we directly compare the widely used small molecule probe FluoZin-3 and a genetically encoded sensor, ZapCY2. We demonstrate that, in contrast to FluoZin-3, ZapCY2 exhibits a well defined cytosolic localization, provides estimates of Zn2+ concentration with little variability, does not perturb cytosolic Zn2+ levels, and exhibits rapid Zn2+ response dynamics. ZapCY2 was used to measure Zn2+ concentrations in 5 different cell types, revealing higher cytosolic Zn2+ levels in prostate cancer cells compared to normal prostate cells (although the total zinc is reduced in prostate cancer cells) , suggesting distinct regulatory mechanisms.

Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for reporting on ions, molecules and biochemical reactions in living cells. Here we describe the development of new sensors for Zn2+based on alternate FRET-pairs that do not involve the traditional CFP and YFP. Zn2+ is an essential micronutrient and plays fundamental roles in cell biology. Consequently there is a pressing need for robust sensors to monitor Zn2+ levels and dynamics in cells with high spatial and temporal resolution. Here we develop a suite of sensors using alternate FRET pairs, including tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE. These sensors were targeted to both the nucleus and cytosol and characterized and validated in living cells. Sensors based on the new FRET pair Clover/mRuby2 displayed a higher dynamic range and better signal-to-noise ratio than the remaining sensors tested and were optimal for monitoring changes in cytosolic and nuclear Zn2+. Using a green-red sensor targeted to the nucleus and cyan-yellow sensor targeted to either the ER, Golgi, or mitochondria, we were able to monitor Zn2+ uptake simultaneously in two compartments, revealing that nuclear Zn2+ rises quickly, whereas the ER, Golgi, and mitochondria all sequester Zn2+ more slowly and with a delay of 600–700 sec. Lastly, these studies provide the first glimpse of nuclear Zn2+ and reveal that nuclear Zn2+ is buffered at a higher level than cytosolic Zn2+.