Search for: All records

Zinc (Zn²⁺) is an essential metal in biology, and its bioavailability is highly regulated. Many cell types exhibit fluctuations in Zn²⁺that appear to play an important role in cellular function. However, the detailed molecular mechanisms by which Zn²⁺dynamics influence cell physiology remain enigmatic. Here, we use a combination of fluorescent biosensors and cell perturbations to define how changes in intracellular Zn²⁺impact kinase signaling pathways. By simultaneously monitoring Zn²⁺dynamics and kinase activity in individual cells, we quantify changes in labile Zn²⁺and directly correlate changes in Zn²⁺with ERK and Akt activity. Under our experimental conditions, Zn²⁺fluctuations are not toxic and do not activate stress-dependent kinase signaling. We demonstrate that while Zn²⁺can nonspecifically inhibit phosphatases leading to sustained kinase activation, ERK and Akt are predominantly activated via upstream signaling and through a common node via Ras. We provide a framework for quantification of Zn²⁺fluctuations and correlate these fluctuations with signaling events in single cells to shed light on the role that Zn²⁺dynamics play in healthy cell signaling.

 

Zinc (Zn²⁺) is an integral component of many proteins and has been shown to act in a regulatory capacity in different mammalian systems, including as a neurotransmitter in neurons throughout the brain. While Zn²⁺plays an important role in modulating neuronal potentiation and synaptic plasticity, little is known about the signaling mechanisms of this regulation. In dissociated rat hippocampal neuron cultures, we used fluorescent Zn²⁺sensors to rigorously define resting Zn²⁺levels and stimulation-dependent intracellular Zn²⁺dynamics, and we performed RNA-Seq to characterize Zn²⁺-dependent transcriptional effects upon stimulation. We found that relatively small changes in cytosolic Zn²⁺during stimulation altered expression levels of 931 genes, and these Zn²⁺dynamics induced transcription of many genes implicated in neurite expansion and synaptic growth. Additionally, while we were unable to verify the presence of synaptic Zn²⁺in these cultures, we did detect the synaptic vesicle Zn²⁺transporter ZnT3 and found it to be substantially upregulated by cytosolic Zn²⁺increases. These results provide the first global sequencing-based examination of Zn²⁺-dependent changes in transcription and identify genes that may mediate Zn²⁺-dependent processes and functions.

 

Green fluorescent proteins (GFP) and their blue, cyan and red counterparts offer unprecedented advantages as biological markers owing to their genetic encodability and straightforward expression in different organisms. Although significant advancements have been made towards engineering the key photo-physical properties of red fluorescent proteins (RFPs), they continue to perform sub-optimally relative to GFP variants. Advanced engineering strategies are needed for further evolution of RFPs in the pursuit of improving their photo-physics. In this report, a microfluidic sorter that discriminates members of a cell-based library based on their excited state lifetime and fluorescence intensity is used for the directed evolution of the photo-physical properties of FusionRed. In-flow measurements of the fluorescence lifetime are performed in a frequency-domain approach with sub-millisecond sampling times. Promising clones are sorted by optical force trapping with an infrared laser. Using this microfluidic sorter, mutants are generated with longer lifetimes than their precursor, FusionRed. This improvement in the excited state lifetime of the mutants leads to an increase in their fluorescence quantum yield up to 1.8-fold. In the course of evolution, we also identified one key mutation (L177M), which generated a mutant (FusionRed-M) that displayed ∼2-fold higher brightness than its precursor upon expression in mammalian (HeLa) cells. Photo-physical and mutational analyses of clones isolated at the different stages of mutagenesis reveal the photo-physical evolution towards higher in vivo brightness.