Amy Palmer

Dissociated Hippocampal Neurons Exhibit Distinct Zn2+ Dynamics in a Stimulation-Method-Dependent Manner

Anonymous — Wed, 26 Feb 2020 20:13:30 +0000

Dissociated Hippocampal Neurons Exhibit Distinct Zn2+ Dynamics in a Stimulation-Method-Dependent Manner Anonymous (not verified) Wed, 02/26/2020 - 13:13

Ionic Zn²⁺ has increasingly been recognized as an important neurotransmitter and signaling ion in glutamatergic neuron pathways. Intracellular Zn²⁺ transiently increases as a result of neuronal excitation, and this Zn²⁺ signal is essential for neuron plasticity, but the source and regulation of the signal is still unclear. In this study, we rigorously quantified Zn²⁺, Ca²⁺, and pH dynamics in dissociated mouse hippocampal neurons stimulated with bath application of high KCl or glutamate. While both stimulation methods yielded Zn²⁺ signals, Ca²⁺ influx, and acidification, glutamate stimulation induced more sustained high intracellular Ca²⁺ and a larger increase in intracellular Zn²⁺. However, the stimulation-induced pH change was similar between conditions, indicating that a different cellular change is responsible for the stimulation-dependent difference in Zn²⁺ signal. This work provides the first robust quantification of Zn²⁺ dynamics in neurons using different methods of stimulation.

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Single cell analysis reveals multiple requirements for zinc in the mammalian cell cycle

Anonymous — Fri, 21 Feb 2020 20:56:15 +0000

Single cell analysis reveals multiple requirements for zinc in the mammalian cell cycle Anonymous (not verified) Fri, 02/21/2020 - 13:56

Zinc is widely recognized as essential for growth and proliferation, yet the mechanisms of how zinc deficiency arrests these processes remain enigmatic. Here we induce subtle zinc perturbations and track asynchronously cycling cells throughout division using fluorescent reporters, high throughput microscopy, and quantitative analysis. Zinc deficiency induces quiescence and resupply stimulates synchronized cell-cycle reentry. Monitoring cells before and after zinc deprivation we found the position of cells within the cell cycle determined whether they either went quiescent or entered another cell cycle but stalled in S-phase. Stalled cells exhibited prolonged S-phase, were defective in DNA synthesis and had increased DNA damage levels, suggesting a role for zinc in maintaining genome integrity. Finally, we demonstrate zinc deficiency-induced quiescence occurs independently of DNA-damage response pathways, and is distinct from mitogen removal and spontaneous quiescence. This suggests a novel pathway to quiescence and reveals essential micronutrients play a role in cell cycle regulation.

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Remodeling of Zn2+ Homeostasis Upon Differentiation of Mammary Epithelial Cells

Anonymous — Thu, 16 Jan 2020 15:32:12 +0000

Remodeling of Zn2+ Homeostasis Upon Differentiation of Mammary Epithelial Cells Anonymous (not verified) Thu, 01/16/2020 - 08:32

Zinc is the second most abundant transition metal in humans and an essential nutrient required for growth and development of newborns. During lactation, mammary epithelial cells differentiate into a secretory phenotype, uptake zinc from blood circulation, and export it into mother’s milk. At the cellular level, many zinc-dependent cellular processes, such as transcription, metabolism of nutrients, and proliferation are involved in the differentiation of mammary epithelial cells. Using mouse mammary epithelial cells as a model system, we investigated the remodeling of zinc homeostasis during differentiation induced by treatment with the lactogenic hormones cortisol and prolactin. RNA-Seq at different stages of differentiation revealed changes in global gene expression, including genes encoding zinc-dependent proteins and regulators of zinc homeostasis. Increases in mRNA levels of three zinc homeostasis genes, Slc39a14 (ZIP14) and metallothioneins (MTs) I and II were induced by cortisol but not by prolactin. The cortisol-induced increase was partially mediated by the nuclear glucocorticoid receptor signaling pathway. An increase in the cytosolic labile Zn2+ pool was also detected in lactating mammary cells, consistent with upregulation of MTs. We found that the zinc transporter ZIP14 was important for the expression of a major milk protein, whey acid protein (WAP), as knockdown of ZIP14 dramatically decreased WAP mRNA levels. In summary, our study demonstrated remodeling of zinc homeostasis upon differentiation of mammary epithelial cells resulting in changes in cytosolic Zn2+ and differential expression of zinc homeostasis genes, and these changes are important for establishing the lactation phenotype.

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Directed evolution of excited state lifetime and brightness in FusionRed using a microfluidic sorter

Anonymous — Mon, 30 Dec 2019 20:37:42 +0000

Directed evolution of excited state lifetime and brightness in FusionRed using a microfluidic sorter Anonymous (not verified) Mon, 12/30/2019 - 13:37

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.

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Intramolecular Fluorescent Protein Association in a Class of Zinc FRET Sensors Leads to Increased Dynamic Range

Anonymous — Tue, 29 Oct 2019 17:44:12 +0000

Intramolecular Fluorescent Protein Association in a Class of Zinc FRET Sensors Leads to Increased Dynamic Range Anonymous (not verified) Tue, 10/29/2019 - 11:44

Genetically encoded Förster resonance energy transfer (FRET) sensors enable the visualization of ions, molecules, and processes in live cells. However, despite their widespread use, the molecular states that determine sensor performance are usually poorly understood, which limits efforts to improve them. We used dynamic light scattering (DLS) and time-resolved fluorescence anisotropy to uncover the sensing mechanism of ZifCV1.173, a Zn2+ FRET sensor. We found that the dynamic range (DR) of ZifCV1.173 was dominated by the high FRET efficiency of the Zn2+-free state, in which the donor and acceptor fluorescent proteins were closely associated. Mutating the donor–acceptor interface revealed that the DR of ZifCV1.173 could be increased or decreased by promoting or disrupting the donor–acceptor interaction, respectively. Adapting the same mutations to a related sensor showed the same pattern of DR tuning, supporting our sensing mechanism and suggesting that DLS and time-resolved fluorescence anisotropy might be generally useful in the biophysical characterization of other FRET sensors.

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Discovery of a ZIP7 inhibitor from a Notch pathway screen.

Anonymous — Tue, 29 Oct 2019 17:40:39 +0000

Discovery of a ZIP7 inhibitor from a Notch pathway screen. Anonymous (not verified) Tue, 10/29/2019 - 11:40

The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.

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Intracellular Zn2+ transients modulate global gene expression in dissociated rat hippocampal neurons

Anonymous — Tue, 29 Oct 2019 17:38:38 +0000

Intracellular Zn2+ transients modulate global gene expression in dissociated rat hippocampal neurons Anonymous (not verified) Tue, 10/29/2019 - 11:38

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.

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