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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Riboglow improves live cell RNA imaging

Anonymous — Wed, 26 Sep 2018 06:00:00 +0000

Riboglow improves live cell RNA imaging Anonymous (not verified) Wed, 09/26/2018 - 00:00

Jessica Miller

In a multidisciplinary study recently published in Nature Chemical Biology, researchers at the University of Colorado Boulder have developed a novel tool for visualizing RNA. This project centered on a collaboration between the Palmer Lab, with expertise in live cell imaging, the Batey Lab, with expertise in RNA, and the Gryko Lab with expertise in chemical synthesis. Researchers from the Parker and Jimenez labs also contributed to the study.

RNA, or ribonucleic acid, is a macromolecule essential to all forms of life. RNA plays a key role in gene expression and regulation, catalyzes the formation of polypeptides, and facilitates the transformation of genetic information from DNA to protein. Considering the many functions of this diverse molecule, visualizing RNA is essential to understanding a wide array of cellular processes.

Visualization of U-bodies in live mammalian cells

“As the community continues to discover new functions for coding and non-coding RNAs, the desire to look at them over time in live cells can provide unique functional insights“, commented Esther Braselmann, the lead author on this study and member of the Palmer Lab at the BioFrontiers Institute. Dr. Braselmann recently won a prestigious NIH K99 award, which helps outstanding postdoctoral researchers transition to tenure-track positions.

Established techniques to fluorescently tag and track RNA have several limitations. These tags are not compatible with all types of RNA, perform poorly in live cell studies, and can interfere with normal RNA activity due to their large size. The authors of this study sought to create a versatile imaging platform applicable to real-time experiments in live cells.

The methodology presented in this study relies on a Cobalamin-fluorophore probe which fluoresces upon binding to riboswitch RNA. This system is highly adaptable, allowing researchers to target diverse types of RNA and customize the probe with fluorophores of different colors.

The authors employed this Riboglow technology to visualize mRNA dynamics in live mammalian cells. They were able to record mRNA localization to stress granules, and visualize U1 snRNA in live cells for the first time. When compared to other imaging techniques, Riboglow was less susceptible to photobleaching and demonstrated a robust fluorescent signal.

“We view Riboglow as a complementary platform to existing tools and an addition to the growing toolbox for labeling RNAs,” remarked Dr. Braselmann. The versatility of the Riboglow platform will allow for widespread application to continue illuminating the many roles of RNA in live cells.

This research was supported by the Human Frontiers Science Project, the National Institute of Health, and the National Science Centre.

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Fluorescent tag allows for real-time imaging of bacterial infection

Anonymous — Wed, 09 May 2018 19:19:34 +0000

Fluorescent tag allows for real-time imaging of bacterial infection Anonymous (not verified) Wed, 05/09/2018 - 13:19

Researchers at the BioFrontiers Institute have developed a fluorescent-based approach to study virulence proteins secreted by the Listeria monocytogenes pathogen. The work was performed by the lab of Dr. Amy Palmer and recently published in Biophysical Journal.

Listeria monocytogenes is a bacterium which causes the foodborne illness listeriosis in approximately 1,600 people per year. Listeria can cross the placental, intestinal, and blood-brain barrier and enter several types of mammalian cells. This makes listeriosis particularly dangerous for pregnant women and individuals with compromised immune systems.

Listeria produces and secretes a series of virulence proteins during infection allowing the pathogen to evade the immune response and spread between cells. “We would like to better understand the mechanism of how Listeria infects human cells and how infection alters biological processes within cells,” said Amy Palmer, an associate professor at the BioFrontiers Institute and senior author of the study.

To study these secreted proteins, researchers in the Palmer Lab leveraged the split-green fluorescent protein (GFP) system. A truncated strand of GFP is genetically fused to the bacterial protein of interest and the remainder of GFP is produced by mammalian cells. The individual components are nonfluorescent, but when infection occurs, the two components fuse and produce green fluorescence. This allows for visualization and tracking of bacterial proteins in real time.

Through this mechanism, the researchers showed accumulation of the Listeria protein Internalin C in host cells. Cells began to fluoresce six hours post infection, and fluorescence continued to increase for several hours. This research demonstrates how the split-fluorescent tagging strategy can be used to quantify protein localization and accumulation throughout infection and indicate differences in how pathogens infect varying cell types.

“This platform is robust and represents a versatile tool to gain insights in Listeria infection biology,” the authors conclude. In the future, this technique will allow for multicolor imaging, in which multiple host and/or pathogen components can be tagged simultaneously.

Pascale Cossart of the Institut Pasteur was a co-author on this study.

This research was supported by the Human Frontiers Science Project and the National Science Foundation.

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Amy Palmer wins NIH Pioneer Award

Anonymous — Thu, 09 Oct 2014 06:00:00 +0000

Amy Palmer wins NIH Pioneer Award Anonymous (not verified) Thu, 10/09/2014 - 00:00

BioFrontiers

Few people think of metals as being vital to our health. Although most people are aware of iron, zinc is just as important, and is involved in a much wider array of biological functions. Ten percent of the proteins used to build our cells, tissues and genes are predicted to bind with zinc. As humans grew in evolutionary complexity, we adopted zinc as a main ingredient to power the creation of our genome. This metal is involved in the susceptibility to illnesses and infections. A lack of it can cause life-threatening diarrhea, a decrease in the ability to heal wounds and delayed growth and maturation in children.

“As a graduate student, I studied copper, which is also an essential metal that plays important roles in biology,” says Associate Professor of Chemistry and Biochemistry and BioFrontiers faculty member, Amy Palmer. “After working with copper, I started reading about zinc in the brain. As far back as the 1950s, doctors studied zinc-rich areas of the brain, and nobody was sure what it was doing there. Metal ions, like zinc, play such an important role in biology. They are essential…we can’t live without them, but the misbalance of metals is central in many diseases.”

Palmer was recently awarded a Pioneer Award from the National Institutes of Health (NIH), which are given to scientists proposing highly innovative approaches to major contemporary challenges in biomedical research. The Pioneer Award, now in its eleventh year, challenges investigators at all career levels to develop groundbreaking approaches that could have an efficacious impact on a broad area of biomedical or behavioral science. The award will span five years and provide a total of $3.7 million dollars in research funding for Palmer’s work.

“It’s a really enabling award,” says Palmer. “It is intentionally designed to allow you to take your research program in a new and different direction. It lets you do pioneering work that is unlikely to be funded by other research grants. It’s for high-risk, high-reward science, and it will allow us to start an entirely new program.”

Zinc availability is highly dynamic and Palmer is hoping to find out how it functions at a basic level – in the cell. She is investigating the prospect that zinc may be a global regulator of protein function, which may help to explain why zinc is involved in so many cellular processes. To accomplish this she’ll develop new technology to map the zinc proteome and define how changes in zinc affect gene expression and cellular metabolism. She hopes to gain an understanding of how zinc changes as certain diseases progress, which may result in biomarkers that could identify illnesses early on in their development. Learning how zinc interacts in the cell may change how we think about cellular regulation and how nutrition affects cells.

Despite the widespread affects of zinc deficiency among humans, it is currently difficult and expensive to test for it in the doctor’s office. The World Health Organization estimates that 30 percent of humans are currently zinc deficient, and as many as 800,000 children die every year because of zinc deficiencies. In fact, zinc supplementation is considered to be as important as providing clean water for the prevention of human death in developing countries. Palmer’s research into this important metal may bring greater understanding as to how it is used by our bodies, and what it can tell us about our health.

"Supporting innovative investigators with the potential to transform scientific fields is a critical element of our mission,”’ said NIH Director Francis S. Collins, M.D., Ph.D. "This program allows researchers to propose highly creative research projects across a broad range of biomedical and behavioral research areas that involve inherent risk but have the potential to lead to dramatic breakthroughs."

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