Biology subjects

Palmer, A. E.

Publications and source records attributed to Palmer, A. E..

13 recordsLinked to original sources

bioRxiv2026

Metal homeostasis is remodeled in response to different quiescence triggers

Cells can enter a reversible non-proliferative state called quiescence either spontaneously or in response to nutrient deprivations. Metal ions are essential nutrients and play wide-ranging regulatory and signaling roles in biological processes. We previously showed that zinc is an essential nutrient for the mammalian cell cycle as zinc deprivation drives cells into quiescence, and this quiescent state is associated with changes in iron, copper, and manganese, suggesting broad remodeling of metal homeostasis. Here we examine whether metal remodeling is a general feature of quiescence by inducing quiescence via different triggers (zinc deficiency, serum starvation, and growth factor withdrawal) in MCF10A cells. Fluorescence microscopy and elemental analysis reveal significant trigger-dependent changes in the labile and total metal pools of quiescent cells. To gain insight into these differences, we carried out RNA sequencing and differential expression analysis, focusing on metal associated, metal regulatory, and metal homeostasis genes. While core quiescence pathways are shared across triggers, quiescence states remain molecularly distinct. A significant percent of 2458 metal homeostasis annotated genes are differentially expressed including 55% in starvation-induced quiescence, 50% in zinc deficiency-induced quiescence and 21% in growth factor withdrawal-induced quiescence. Our results also showed unique alteration of genes involved in major metal dependent processes including antioxidant activity, oxidative phosphorylation, heme metabolism, and chromatin accessibility in different quiescence states. Overall, this work demonstrates that metal homeostasis is systematically rewired during cellular quiescence with associated effects on genes that regulate critical biological processes. Significance StatementCells constantly evaluate their nutrient and energy status and integrate these signals into proliferation-quiescence decisions. Quiescence prevents cells from passing damage to daughter cells. Metals are essential micronutrients for biological processes. While limitation of zinc can drive cells into quiescence and alter other metals, how metals are remodeled and whether this is a common feature of quiescence was unknown. Here we examined three quiescence triggers by modifying growth media and serum, the primary source of metals. We report significant changes in labile and total Cu, Fe, Zn and Mn pools, and metal associated genes in response to distinct quiescence triggers. These changes converge on mitochondrial function, cellular antioxidant activity, and heme biosynthesis in a trigger specific manner.

cell biology↗

bioRxiv2025

Systematic characterization of zinc in a series of breast cancer cell lines reveals significant changes in zinc homeostasis

An optimal amount of labile zinc (Zn2+) is essential for proliferation of human cells, where Zn2+ levels that are too high or too low cause cell cycle exit. Tumors of the breast have been characterized by high levels of total Zn2+. Given the role of Zn2+ in proliferation of human cells and elevation of zinc in breast cancer tumors, we examined the concentration of total and labile Zn2+ across a panel of 5 breast cancer cell lines, compared to the normal MCF10A cell line. We found that three cell lines (MDA-MB-231, MDA-MB-157, and SK-Br-3) showed elevated labile Zn2+ in the cytosol, while T-47D showed significantly lower Zn2+, and MCF7 showed no change compared to MCF10A cells. There was no change in total Zn2+ across the cell lines, as measured by ICP-MS, but we did observe a difference in the cells ability to accumulate Zn2+ when Zn2+ in the media was elevated. Therefore, we examined how proliferation of each cell line was affected by increases and decreases in the media. We found striking differences, where three cancer cell lines (MDA-MB-231, MDA-MB-157, and MCF7) showed robust proliferation in high Zn2+ at concentrations that killed MCF10A, T-47D, and SK-Br-3 cells. We also discovered that 4 of the 5 cancer cell lines demonstrate compromised proliferation and increased cell death in low Zn2+, suggesting these cells may be addicted to Zn2+. Overall, our study suggests significant differences in Zn2+ homeostasis and regulation in different types of breast cancer cells, with consequences for both proliferation and cell viability.

cell biology↗

bioRxiv2024

Structural basis for ring-opening fluorescence by the RhoBAST RNA aptamer.

Tagging RNAs with fluorogenic aptamers has enabled imaging of transcripts in living cells, thereby revealing novel aspects of RNA metabolism and dynamics. While a diverse set of fluorogenic aptamers has been developed, a new generation of aptamers are beginning to exploit the ring-opening of spirocyclic rhodamine dyes to achieve robust performance in live mammalian cells. These fluorophores have two chemical states: a colorless, cell-permeable spirocyclic state and a fluorescent zwitterionic state. Recently, the developed dye SpyRho555 almost exclusively adopts the closed state in solution and becomes fluorescent in complex with the RhoBAST aptamer. To understand the basis for RhoBAST-SpyRho555 fluorogenicity, we have determined crystal structures of RhoBAST in complex with 5-carboxytetramethylrhodamine and a SpyRho555 analogue, MaP555. RhoBAST is organized by a perfect four-way junction that positions two loops to form the dye-binding pocket. The core of the ligand resides between a tri-adenine floor and a single guanine base, largely driven by {pi}-stacking interactions. Importantly, the unpaired guanine interacts with the 3-position group of MaP555 to stabilize the open conformation, supported by mutagenesis data, and may play an active role in promoting the open conformation of the dye. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/630784v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1daaee9org.highwire.dtl.DTLVardef@11773corg.highwire.dtl.DTLVardef@ab41b9org.highwire.dtl.DTLVardef@167fae0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Unveiling the Promise of Peptide Nucleic Acids as Functional Linkers for Riboglow RNA Imaging Platform

Linkers in chemical biology provide more than just connectivity between molecules; their intrinsic properties can be harnessed to enhance the stability and functionality of chemical probes. In this study, we explored the incorporation of a peptide nucleic acid (PNA)-based linker into RNA-targeting probes to improve their affinity and specificity. By integrating a PNA linker into a small molecule probe of Riboglow platform, we enabled dual binding events: cobalamin (Cbl)-RNA structure-based recognition and sequence-specific PNA-RNA interaction. We show that incorporating a six-nucleotide PNA sequence complementary to the region of wild type RNA aptamer (env8) results in a 30-fold improvement in binding affinity compared to the probe with nonfunctional PEG linker. Even greater improvements are observed when the PNA probe was tested against truncated versions of the RNA aptamer, with affinity increasing by up to 280-fold. Additionally, the PNA linker is able to rescue Cbl-RNA interaction even when the cobalamin binding pocket is compromised. We demonstrated that PNA probes effectively bind RNA both in vitro and in live cells, enhancing visualization of RNA in stress granules and U-bodies at low concentrations. The modular nature of the Riboglow platform allows for flexible modifications of the PNA linker, fluorophore and RNA tag, while maintaining high specificity and affinity. This work establishes a new approach for enhancing RNA imaging platforms through the use of PNA linkers, highlighting the potential of combining short oligonucleotides with small molecules to improve the affinity and specificity of RNA-targeting probes. Furthermore, this dual-binding approach presents a promising strategy for driving advancements in RNA-targeted drug development. Table of Contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/616516v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@da5652org.highwire.dtl.DTLVardef@5923fborg.highwire.dtl.DTLVardef@494140org.highwire.dtl.DTLVardef@15afaf4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Transient Zn2+ deficiency induces replication stress and compromises daughter cell proliferation

Cells must replicate their genome quickly and accurately, and they require metabolites and cofactors to do so. Ionic zinc (Zn2+) is an essential micronutrient that is required for hundreds of cellular processes, including DNA synthesis and adequate proliferation. Deficiency in this micronutrient impairs DNA synthesis and inhibits proliferation, but the mechanism is unknown. Using fluorescent reporters to track single cells via long-term live-cell imaging, we find that Zn2+ is required at the G1/S transition and during S-phase for timely completion of S-phase. A short pulse of Zn2+ deficiency impairs DNA synthesis and increases markers of replication stress. These markers of replication stress are reversed upon resupply of Zn2+. Finally, we find that if Zn2+ is removed during the mother cells S-phase, daughter cells enter a transient quiescent state, maintained by sustained expression of p21, which disappears upon reentry into the cell cycle. In summary, short pulses of mild Zn2+ deficiency in S-phase specifically induce replication stress, which causes downstream proliferation impairments in daughter cells. SignificanceZinc is an essential micronutrient required for cells to grow and proliferate. However, the mechanism of how zinc influences proliferation is unknown. We show that short exposure to mild zinc deficiency in S-phase impairs DNA synthesis and induces replication stress, leading to pauses in daughter cell proliferation. However, pulses of low zinc during other phases of the cell cycle dont affect mother cell cycle progression or daughter cell proliferation. These results indicate that while zinc is important for many proteins, during the cell cycle short pulses of mild zinc deficiency have the biggest impact on a cells ability to synthesize DNA, suggesting that DNA polymerase complex acts as a gate keeper, sensing zinc status in the cell.

cell biology↗

bioRxiv2023

Cellular zinc status alters chromatin accessibility and binding of transcription factor TP53 to genomic sites

Zinc (Zn2+) is an essential metal required by approximately 2500 proteins. Nearly half of these proteins act on DNA, including > 850 human transcription factors, polymerases, DNA damage response factors, and proteins involved in chromatin architecture. How these proteins acquire their essential Zn2+ cofactor and whether they are sensitive to changes in the labile Zn2+ pool in cells remain open questions. Here, we examine how changes in the labile Zn2+ pool affect chromatin accessibility and transcription factor binding to DNA. We observed both increases and decreases in accessibility in different chromatin regions via ATAC-seq upon treating MCF10A cells with elevated Zn2+ or the Zn2+-specific chelator tris(2-pyridylmethyl)amine (TPA). Transcription factor enrichment analysis was used to correlate changes in chromatin accessibility with transcription factor motifs, revealing 477 transcription factor motifs that were differentially enriched upon Zn2+ perturbation. 186 of these transcription factor motifs were enriched in Zn2+ and depleted in TPA, and the majority correspond to Zn2+ finger transcription factors. We selected TP53 as a candidate to examine how changes in motif enrichment correlate with changes in transcription factor occupancy by ChIP-qPCR. Using publicly available ChIP-seq and nascent transcription datasets, we narrowed the 50,000+ ATAC-seq peaks to 2164 TP53 targets and subsequently selected 6 high-probability TP53 binding sites for testing. ChIP-qPCR revealed that for 5 of the 6 targets, TP53 binding correlates with the local accessibility determined by ATAC-seq. These results demonstrate that changes in labile zinc directly alter chromatin accessibility and transcription factor binding to DNA.

genomics↗

bioRxiv2022

Targeting riboswitches with beta-axial substituted cobalamins

RNA-targeting small molecule therapeutics an emerging field hindered by an incomplete understanding of the basic principles governing RNA-ligand interactions. One way to advance our knowledge in this area is to study model systems where these interactions are better understood, such as riboswitches. Riboswitches bind a wide array of small molecules with high affinity and selectivity, providing a wealth of information on how RNA recognizes ligands through diverse structures. The cobalamin-sensing riboswitch is a particularly useful model system as similar sequences show highly specialized binding preferences for different biological forms of cobalamin. This riboswitch is also widely dispersed across bacteria and therefore holds strong potential as an antibiotic target. Many synthetic cobalamin forms have been developed for various purposes including therapeutics, but their interaction with cobalamin riboswitches is yet to be explored. In this study, we characterize the interactions of eleven cobalamin derivatives with three representative cobalamin riboswitches using in vitro binding experiments (both chemical footprinting and a fluorescence-based assay) and a cell-based reporter assay. The derivatives show productive interactions with two of the three riboswitches, demonstrating simultaneously plasticity and selectivity within these RNAs. The observed plasticity is partially achieved through a novel structural rearrangement within the ligand binding pocket, providing insight into how similar RNA structures can be targeted in the future. As the derivatives also show in vivo functionality, they serve as several potential lead compounds for further drug development.

biochemistry↗

bioRxiv2022

Single molecule microscopy to profile the effect of zinc status of transcription factor dynamics

Transcription factors (TFs) are DNA binding proteins that control the expression of genes. The regulation of transcription is a complex process that involves binding of TFs to specific sequences, recruitment of cofactors and chromatin remodelers, assembly of the pre-initiation complex and ultimately the recruitment of RNA polymerase II. Increasing evidence suggests that TFs are highly dynamic and interact only transiently with DNA. Single molecule microscopy techniques are powerful approaches for visualizing and tracking individual TF molecules as they diffuse in the nucleus and interact with DNA. In this work, we employ multifocus microscopy and highly inclined and laminated optical sheet microscopy to track TF dynamics in response to perturbations in labile zinc inside cells. We sought to define whether zinc-dependent TFs sense changes in the labile zinc pool by determining whether their dynamics and DNA binding can be modulated by zinc. While it is widely appreciated that TFs need zinc to bind DNA, whether zinc occupancy and hence TF function are sensitive to changes in cellular zinc remain open questions. We utilized fluorescently tagged versions of the glucocorticoid receptor (GR), with two C4 zinc finger domains, and CCCTC-binding factor (CTCF), with eleven C2H2 zinc finger domains. We found that the biophysical dynamics of both TFs are susceptible to changes in zinc, but in subtly different ways. These results indicate that at least some transcription factors are sensitive to zinc dynamics, revealing a potential new layer of transcriptional regulation.

biophysics↗

bioRxiv2020

Zn2+ influx activates ERK and Akt signaling pathways through a common mechanism

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 non-specifically 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. Significance StatementWhile zinc (Zn2+) is a vital ion for cell function and human health, little is known about the role it plays in regulating cell signaling. Here, we use fluorescent tools to study the interaction between Zn2+ and cell signaling pathways that play a role in cell growth and proliferation. Importantly, we use small, non-toxic Zn2+concentrations to ensure that our Zn2+ changes are closer to what cells would experience in the body and not stress-inducing. We also demonstrate that these signaling changes are driven by Ras activation, which contradicts one of the major hypotheses in the field. Our sensors shed light on how cells respond to a very important micronutrient in real time.

cell biology↗

bioRxiv2020

Structure-guided point mutations on FusionRed produce a brighter red fluorescent protein

The development of fluorescent proteins (FPs) has revolutionized biological imaging. FusionRed, a monomeric red FP (RFP), is known for its low cytotoxicity and appropriate localization of target fusion proteins in mammalian cells but is limited in application by low fluorescence brightness. We report a brighter variant of FusionRed, FusionRed-MQV, which exhibits an extended fluorescence lifetime (2.8 ns), enhanced quantum yield (0.53), higher extinction coefficient (~140,000 M-1cm-1), increased radiative rate constant and reduced non-radiative rate constant with respect to its precursor. The properties of FusionRed-MQV derive from three mutations - M42Q, C159V and the previously identified L175M. A structure-guided approach was used to identify and mutate candidate residues around the phenol and the acylimine ends of the chromophore. The C159V mutation was identified via lifetime-based flow cytometry screening of a library in which multiple residues adjacent to the phenol end of the chromophore were mutated. The M42Q mutation is located near the acylimine end of the chromophore and was discovered using site-directed mutagenesis guided by x-ray crystal structures. FusionRed-MQV exhibits 3.4-fold higher molecular brightness and a 5-fold increase in the cellular brightness in HeLa cells (based on FACS) compared to FusionRed. It also retains the low cytotoxicity and high-fidelity localization of FusionRed, as demonstrated through assays in mammalian cells.

biophysics↗

bioRxiv2020

Dissociated hippocampal neurons exhibit distinct Zn2+ dynamics in a stimulation method-dependent manner

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.

neuroscience↗

bioRxiv2019

Single Cell Analysis Reveals Multiple Requirements for Zinc in the Mammalian Cell Cycle

Despite recognition of the fundamental role of zinc (Zn2+) for growth and proliferation, mechanisms of how Zn2+ deficiency arrests these processes remain enigmatic. We induced subtle intracellular Zn2+ perturbations and tracked asynchronously cycling cells throughout division using fluorescent reporters, high throughput microscopy, and quantitative analysis. We found that Zn2+ deficiency induces quiescence and Zn2+ resupply stimulates cell-cycle reentry. By monitoring single cells after Zn2+ deprivation, we found that depending on where cells were in the cell cycle, they either went quiescent or entered the cell cycle but stalled in S phase. Stalled cells were defective in DNA synthesis and had increased DNA damage levels, suggesting a role for Zn2+ in maintaining genome integrity. Finally, we found that Zn2+ deficiency-induced quiescence does not require the cell-cycle inhibitor p21. Overall, our study provides new insights into when Zn2+ is required during the mammalian cell cycle and the consequences Zn2+ deficiency.

cell biology↗

bioRxiv2017

Development of a riboswitch-based platform for live cell imaging of RNAs in mammalian cells

RNAs directly regulate a vast array of critical cellular processes, emphasizing the need for robust approaches to fluorescently tag and track RNAs in living cells. Here, we develop an RNA imaging platform using the cobalamin riboswitch as an RNA tag and a series of probes containing cobalamin as a fluorescence quencher. This highly modular Riboglow platform leverages different color fluorescent dyes, linkers and riboswitch RNA tags to elicit fluorescent turn-on upon binding RNA. We demonstrate the ability of two different Riboglow probes to track mRNA and small non-coding U RNA in live mammalian cells. A direct side-by-side comparison revealed that Riboglow outperformed the dye binding aptamer Broccoli and performed on par with the current gold standard RNA imaging system, the MS2-fluorescent protein system, while featuring a much smaller RNA tag. Together, the versatility of the Riboglow platform and ability to track diverse RNAs suggest broad applicability for a variety of imaging approaches.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC=\"FIGDIR/small/199240_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (29K):\norg.highwire.dtl.DTLVardef@11b5e3borg.highwire.dtl.DTLVardef@1c78fc0org.highwire.dtl.DTLVardef@aeba74org.highwire.dtl.DTLVardef@1531e3a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗