Cell-Permeable Fluorescent Sensors Enable Rapid Live Cell Visualization of Plasma Membrane and Nuclear PIP3 Pools.

Phosphoinositides, phospholipids that are key cell-signal mediators, are present at very low levels in cellular membranes and within nuclei. Phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), a phosphoinositide barely present in resting cell membranes, is produced when cells receive either growth, proliferation, or movement signals. Aberrant PIP3 levels are associated with the formation of cancers. PIP3 pools are also present in the nucleus, specifically in the nucleolus. However, questions related to the organization and function of this lipid in such membraneless intranuclear structures remain unanswered. Therefore, chemical sensors for tracking cellular PIP3 are invaluable not only for timing signal initiation in membranes but also for identifying the organization and function of membraneless nuclear PIP3 pools. Because PIP3 is present in the inner leaflet of cell membranes and in the nucleus, cell-permeable, rapid-response fluorescent sensors would be ideal. We have designed two peptide-based, water-soluble, cell-permeable, ratiometric PIP3 sensors named as MFR-K17H and DAN-NG-H12G. MFR-K17H rapidly entered into the cell cytoplasm, distinctly reporting rapid (<1 min) time scales of growth factor-stimulated PIP3 generation and depletion within cell membranes in living cells. Importantly, MFR-K17H lighted up inherently high levels of PIP3 in triple-negative breast cancer cell membranes, implying future applications in the detection of enhanced PIP3 levels in cancerous cells. On the other hand, DAN-NG-H12G targeted intranuclear PIP3 pools, revealing that within membraneless structures, PIP3 resided in a hydrophobic environment. Together, both probes form a unique orthogonally targeted combination of cell-permeable, ratiometric probes that, unlike previous cell-impermeable protein-based sensors, are easy to apply and provide an unprecedented handle into PIP3-mediated cellular processes.

Correction: An activity-based fluorescent sensor with a penta-coordinate N-donor binding site detects Cu ions in living systems.

Correction for 'An activity-based fluorescent sensor with a penta-coordinate N-donor binding site detects Cu ions in living systems' by Kunika Gupta et al., Chem. Commun., 2023, 59, 8282-8285, https://doi.org/10.1039/D3CC02201C.

An activity-based fluorescent sensor with a penta-coordinate N-donor binding site detects Cu ions in living systems.

An activity-based sensor afforded a 63 times fluorescence-enhancement with Cu2+/Cu+ ions and could image Cu2+/Cu+ in living cells and in a multicellular organism. The sensor functioned only in the presence of ambient dioxygen and glutathione, and the characterization of intermediates and products hinted toward a sensing mechanism involving a CuII hydroperoxo species.

A Peptide-Based Fluorescent Sensor for Anionic Phospholipids.

Anionic phospholipids are key cell signal mediators. The distribution of these lipids on the cell membrane and intracellular organelle membranes guides the recruitment of signaling proteins leading to the regulation of cellular processes. Hence, fluorescent sensors that can detect anionic phospholipids within living cells can provide a handle into revealing molecular mechanisms underlying lipid-mediated signal regulation. A major challenge in the detection of anionic phospholipids is related to the presence of these phospholipids mostly in the inner leaflet of the plasma membrane and in the membranes of intracellular organelles. Hence, cell-permeable sensors would provide an advantage by enabling the rapid detection and tracking of intracellular pools of anionic phospholipids. We have developed a peptide-based, cell-permeable, water-soluble, and ratiometric fluorescent sensor that entered cells within 15 min of incubation via the endosomal machinery and showed punctate labeling in the cytoplasm. The probe could also be introduced into living cells via lipofection, which allows bypassing of endosomal uptake, to image anionic phospholipids in the cell membrane. We validated the ability of the sensor toward detection of intracellular anionic phospholipids by colocalization studies with a fluorescently tagged lipid and a protein-based anionic phospholipid sensor. Further, the sensor could image the externalization of anionic phospholipids during programmed cell death, indicating the ability of the probe toward detection of both intra- and extracellular anionic phospholipids based on the biological context.

Spatio-Temporal Autophagy Tracking with a Cell-Permeable, Water-Soluble, Peptide-Based, Autophagic Vesicle-Targeted Sensor.

Autophagy is an essential cellular degradation process. Impaired autophagy has been linked to multiple disorders, including cancer and neurodegeneration. Tracking the autophagic flux in living cells will provide mechanistic insights into autophagy and will allow rapid screening of autophagy modulators as potential therapeutics. Imaging autophagy to track the autophagic flux demands a cell-permeable probe that can specifically target autophagic vesicles and report on the extent of autophagy. Existing fluorescent protein-based probes for imaging autophagy target autophagic vesicles but are cell-impermeable and degrade with the progress of autophagy resulting in ambiguous information on the later stages of autophagy. Although small-molecule-based autophagy probes can be cell-permeable, they are mostly water-insoluble and often target lysosomes instead of autophagic vesicles leading to incomplete evidence of the early stages of the process. Hence, there is a major gap in the ability to link the imaging data obtained by applying fluorescent sensors to the real extent of autophagy in living cells. To address these challenges, we have combined the desirable features of targetability and cell permeability to develop a novel water-soluble, cell-permeable, visible-light excitable, peptide-based, fluorescent sensor, HCFP, for imaging autophagy and tracking the autophagic flux. The probe readily enters living cells within 30 min of incubation, distinctly targets autophagic vesicles, and spatio-temporally tracks the entire autophagy pathway in living cells via a ratiometric pH-sensitive detection scheme. The salient features of the probe combining targetability with cell permeability should provide an edge in high-throughput screening of autophagy modulators by tracking autophagy live.

Manganese co-localizes with calcium and phosphorus in Chlamydomonas acidocalcisomes and is mobilized in manganese-deficient conditions.

Exposing cells to excess metal concentrations well beyond the cellular quota is a powerful tool for understanding the molecular mechanisms of metal homeostasis. Such improved understanding may enable bioengineering of organisms with improved nutrition and bioremediation capacity. We report here that Chlamydomonas reinhardtii can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) content, indicated by 1) a consistent failure of C. reinhardtii vtc1 mutant strains, which are deficient in polyphosphate synthesis, to accumulate Mn and 2) a drastic reduction of the Mn storage capacity in P-deficient cells. Rather surprisingly, X-ray absorption spectroscopy, EPR, and electron nuclear double resonance revealed that only little Mn2+ is stably complexed with polyP, indicating that polyP is not the final Mn ligand. We propose that polyPs are a critical component of Mn accumulation in Chlamydomonas by driving Mn relocation from the cytosol to acidocalcisomes. Within these structures, polyP may, in turn, escort vacuolar Mn to a number of storage ligands, including phosphate and phytate, and other, yet unidentified, compounds.

Manganese Mapping Using a Fluorescent Mn2+ Sensor and Nanosynchrotron X-ray Fluorescence Reveals the Role of the Golgi Apparatus as a Manganese Storage Site.

Elucidating dynamics in transition-metal distribution and localization under physiological and pathophysiological conditions is central to our understanding of metal-ion regulation. In this Forum Article, we focus on manganese and specifically recent developments that point to the relevance of the Golgi apparatus in manganese detoxification when this essential metal ion is overaccumulated because of either environmental exposure or mutations in manganese efflux transporters. In order to further evaluate the role of the Golgi apparatus as a manganese-ion storage compartment under subcytotoxic manganese levels, we use a combination of confocal microscopy using a sensitive "turn-on" fluorescent manganese sensor, M1, and nanosynchrotron X-ray fluorescence imaging to show that manganese ions are stored in the Golgi apparatus under micromolar manganese exposure concentrations. Our results, along with previous reports on manganese accumulation, now indicate a central role of the Golgi apparatus in manganese storage and trafficking under subcytotoxic manganese levels and hint toward a possible role of the Golgi apparatus in manganese storage even under physiological conditions.

Emerging chemical tools and techniques for tracking biological manganese.

Recent developments in Mn biology have added new physiological and pathophysiological roles of this essential metal ion to the already existing repertoire of indispensable biological roles of Mn ions. Notably, the discovery of Mn2+ specific transporters, maladies related to mutations in these transporters, and evidence of the role of labile Mn2+ species as anti-oxidants have initiated studies targeted at elucidating Mn ion regulation and pathways implicated in pathological conditions. Closely inter-linked with the quest for understanding metal ion homeostasis are basic questions like "How are metal ions installed in their correct biological addresses where they need to function?" and "Are dynamic changes in metal ion distribution functionally relevant?" These questions become more critical in the context of Mn2+ ions, which have inherently low binding affinities toward most ligands and hence would always face competing metal ions in the biological milieu. In the emerging context of functional roles of the labile Mn2+ ion pool, the development of chemical tools and techniques that can provide information on the location, distribution and dynamic changes in these parameters under physiological and pathophysiological conditions becomes imperative. In this frontier article, we discuss the challenges that had left Mn2+ ions lagging behind in the race for the development of chemical tools and recent approaches that addressed these challenges to develop tools and techniques that can illuminate Mn ions in living systems.

Cu2+ selective chelators relieve copper-induced oxidative stress in vivo.

Copper ions are essential for biological function yet are severely detrimental when present in excess. At the molecular level, copper ions catalyze the production of hydroxyl radicals that can irreversibly alter essential bio-molecules. Hence, selective copper chelators that can remove excess copper ions and alleviate oxidative stress will help assuage copper-overload diseases. However, most currently available chelators are non-specific leading to multiple undesirable side-effects. The challenge is to build chelators that can bind to copper ions with high affinity but leave the levels of essential metal ions unaltered. Here we report the design and development of redox-state selective Cu ion chelators that have 108 times higher conditional stability constants toward Cu2+ compared to both Cu+ and other biologically relevant metal ions. This unique selectivity allows the specific removal of Cu2+ ions that would be available only under pathophysiological metal overload and oxidative stress conditions and provides access to effective removal of the aberrant redox-cycling Cu ion pool without affecting the essential non-redox cycling Cu+ labile pool. We have shown that the chelators provide distinct protection against copper-induced oxidative stress in vitro and in live cells via selective Cu2+ ion chelation. Notably, the chelators afford significant reduction in Cu-induced oxidative damage in Atp7a-/- Menkes disease model cells that have endogenously high levels of Cu ions. Finally, in vivo testing of our chelators in a live zebrafish larval model demonstrate their protective properties against copper-induced oxidative stress.

Manganese is a Deinococcus radiodurans growth limiting factor in rich culture medium.

To understand the effects triggered by Mn2+ on Deinococcus radiodurans, the proteome patterns associated with different growth phases were investigated. In particular, under physiological conditions we tested the growth rate and the biomass yield of D. radiodurans cultured in rich medium supplemented or not with MnCl2. The addition of 2.5-5.0 µM MnCl2 to the medium neither altered the growth rate nor the lag phase, but significantly increased the biomass yield. When higher MnCl2 concentrations were used (10-250 µM), biomass was again found to be positively affected, although we did observe a concentration-dependent lag phase increase. The in vivo concentration of Mn2+ was determined in cells grown in rich medium supplemented or not with 5 µM MnCl2. By atomic absorption spectroscopy, we estimated 0.2 and 0.75 mM Mn2+ concentrations in cells grown in control and enriched medium, respectively. We qualitatively confirmed this observation using a fluorescent turn-on sensor designed to selectively detect Mn2+in vivo. Finally, we investigated the proteome composition of cells grown for 15 or 19 h in medium to which 5 µM MnCl2 was added, and we compared these proteomes with those of cells grown in the control medium. The presence of 5 µM MnCl2 in the culture medium was found to alter the pI of some proteins, suggesting that manganese affects post-translational modifications. Further, we observed that Mn2+ represses enzymes linked to nucleotide recycling, and triggers overexpression of proteases and enzymes linked to the metabolism of amino acids.

A Sensitive Water-Soluble Reversible Optical Probe for Hg2+ Detection.

We report the serendipitous discovery of an optical mercury sensor while trying to develop a water-soluble manganese probe. The sensor is based on a pentaaza macrocycle conjugated to a hemicyanine dye. The pentaaza macrocycle earlier designed in our group was used to develop photoinduced electron transfer (PET)-based "turn-on" fluorescent sensors for manganese. (1) In an attempt to increase the water-solubility of the manganese sensors we changed the dye from BODIPY to hemicyanine. The resultant molecule qHCM afforded a distinct reversible change in the absorption features and a concomitant visible color change upon binding to Hg2+ ions, leading to a highly water-soluble mercury sensor with a 10 ppb detection limit. The molecule acts as a reversible "ON-OFF" fluorescent sensor for Hg2+ with a 35 times decrease in the emission intensity in the presence of 1 equiv of Hg2+ ions. We have demonstrated the applicability of the probe for detecting Hg2+ ions in living cells and in live zebrafish larvae using confocal fluorescence microscopy with visible excitation. High selectivity and sensitivity toward Hg2+ detection make qHCM an attractive probe for detecting Hg2+ in contaminated water sources, which is a major environmental toxicity concern. We have scrutinized the altered metal-ion selectivity of the probe using density functional theory (DFT) and time-dependent DFT calculations, which show that a PET-based metal-sensing scheme is not operational in qHCM. 1H NMR studies and DFT calculations indicate that Hg2+ ions coordinate to oxygen-donor atoms from both the chromophore and macrocycle, leading to sensitive mercury detection.

Optically sensing phospholipid induced coil-helix transitions in the phosphoinositide-binding motif of gelsolin.

We present a systematic experimental and computational study of phospholipid induced peptide coil-helix transitions which are relevant in the context of proteins mediating cytoskeletal rearrangement via membrane binding. We developed a sensitive Förster resonance energy transfer (FRET) based assay to address whether coil-helix transitions in phospholipid binding motifs of actin-binding proteins can be induced by physiologically-relevant concentrations (1-20 µM) of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) phospholipids. Based on inter-residue distance constraints obtained from Molecular Dynamics (MD) simulations of a 20 residue peptide (Gel 150-169) from the actin-severing protein gelsolin, we synthetized and labeled the peptide with a tryptophan donor and IAEDANS acceptor pair. Upon addition of PI(4,5)P2 micelles and mixed vesicles containing PI(4,5)P2 and phosphatidylcholine to the peptide, we observed a decrease in the tryptophan emission intensity with increasing concentrations of PI(4,5)P2. The IAEDANS emission spectra showed a more complex profile exhibiting a blue shift of the emission peak and non-monotonic changes in the intensity profile with increasing concentrations of PI(4,5)P2. We showed that the IAEDANS acceptor emission response is a result of both intrinsic polarity sensitivity of the acceptor in the vicinity of the membrane surface and fluorescence energy transfer from the donor. Importantly, the fluorescence lifetime of the donor (tryptophan) showed a monotonous decrease with increasing mol% of PI(4,5)P2 from 1.13 ± 0.10 ns in the absence of phospholipids to 0.25 ± 0.03 ns in the presence of 100% PI(4,5)P2 micelles. We also showed a concomitant increase in FRET efficiency with increasing PI(4,5)P2 levels indicating a PI(4,5)P2 concentration dependent coil-helix transition. Our studies demonstrate that membrane PI(4,5)P2 concentrations as low as 2.5-5 µM can trigger helix-coil conformational changes in gelsolin relevant for triggering regulatory processes in the cell.

Cell Permeable Ratiometric Fluorescent Sensors for Imaging Phosphoinositides.

Phosphoinositides are critical cell-signal mediators present on the plasma membrane. The dynamic change of phosphoinositide concentrations on the membrane including clustering and declustering mediates signal transduction. The importance of phosphoinositides is scored by the fact that they participate in almost all cell-signaling events, and a defect in phosphoinositide metabolism is linked to multiple diseases including cancer, bipolar disorder, and type-2 diabetes. Optical sensors for visualizing phosphoinositide distribution can provide information on phosphoinositide dynamics. This exercise will ultimately afford a handle into understanding and manipulating cell-signaling processes. The major requirement in phosphoinositide sensor development is a selective, cell permeable probe that can quantify phosphoinositides. To address this requirement, we have developed short peptide-based ratiometric fluorescent sensors for imaging phosphoinositides. The sensors afford a selective response toward two crucial signaling phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol-4-phosphate (PI4P), over other anionic membrane phospholipids and soluble inositol phosphates. Dissociation constant values indicate up to 4 times higher probe affinity toward PI(4,5)P2 when compared to PI4P. Significantly, the sensors are readily cell-permeable and enter cells within 15 min of incubation as indicated by multiphoton excitation confocal microscopy. Furthermore, the sensors light up signaling phosphoinositides present both on the cell membrane and on organelle membranes near the perinuclear space, opening avenues for quantifying and monitoring phosphoinositide signaling.