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.
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 over-accumulated either due to 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 sub-cytotoxic Mn levels, we use a combination of confocal microscopy using a sensitive 'turn-on' fluorescent manganese sensor and nano-synchrotron Xray fluorescence imaging to show that manganese ions are also stored in the Golgi under physiological conditions. Our results along with previous reports on manganese accumulation now imply a central role of the Golgi apparatus in manganese storage and trafficking under subcytotoxic Mn levels.
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 Hg 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 Hg with a 35 times decrease in the emission intensity in the presence of 1 equiv of Hg ions. We have demonstrated the applicability of the probe for detecting Hg ions in living cells and in live zebrafish larvae using confocal fluorescence microscopy with visible excitation. High selectivity and sensitivity toward Hg detection make qHCM an attractive probe for detecting Hg 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. H NMR studies and DFT calculations indicate that Hg ions coordinate to oxygen-donor atoms from both the chromophore and macrocycle, leading to sensitive mercury detection.
Many viruses target host cell mitochondria to create a microenvironment conducive to viral dissemination. Dengue virus also exploits host cell mitochondria to facilitate its viral life cycle.
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