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

Abstract

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.

Cytoplasmic aggregation of uranium in human dopaminergic cells after continuous exposure to soluble uranyl at non-cytotoxic concentrations

“…Complete cell medium containing 36 µg.mL -1 of fluorescently labeled Alexa Fluor® 488 fetuin-A was used to expose cells for 1.5 h before the live cell images acquisition. All cells, either labeled for organelles or for fetuin-A, were additionally labeled with a blue fluorescent nuclear dye, Hoechst 33342, just prior to fluorescent live imaging acquisition at 5-10 µg.mL -1 for 10 minutes at 37°C (Roudeau et al, 2014;Carmona et al, 2019;Das et al, 2019). Live cell imaging of the fluorescent labels was recorded with an epifluorescence microscope (Olympus BX51).…”

“…The protocol for sample preparation was adapted from optimized methods for element imaging in single cells using SXRF and micro-PIXE (Perrin et al, 2015;Carmona et al, 2019;Das et al, 2019). Immediately after live cell microscopy, cells were rinsed with a 150 mM ammonium acetate buffer solution, pH 7.4, prepared in ultrapure water (Fisher) to remove traces of extracellular elements.…”

“…1). The stability of Hoechst 33342 nuclear dye fluorescence after freeze drying allows the localization of the nuclear areas on freeze-dried cells (Roudeau et al, 2014;Das et al, 2019). Finally, samples were stored into a desiccator box until SXRF analysis.…”

“…We applied Synchrotron X-ray Fluorescence (SXRF) imaging, a technique with high spatial resolution (300 nm at Nanoscopium beamline, SOLEIL synchrotron) and high analytical element sensitivity (< 1 µg.g -1 per 300 nm pixel). We performed a cellular correlative microscopy approach by coupling SXRF imaging to epifluorescence microscopy for the identification of organelles and/or proteins fluorescently labeled (Roudeau et al, 2014;Carmona et al, 2019;Das et al, 2019). Previous studies using transmission electron microscopy (TEM) indicated that after acute exposure to high uranium concentrations (> 300 µM) of alveolar macrophages, renal, or bone cells, uranium precipitates forming large needle-shape structures within cytoplasmic vesicles such as lysosomes (Hengé-Napoli et al, 1996;Mirto et al, 1999;Carrière et al, 2008;Pierrefite-Carle et al, 2017).…”

Manganese matters: feeding manganese into the secretory system for cell wall synthesis

“…This model is inferred from genetic and biochemical evidence. However, it would need to be substantiated by direct detection of Mn in organelles using Mn specific fluorescent sensors or high‐resolution X‐ray fluorescence (XRF) microscopy (Das et al ., 2019). These approaches may allow the visualization of a Mn concentration gradient through the secretory pathway and how it is spatially modulated by the activities of PML3, MTP11, ECA3 and NRAMP2.…”