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Summary: In the finely regulated process of mammalian erythropoiesis, the path of the labile iron pool into mitochondria for heme production is not well understood. Existing models for erythropoiesis do not include a central role for the ubiquitous iron storage protein ferritin; one model proposes that incoming endosomal Fe3+ bound to transferrin enters the cytoplasm through an ion transporter after reduction to Fe2+ and is taken up into mitochondria through mitoferrin-1 transporter. Here, we apply a dual three-dimensional imaging and spectroscopic technique, based on scanned electron probes, to measure Fe3+ in ex vivo human hematopoietic stem cells. After seven days in culture, we observe cells displaying a highly specialized architecture with anchored clustering of mitochondria and massive accumulation of nanoparticles containing high iron concentrations localized to lysosomal storage depots, identified as ferritin. We hypothesize that lysosomal ferritin iron depots enable continued heme production after expulsion of most of the cellular machinery.

Summary Imaging of cells and tissues has improved significantly over the last decade. Dual-beam instruments with a focused ion beam mounted on a scanning electron microscope (FIB-SEM), offering high-resolution 3D imaging of large volumes and fields-of-view are becoming widely used in the life sciences. FIB-SEM has most recently been implemented on fully hydrated, cryo-immobilized, biological samples. Correlative light and electron microscopy workflows combining fluorescence microscopy (FM) with FIB-SEM imaging exist, whereas workflows combining cryo-FM and cryo-FIB-SEM imaging are not yet commonly available. Here, we demonstrate that fluorescently labeled lipid droplets can serve as in situ fiducial markers for correlating cryo-FM and FIB-SEM datasets and that this approach can be used to target the acquisition of large FIB-SEM stacks spanning tens of microns under cryogenic conditions. We also show that cryo-FIB-SEM imaging is particularly informative for questions related to organelle structure and inter-organellar contacts, nuclear organization, and mineral deposits in cells., Graphical abstract, Highlights • 3D CLEM of cryo-FM and FIB-SEM datasets using fluorescently labeled lipid droplets • Cryo-FIB-SEM imaging of organelle-organelle interactions and nuclear organization, Cell biology; Technical aspects of cell biology; Structural biology

Summary In the finely regulated process of mammalian erythropoiesis, the path of the labile iron pool into mitochondria for heme production is not well understood. Existing models for erythropoiesis do not include a central role for the ubiquitous iron storage protein ferritin; one model proposes that incoming endosomal Fe3+ bound to transferrin enters the cytoplasm through an ion transporter after reduction to Fe2+ and is taken up into mitochondria through mitoferrin-1 transporter. Here, we apply a dual three-dimensional imaging and spectroscopic technique, based on scanned electron probes, to measure Fe3+ in ex vivo human hematopoietic stem cells. After seven days in culture, we observe cells displaying a highly specialized architecture with anchored clustering of mitochondria and massive accumulation of nanoparticles containing high iron concentrations localized to lysosomal storage depots, identified as ferritin. We hypothesize that lysosomal ferritin iron depots enable continued heme production after expulsion of most of the cellular machinery., Graphical abstract, Highlights • Erythroblasts maintain highly organized architecture as revealed by SB-SEM • STEM/EELS reveal ferritin-containing lysosomal vesicles surrounded by mitochondria • In erythropoiesis, ferritin is attributed to be an iron depot used in heme synthesis, Biological sciences; Biochemistry; Cell biology; Technical aspects of cell biology

Summary pH and Ca2+ play important roles in regulating lysosomal activity and lysosome-mediated physiological and pathological processes. However, effective methods for simultaneous determination of pH and Ca2+ is the bottleneck. Herein, a single DNA-based FLIM reporter was developed for real-time imaging and simultaneous quantification of pH and Ca2+ in lysosomes with high affinity, in which a specific probe for recognition of Ca2+ was assembled onto a DNA nanostructure together with pH-responsive and lysosome-targeted molecules. The developed DNA reporter showed excellent biocompatibility and long-term stability up to ∼56 h in lysosomes. Using this powerful tool, it was discovered that pH was closely related to Ca2+ concentration in lysosome, whereas autophagy can be regulated by lysosomal pH and Ca2+. Furthermore, Aβ-induced neuronal death resulted from autophagy abnormal through lysosomal pH and Ca2+ changes. In addition, lysosomal pH and Ca2+ were found to regulate the transformation of NSCs, resulting in Rapamycin-induced antiaging., Graphical Abstract, Highlights • A DNA-based FLIM reporter was developed for tracking lysosomal pH and Ca2+ • It was found that autophagy could be induced by lysosomal pH and Ca2+ • Aβ-induced neuronal death was due to pHly- and [Ca2+]ly-mediated autophagy abnormal • Antiaging-related transformation of qNSCs can be regulated by pHly and [Ca2+]ly, Optical Imaging; Cellular Neuroscience; Technical Aspects of Cell Biology

Summary A major unresolved challenge in cell-based regenerative medicine is the absence of non-invasive technologies for tracking cell fate in deep tissue and with high spatial resolution over an extended interval. MRI is highly suited for this task, but current methods fail to provide longitudinal monitoring or high sensitivity, or both. In this study, we fill this technological gap with the first discovery and demonstration of in vivo cellular production of endogenous bright contrast via an MRI genetic reporter system that forms manganese-ferritin nanoparticles. We demonstrate this technology in human embryonic kidney cells genetically modified to stably overexpress ferritin and show that, in the presence of manganese, these cells produce far greater contrast than conventional ferritin overexpression with iron or manganese-permeable cells. In living mice, diffusely implanted bright-ferritin cells produce the highest and most sustained contrast in skeletal muscle. The bright-ferritin platform has potential for on-demand, longitudinal, and sensitive cell tracking in vivo., Graphical Abstract, Highlights • First bright-ferritin MRI gene reporter platform for longitudinal cell tracking • In vivo assembly of manganese nanoparticles for bright MRI contrast • On-demand, sensitive, non-invasive in vivo deep imaging of proliferating cells, Medical Imaging; Technical Aspects of Cell Biology; Nanomaterials

Summary We present here PhotoGal4, a phytochrome B-based optogenetic switch for fine-tuned spatiotemporal control of gene expression in Drosophila explants. This switch integrates the light-dependent interaction between phytochrome B and PIF6 from plants with regulatory elements from the yeast Gal4/UAS system. We found that PhotoGal4 efficiently activates and deactivates gene expression upon red- or far-red-light irradiation, respectively. In addition, this optogenetic tool reacts to different illumination conditions, allowing for fine modulation of the light-dependent response. Importantly, by simply focusing a laser beam, PhotoGal4 induces intricate patterns of expression in a customized manner. For instance, we successfully sketched personalized patterns of GFP fluorescence such as emoji-like shapes or letterform logos in Drosophila explants, which illustrates the exquisite precision and versatility of this tool. Hence, we anticipate that PhotoGal4 will expand the powerful Drosophila toolbox and will provide a new avenue to investigate intricate and complex problems in biomedical research., Graphical Abstract, Highlights • PhotoGal4 activates gene expression in response to red light in Drosophila explants • Exposure to far-red light allows for PhotoGal4 deactivation • PhotoGal4 can activate gene expression in subgroups of cells within a single tissue, Optical Imaging; Genetics; Cell Biology; Technical Aspects of Cell Biology

Summary The immortalized cell is an essential research tool that uses robust growth properties for the functional investigation of gene products. Immortalized mammalian cells have mainly been established using three methods: expression of simian vacuolating virus 40 T antigen (the SV40 method); human papilloma virus-derived oncoprotein E6/E7 (the E6/E7 method); or combinatorial expression of R24C mutant cyclin-dependent kinase 4, cyclin D1, and telomerase reverse transcriptase (the K4DT method). However, it is unclear as to which method is optimal for an in vitro model. Here, we compared the biological characteristics and genome-wide expression profiles of immortalized human dermal papilla cells generated by the SV40, E6/E7, or K4DT method. To our knowledge, this is the first study to comprehensively compare expression profiles to determine the optimal immortalization method for maintaining the original nature of the wild-type cells. These data would be valuable to scientists aiming to establish new immortalized cell lines., Graphical Abstract, Highlights • Expression profiles of immortalized dermal papilla cells by the SV40, E6/E7, K4DT method were analyzed • The distance from wild type to K4DT was close, E6E7 was middle, SV40 was most distant • K4DT method showed the most conservative profile when it compared with SV40 and E6E7 method, Cell Biology; Stem Cell Research; Technical Aspects of Cell Biology

Summary The role of maternal and embryonic leucine zipper kinase (MELK) in cancer cell proliferation has been contentious, with recent studies arriving at disparate conclusions. We investigated the in vitro dependency of cancer cells on MELK under a range of assay conditions. Abrogation of MELK expression has little effect under common culture conditions, in which cells are seeded at high densities and reach confluence in 3–5 days. However, MELK dependency becomes clearly apparent in clonogenic growth assays using either RNAi or CRISPR technologies to modulate MELK expression. This dependency is in sharp contrast to that of essential genes, such as those encoding classic mitotic kinases, but is similar to that of other oncogenes including MYC and KRAS. Our study provides an example demonstrating some of the challenges encountered in cancer target validation, and reveals how subtle, but important, technical variations can ultimately lead to divergent outcomes and conclusions., Graphical Abstract, Highlights • Inhibiting MELK expression compromises clonogenic growth of cancer cells • MELK depletion minimally affects non-clonogenic cell growth • MELK depletion by RNAi or CRISPR has similar effects on cell growth • Cancer cell dependency on MELK is similar to that on classic oncogenes, Techniques in Genetics; Technical Aspects of Cell Biology; Cancer

Summary: To understand various intranuclear functions, it is important to know when, what, and how proteins enter the nucleus. Although many methods and commercial kits for nuclear fractionation have been developed, there are still no methods for obtaining a complete nuclear proteome. Soluble nuclear proteins are often lost during fractionation. We developed remarkably improved methods to obtain nuclear soluble fractions by optimizing the conditions of selective permeabilization of the plasma membrane. As a result, 10 million cells could be separated into the cytoplasmic and nuclear soluble fractions more precisely in a 1.5-mL test tube. Moreover, the addition of an inhibitor to prevent leakage from the nucleus retained small proteins in the nucleus. Because of the simple protocols and easy application for multiple samples, our methods are expected to be applied to various studies on spatiotemporal changes of dynamic nuclear proteins, such as signal transduction.

Summary Precise measurement of action potentials (APs) is needed to observe electrical activity and cellular communication within cardiac tissue. Voltage-sensitive dyes (VSDs) are traditionally used to measure cardiac APs; however, they require acute chemical addition that prevents chronic imaging. Genetically encoded voltage indicators (GEVIs) enable long-term studies of APs without the need of chemical additions, but current GEVIs used in cardiac tissue exhibit poor kinetics and/or low signal to noise (SNR). Here, we demonstrate the use of Archon1, a recently developed GEVI, in hiPSC-derived cardiomyocytes (CMs). When expressed in CMs, Archon1 demonstrated fast kinetics comparable with patch-clamp electrophysiology and high SNR significantly greater than the VSD Di-8-ANEPPS. Additionally, Archon1 enabled monitoring of APs across multiple cells simultaneously in 3D cardiac tissues. These results highlight Archon1's capability to investigate the electrical activity of CMs in a variety of applications and its potential to probe functionally complex in vitro models, as well as in vivo systems., Graphical Abstract, Highlights • Genetic sensor Archon1 reports membrane voltage in hiPSC-derived cardiomyocytes • Archon1 monitors action potentials in 2D and 3D cardiac tissue with high sensitivity • Archon1 repeatedly monitored voltage in the same cells and over extended time periods • Voltage dynamics of multiple cells were recorded simultaneously with Archon1, Biotechnology; Bioelectronics; Technical Aspects of Cell Biology; Electronic Materials

Summary Misfolded or unfolded proteins in the ER are said to be degraded only after translocation or isolation from the ER. Here, we describe a mechanism by which mutant proteins are degraded within the ER. Aggregates of mutant arginine vasopressin (AVP) precursor were confined to ER-associated compartments (ERACs) connected to the ER in AVP neurons of a mouse model of familial neurohypophysial diabetes insipidus. The ERACs were enclosed by membranes, an ER chaperone and marker protein of phagophores and autophagosomes were expressed around the aggregates, and lysosomes fused with the ERACs. Moreover, lysosome-related molecules were present within the ERACs, and aggregate degradation within the ERACs was dependent on autophagic-lysosomal activity. Thus, we demonstrate that protein aggregates can be degraded by autophagic-lysosomal machinery within specialized compartments of the ER., Graphical Abstract, Highlights • Mutant AVP precursors are confined to ERACs connected to the ER of FNDI AVP neurons • Lysosomes fuse with ERACs surrounded by phagophore-like membranes • Lysosome-related molecules are localized within ERACs • Rapamycin reduces and chloroquine increases protein aggregate accumulation in ERACs, neuroscience; cell biology; technical aspects of cell biology