The weight of synaptic connections, which is controlled not only postsynaptically but also presynaptically, is a key determinant in neuronal network dynamics. The mechanisms controlling synaptic weight, especially on the presynaptic side, remain elusive. Using single-synapse imaging of the neurotransmitter glutamate combined with super-resolution imaging of presynaptic proteins, we identify a presynaptic mechanism for setting weight in central glutamatergic synapses. In the presynaptic terminal, Munc13-1 molecules form multiple and discrete supramolecular self-assemblies that serve as independent vesicular release sites by recruiting syntaxin-1, a soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) protein essential for synaptic vesicle exocytosis. The multiplicity of these Munc13-1 assemblies affords multiple stable states conferring presynaptic weight, potentially encoding several bits of information at individual synapses. Supramolecular assembling enables a stable synaptic weight, which confers robustness of synaptic computation on neuronal circuits and may be a general mechanism by which biological processes operate despite the presence of molecular noise.

Mitochondrial Ca 2+ dynamics are involved in the regulation of multifarious cellular processes, including intracellular Ca 2+ signalling, cell metabolism and cell death. Use of mitochondria-targeted genetically encoded Ca 2+ indicators has revealed intercellular and subcellular heterogeneity of mitochondrial Ca 2+ dynamics, which are assumed to be determined by distinct thresholds of Ca 2+ increases at each subcellular mitochondrial domain. The balance between Ca 2+ influx through the mitochondrial calcium uniporter and extrusion by cation exchangers across the inner mitochondrial membrane may define the threshold; however, the precise mechanisms remain to be further explored. We here report the new red fluorescent genetically encoded Ca 2+ indicators, R-CEPIA3mt and R-CEPIA4mt, which are targeted to mitochondria and their Ca 2+ affinities are engineered to match the intramitochondrial Ca 2+ concentrations. They enable visualization of mitochondrial Ca 2+ dynamics with high spatiotemporal resolution in parallel with the use of green fluorescent probes and optogenetic tools. Thus, R-CEPIA3mt and R-CEPIA4mt are expected to be a useful tool for elucidating the mechanisms of the complex mitochondrial Ca 2+ dynamics and their functions.Mitochondrial Ca 2+ dynamics contribute to the control of various cellular functions such as formation of spatiotemporal patterns of cytosolic Ca 2+ dynamics, cellular metabolism and cell survival 1 . Ca 2+ concentrations in the mitochondrial matrix are regulated by the balance between the influx of Ca 2+ through the mitochondrial Ca 2+ uniporter (MCU) and the efflux of Ca 2+ by Na + /Ca 2+ or H + /Ca 2+ exchangers 1-3 . Recent studies have elucidated the molecular identity of the channel and regulatory components of MCU 4-11 as well as Na + -Ca 2+ -Li + exchanger in the mitochondrial inner membrane 12,13 . Furthermore, the advent of genetically encoded Ca 2+ indicators (GECIs) that are targeted to the mitochondrial matrix has enabled monitoring mitochondrial Ca 2+ dynamics with high spatiotemporal resolution, revealing both the subcellular and intercellular heterogeneity of mitochondrial Ca 2+ responses upon agonist-induced increases in the cytosolic Ca 2+ concentration [14][15][16] . These imaging results suggest that the threshold of the net Ca 2+ flux into the mitochondrial matrix is differentially determined in individual cells or even in each subcellular mitochondrial domain. However, the mechanism of these heterogeneous mitochondrial Ca 2+ dynamics and their functional significance remains to be clarified. Thus, further analyses combined with high-resolution mitochondrial Ca 2+ imaging are required.We have previously developed a Ca 2+ indicator protein family of Calcium-measuring organelle-Entrapped Protein IndicAtors (CEPIA) to visualize Ca 2+ signals in both the endoplasmic reticulum (ER) and mitochondria 15 . ER-targeted CEPIAs have K d values for Ca 2+ ranging between 558 and 672 µM, which are higher than those of other ER-targeted GECIs such as D1ER 17 , GCaMPer 18 , ER-GCaM...

Human amniotic epithelial cells (hAECs), which are a type of placental stem cell, express stem cell marker genes and are capable of differentiating into all three germ layers under appropriate culture conditions. hAECs are known to undergo TGF-β-dependent epithelial-mesenchymal transition (EMT); however, the impact of EMT on the stemness or differentiation of hAECs has not yet been determined. Here, we first confirmed that hAECs undergo EMT immediately after starting primary culture. Comprehensive transcriptome analysis using RNA-seq revealed that inhibition of TGF-β-dependent EMT maintained the expression of stemness-related genes, including NANOG and POU5F1, in hAECs. Moreover, the maintenance of stemness did not affect the nontumorigenic characteristics of hAECs. We showed for the first time that TGF-β-dependent EMT negatively affected the stemness of hAECs, providing novel insight into cellular processes of placental stem cells. Graphical abstract

The phenomenon of intercellular mitochondrial transfer has attracted great attention in various fields of research, including stem cell biology. Elucidating the mechanism of mitochondrial transfer from healthy stem cells to cells with mitochondrial dysfunction may lead to the development of novel stem cell therapies to treat mitochondrial diseases, among other advances. To visually evaluate and analyze the mitochondrial transfer process, dual fluorescent labeling systems are often used to distinguish the mitochondria of donor and recipient cells. Although enhanced green fluorescent protein (EGFP) has been well-characterized for labeling mitochondria, other colors of fluorescent protein have been less extensively evaluated in the context of mitochondrial transfer. Here, we generated different lentiviral vectors with mitochondria-targeted red fluorescent proteins (RFPs), including DsRed, mCherry (both from Discosoma sp.) Kusabira orange (mKOκ, from Verrillofungia concinna), and TurboRFP (from Entacmaea quadricolor). Among these proteins, mitochondria-targeted DsRed and its variant mCherry often generated bright aggregates in the lysosome while other proteins did not. We further validated that TurboRFP-labeled mitochondria were successfully transferred from amniotic epithelial cells, one of the candidates for donor stem cells, to mitochondria-damaged recipient cells without losing the membrane potential. Our study provides new insight into the genetic labeling of mitochondria with red fluorescent proteins, which may be utilized to analyze the mechanism of intercellular mitochondrial transfer.

Pancreatic β-cells release insulin in a Ca 2+ -dependent pulsatile manner to control blood glucose levels. Although the Ca 2+ signaling mechanism in β-cells has been extensively studied using in vitro and ex vivo preparations, its analysis in living animals remains challenging. Therefore, while β-cell activities in vivo are under the influence of the autonomic nervous system, various hormones and other bioactive substances, Ca 2+ responses of β-cells under physiological conditions have not been clarified. We here report a method to monitor and analyze in vivo β-cell Ca 2+ activities using a transgenic mouse line expressing a genetically encoded ratiometric Ca 2+ indicator, YC-Nano50.Using the method, we visualized β-cell Ca 2+ signals in laparotomized mice under anesthesia, and observed synchronized Ca 2+ oscillations in β-cells within individual islets. Furthermore, we succeeded in monitoring Ca 2+ activities in multiple islets simultaneously, which may clarify the basis for a pulsatile insulin secretion. Further studies in living animals using the new method is expected to help elucidate the mechanism of insulin secretion and the etiology of diabetes.