Biology subjects

Nasu, Y.

Publications and source records attributed to Nasu, Y..

6 recordsLinked to original sources

bioRxiv2024

Synthesis and application of a photocaged L-lactate

O_SCPLOWLC_SCPLOW-Lactate, once considered a metabolic waste product of glycolysis, is now recognized as a vitally important metabolite and signaling molecule in multiple biological pathways. However, exploring O_SCPLOWLC_SCPLOW-lactates emerging intra- and extra-cellular roles is hindered by a lack of tools to locally perturb O_SCPLOWLC_SCPLOW-lactate concentration intracellularly and extracellularly. Photocaged compounds are a powerful way to introduce bioactive molecules with spatial and temporal precision using illumination. Here, we report the development of a photocaged derivative of O_SCPLOWLC_SCPLOW-lactate, 4-methoxy-7-nitroindolinyl O_SCPLOWLC_SCPLOW-lactate (MNI-O_SCPLOWLC_SCPLOW-lac), that releases O_SCPLOWLC_SCPLOW-lactate upon UV illumination. We validated MNI-O_SCPLOWLC_SCPLOW-lac in cell culture by demonstrating that the photorelease of O_SCPLOWLC_SCPLOW-lactate elicits a response from genetically encoded extra- and intracellular O_SCPLOWLC_SCPLOW-lactate biosensors. These results indicate that MNI-O_SCPLOWLC_SCPLOW-lac may be useful for perturbing the concentration of endogenous O_SCPLOWLC_SCPLOW-lactate in order to investigate O_SCPLOWLC_SCPLOW-lactates roles in metabolism and signaling pathways.

biochemistry↗

bioRxiv2023

A lactate-dependent shift of glycolysis mediates synaptic and cognitive processes

Control of brain energy metabolism and regulation of synaptic activity through gliotransmission are two important ways, through which astrocytes contribute to mental functions. However, the potential functional and molecular links between these two astrocyte-dependent processes have been scantly explored. Here we show that a lactate-dependent shift of glycolysis underlies the production of the gliotransmitter D-serine by acute activation of astrocyte type-1 cannabinoid (CB1) receptors, thereby gating synaptic and cognitive processes. Acute cannabinoid application causes a CB1 receptor-dependent rapid and reversible increase of lactate production and release in primary astrocyte cultures. As shown before, mutant mice lacking the CB1 receptor gene in astrocytes (GFAP-CB1-KO) were impaired in a novel object recognition (NOR) memory task. This phenotype was rescued not only by the gliotransmitter D-serine, but also by its precursor L-serine. Surprisingly, the administration of lactate and of an agonist of the lactate receptor HCAR1 also reverted the memory impairment of GFAP-CB1-KO mice. This rescue effect was abolished by in vivo blockade of the astrocyte-specific phosphorylated pathway (PP), which diverts glycolysis towards L-serine synthesis, suggesting that lactate signaling might promote the accumulation of this amino acid. Consistent with this idea, lactate and HCAR1 agonism increased the co-agonist occupancy of CA1 post-synaptic hippocampal NMDA receptors. This effect of lactate was abolished by blockade of PP. By establishing a mechanistic link between lactate production and signaling, serine availability, synaptic activity and behavior, these results reveal an unforeseen functional connection between energy metabolism and gliotransmission to control cognitive processes.

neuroscience↗

bioRxiv2022

Improved genetically encoded fluorescent biosensors for monitoring of intra- and extracellular L-lactate

O_SCPLOWLC_SCPLOW-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. To enable investigations of both the inter- and intra-cellular dynamics of O_SCPLOWLC_SCPLOW-Lactate, we develop a second-generation green fluorescent extracellular O_SCPLOWLC_SCPLOW-Lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular O_SCPLOWLC_SCPLOW-Lactate biosensor, designated R-iLACCO1. Compared to the first-generation eLACCO1.1 ({Delta}F/F = 1.5 in cultured neurons), eLACCO2.1 exhibits better membrane localization and fluorescence response ({Delta}F/F = 8.1 in cultured neurons) with faster response kinetics to extracellular O_SCPLOWLC_SCPLOW-Lactate on the surface of live mammalian cells. R-iLACCO1 and its affinity variants exhibit large fluorescence responses to changes in O_SCPLOWLC_SCPLOW-Lactate concentration in vitro ({Delta}F/F = 15 to 22) and in live mammalian cells ({Delta}F/F = 5.5 to 11). We demonstrate that these biosensors enable cellular-resolution imaging of extracellular and intracellular O_SCPLOWLC_SCPLOW-Lactate in cultured mammalian cells.

molecular biology↗

bioRxiv2022

High performance genetically-encoded green fluorescent biosensors for intracellular L-lactate

L-Lactate is a monocarboxylate produced during the process of cellular glycolysis and has long been generally considered a waste product. However, studies in recent decades have provided new perspectives on the physiological roles of L-lactate as a major energy substrate and a signaling molecule. To enable further investigations of the physiological roles of L-lactate, we have developed a series of high-performance ({Delta}F/F = 15 to 30 in vitro), intensiometric, genetically-encoded green fluorescent protein (GFP)-based intracellular L-lactate biosensors with a range of affinities. We evaluated the performance of these biosensors by in vitro and live-cell characterization and demonstrated the utility with imaging applications in several cell lines.

bioengineering↗

bioRxiv2022

A red fluorescent genetically encoded biosensor for extracellular L-lactate

L-Lactate, traditionally recognized as a waste product of metabolism, is now appreciated as a key intercellular energy currency in mammals. To enable investigations of intercellular shuttling of L-lactate, we have previously reported eLACCO1.1, a green fluorescent genetically encoded biosensor for extracellular L-lactate. eLACCO1.1 enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue. However, eLACCO1.1 spectrally overlaps with commonly used optical biosensors and actuators, limiting its application for multiplexed imaging or combined use with optogenetic actuators. Here, we report a red fluorescent extracellular L-lactate biosensor, designated R-eLACCO2. R-eLACCO2 is the end-product of extensive directed evolution and exhibits a large fluorescence response to L-lactate with high molecular specificity in vitro. We demonstrate that R-eLACCO2 with optimized leader and anchor sequences shows a large fluorescence change in response to extracellular L-lactate on the membrane of live mammalian cells. R-eLACCO2 should enable multicolor imaging of extracellular L-lactate in combination with other fluorescent probes and optogenetic actuators.

bioengineering↗

bioRxiv2022

Pacsin 2-dependent N-cadherin internalization regulates the migration behaviour of malignant cancer cells

Cell migration is essential for both physiological and pathological processes such as embryonic morphogenesis, wound repair and metastasis of cancer cells. Collective cell migration is the coordinated movement of multiple cells connected with cadherin-based adherence junctions. Cadherins undergo dynamic intracellular trafficking and their surface level is determined by a balance between endocytosis, recycling and degradation. However, regulatory mechanisms of cadherin turnover in the collective cell migration remain to be elucidated. In this study, we show that a BAR domain protein pacsin 2 plays an essential role in collective cell migration by regulating the internalization of N-cadherin in human bladder cancer cells T24. Pacsin 2 and its associating GTPase dynamin 2 colocalized with N-cadherin at the cell periphery in T24 cells. Depletion of either pacsin 2 or dynamin 2 induced interdigitating cell-cell contacts enriched with N-cadherin. Imaging analyses of the wound healing assay showed that pacsin 2-depleted T24 cells migrated in a collective and directed manner in contrast with randomly migrating control cells. Furthermore, cell-surface biotinylation assay showed that internalization of N-cadherin is attenuated in pacsin 2-depleted cells. Interestingly, the GST-pulldown assay demonstrated that the SH3 domain of pacsin 2 binds to the cytoplasmic domain of N-cadherin, suggesting that surface levels of N-cadherin are regulated by pacsin 2-mediated endocytosis. These data support new insights into a novel endocytic route of N-cadherin in collective cell migration providing pacsin 2 as a possible therapeutic target for cancer metastasis.

cell biology↗