Post-click labeling enables highly accurate single cell analyses of glucose uptake ex vivo and in vivo.

Cellular glucose uptake is a key feature reflecting metabolic demand of cells in physiopathological conditions. Fluorophore-conjugated sugar derivatives are widely used for monitoring glucose transporter (GLUT) activity at the single-cell level, but have limitations in in vivo applications. Here, we develop a click chemistry-based post-labeling method for flow cytometric measurement of glucose uptake with low background adsorption. This strategy relies on GLUT-mediated uptake of azide-tagged sugars, and subsequent intracellular labeling with a cell-permeable fluorescent reagent via a copper-free click reaction. Screening a library of azide-substituted monosaccharides, we discover 6-azido-6-deoxy-D-galactose (6AzGal) as a suitable substrate of GLUTs. 6AzGal displays glucose-like physicochemical properties and reproduces in vivo dynamics similar to 18F-FDG. Combining this method with multi-parametric immunophenotyping, we demonstrate the ability to precisely resolve metabolically-activated cells with various GLUT activities in ex vivo and in vivo models. Overall, this method provides opportunities to dissect the heterogenous metabolic landscape in complex tissue environments.

Flow cytometric analysis of phosphatidylcholine metabolism using organelle-selective click labeling.

Here, we present a protocol to analyze phosphatidylcholine (PC) metabolism in mammalian cells using organelle-selective click labeling coupled with flow cytometry (O-ClickFC). We describe steps for the metabolic incorporation of azide-choline into PC. We then detail fluorescent labeling of the azide-modified PC with organelle-targeting clickable dyes in the ER-Golgi, plasma membrane, and mitochondria, and by flow cytometry. This protocol is optimized for flow cytometric quantification of the labeled PC at the organelle level within single live cells. For complete details on the use and execution of this protocol, please refer to Tsuchiya et al. (2023).1.

Organelle-selective click labeling coupled with flow cytometry allows pooled CRISPR screening of genes involved in phosphatidylcholine metabolism.

Cellular lipid synthesis and transport are governed by intricate protein networks. Although genetic screening should contribute to deciphering the regulatory networks of lipid metabolism, technical challenges remain-especially for high-throughput readouts of lipid phenotypes. Here, we coupled organelle-selective click labeling of phosphatidylcholine (PC) with flow cytometry-based CRISPR screening technologies to convert organellar PC phenotypes into a simple fluorescence readout for genome-wide screening. This technique, named O-ClickFC, was successfully applied in genome-scale CRISPR-knockout screens to identify previously reported genes associated with PC synthesis (PCYT1A, ACACA), vesicular membrane trafficking (SEC23B, RAB5C), and non-vesicular transport (PITPNB, STARD7). Moreover, we revealed previously uncharacterized roles of FLVCR1 as a choline uptake facilitator, CHEK1 as a post-translational regulator of the PC-synthetic pathway, and CDC50A as responsible for the translocation of PC to the outside of the plasma membrane bilayer. These findings demonstrate the versatility of O-ClickFC as an unprecedented platform for genetic dissection of cellular lipid metabolism.

The mechanosensitive ion channel PIEZO1 promotes satellite cell function in muscle regeneration.

Muscle satellite cells (MuSCs), myogenic stem cells in skeletal muscles, play an essential role in muscle regeneration. After skeletal muscle injury, quiescent MuSCs are activated to enter the cell cycle and proliferate, thereby initiating regeneration; however, the mechanisms that ensure successful MuSC division, including chromosome segregation, remain unclear. Here, we show that PIEZO1, a calcium ion (Ca2+)-permeable cation channel activated by membrane tension, mediates spontaneous Ca2+ influx to control the regenerative function of MuSCs. Our genetic engineering approach in mice revealed that PIEZO1 is functionally expressed in MuSCs and that Piezo1 deletion in these cells delays myofibre regeneration after injury. These results are, at least in part, due to a mitotic defect in MuSCs. Mechanistically, this phenotype is caused by impaired PIEZO1-Rho signalling during myogenesis. Thus, we provide the first concrete evidence that PIEZO1, a bona fide mechanosensitive ion channel, promotes proliferation and regenerative functions of MuSCs through precise control of cell division.

Extreme deformability of insect cell membranes is governed by phospholipid scrambling.

Organization of dynamic cellular structure is crucial for a variety of cellular functions. In this study, we report that Drosophila and Aedes have highly elastic cell membranes with extremely low membrane tension and high resistance to mechanical stress. In contrast to other eukaryotic cells, phospholipids are symmetrically distributed between the bilayer leaflets of the insect plasma membrane, where phospholipid scramblase (XKR) that disrupts the lipid asymmetry is constitutively active. We also demonstrate that XKR-facilitated phospholipid scrambling promotes the deformability of cell membranes by regulating both actin cortex dynamics and mechanical properties of the phospholipid bilayer. Moreover, XKR-mediated construction of elastic cell membranes is essential for hemocyte circulation in the Drosophila cardiovascular system. Deformation of mammalian cells is also enhanced by the expression of Aedes XKR, and thus phospholipid scrambling may contribute to formation of highly deformable cell membranes in a variety of living eukaryotic cells.

Organelle-Selective Labeling of Choline-Containing Phospholipids (CPLs) and Real-Time Imaging in Living Cells.

Choline-containing phospholipids (CPLs), including phosphatidylcholine (PC) and sphingomyelin (SM), are the major components of mammalian cell membranes and play critical roles during a variety of cellular processes. However, intracellular dynamics of CPLs is poorly understood due to a lack of methods to trace CPL trafficking at organelle resolution. Here, we describe protocols that make it possible to fluorescently label CPLs at the targeted organelles and to monitor their movement within living cells using confocal microscopy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: ER-Golgi-selective labeling of azide-tagged CPLs for confocal imaging Basic Protocol 2: Mitochondria-selective labeling of azide-tagged CPLs for confocal imaging.

Organelle membrane-specific chemical labeling and dynamic imaging in living cells.

Lipids play crucial roles as structural elements, signaling molecules and material transporters in cells. However, the functions and dynamics of lipids within cells remain unclear because of a lack of methods to selectively label lipids in specific organelles and trace their movement by live-cell imaging. We describe here a technology for the selective labeling and fluorescence imaging (microscopic or nanoscopic) of phosphatidylcholine in target organelles. This approach involves the metabolic incorporation of azido-choline, followed by a spatially limited bioorthogonal reaction that enables the visualization and quantitative analysis of interorganelle lipid transport in live cells. More importantly, with live-cell imaging, we obtained direct evidence that the autophagosomal membrane originates from the endoplasmic reticulum. This method is simple and robust and is thus powerful for real-time tracing of interorganelle lipid trafficking.

Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation.

Myotube formation by fusion of myoblasts and subsequent elongation of the syncytia is essential for skeletal muscle formation. However, molecules that regulate myotube formation remain elusive. Here we identify PIEZO1, a mechanosensitive Ca2+ channel, as a key regulator of myotube formation. During myotube formation, phosphatidylserine, a phospholipid that resides in the inner leaflet of the plasma membrane, is transiently exposed to cell surface and promotes myoblast fusion. We show that cell surface phosphatidylserine inhibits PIEZO1 and that the inward translocation of phosphatidylserine, which is driven by the phospholipid flippase complex of ATP11A and CDC50A, is required for PIEZO1 activation. PIEZO1-mediated Ca2+ influx promotes RhoA/ROCK-mediated actomyosin assemblies at the lateral cortex of myotubes, thus preventing uncontrolled fusion of myotubes and leading to polarized elongation during myotube formation. These results suggest that cell surface flip-flop of phosphatidylserine acts as a molecular switch for PIEZO1 activation that governs proper morphogenesis during myotube formation.

Development of a Novel Tetravalent Synthetic Peptide That Binds to Phosphatidic Acid.

We employed a multivalent peptide-library screening technique to identify a peptide motif that binds to phosphatidic acid (PA), but not to other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). A tetravalent peptide with the sequence motif of MARWHRHHH, designated as PAB-TP (phosphatidic acid-binding tetravalent peptide), was shown to bind as low as 1 mol% of PA in the bilayer membrane composed of PC and cholesterol. Kinetic analysis of the interaction between PAB-TP and the membranes containing 10 mol% of PA showed that PAB-TP associated with PA with a low dissociation constant of KD = 38 ± 5 nM. Coexistence of cholesterol or PE with PA in the membrane enhanced the PAB-TP binding to PA by increasing the ionization of the phosphomonoester head group as well as by changing the microenvironment of PA molecules in the membrane. Amino acid replacement analysis demonstrated that the tryptophan residue at position 4 of PAB-TP was involved in the interaction with PA. Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA. Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane. The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

Surface treatment of flow channels in microfluidic devices fabricated by stereolithography.

A microfluidic device with three-dimensional flow channels was fabricated by stereolithography, and hydrophilic surface treatment of the flow channel was performed by coating the wall of the channel with a silica layer. After the treatment, the device produced monodisperse oil-in-water (O/W) emulsions. The silica layer on the channel surface was then coated with a fluorinated silane coupling agent to make it hydrophobic, thus enabling the treated device to produce monodisperse inverted water-in-oil (W/O) emulsions.

Breast cancer in first-degree relatives and risk of lung cancer: assessment of the existence of gene sex interactions.

BACKGROUND: Previous studies have shown the sex differences in lung cancer and the associations between estrogen-related genes and non-small cell lung cancer. In the present study, we assumed the existence of shared candidate genes that are common in lung and breast cancers, and examined whether women with a family history of breast cancer are at increased risk of lung cancer compared with men, especially adenocarcinoma, in a case-only study. METHODS: This case-only study was conducted based on the Lung Cancer Database Project at the National Cancer Center Hospital East. A total of 1566 patients with newly diagnosed primary lung cancer were consecutively recruited between 1999 and 2003. Information on their family history of cancer and smoking habit was obtained from a self-administered questionnaire. To assess an interactions between two factors, odds ratios for interaction (ORis) and 95% confidence intervals (CIs) were calculated by case-only contingency table. RESULTS: A statistically significant ORi was observed between a family history of breast cancer in first-degree relatives (parent and siblings, not including children) and the sex of a patient (ORi: 2.22, 95% CI: 1.02-4.81). A stratified analysis by histologic subtypes showed a statistically significant ORi only for adenocarcinoma (ORi: 3.27, 95% CI: 1.19-8.98). No other family history of cancer, such as stomach, colon and lung cancer, showed a statistically significant ORi. CONCLUSION: This study suggests the possibility of gene-sex interaction in lung cancer.

Effect of soy isoflavones on endometriosis: interaction with estrogen receptor 2 gene polymorphism.

BACKGROUND: Progression of endometriosis is considered estrogen-dependent. Dietary soy isoflavones may affect the risk of endometriosis, and polymorphisms in estrogen receptor genes may modify this association. We examined associations among soy isoflavone intake, estrogen receptor 2 (ESR2) gene polymorphisms and risk of endometriosis. METHODS: We recruited women age 20-45 years old who had consulted a university hospital for infertility in Tokyo, Japan in 1999 or 2000. A total of 138 eligible women were diagnosed laparoscopically and classified into 3 subgroups: control (no endometriosis), early endometriosis (stage I-II) and advanced endometriosis (stage III-IV). We measured urinary levels of genistein and daidzein as markers for dietary intake of soy isoflavones, and genotyped ESR2 gene RsaI polymorphisms. RESULTS: Higher levels of urinary genistein and daidzein were associated with decreased risk of advanced endometriosis (P for trend = 0.01 and 0.06, respectively) but not early endometriosis. For advanced endometriosis, the adjusted odds ratio for the highest quartile group was 0.21 (95% confidence interval = 0.06-0.76) for genistein and 0.29 (0.08-1.03) for daidzein, when compared with the lowest group. Inverse associations were also noted between urinary isoflavones and the severity of endometriosis (P for trend = 0.01 for genistein and 0.07 for daidzein). For advanced endometriosis, ESR2 gene RsaI polymorphism appeared to modify the effects of genistein (P for interaction = 0.03). CONCLUSIONS: Dietary isoflavones may reduce the risk of endometriosis among Japanese women.

Interaction between cytochrome P450 gene polymorphisms and serum organochlorine TEQ levels in the risk of endometriosis.

Exposure to dioxins and polychlorinated biphenyls (PCBs) has been suggested as a possible etiologic factor for endometriosis, but the association remains highly controversial. To assess whether cytochrome P450 (CYP) gene polymorphisms modulate the effect of dioxins and/or PCBs in endometriosis risk, we conducted a case-control study among infertile Japanese women. A total of 138 eligible women aged 20-45 were diagnosed laparoscopically and classified into three subgroups: control (no endometriosis), early endometriosis (stages I-II) and advanced endometriosis (stages III-IV). Neither CYP1A1 Ile462Val and CYP1B1 Leu432Val polymorphisms (genotypes with versus genotypes without the minor allele) nor serum dioxin and PCB toxic equivalency (TEQ) levels (low versus high) were independently associated with either early or advanced endometriosis risk. However, genotypes with the CYP1A1 462Val allele showed a statistically significant reduced risk of advanced endometriosis in combination with high serum dioxin TEQ levels (adjusted odds ratio = 0.13, 95% confidence interval: 0.02-0.76) (P for interaction = 0.08). Although no association was found between serum PCB TEQ level and advanced endometriosis in any stratum of CYP1B1 Leu432Val polymorphism, a statistically significant interaction was found (P for interaction = 0.05). CYP1A1 and CYP1B1 polymorphisms may modify the relation between environmental exposure to organochlorine and advanced endometriosis risk.

Analysis of the AhR, ARNT, and AhRR gene polymorphisms: genetic contribution to endometriosis susceptibility and severity.

OBJECTIVE: To explore whether polymorphisms in AhR, ARNT, and AhRR contribute to endometriosis susceptibility and severity. DESIGN: Case control study. SETTING: Hospital. PATIENT(S): One hundred thirty-eight Japanese women with or without endometriosis, diagnosed endoscopically. INTERVENTION(S): Endoscopic laparoscopy, with blood samples for genotyping obtained before the laparoscopic examination for genomic DNA extraction from peripheral leukocytes. MAIN OUTCOME MEASURE(S): AhR, ARNT, and AhRR polymorphisms were genotyped using real-time polymerase chain reaction (PCR) analysis. Odds ratios and 95% confidence intervals were calculated for AhR, ARNT, and AhRR genotypes to evaluate the risk of endometriosis. Associations between these polymorphisms and stage of endometriosis were also examined. RESULT(S): The C/G + G/G genotypes at codon 185 of AhRR showed a statistically significant association with risk of endometriosis (adjusted odds ratio, 2.53; 95% confidence interval, 1.16-5.55). Furthermore, a statistically significant trend associated the C/G + G/G genotypes with the clinical stage of endometriosis. No statistically significant association was observed between AhR codon 554 or ARNT codon 189 polymorphisms and endometriosis. CONCLUSION(S): AhRR codon 185 polymorphism was associated with susceptibility to and severity of endometriosis in Japanese women.

[Potential links between endocrine disrupting compounds and endometriosis].

There is an increasing awareness of potential links between human health and environmental pollution. Endocrine disrupting compounds(EDCs), such as dioxins, are exogenous chemicals that either imitate endogenous hormones, interfere with pharmacokinetics or act by other mechanisms. It is generally assumed that the number of patients with endometriosis have been increasing. Endometriosis is estimated to occur in approximately 7-10% of reproductive age women. Recently, much more attention has been paid to the relationship between endometriosis and EDCs, especially dioxins. Endometriosis is a common disease that can affect any menstruating woman, but the pathogenesis and pathophysiology are still poorly understood and there is no strategy for prevention. However, there are some new studies that have investigated biological activity and consequences of exposure to EDCs. Endometriosis is the disease in which genetic and environmental factors act together to produce the phenotypes. Findings for environmental factors as well as genetic variants have been reviewed, but the issue remains controversial. The findings of these studies would be a great help to understand the mechanisms of endometriosis.