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1. Dynamic single-cell intracellular pH sensing using a SERS-active nanopipette

   https://doi.org/10.1039/D0AN00838A  

   Guo, Jing; Sesena Rubfiaro, Alberto; Lai, Yanhao; Moscoso, Joseph; Chen, Feng; Liu, Yuan; Wang, Xuewen; He, Jin (July 2020, The Analyst)

   null (Ed.)

   Glass nanopipettes have shown promise for applications in single-cell manipulation, analysis, and imaging. In recent years, plasmonic nanopipettes have been developed to enable surface-enhanced Raman spectroscopy (SERS) measurements for single-cell analysis. In this work, we developed a SERS-active nanopipette that can be used to perform long-term and reliable intracellular analysis of single living cells with minimal damage, which is achieved by optimizing the nanopipette geometry and the surface density of the gold nanoparticle (AuNP) layer at the nanopipette tip. To demonstrate its ability in single-cell analysis, we used the nanopipette for intracellular pH sensing. Intracellular pH (pH i ) is vital to cells as it influences cell function and behavior and pathological conditions. The pH sensitivity was realized by simply modifying the AuNP layer with the pH reporter molecule 4-mercaptobenzoic acid. With a response time of less than 5 seconds, the pH sensing range is from 6.0 to 8.0 and the maximum sensitivity is 0.2 pH units. We monitored the pH i change of individual HeLa and fibroblast cells, triggered by the extracellular pH (pH e ) change. The HeLa cancer cells can better resist pH e change and adapt to the weak acidic environment. Plasmonic nanopipettes can be further developed to monitor other intracellular biomarkers. 

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2. Soluble adenylyl cyclase coordinates intracellular pH homeostasis and biomineralization in calcifying cells of a marine animal

   https://doi.org/10.1152/ajpcell.00524.2022  

   Chang, William Weijen; Thies, Angus B.; Tresguerres, Martin; Hu, Marian Y. (March 2023, American Journal of Physiology-Cell Physiology)

   Biomineralizing cells concentrate dissolved inorganic carbon (DIC) and remove protons from the site of mineral precipitation. However, the molecular regulatory mechanisms that orchestrate pH homeostasis and biomineralization of calcifying cells are poorly understood. Here, we report that the acid-base sensing enzyme soluble adenylyl cyclase (sAC) coordinates intracellular pH (pH i ) regulation in the calcifying primary mesenchyme cells (PMCs) of sea urchin larvae. Single-cell transcriptomics, in situ hybridization, and immunocytochemistry elucidated the spatiotemporal expression of sAC during skeletogenesis. Live pH i imaging of PMCs revealed that the downregulation of sAC activity with two structurally unrelated small molecules inhibited pH i regulation of PMCs, an effect that was rescued by the addition of cell-permeable cAMP. Pharmacological sAC inhibition also significantly reduced normal spicule growth and spicule regeneration, establishing a link between PMC pH i regulation and biomineralization. Finally, increased expression of sAC mRNA was detected during skeleton remineralization and exposure to CO 2 -induced acidification. These findings suggest that transcriptional regulation of sAC is required to promote remineralization and to compensate for acidic stress. This work highlights the central role of sAC in coordinating acid-base regulation and biomineralization in calcifying cells of a marine animal. 

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3. Differential Impacts on Tensional Homeostasis of Gastric Cancer Cells Due to Distinct Domain Variants of E-Cadherin

   https://doi.org/10.3390/cancers14112690  

   Xu, Han; Bunde, Katie A.; Figueiredo, Joana; Seruca, Raquel; Smith, Michael L.; Stamenović, Dimitrije (June 2022, Cancers)

   In epithelia, breakdown of tensional homeostasis is closely associated with E-cadherin dysfunction and disruption of tissue function and integrity. In this study, we investigated the effect of E-cadherin mutations affecting distinct protein domains on tensional homeostasis of gastric cancer cells. We used micropattern traction microscopy to measure temporal fluctuations of cellular traction forces in AGS cells transfected with the wild-type E-cadherin or with variants affecting the extracellular, the juxtamembrane, and the intracellular domains of the protein. We focused on the dynamic aspect of tensional homeostasis, namely the ability of cells to maintain a consistent level of tension, with low temporal variability around a set point. Cells were cultured on hydrogels micropatterned with different extracellular matrix (ECM) proteins to test whether the ECM adhesion impacts cell behavior. A combination of Fibronectin and Vitronectin was used as a substrate that promotes the adhesive ability of E-cadherin dysfunctional cells, whereas Collagen VI was used to test an unfavorable ECM condition. Our results showed that mutations affecting distinct E-cadherin domains influenced differently cell tensional homeostasis, and pinpointed the juxtamembrane and intracellular regions of E-cadherin as the key players in this process. Furthermore, Fibronectin and Vitronectin might modulate cancer cell behavior towards tensional homeostasis. 

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4. Marine heatwaves depress metabolic activity and impair cellular acid–base homeostasis in reef‐building corals regardless of bleaching susceptibility

   https://doi.org/10.1111/gcb.15622  

   Innis, Teegan; Allen‐Waller, Luella; Brown, Kristen T.; Sparagon, Wesley; Carlson, Christopher; Kruse, Elisa; Huffmyer, Ariana S.; Nelson, Craig E.; Putnam, Hollie M.; Barott, Katie L. (April 2021, Global Change Biology)

   Abstract Ocean warming is causing global coral bleaching events to increase in frequency, resulting in widespread coral mortality and disrupting the function of coral reef ecosystems. However, even during mass bleaching events, many corals resist bleaching despite exposure to abnormally high temperatures. While the physiological effects of bleaching have been well documented, the consequences of heat stress for bleaching‐resistant individuals are not well understood. In addition, much remains to be learned about how heat stress affects cellular‐level processes that may be overlooked at the organismal level, yet are crucial for coral performance in the short term and ecological success over the long term. Here we compared the physiological and cellular responses of bleaching‐resistant and bleaching‐susceptible corals throughout the 2019 marine heatwave in Hawai'i, a repeat bleaching event that occurred 4 years after the previous regional event. Relative bleaching susceptibility within species was consistent between the two bleaching events, yet corals of both resistant and susceptible phenotypes exhibited pronounced metabolic depression during the heatwave. At the cellular level, bleaching‐susceptible corals had lower intracellular pH than bleaching‐resistant corals at the peak of bleaching for both symbiont‐hosting and symbiont‐free cells, indicating greater disruption of acid–base homeostasis in bleaching‐susceptible individuals. Notably, cells from both phenotypes were unable to compensate for experimentally induced cellular acidosis, indicating that acid–base regulation was significantly impaired at the cellular level even in bleaching‐resistant corals and in cells containing symbionts. Thermal disturbances may thus have substantial ecological consequences, as even small reallocations in energy budgets to maintain homeostasis during stress can negatively affect fitness. These results suggest concern is warranted for corals coping with ocean acidification alongside ocean warming, as the feedback between temperature stress and acid–base regulation may further exacerbate the physiological effects of climate change. 

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5. Intracellular pH dynamics regulates intestinal stem cell lineage specification

   https://doi.org/10.1038/s41467-023-39312-9  

   Liu, Yi; Reyes, Efren; Castillo-Azofeifa, David; Klein, Ophir D.; Nystul, Todd; Barber, Diane L. (June 2023, Nature Communications)

   Abstract Intracellular pH dynamics is increasingly recognized to regulate myriad cell behaviors. We report a finding that intracellular pH dynamics also regulates adult stem cell lineage specification. We identify an intracellular pH gradient in mouse small intestinal crypts, lowest in crypt stem cells and increasing along the crypt column. Disrupting this gradient by inhibiting H⁺efflux by Na⁺/H⁺exchanger 1 abolishes crypt budding and blocks differentiation of Paneth cells, which are rescued with exogenous WNT. Using single-cell RNA sequencing and lineage tracing we demonstrate that intracellular pH dynamics acts downstream of ATOH1, with increased pH promoting differentiation toward the secretory lineage. Our findings indicate that an increase in pH is required for the lineage specification that contributes to crypt maintenance, establishing a role for intracellular pH dynamics in cell fate decisions within an adult stem cell lineage. 

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