Zn 2+ plays essential roles in biology, and cells have adopted exquisite mechanisms for regulating steady-state Zn 2+ levels. Although much is known about total Zn 2+ in cells, very little is known about its subcellular distribution. Yet defining the location of Zn 2+ and how it changes with signaling events is essential for elucidating how cells regulate this essential ion. Here we create fluorescent sensors genetically targeted to the endoplasmic reticulum (ER) and Golgi to monitor steady-state Zn 2+ levels as well as flux of Zn 2+ into and out of these organelles. These studies reveal that ER and Golgi contain a concentration of free Zn 2+ that is 100 times lower than the cytosol. Both organelles take up Zn 2+ when cytosolic levels are elevated, suggesting that the ER and Golgi can sequester elevated cytosolic Zn 2+ and thus have the potential to play a role in influencing Zn 2+ toxicity. ER Zn 2+ homeostasis is perturbed by small molecule antagonists of Ca 2+ homeostasis and ER Zn 2+ is released upon elevation of cytosolic Ca 2+ pointing to potential exchange of these two ions across the ER. This study provides direct evidence that Ca 2+ signaling can influence Zn 2+ homeostasis and vice versa, that Zn 2+ dynamics may modulate Ca 2+ signaling.

In the past 5–10 years, the power of the green fluorescent protein (GFP) and its numerous derivatives has been harnessed toward the development of genetically encoded fluorescent biosensors. These sensors are incorporated into cells or organisms as plasmid DNA, which leads the transcriptional and translational machinery of the cell to express a functional sensor. To date, over 100 different genetically encoded biosensors have been developed for targets as diverse as ions, molecules and enzymes. Such sensors are instrumental in providing a window into the real-time biochemistry of living cells and whole organisms, and are providing unprecedented insight into the inner workings of a cell.

Zinc (Zn2+) homeostasis plays a vital role in cell function, and the dysregulation of intracellular Zn2+ is associated with mitochondrial dysfunction. Few tools exist to quantitatively monitor the buffered, free Zn2+ concentration in mitochondria of living cells ([Zn2+]mito). We have validated three high dynamic range, ratiometric, genetically encoded, fluorescent Zn2+ sensors that we have successfully used to precisely measure and monitor [Zn2+]mito in several cell types. Using one of these sensors, called mito-ZapCY1, we report observations that free Zn2+ is buffered at concentrations about 3 orders of magnitude lower in mitochondria than in the cytosol, and that HeLa cells expressing mito-ZapCY1 have an average [Zn2+]mito of 0.14 pM, which differs significantly from other cell types. These optimized mitochondrial Zn2+ sensors could improve our understanding of the relationship between Zn2+ homeostasis and mitochondrial function.

No abstract

Fluorescent sensors are powerful tools for visualizing and quantifying molecules and ions in living cells. A variety of small molecule and genetically encoded sensors have been developed for studying intracellular Zn2+ homeostasis and signaling, but no direct comparisons exist making it challenging for researchers to identify the appropriate sensor for a given application. Here we directly compare the widely used small molecule probe FluoZin-3 and a genetically encoded sensor, ZapCY2. We demonstrate that, in contrast to FluoZin-3, ZapCY2 exhibits a well defined cytosolic localization, provides estimates of Zn2+ concentration with little variability, does not perturb cytosolic Zn2+ levels, and exhibits rapid Zn2+ response dynamics. ZapCY2 was used to measure Zn2+ concentrations in 5 different cell types, revealing higher cytosolic Zn2+ levels in prostate cancer cells compared to normal prostate cells (although the total zinc is reduced in prostate cancer cells) , suggesting distinct regulatory mechanisms.

Highlights Tocilizumab can significantly improve COVID-19’s pulmonary inflammation. Tocilizumab can significantly improve the clinical symptoms of COVID-19 patients. Tocilizumab combined with or without favipiravir can effectively reduce the mortality of COVID-19.

Infection with the hepatitis B virus (HBV) promotes the development of hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) and is a leading cause of morbidity and mortality worldwide. HBV X protein (HBx) is an important effector for HBV pathogenesis, but its cellular targets and acting mechanisms remain elusive. We show here that HBx interacts with the anti-apoptotic proteins Bcl-2 and Bcl-xL through a Bcl-2 homology 3 (BH3)-like motif in mammalian cells. Importantly, mutations in the BH3-like motif that prevent HBx binding to Bcl-2 and Bcl-xL abrogate cytosolic calcium elevation and cell death induced by HBx expression in hepatocytes and severely impair HBV viral replication, which can be substantially rescued by restoring cytosolic calcium. These results suggest that HBx binding to Bcl-2 family members and subsequent elevation of cytosolic calcium are important for HBV viral replication. Consistently, RNAi knockdown of Bcl-2 or Bcl-xL results in reduced calcium elevation by HBx and decreased viral replication in hepatocytes. Our results suggest that HBx targets Bcl-2 proteins through its BH3-like motif to promote cytosolic calcium elevation, cell death, and viral replication during HBV pathogenesis, which presents an excellent therapeutic intervention point in treating patients with chronic HBV.calcium signaling | apoptosis | necrosis

Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for reporting on ions, molecules and biochemical reactions in living cells. Here we describe the development of new sensors for Zn2+based on alternate FRET-pairs that do not involve the traditional CFP and YFP. Zn2+ is an essential micronutrient and plays fundamental roles in cell biology. Consequently there is a pressing need for robust sensors to monitor Zn2+ levels and dynamics in cells with high spatial and temporal resolution. Here we develop a suite of sensors using alternate FRET pairs, including tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE. These sensors were targeted to both the nucleus and cytosol and characterized and validated in living cells. Sensors based on the new FRET pair Clover/mRuby2 displayed a higher dynamic range and better signal-to-noise ratio than the remaining sensors tested and were optimal for monitoring changes in cytosolic and nuclear Zn2+. Using a green-red sensor targeted to the nucleus and cyan-yellow sensor targeted to either the ER, Golgi, or mitochondria, we were able to monitor Zn2+ uptake simultaneously in two compartments, revealing that nuclear Zn2+ rises quickly, whereas the ER, Golgi, and mitochondria all sequester Zn2+ more slowly and with a delay of 600–700 sec. Lastly, these studies provide the first glimpse of nuclear Zn2+ and reveal that nuclear Zn2+ is buffered at a higher level than cytosolic Zn2+.