Deciphering cell signaling networks with massively multiplexed biosensor barcoding.

Genetically encoded fluorescent biosensors are powerful tools for monitoring biochemical activities in live cells, but their multiplexing capacity is limited by the available spectral space. We overcome this problem by developing a set of barcoding proteins that can generate over 100 barcodes and are spectrally separable from commonly used biosensors. Mixtures of barcoded cells expressing different biosensors are simultaneously imaged and analyzed by deep learning models to achieve massively multiplexed tracking of signaling events. Importantly, different biosensors in cell mixtures show highly coordinated activities, thus facilitating the delineation of their temporal relationship. Simultaneous tracking of multiple biosensors in the receptor tyrosine kinase signaling network reveals distinct mechanisms of effector adaptation, cell autonomous and non-autonomous effects of KRAS mutations, as well as complex interactions in the network. Biosensor barcoding presents a scalable method to expand multiplexing capabilities for deciphering the complexity of signaling networks and their interactions between cells.

ASKA technology-based pull-down method reveals a suppressive effect of ASK1 on the inflammatory NOD-RIPK2 pathway in brown adipocytes.

Recent studies have shown that adipose tissue is an immunological organ. While inflammation in energy-storing white adipose tissues has been the focus of intense research, the regulatory mechanisms of inflammation in heat-producing brown adipose tissues remain largely unknown. We previously identified apoptosis signal-regulating kinase 1 (ASK1) as a critical regulator of brown adipocyte maturation; the PKA-ASK1-p38 axis facilitates uncoupling protein 1 (UCP1) induction cell-autonomously. Here, we show that ASK1 suppresses an innate immune pathway and contributes to maintenance of brown adipocytes. We report a novel chemical pull-down method for endogenous kinases using analog sensitive kinase allele (ASKA) technology and identify an ASK1 interactor in brown adipocytes, receptor-interacting serine/threonine-protein kinase 2 (RIPK2). ASK1 disrupts the RIPK2 signaling complex and inhibits the NOD-RIPK2 pathway to downregulate the production of inflammatory cytokines. As a potential biological significance, an in vitro model for intercellular regulation suggests that ASK1 facilitates the expression of UCP1 through the suppression of inflammatory cytokine production. In parallel to our previous report on the PKA-ASK1-p38 axis, our work raises the possibility of an auxiliary role of ASK1 in brown adipocyte maintenance through neutralizing the thermogenesis-suppressive effect of the NOD-RIPK2 pathway.

The mitochondrial Ca2+ uptake regulator, MICU1, is involved in cold stress-induced ferroptosis.

Ferroptosis has recently attracted much interest because of its relevance to human diseases such as cancer and ischemia-reperfusion injury. We have reported that prolonged severe cold stress induces lipid peroxidation-dependent ferroptosis, but the upstream mechanism remains unknown. Here, using genome-wide CRISPR screening, we found that a mitochondrial Ca2+ uptake regulator, mitochondrial calcium uptake 1 (MICU1), is required for generating lipid peroxide and subsequent ferroptosis under cold stress. Furthermore, the gatekeeping activity of MICU1 through mitochondrial calcium uniporter (MCU) is suggested to be indispensable for cold stress-induced ferroptosis. MICU1 is required for mitochondrial Ca2+ increase, hyperpolarization of the mitochondrial membrane potential (MMP), and subsequent lipid peroxidation under cold stress. Collectively, these findings suggest that the MICU1-dependent mitochondrial Ca2+ homeostasis-MMP hyperpolarization axis is involved in cold stress-induced lipid peroxidation and ferroptosis.

Cold stress-induced ferroptosis involves the ASK1-p38 pathway.

A wide variety of cell death mechanisms, such as ferroptosis, have been proposed in mammalian cells, and the classification of cell death attracts global attention because each type of cell death has the potential to play causative roles in specific diseases. However, the precise molecular mechanisms leading to cell death are poorly understood, particularly in ferroptosis. Here, we show that continuous severe cold stress induces ferroptosis and the ASK1-p38 MAPK pathway in multiple cell lines. The activation of the ASK1-p38 pathway is mediated by critical determinants of ferroptosis: MEK activity, iron ions, and lipid peroxide. The chemical compound erastin, a potent ferroptosis inducer, also activates the ASK1-p38 axis downstream of lipid peroxide accumulation and leads to ASK1-dependent cell death in a cell type-specific manner. These lines of evidence provide mechanistic insight into ferroptosis, a type of regulated necrosis.

Growth and size control in anti-solvent crystallization of glycine with high frequency ultrasound.

Antisolvent crystallization of glycine was performed under ultrasonic irradiation of 1.6 MHz. The irradiation enhanced both the growth of α-glycine crystal and the uniformity in the crystal size. The degree of both enhancement effects increased with increasing ultrasonic power. While under the irradiation of 20 kHz ultrasound, no growth enhancement was observed, but the crystal size reduced as was reported in the literature. To elucidate the mechanism of growth enhancement, another experiment was designed and conducted to avoid the effect of nucleation from the sonocrystallization. The result suggests that the ultrasound enhances the incorporation of microcrystals to larger crystals. Probably, the collision between solid particles is intensified by the disturbance characterized by the high frequency ultrasound. The crystal growth was modeled with an apparent reaction of microcrystal and larger crystal. The result of the growth experiment was successfully predicted with a rate equation for pseudo first order reaction with a single parameter of rate constant. The rate constant linearly increased with the ultrasonic power. The analysis enables quantitative evaluation of the ultrasonic effect on the crystal growth.