Ting, K. K. Y. | Search bioRxiv

bioRxiv2026

Apollo-IRE1: A Genetically Encoded Sensor for Live Cell and Multiplexed Imaging of ER Stress

Pancreatic beta cells face exceptional protein folding demands from high insulin production requirements, placing extraordinary stress on the ER and contributing to dysfunction in diabetes pathogenesis. Monitoring ER stress dynamics in living cells remains challenging due to the destructive nature of traditional biochemical methods and the limitations of existing fluorescent sensors. Here, we present Apollo-IRE1, a genetically encoded sensor that reports on stress-induced IRE1 oligomerization and associated change in homoFRET via changes in fluorescence anisotropy. Apollo-IRE1 provides a ratiometric, intensity-independent readout, resulting in low day-to-day variability and a minimal spectral bandwidth, enabling multiplexed imaging alongside other cellular parameters. Photobleaching and enhancement curve analysis show that Apollo-IRE1 exists in apparent monomeric, dimeric, and oligomeric states corresponding to baseline, moderate, and terminal ER stress conditions. The sensor also responds rapidly to chemical and physiological ER stressors in both immortalized beta-cell lines and primary mouse islet cells. These data establish Apollo-IRE1 as a practical tool for investigating ER stress dynamics in beta cells and other contexts where longitudinal single-cell measurements are essential.

bioengineering↗

bioRxiv2024

Cholesterol accumulation-induced impairment of AKT signaling in LPS-stimulated macrophages play a dispensable role in suppressing HIF-1α-dependent glycolysis

The formation of lipid-laden macrophages (M{varphi}s) is a hallmark of atherosclerosis, yet how the accumulation of cholesterol in M{varphi}s underlies the inflammatory process of atherogenesis remains unclear. It is well recognized that the reprogramming of metabolism in M{varphi}s is critical for supporting their inflammatory responses, which may shed light on how the metabolism of M{varphi} foam cells is linked to inflammation. Indeed, recent research has now revealed M{varphi}s that accumulate excess cholesterol adopt a distinct metabolic adaptation, a metabolic profile that is unexpectedly associated with a deactivated inflammatory response. Mechanistically, our group has previously shown that upon LPS stimulation, excess cholesterol accumulation in M{varphi}s impaired their induction of AKT-dependent early glycolytic reprogramming and HIF-1-dependent late glycolytic reprogramming. However, it remains unclear if these events are interconnected and synergistically contribute to the suppression of inflammation observed in these M{varphi}s. Here, we demonstrated that cholesterol loading of M{varphi}s impaired LPS-induced early glycolysis by reducing the phosphorylation of hexokinases, yet complete inhibition of AKT only modestly impaired HIF-1-dependent glycolytic reprogramming. On the other hand, we confirmed that HIF-1 degradation, but not its reduced synthesis, is the primary mechanism that underlies its impaired expression in cholesterol loaded M{varphi}s. Finally, we showed that cholesterol loading of M{varphi}s alone was sufficient to induce oxidative stress, such as the production of 4-HNE, and deplete the levels of reduced KEAP1 proteins. M{varphi}s lacking NRF2 resisted the effects of cholesterol loading on suppressing the expression of glycolytic and pro-inflammatory proteins.

immunology↗