Starvation Stimulation Research Articles

A new organic molecular probe as a powerful tool for fluorescence imaging and biological study of lipid droplets.

Background: Lipid droplets (LDs) are critical organelles associated with many physiological processes in eukaryotic cells. To visualize and study LDs, fluorescence imaging techniques including the confocal imaging as well as the emerging super-resolution imaging of stimulated emission depletion (STED), have been regarded as the most useful methods. However, directly limited by the availability of advanced LDs fluorescent probes, the performances of LDs fluorescence imaging are increasingly unsatisfied with respect to the fast research progress of LDs. Methods: We herein newly developed a superior LDs fluorescent probe named Lipi-QA as a powerful tool for LDs fluorescence imaging and biological study. Colocalization imaging of Lipi-QA and LDs fluorescent probe Ph-Red was conducted in four cell lines. The LDs staining selectivity and the photostability of Lipi-QA were also evaluated by comparing with the commercial LDs probe Nile Red. The in-situ fluorescence lifetime of Lipi-QA in LDs was determined by time-gated detection. The cytotoxicity of Lipi-QA was assessed by MTT assay. The STED saturation intensity as well as the power- and gate time-dependent resolution were tested by Leica SP8 STED super-resolution nanoscopy. The time-lapse 3D confocal imaging and time-lapse STED super-resolution imaging were then designed to study the complex physiological functions of LDs. Results: Featuring with the advantages of the super-photostability, high LDs selectivity, long fluorescence lifetime and low STED saturation intensity, the fluorescent probe Lipi-QA was capable of the long-term time-lapse three-dimensional (3D) confocal imaging to in-situ monitor LDs in 3D space and the time-lapse STED super-resolution imaging (up to 500 STED frames) to track the dynamics of LDs with nanoscale resolution (37 nm). Conclusions: Based on the state-of-the-art fluorescence imaging results, some new biological insights into LDs have been successfully provided. For instance, the long-term time-lapse 3D confocal imaging has surely answered an important and controversial question that the number of LDs would significantly decrease rather than increase upon starvation stimulation; the time-lapse STED super-resolution imaging with the highest resolution has impressively uncovered the fission process of nanoscale LDs for the first time; the starvation-induced change of LDs in size and in speed has been further revealed at nanoscale by the STED super-resolution imaging. All of these results not only highlight the utility of the newly developed fluorescent probe but also significantly promote the biological study of LDs.

Polysiloxane-based ratiometric fluorescent probe for monitoring lysosomal autophagy pathway in three distinguishable channels

Autophagy is an essential process for the degradation of malfunctional organelles and the response to cancer immunotherapy. The occurrence of defective autophagy is correlated to obesity, aging, neurological diseases, inflammation and tumors. Therefore, it is urgent to accurately depict the autophagic process. We herein architecture a polysiloxane-based probe (PA-1) that was lysosome targeted and pH-responsive for discerning autophagy process. We have demonstrated that PA-1 enabled specific visualization of lysosomal autophagy. PA-1 also detected the down-regulation of the pH value that accompanied the autophagy process under starvation stimulation. During autophagy, the green fluorescence of some spots in the lysosomes gradually decreased along with the emergence of strong agglomerated red fluorescence, which might be caused by the membrane fusion of lysosomes and autophagosomes. The location of the autolysosome could be precisely displayed by the ratio image. In addition, we observed that the autophagy inhibitor CQ could increase the pH of the intracellular environment and inhibit autophagy. The design strategy will broaden the research field of polysiloxane in cellular physiological processes and serves as a candidate for basic research on autophagic processes and effective therapeutic strategies for autophagy-related diseases.