Intramolecular Spirocyclization Enables Design of a Single Fluorescent Probe for Monitoring the Interplay between Mitochondria and Lipid Droplets

The interplay between mitochondria and lipid droplets (LDs) plays a central role in regulating the β-oxidation and storage of fatty acids (FA) and is also engaged in responding to external stimuli such as nutrient deficiency. However, a single fluorescent probe enabling the discriminative and simultaneous visualization of the two organelles has not been reported yet, which brings limitation for the in-depth study on their interplay. In this work, utilizing the intramolecular spirocyclization reaction of rhodamine dyes that can dramatically change the optical and soluble properties, we have designed a new single fluorescent probe for labeling LDs and mitochondria in clearly separated dual-emission channels. The newly designed "biform" probe, MT-LD, presented in a ring-opened form in mitochondria to give a strong red emission, while it underwent the intramolecular spirocyclization reaction to target LDs showing an intense blue fluorescence. In this manner, MT-LD can label LDs and mitochondria in blue and red fluorescence, respectively. With this robust probe, the increase of mitochondria-LD contact and peridroplet mitochondria (PDM) amount during oleic acid treatment and starvation-induced autophagy has been successfully revealed. The interaction between the two organelles was also visualized in different tissues, which revealed an obviously higher level of mitochondria-LD contact and PDM amount in brown adipose tissue and lung tissue. This work provides a promising molecular tool to investigate the interplay between mitochondria and LDs and promotes studies on FA metabolism and autophagy.