细胞生物学

Mitophagy is a highly conserved process among eukaryotic cells, playing a primordial role in mitochondrial quality control and overall cellular homeostasis. In Saccharomyces cerevisiae, Atg32 is the only identified mitophagy receptor localized to the mitochondrial outer membrane, making this yeast a particularly powerful model for molecular studies of mitophagy that require the isolation of intact mitochondria. However, traditional methods for isolating mitochondria from yeast often rely on enzymatic cell wall digestion and homogenization, which can compromise the stability of mitochondrial surface proteins such as Atg32. In this protocol, we describe an optimized mechanical approach for yeast cell disruption using glass beads in a cold, protease-inhibited buffer to preserve mitochondrial integrity and facilitate the detection of Atg32. Subsequent differential centrifugation and washing steps yield mitochondrial fractions suitable for downstream biochemical analyses. This workflow eliminates enzymatic digestion steps, reduces sample variability, and allows parallel processing of multiple strains or experimental conditions. Overall, this method offers a rapid, low-cost, and reproducible alternative for crude mitochondrial isolation, ensuring excellent preservation of Atg32 and broad compatibility with quantitative and comparative studies.

Ultra-precise stimulation solely to individual mitochondria, without any influence to the whole cell, is quite difficult by traditional biochemical reagents. In mitophagy research, the mitochondria and even the whole cell usually suffer irreversible and great damage caused by treatment with potent chemicals. In this protocol, we present the technical procedures of our developed noninvasive ultra-precise laser stimulation (UPLaS) technology, which introduces precise stimulation to individual mitochondria, to excite mitochondrial Ca²⁺ (mitoCa²⁺) oscillations, with little perturbation to mitochondrial membrane potential (MMP), or mitochondrial reactive oxygen species (mitoROS). The mitoCa²⁺ oscillation by UPLaS was able to initiate the PINK1/Parkin pathway for mitophagy. This protocol has good potential to benefit researches on mitophagy and mitochondrial diseases.

Graphic abstract:

Figure 1. Flowchart of the UPLaS technology. The femtosecond laser (1030 nm, 1 MHz, 220 fs) can stimulate individual mitochondria (1 μm²) for a short period (0.1 s), whereas confocal microscopy (CM) provides continuous cell imaging to monitor molecular dynamics in real time, before and after UPLaS.

Autophagy is a dynamic cellular event that is involved in the degradation of long lived proteins and organelles in cells. Biochemical methods such as western blot to measure autophagic proteins have been increasingly used in autophagy studies because it is convenient and objective. Among them, the total amount of Microtubule-associated protein light chain 3 (LC3), the mammalian homologue of the autophagy-related Atg8 in yeast, is a very useful and most commonly used tool in autophagy studies. Other methods such as electron microscopy and immunofluorescence are also available to measure autophagy. The following protocol describes three simple and commonly used protocols for measuring autophagy in cells: LC3B immunofluorescence, western blot and acridine orange assay. Although these three methods are frequently used to provide basic information about autophagy, people should keep in mind that they are not enough to give the exact details about the autophagy flux due to the complexity of this dynamic process. In addition, acridine orange assay is only a supplementary method to detect autophagy because it also has high affinity to other organelles such as lysosomes. For further investigation about a compound’s effect on autophagy flux, additional and more complicated assays are recommended. Protocols here provide a starting point for people to get a snapshot of whether a compound can affect autophagy in tissue culture cells.