Published papers

During myoblast differentiation, mitochondria undergo dynamic changes in their morphology and function. Although the mitochondrial membrane lipid environment is closely related to mitochondrial integrity, how mitochondrial lipid composition changes during myoblast differentiation and whether it is involved in efficient differentiation remains unclear. In this study, we applied LC-MS/MS-based untargeted lipidomics to the mitochondria isolated from C2C12 murine myoblasts and found that the proportion of linoleic acid (C18:2)containing cardiolipin (CL) increased during the early stages of differentiation. In parallel, the expression of tafazzin, a mitochondrial CL remodeling enzyme, increased in line with myoblast differentiation. Notably, the increase in C18:2-containing CL was not suppressed by the knockdown of MyoD (myoblast determination protein 1), a master transcription factor for myoblast differentiation. In contrast, the inhibition of CL biosynthesis and remodeling significantly suppressed differentiation progression, which was partially rescued by exogenous supplementation with C18:2. Similar trends in CL remodeling were observed when primary stem cells isolated from mouse skeletal muscle differentiated into myotubes. These results demonstrate that mitochondrial CL remodeling at an early stage is required to promote efficient myoblast differentiation.

Mitochondria adapt to different energetic demands reshaping their proteome. Mitochondrial proteases are emerging as key regulators of these adaptive processes. Here, we use a multiproteomic approach to demonstrate the regulation of the m-AAA protease AFG3L2 by the mitochondrial proton gradient, coupling mitochondrial protein turnover to the energetic status of mitochondria. We identify TMBIM5 (previously also known as GHITM or MICS1) as a Ca2+/H+ exchanger in the mitochondrial inner membrane, which binds to and inhibits the m-AAA protease. TMBIM5 ensures cell survival and respiration, allowing Ca2+ efflux from mitochondria and limiting mitochondrial hyperpolarization. Persistent hyperpolarization, however, triggers degradation of TMBIM5 and activation of the m-AAA protease. The m-AAA protease broadly remodels the mitochondrial proteome and mediates the proteolytic breakdown of respiratory complex I to confine ROS production and oxidative damage in hyperpolarized mitochondria. TMBIM5 thus integrates mitochondrial Ca2+ signaling and the energetic status of mitochondria with protein turnover rates to reshape the mitochondrial proteome and adjust the cellular metabolism.

The inner mitochondrial membrane (IMM) undergoes dynamic morphological changes, which are crucial for the maintenance of mitochondrial functions as well as cell survival. As the dynamics of the membrane are governed by its lipid components, a fluorescent probe that can sense spatiotemporal alterations in the lipid properties of the IMM over long periods of time is required to understand mitochondrial physiological functions in detail. Herein, we report a red-emissive IMM-labeling reagent with excellent photostability and sensitivity to its environment, which enables the visualization of the IMM ultrastructure using super-resolution microscopy as well as of the lipid heterogeneity based on the fluorescence lifetime at the single mitochondrion level. Combining the probe and fluorescence lifetime imaging microscopy (FLIM) showed that peroxidation of unsaturated lipids in the IMM by reactive oxygen species caused an increase in the membrane order, which took place prior to mitochondrial swelling.

Monogalactosyl diacylglycerol (MGDG) is a major membrane lipid component in plants and is crucial for proper thylakoid functioning. However, MGDG in mammals has not received much attention, partly because of its relative scarcity in mammalian tissues. In addition, the biosynthetic pathway of MGDG in mammals has not been thoroughly analysed, although some reports have suggested that UGT8, a ceramide galactosyltransferase, has the potential to catalyse MGDG biosynthesis. Here, we successfully captured the endogenous levels of MGDG in HeLa cells using liquid chromatography quadrupole time-of-flight mass spectrometry (LC–QTOF-MS)-based lipidomics. Cellular MGDG was completely depleted in CRISPR/Cas9-mediated UGT8 knockout (KO) HeLa cells. Transient overexpression of UGT8 enhanced MGDG production in HeLa cells, and the corresponding cell lysates displayed MGDG biosynthetic activity in vitro. Site-directed mutagenesis revealed that His358 within the UGT signature sequence was important for its activity. UGT8 was localized in the endoplasmic reticulum and activation of the unfolded protein response by membrane lipid saturation was impaired in UGT8 KO cells. These results demonstrate that UGT8 is an MGDG synthase in mammals and that UGT8 regulates membrane lipid saturation signals in cells.