A novel super-resolution STED microscopy analysis approach to observe spatial MCU and MICU1 distribution dynamics in cells.

The uptake of Ca by mitochondria is an important and tightly controlled process in various tissues. Even small changes in the key proteins involved in this process can lead to significant cellular dysfunction and, ultimately, cell death. In this study, we used stimulated emission depletion (STED) microscopy and developed an unbiased approach to monitor the sub-mitochondrial distribution and dynamics of the mitochondrial calcium uniporter (MCU) and mitochondrial calcium uptake 1 (MICU1) under resting and stimulated conditions.

Visualizing VDAC1 in live cells using a tetracysteine tag.

The voltage-dependent anion channel 1 (VDAC1) is a crucial gatekeeper in the outer mitochondrial membrane, controlling metabolic and energy homeostasis. The available methodological approaches fell short of accurate visualization of VDAC1 in living cells. To permit precise VDAC1 imaging, we utilized the tetracysteine (TC)-tag and visualized VDAC1 dynamics in living cells.

Development of a Dual Reporter System to Simultaneously Visualize Ca Signals and AMPK Activity.

In this study, we introduce a new separation of phases-based activity reporter of kinase (SPARK) for AMP-activated kinase (AMPK), named AMPK-SPARK, which reports the AMPK activation by forming bright fluorescent clusters. Furthermore, we introduce a dual reporter system, named GCaMP-AMPK-SPARK, by incorporating a single-fluorescent protein (FP)-based Ca biosensor, GCaMP6f, into our initial design, enabling simultaneous monitoring of Ca levels and AMPK activity. This system offers the essential quality of information by single-channel fluorescence microscopy without the need for coexpression of different biosensors and elaborate filter layouts to overcome spectral limitations.

Hexokinase 1 forms rings that regulate mitochondrial fission during energy stress.

Metabolic enzymes can adapt during energy stress, but the consequences of these adaptations remain understudied. Here, we discovered that hexokinase 1 (HK1), a key glycolytic enzyme, forms rings around mitochondria during energy stress. These HK1-rings constrict mitochondria at contact sites with the endoplasmic reticulum (ER) and mitochondrial dynamics protein (MiD51).

Implications of mitochondrial membrane potential gradients on signaling and ATP production analyzed by correlative multi-parameter microscopy.

The complex architecture and biochemistry of the inner mitochondrial membrane generate ultra-structures with different phospholipid and protein compositions, shapes, characteristics, and functions. The crista junction (CJ) serves as an important barrier separating the cristae (CM) and inner boundary membranes (IBM). Thereby CJ regulates the movement of ions and ensures distinct electrical potentials across the cristae (ΔΨ) and inner boundary (ΔΨ) membranes.