How cytoskeletal proteins regulate mitochondrial energetics in cell physiology and diseases.

Mitochondria are ubiquitous organelles that play a pivotal role in the supply of energy through the production of adenosine triphosphate in all eukaryotic cells. The importance of mitochondria in cells is demonstrated in the poor survival outcomes observed in patients with defects in mitochondrial gene or RNA expression. Studies have identified that mitochondria are influenced by the cell's cytoskeletal environment.

Restricting α-synuclein transport into mitochondria by inhibition of α-synuclein-VDAC complexation as a potential therapeutic target for Parkinson's disease treatment.

Involvement of alpha-synuclein (αSyn) in Parkinson's disease (PD) is complicated and difficult to trace on cellular and molecular levels. Recently, we established that αSyn can regulate mitochondrial function by voltage-activated complexation with the voltage-dependent anion channel (VDAC) on the mitochondrial outer membrane. When complexed with αSyn, the VDAC pore is partially blocked, reducing the transport of ATP/ADP and other metabolites.

VDAC regulation of mitochondrial calcium flux: From channel biophysics to disease.

Voltage-dependent anion channel (VDAC), the most abundant mitochondrial outer membrane protein, is important for a variety of mitochondrial functions including metabolite exchange, calcium transport, and apoptosis. While VDAC's role in shuttling metabolites between the cytosol and mitochondria is well established, there is a growing interest in understanding the mechanisms of its regulation of mitochondrial calcium transport. Here we review the current literature on VDAC's role in calcium signaling, its biophysical properties, physiological function, and pathology focusing on its importance in cardiac diseases.

Quantifying Acute Fuel and Respiration Dependent pH Homeostasis in Live Cells Using the mCherryTYG Mutant as a Fluorescence Lifetime Sensor.

Intracellular pH plays a key role in physiology, and its measurement in living specimens remains a crucial task in biology. Fluorescent protein-based pH sensors have gained widespread use, but there is limited spectral diversity for multicolor detection, and it remains a challenge to measure absolute pH values. Here we demonstrate that mCherryTYG is an excellent fluorescence lifetime pH sensor that significantly expands the modalities available for pH quantification in live cells.

Imaging pH Dynamics Simultaneously in Two Cellular Compartments Using a Ratiometric pH-Sensitive Mutant of mCherry.

The regulation of pH is essential for proper organelle function, and organelle-specific changes in pH often reflect the dynamics of physiological signaling and metabolism. For example, mitochondrial energy production depends on the proton gradient maintained between the alkaline mitochondrial matrix and neutral cytosol. However, we still lack a quantitative understanding of how pH dynamics are coupled between compartments and how pH gradients are regulated at organelle boundaries.

Imaging Adenosine Triphosphate (ATP).

Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provide valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells.

Evaluating the performance of time-gated live-cell microscopy with lanthanide probes.

Probes and biosensors that incorporate luminescent Tb(III) or Eu(III) complexes are promising for cellular imaging because time-gated microscopes can detect their long-lifetime (approximately milliseconds) emission without interference from short-lifetime (approximately nanoseconds) fluorescence background. Moreover, the discrete, narrow emission bands of Tb(III) complexes make them uniquely suited for multiplexed imaging applications because they can serve as Förster resonance energy transfer (FRET) donors to two or more differently colored acceptors. However, lanthanide complexes have low photon emission rates that can limit the image signal/noise ratio, which has a square-root dependence on photon counts.

Cytoplasmic delivery and selective, multicomponent labeling with oligoarginine-linked protein tags.

Strategies that leverage bio-orthogonal interactions between small molecule ligands and genetically encoded amino acid sequences can be used to attach high-performance fluorophores to proteins in living cells. However, a major limitation of chemical protein labeling is that cells' plasma membranes are impermeable to many useful probes and biolabels. Here, we show that conjugation to nonaarginine, a cell penetrating peptide (CPP), enables passive cytoplasmic delivery of otherwise membrane-impermeant, small molecule protein labels.

How to build a time-gated luminescence microscope.

The sensitivity of filter-based fluorescence microscopy techniques is limited by autofluorescence background. Time-gated detection is a practical way to suppress autofluorescence, enabling higher contrast and improved sensitivity. In the past few years, three groups of authors have demonstrated independent approaches to build robust versions of time-gated luminescence microscopes.

Lanthanide-based imaging of protein-protein interactions in live cells.

In order to deduce the molecular mechanisms of biological function, it is necessary to monitor changes in the subcellular location, activation, and interaction of proteins within living cells in real time. Förster resonance energy-transfer (FRET)-based biosensors that incorporate genetically encoded, fluorescent proteins permit high spatial resolution imaging of protein-protein interactions or protein conformational dynamics. However, a nonspecific fluorescence background often obscures small FRET signal changes, and intensity-based biosensor measurements require careful interpretation and several control experiments.

Cell-penetrating peptides as delivery vehicles for a protein-targeted terbium complex.

Release after transmission: Arginine-rich, cell-penetrating peptides (CPPs) mediate cytoplasmic delivery of trimethoprim (TMP)-terbium complex conjugates and selective, intracellular labeling of E. coli dihydrofolate reductase (eDHFR) fusion proteins. A disulfide bond linking CPP and cargo is reduced following uptake.

Applications of propargyl esters of amino acids in solution-phase Peptide synthesis.

Propargyl esters are employed as effective protecting groups for the carboxyl group during solution-phase peptide synthesis. The propargyl ester groups can be introduced onto free amino acids by treating them with propargyl alcohol saturated with HCl. The reaction between propargyl groups and tetrathiomolybdate is exploited to deblock the propargyl esters.