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Protein sequences, directly translated from genomic data, need functional and structural annotation. Together with molecular function and biological process, subcellular localization is an important feature necessary for understanding the protein role and the compartment where the mature protein is active. In the case of mitochondrial proteins, their precursor sequences translated by the ribosome machinery include specific patterns from which it is possible not only to recognize their final destination within the organelle but also which of the mitochondrial subcompartments the protein is intended for. Four compartments are routinely discriminated, including the inner and the outer membranes, the intermembrane space, and the matrix. Here we discuss to which extent it is feasible to develop computational methods for detecting mitochondrial targeting peptides in the precursor sequence and to discriminate their final destination in the organelle. We benchmark two of our methods on the general task of recognizing human mitochondrial proteins endowed with an experimentally characterized targeting peptide (TPpred3) and predicting which submitochondrial compartment is the final destination (DeepMito). CDK activity We describe how to adopt our web servers in order to discriminate which human proteins are endowed with mitochondrial targeting peptides, the position of cleavage sites, and which submitochondrial compartment are intended for. By this, we add some other 1788 human proteins to the 450 ones already manually annotated in UniProt with a mitochondrial targeting peptide, providing for each of them also the characterization of the suborganellar localization.The cross talk between mitochondrial dynamic structure, determined primarily by mitochondrial fission and fusion events, and mitochondrial function of energetics, primarily ATP and ROS production, is widely appreciated. Understanding the mechanistic details of such cross talk between mitochondrial structure and function needs integrated quantitative analyses between mitochondrial dynamics and energetics. Here we describe our recently designed approach of mito-SinCe2 that involves high resolution confocal microscopy of genetically expressed ratiometric fluorescent probes targeted to mitochondria, and its quantitative analyses. Mito-SinCe2 analyses allows for quantitative analyses of mitochondrial structure-function relationship in single cells toward understanding the role of mitochondria and their heterogeneity in various physiological and pathological conditions.Time-resolved fluorescence spectrometry is a highly valuable technological tool to detect and characterize mitochondrial metabolic oxidative changes by means of endogenous fluorescence. Here, we describe detection and measurement of endogenous mitochondrial flavin fluorescence directly in living cardiac cells using fluorescence lifetime imaging microscopy (FLIM) after excitation with 473 nm picoseconds (ps) laser. Time-correlated single photon counting (TCSPC) method is employed.Kidneys are highly aerobic organs and their function is tightly coupled to mitochondrial energy production. Renal tubular cells, particularly the proximal tubule (PT), require an abundance of mitochondria to provide sufficient energy for regulating fluid and electrolyte balance. Meanwhile, mitochondrial defects are implicated in a range of different kidney diseases. Multiphoton microscopy (MP) is a powerful tool that allows detailed study of mitochondrial morphology, dynamics, and function in kidney tissue. Here, we describe how MP can be used to image mitochondria in kidney tubular cells, either ex vivo in tissue slices or in vivo in living rodents, using both endogenous and exogenous fluorescent molecules. Moreover, changes in mitochondrial signals can be followed in real time in response to different insults or stimuli, in parallel with other important readouts of kidney tubular function, such as solute uptake and trafficking.Untargeted lipidomics profiling by liquid chromatography -mass spectrometry (LC-MS) allows researchers to observe the occurrences of lipids in a biological sample without showing intentional bias to any specific class of lipids and allows retrospective reanalysis of data collected. Typically, and in the specific method described, a general extraction method followed by LC separation is used to achieve nonspecific class coverage of the lipidome prior to high resolution accurate mass (HRAM) MS detection . Here we describe a workflow including the isolation of mitochondria from liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for data acquisition. We also highlight how, in this method, all ion fragmentation can be used to identify species of lower abundances, often missed by data dependent fragmentation techniques. Here we describe the isolation of mitochondria from liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for data acquisition.In the last decades, membrane contact sites (MCSs) have been the object of intense investigation in different fields of cell physiology and pathology and their importance for the correct functioning of the cell is now widely recognized. MCS between any known intercellular organelles, including endoplasmic reticulum (ER), mitochondria, Golgi, endosomes, peroxisomes, lysosomes, lipid droplets, and the plasma membrane (PM), have been largely documented and in some cases the molecules responsible for the tethering also identified. They represent specific membrane hubs where a tightly coordinated exchange of ions, lipids, nutrients, and factors required to maintain proper cellular homeostasis takes place. Their delicate, dynamic, and sometimes elusive nature prevented and/or delayed the development of tools to easily image interorganelle proximity under physiological conditions and in living organisms. Nowadays, this aspect received great momentum due to the finding that MCSs’ dysregulation is involved in several pathological conditions. We have recently developed modular, split-GFP-based contact site sensors (SPLICS) engineered to fluoresce when homo- and heterotypic juxtapositions between ER and mitochondria occur over a range of specific distances. Here we describe in detail, by highlighting strengths and weaknesses, the use and the application of these novel genetically encoded SPLICS sensors and how to properly quantify short- and long-range ER-mitochondria interactions.