Subcellular functions of proteins under fluorescence single-cell microscopy

Abstract

A cell is a highly organized, dynamic, and intricate biological entity orchestrated by a myriad of proteins and their self-assemblies. Because a protein’s actions depend on its coordination in both space and time, our curiosity about protein functions has extended from the test tube into the intracellular space of the cell. Accordingly, modern technological developments and advances in enzymology have been geared towards analyzing protein functions within intact single cells. We discuss here how fluorescence single-cell microscopy has been employed to identify subcellular locations of proteins, detect reversible protein–protein interactions, and measure protein activity and kinetics in living cells. Considering that fluorescence single-cell microscopy has been only recently recognized as a primary technique in enzymology, its potentials and outcomes in studying intracellular protein functions are projected to be immensely useful and enlightening. We anticipate that this review would inspire many investigators to study their proteins of interest beyond the conventional boundary of specific disciplines. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.

A New View into the Regulation of Purine Metabolism: The Purinosome

“…The purinosome presents itself as a new level of metabolic enzyme organization and regulation in purine biosynthesis and is largely contributed to the advancements in in-cell enzymology by fluorescence microscopy [78]. The emergence of super-resolution imaging techniques such as Stochastic Optical Reconstruction Microscopy (STORM) in characterizing the subcellular localization of de novo purine biosynthetic enzymes has renewed the importance mitochondria play in the control of purine metabolism [61].…”

Ubiquitously Expressed Proteins and Restricted Phenotypes: Exploring Cell-Specific Sensitivities to Impaired tRNA Charging

“…Additionally, knockdown of CARS1 has been reported to inhibit ferroptosis induced by cysteine deprivation and to induce the transsulfuration pathway [98]; studies investigating components of these pathways in the environment of pathogenic CARS1 variants would be informative. To further investigate the two widely expressed CARS1 isoforms and their potential functions, subcellular fractionation [101] and fluorescence imaging [102] experiments would indicate if the isoforms have different subcellular localizations. Coimmunoprecipitation and mass spectrometry experiments [103] would also indicate if the isoforms have differential binding partners.…”

Quantitative Imaging of Biochemistry in Situ and at the Nanoscale

“…Organelle-targeted photoactivatable FPs provide information on their biogenesis and maturation, 44 , 45 but measures of absolute protein activity within organelles have not yet been achieved. 46 Most FPs are pH sensitive in the acidic regime and therefore have been used as effective reporters of acidic pH by utilizing a second fluorescent protein that is less sensitive to pH as a ratiometric signal. 47 Genetically encoded Ca 2+ indicators and genetically encoded voltage indicators have also been realized, but these cannot yet report on absolute values of calcium or membrane potential.…”