Imaging Complex Protein Metabolism in Live Organisms by Stimulated Raman Scattering Microscopy with Isotope Labeling

Protein metabolism, consisting of both synthesis and degradation, is highly complex, playing an indispensable regulatory role throughout physiological and pathological processes. Over recent decades, extensive efforts, using approaches such as autoradiography, mass spectrometry, and fluorescence microscopy, have been devoted to the study of protein metabolism. However, noninvasive and global visualization of protein metabolism has proven to be highly challenging, especially in live systems. Recently, stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (D-AAs) was demonstrated for use in imaging newly synthesized proteins in cultured cell lines. Herein, we significantly generalize this notion to develop a comprehensive labeling and imaging platform for live visualization of complex protein metabolism, including synthesis, degradation, and pulse–chase analysis of two temporally defined populations. First, the deuterium labeling efficiency was optimized, allowing time-lapse imaging of protein synthesis dynamics within individual live cells with high spatial–temporal resolution. Second, by tracking the methyl group (CH₃) distribution attributed to pre-existing proteins, this platform also enables us to map protein degradation inside live cells. Third, using two subsets of structurally and spectroscopically distinct D-AAs, we achieved two-color pulse–chase imaging, as demonstrated by observing aggregate formation of mutant hungtingtin proteins. Finally, going beyond simple cell lines, we demonstrated the imaging ability of protein synthesis in brain tissues, zebrafish, and mice <i>in vivo</i>. Hence, the presented labeling and imaging platform would be a valuable tool to study complex protein metabolism with high sensitivity, resolution, and biocompatibility for a broad spectrum of systems ranging from cells to model animals and possibly to humans

Correlative Genetic Variation in Natural Populations of Cats, Mice and Men

The study of the extent and basis of gene-enzyme variation has long been a principal concern of population genetics. Numerous surveys have indicated considerable amounts of genetic variation detectable in natural populations, with few exceptions. The variances of average heterozygosities (H) between species and among populations within species are large, prompting Lewontin to emphasize the importance of large gene sample sizes and Selander to encourage analysis of variation of homologous gene-enzyme systems when making species comparisons. We present here a comparative genetic analysis of electrophoretic variation at 57 homologous biochemical loci of cats, mice and men. The distribution of polymorphism among the sampled loci in the three species was nonrandom. A large group of sampled loci (60%) were monomorphic in all three species, whereas a second group (30%) of the loci were polymorphic in two or more species. This conservation of the tolerance of genetic polymorphism is apparently more a characteristic of a particular locus than of the vertebrate species or of the genome. The current hypotheses for classifying polymorphic and monomorphic loci in terms of physiological and physical enzyme characteristics have been re-examined