Using enhanced number and brightness to measure protein oligomerization dynamics in live cells

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour

Hypoxia induces heart regeneration in adult mice

The adult mammalian heart is incapable of regeneration following cardiomyocyte loss, which underpins the lasting and severe effects of cardiomyopathy. Recently, it has become clear that the mammalian heart is not a post-mitotic organ. For example, the neonatal heart is capable of regenerating lost myocardium, and the adult heart is capable of modest self-renewal. In both of these scenarios, cardiomyocyte renewal occurs via the proliferation of pre-existing cardiomyocytes, and is regulated by aerobic-respiration-mediated oxidative DNA damage. Therefore, we reasoned that inhibiting aerobic respiration by inducing systemic hypoxaemia would alleviate oxidative DNA damage, thereby inducing cardiomyocyte proliferation in adult mammals. Here we report that, in mice, gradual exposure to severe systemic hypoxaemia, in which inspired oxygen is gradually decreased by 1% and maintained at 7% for 2 weeks, results in inhibition of oxidative metabolism, decreased reactive oxygen species production and oxidative DNA damage, and reactivation of cardiomyocyte mitosis. Notably, we find that exposure to hypoxaemia 1 week after induction of myocardial infarction induces a robust regenerative response with decreased myocardial fibrosis and improvement of left ventricular systolic function. Genetic fate-mapping analysis confirms that the newly formed myocardium is derived from pre-existing cardiomyocytes. These results demonstrate that the endogenous regenerative properties of the adult mammalian heart can be reactivated by exposure to gradual systemic hypoxaemia, and highlight the potential therapeutic role of hypoxia in regenerative medicine