Expression of photoactivatable molecules enables FCS in live cells by controlling fluorescence intensity

Abstract

Cellular systems must act robustly to maintain organismal health, including maintaining biophysical properties that allow for appropriate cellular function, and adapting these properties through changes such as those that occur during cell division. However, we still lack tools to measure many of these physical properties with precision in the living cell. For example, the mechanical properties of the nucleoplasm, the fluid-like substance that fills the nucleus, have not been fully characterized. To investigate these properties, we have turned to the fission yeast Schizosaccharomyces pombe (S. pombe), a well-established, genetically tractable model organism that has been used extensively for studying a variety of cell biophysical processes and structures, including the cytoskeleton and cell division. It is an apt system for studying how the nucleus adapts over the course of the cell cycle, since it undergoes closed mitosis, where the nuclear envelope remains intact during cell division. Studying nucleoplasm properties over the course of closed mitosis may help reveal how nuclear volume, shape, surface area expansion, and chromosome segregation are linked and coordinated. To measure nucleoplasm material properties in S. pombe, we have paired Fluorescence Correlation Spectroscopy (FCS) with a photoswitchable fluorophore, enabling fine control over fluorescent intensity inside live cells. We infer material properties from FCS measurements, while the photoswitchable probe enables confocal imaging in conjunction with these measurements, yielding corresponding information about cellular state and dynamics. Interestingly, we find that nucleoplasm material properties do not vary significantly over the cell cycle. Future studies will use this tool to examine how diverse molecular and genetic perturbations alter nucleoplasmic properties, providing insight into how these properties maintain nuclear function and protect genomic integrity over the cell cycle and during development.