Abstract
Since the first schematic illustrations of dividing cells, we have come a long way in characterising eukaryotic cells and defining their cell-cycle status thanks to a number of complementary approaches. Although most of these approaches rely on cell-fixation procedures to identify molecular components in cell lysates, cultured cells or tissues, the development of GFP technology has enabled visualisation of virtually any fusion protein in cellulo and in vivo, and the exploitation of functional elements with well-defined spatiotemporal characteristics has enabled the development of genetically encoded fluorescent markers of cell-cycle phases, thus providing novel means of characterising the status of living cells in real time with high resolution. Together with technological advances in fluorescence chemistry and imaging approaches, the more recent development of fluorescent biosensors has provided direct means of probing cell-cycle regulators and of studying their dynamics with high spatial and temporal resolution. Here we review classical approaches that rely on cell fixation to characterise the cell-cycle status and its regulatory enzymes, and we describe the more recent development of cell-cycle markers based on genetically encoded fusions of fluorescent proteins with characteristic cell-cycle features, and of fluorescent biosensor technology to probe cell-cycle regulators in living cells. Biosensors not only provide a means of characterising the behaviour of cell-cycle regulators in their natural environment, they are also very useful for comparative studies of biological processes in healthy and pathological conditions, and can be further applied to diagnostic approaches to assess the status of a specific target, and to monitor response to therapeutic intervention.