Core Clock Protein Subcellular Dynamics Coordinate Local and Global Circadian Control in Syncytia

Circadian rhythms are pervasive among eukaryotes, and the underlying clocks share a common regulatory architecture - a negative feedback loop. A wealth of genetic and biochemical data underpin current perceptions of circadian oscillators but aspects of their cell biology remain cryptic, especially in syncytial systems. We employed novel microfluidic systems and a blind mutant that retains circadian function to simultaneously track multiple clock components in vivo across circadian cycles, revealing remarkable subcellular and subnuclear dynamics of clock proteins and providing insights into spatiotemporal regulation in a multinucleated system. Despite heterogeneity of clock gene (frq) expression, we find robust cycles in FRQ nuclear localization among all nuclei and document free diffusion of multiple clock components among nuclei. Within nuclei, clock components form distinct, small, highly dynamic nuclear bodies that persist throughout the cycle, occasionally co-localizing for circadian regulatory functions. This rich context of in vivo spatiotemporal information illustrates how separate nuclear clocks ensure synchronous regulation of cellular activities across a macroscopic syncytium.