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 like Neurospora crassa. We employed novel microfluidic systems and a light-blind mutant that retains circadian function to simultaneously track multiple clock components in vivo across circadian cycles in Neurospora. Despite heterogeneity of clock gene (frq) expression, we find robust, synchronous cycles in FRQ nuclear localization among all nuclei and document free diffusion of multiple clock components among nuclei. Within nuclei, clock components form small, highly dynamic nuclear bodies that persist throughout the cycle and exhibit time-dependent changes in composition, including transient colocalization between the positive and negative components for circadian regulatory functions. This rich context of in vivo spatiotemporal information illustrates how dynamic subnuclear organization and internuclear exchange of clock proteins ensure synchronous regulation of cellular activities across a macroscopic, multinucleated syncytium.