The RHO1 signaling pathway acts in circadian clock neurons to control behavioral rhythms. Herman Wijnen^1,2,3, Neethi Rao³, Rachel Siegmund³, Laura Thomason³, Ariel Talts³, Emmanuel Anyetei-Anum³. 1) Centre for Biological Sciences, University of Southampton, Southampton, United Kingdom; 2) Institute for Life Sciences, University of Southampton, Southampton, United Kingdom; 3) Deparment of Biology, University of Virginia, Charlottesville, VA.

   Circadian clocks in animals involve feedback loops of gene expression that organize daily rhythms in physiology and behavior. It remains largely unclear, however, what signaling mechanisms link circadian gene expression to rhythmic behavior. As a result of a systematic genetic screen we discovered that components of the RHO signaling pathway that is known to regulate the actin cytoskeleton in many eukaryotes affect Drosophila circadian behavior . Although a functional RHO1 pathway is essential for basic cellular functions, we found that the observed circadian phenotypes were not generally associated with cell death, abnormal morphology, or developmental defects, but appeared to specifically affect the molecular clock circuits. Specifically, RHO1 signaling in the adult small ventral lateral (s-LN_v) clock neurons controls daily locomotor activity rhythms. Knockdown of the downstream effector ROK (RHO-KINASE) slows rhythms in the accumulation of the clock components TIMELESS (TIM) and PAR-DOMAIN PROTEIN 1 (PDP1) and selectively delays nuclear entry of PERIOD (PER) and TIM. ROK could act on the molecular circadian oscillator in the s-LN_v clock neurons cell-autonomously or via synaptic or peptidergic intercellular signaling. Experiments addressing the latter possibilities revealed that regulation of molecular oscillations by ROK in clock neurons does not require active neurotransmission or the neuropeptide PIGMENT-DISPERSING FACTOR (PDF). In addition, rhythms in the morphology of the s-LN_v dorsal projections are unaffected by rok knockdown. Taken together, these results suggest that conserved RHO-GTPase pathways acts in s-LN_v circadian pacemaker cells to link modulation of the actin cytoskeleton to molecular and behavioral circadian rhythms.