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Actin's Role in Signaling Light-induced Phase Shifts in the Mammalian Circadian Clock

出版	University of Illinois at Urbana-Champaign, 2012
URL	http://books.google.com.hk/books?id=Z5VMjwEACAAJ&hl=&source=gbs_api

註釋Based on responses to glutamate in vitro, I hypothesized that activation of the early night light signaling pathway results in a rapid and transient decrease in actin polymerization, which is required for the phase delay to occur. I first characterized actin state around a day-night cycle, in order to determine whether sensitivity to actin depolymerization changes over this period. To this end, I showed that polymerized actin peaks in early night, but this oscillation in actin state is abolished in mice rendered genetically arrhythmic, the BMAL1 -/- mice. Secondly, I measured actin state after light exposure, and found that brief light pulses induced a breakdown of polymerized actin. By injecting the actin-stabilizing chemical jasplakinolide directly into the mouse SCN, I demonstrated a requirement for actin breakdown in mediating the phase delay, while conversely I induced a delay by injecting the actin depolymerzing agent latrunculin A. Thirdly, I measured induction of potential downstream effectors upon actin depolymerization, to analyze the effect of actin dynamics on transcription. Breaking down the cytoskeleton with latrunculin A resulted in activation of the MAP kinase component PERK, as well as a potential activation of PCREB. The importance of actin dynamics on CRE-mediated transcription was demonstrated using electrophysiological recordings, in which inhibition of CRE-mediated transcription abolished the effects of latrunculin A on clock state. Finally, I showed that actin depolymerization induced the clock gene Period 2, providing a potential link between cytoplasmic signaling events that facilitate actin remodeling and nuclear mechanisms of transcription. This work, together with previous in vitro studies, places actin dynamics firmly within the light signaling pathway. This not only contributes to knowledge about circadian cell signaling, but provides a new important role for actin in mediating cellular physiological processes.