Brugia Malayi - Clark Science Center - Smith College

Circadian Synchronization in Per2:Luc Transgenic Mice Hepatocyte
Cultures
Lorna Pyle
The suprachiasmatic nucleus (SCN) of the anterior hypothalamus is known as the master pacemaker because of its control of
the endogenous rhythms of the mammalian circadian clock. The SCN coordinates the molecular clocks within every cell in the
body to maintain systemic synchronization. However, recent studies of the circadian rhythms of the liver counter this notion of
complete neural control of the peripheral oscillators. For instance, abnormal meal times phase-shifted the biological rhythms
of the liver without changing the rhythms of the SCN. 1 Additionally, in the absence of input from the SCN, the liver remains
rhythmic. 2 Whereas cultured fibroblasts lose their rhythmicity in a matter of days, hepatocytes, the primary cells of the liver, retain
robust rhythms in vitro for over 60 days. 3 Our research aims to show that hepatocytes synchronize to express robust circadian
rhythms without neural input in order to further understand the mechanism of circadian synchrony.
Hepatocytes are isolated from Period2: Luciferase (PER2:LUC) mice. In these transgenic mice, the expression of the clock
gene PER2 produces bioluminescence. 4 We capture the rhythmic bioluminescence of individual hepatocytes with a chilled
CCD camera. Due to construction, this study is still in its early stages. We are still in the process of acquiring data. This data
will then be quantified with MATLAB to determine the phase and period of each hepatocyte. We eventually will compare the
rhythms of neighboring hepatocytes. Localized synchronization will indicate whether the circadian rhythms of the liver can
remain synchronized through local communication and without the input from the SCN. (Supported by the National Science
Foundation)
Advisor: Mary Harrington
References:
1
Damiola F, Minh NL, Preitner N, Kornmann B, Fleury-Olela F, Schibler U (2001) Restricted feeding uncouples circadian oscillators in peripheral tissues from the
central pacemaker in the suprachiasmatic nucleus. Genes & Dev 14: 2950-2961.
2
Pezuk P, Mohawk J, Yoshikawa T, Sellix M, Menaker M (2010) Circadian organization is governed by extra-SCN pacemakers. J Biol Rhythms 25: 432-441.
3
Luitje M.E. 2011. Circadian rhythms and coupling in hepatocytes. Smith College Honors Thesis.
4
Yoo S-H, Yamazaki S, Lowrey PL, Shimomura K, Ko CH, Buhr ED, Siepka SM, Hong H-K, Oh WJ, Yoo OJ, Menaker M and Takahashi JS (2004)
PERIOD2:LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. PNAS 101:5339-5346.
Isolated PER2:LUC hepatocytes
(Nikon Eclipse Ti-U, 10X objective;
taken with the iKon-M Andor camera)
2012
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