TY - JOUR
T1 - Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression
AU - Welsh, David K.
AU - Yoo, Seung Hee
AU - Liu, Andrew C.
AU - Takahashi, Joseph S.
AU - Kay, Steve A.
N1 - Funding Information:
We thank Kathy Spencer for expert microscopy support, Camilo Orozco for computer programming, Trey Sato for supplying the mBmal1::luc plasmid, Rie Yasuda for culturing primary fibroblasts, Hien Tran for earlier work on transfection of Rat-1 cells, and Tom Schultz for thoughtful comments on the manuscript. Supported in part by K08 MH067657 (D.K.W.) and MH51573 (S.A.K.). J.S.T. is an Investigator in the Howard Hughes Medical Institute.
PY - 2004/12/29
Y1 - 2004/12/29
N2 - Circadian (ca. 24 hr) oscillations in expression of mammalian "clock genes" are found not only in the suprachiasmatic nucleus (SCN), the central circadian pacemaker, but also in peripheral tissues [1]. Under constant conditions in vitro, however, rhythms of peripheral tissue explants [2] or immortalized cells [3] damp partially or completely. It is unknown whether this reflects an inability of peripheral cells to sustain rhythms, as SCN neurons can, or a loss of synchrony among cells. Using bioluminescence imaging of Rat-1 fibroblasts transfected with a Bmal1::luc plasmid and primary fibroblasts dissociated from mPer2Luciferase-SV40 knockin mice, we monitored single-cell circadian rhythms of clock gene expression for 1-2 weeks. We found that single fibroblasts can oscillate robustly and independently with undiminished amplitude and diverse circadian periods. Cells were partially synchronized by medium changes at the start of an experiment, but due to different intrinsic periods, their phases became randomly distributed after several days. Closely spaced cells in the same culture did not have similar phases, implying a lack of functional coupling among cells. Thus, like SCN neurons, single fibroblasts can function as independent circadian oscillators; however, lack of oscillator coupling in dissociated cell cultures leads to a loss of synchrony among individual cells and damping of the ensemble rhythm at the population level.
AB - Circadian (ca. 24 hr) oscillations in expression of mammalian "clock genes" are found not only in the suprachiasmatic nucleus (SCN), the central circadian pacemaker, but also in peripheral tissues [1]. Under constant conditions in vitro, however, rhythms of peripheral tissue explants [2] or immortalized cells [3] damp partially or completely. It is unknown whether this reflects an inability of peripheral cells to sustain rhythms, as SCN neurons can, or a loss of synchrony among cells. Using bioluminescence imaging of Rat-1 fibroblasts transfected with a Bmal1::luc plasmid and primary fibroblasts dissociated from mPer2Luciferase-SV40 knockin mice, we monitored single-cell circadian rhythms of clock gene expression for 1-2 weeks. We found that single fibroblasts can oscillate robustly and independently with undiminished amplitude and diverse circadian periods. Cells were partially synchronized by medium changes at the start of an experiment, but due to different intrinsic periods, their phases became randomly distributed after several days. Closely spaced cells in the same culture did not have similar phases, implying a lack of functional coupling among cells. Thus, like SCN neurons, single fibroblasts can function as independent circadian oscillators; however, lack of oscillator coupling in dissociated cell cultures leads to a loss of synchrony among individual cells and damping of the ensemble rhythm at the population level.
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U2 - 10.1016/j.cub.2004.11.057
DO - 10.1016/j.cub.2004.11.057
M3 - Article
C2 - 15620658
AN - SCOPUS:11144311974
SN - 0960-9822
VL - 14
SP - 2289
EP - 2295
JO - Current Biology
JF - Current Biology
IS - 24
ER -