Immunocytochemical analysis of the circadian clock protein in mouse hepatocytes.
Many biochemical, physiological, and behavioral processes in organisms ranging from prokaryotes to humans exhibit circadian rhythms, defined as cyclic oscillations of about 24 hours. The mechanism of the cellular circadian clock relies on interlocking positive and negative transcriptional/translational feedback loops based on the regulated expression of several genes. Clock is one of these genes and its transcript, CLOCK protein, is a transcription factor belonging to the bHLH-PAS family. In mammals the clock gene is expressed in several tissues, including the liver. In the present study, we analyzed by means of quali-quantitative immunoelectron microscopy the fine intracellular distribution of the CLOCK protein in mouse hepatocytes during the daily cycle. We demonstrated that CLOCK protein is mostly located in the cell nucleus, where it accumulates on perichromatin fibrils, representing the in situ form of nascent pre-mRNA, while condensed chromatin and nucleoli contain lower amounts of protein. Moreover, we found that CLOCK protein shows circadian oscillations in these nuclear compartments, peaking in late afternoon. At this time the hepatic transcriptional rate reaches the maximal level, thus suggesting an important role of CLOCK protein in the regulation of liver gene expression.
Malatesta
Manuela
M
Istituto di Istologia ed Analisi di Laboratorio, University of Urbino, I-61029 Urbino, Italy. malatesta@uniurb.it
Baldelli
Beatrice
B
Marcheggiani
Francesco
F
Gazzanelli
Giancarlo
G
D000818:Animals; D056926:CLOCK Proteins; D002467:Cell Nucleus / Q000378:metabolism; D002940:Circadian Rhythm / Q000502:physiology*; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D022781:Hepatocytes / Q000502:physiology* / Q000648:ultrastructure; D007150:Immunohistochemistry; D051379:Mice; D016253:Microscopy, Immunoelectron; D015534:Trans-Activators / Q000096:biosynthesis*
D015534:Trans-Activators; D056926:CLOCK Proteins; C538943:CLOCK protein, human; C538944:Clock protein, mouse