Dynamic monitoring and quantification of gene expression in single, living cells: a molecular basis for secretory cell heterogeneity.
Progress in understanding the dynamics of gene expression has been hampered by lack of a strategy for continuously monitoring this process within normal, living cells. Here, we employed a modification of conventional luciferase technology to make single and repeated real-time measurements of PRL gene expression from individual, living lactotropes from nursing rats. Cells were individually transfected by microinjection with a PRL promoter/luciferase reporter construct. Levels of PRL gene transcription were quantified by photonic imaging in the same cells before and after 24 h of culture in the presence or absence of the dopamine agonist bromocryptine or epidermal growth factor, two well known regulators of PRL gene transcription. We found these cells to be remarkably heterogeneous with respect to basal PRL gene expression and that the degree of activity within a single cell could fluctuate greatly over time under basal culture conditions. Treatment with bromocryptine or epidermal growth factor induced predictable and reversible changes in the average responses observed, yet individual cells displayed marked differences in response to these agents. These findings demonstrate the utility of this paradigm for monitoring dynamics of gene expression within normal, living cells of any type. Moreover, they provide a molecular basis for the secretory heterogeneity and plasticity that have come to be known as hallmarks of lactotrope cell function.
Castaño
J P
JP
Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston 29425, USA.
Kineman
R D
RD
Frawley
L S
LS
D000818:Animals; D001971:Bromocriptine / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter; D007774:Lactation; D008156:Luciferases / Q000235:genetics; D008845:Microinjections; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D001971:Bromocriptine; D004815:Epidermal Growth Factor; D011388:Prolactin; D008156:Luciferases