Imaging native beta-actin mRNA in motile fibroblasts.
Nuclease-resistant, cytoplasmically resident molecular beacons were used to specifically label beta-actin mRNA in living and motile chicken embryonic fibroblasts. beta-actin mRNA signals were most abundant in active lamellipodia, which are protrusions that cells extend to adhere to surfaces. Time-lapse images show that the immediate sources of beta-actin mRNA for nascent lamellipodia are adjacent older protrusions. During the development of this method, we observed that conventional molecular beacons are rapidly sequestered in cell nuclei, leaving little time for them to find and bind to their cytoplasmic mRNA targets. By linking molecular beacons to a protein that tends to stay within the cytoplasm, nuclear sequestration was prevented, enabling cytoplasmic mRNAs to be detected and imaged. Probing beta-actin mRNA with these cytoplasmically resident molecular beacons did not affect the motility of the fibroblasts. Furthermore, mRNAs bound to these probes undergo translation within the cell. The use of cytoplasmically resident molecular beacons will enable further studies of the mechanism of beta-actin mRNA localization, and will be useful for understanding the dynamics of mRNA distribution in other living cells.
Tyagi
Sanjay
S
Department of Molecular Genetics, Public Health Research Institute, Newark, New Jersey 07103, USA. sanjay@phri.org
Alsmadi
Osama
O
D000199:Actins / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D002642:Chick Embryo; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015336:Molecular Probe Techniques; D015345:Oligonucleotide Probes / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism* / Q000648:ultrastructure
D000199:Actins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger