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.

[1]  G. Borisy,et al.  Cell Migration: Integrating Signals from Front to Back , 2003, Science.

[2]  Diana P Bratu,et al.  Visualizing the distribution and transport of mRNAs in living cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[3]  R. Singer,et al.  Real-Time Visualization of ZBP1 Association with β-Actin mRNA during Transcription and Localization , 2003, Current Biology.

[4]  Gang Bao,et al.  Hybridization of 2'-O-methyl and 2'-deoxy molecular beacons to RNA and DNA targets. , 2002, Nucleic acids research.

[5]  N. Dias,et al.  Antisense oligonucleotides: basic concepts and mechanisms. , 2002, Molecular cancer therapeutics.

[6]  J. Condeelis,et al.  The physiological significance of β-actin mRNA localization in determining cell polarity and directional motility , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[7]  K Ribbeck,et al.  Kinetic analysis of translocation through nuclear pore complexes , 2001, The EMBO journal.

[8]  D. Weil,et al.  Mature mRNAs accumulated in the nucleus are neither the molecules in transit to the cytoplasm nor constitute a stockpile for gene expression. , 2000, RNA.

[9]  A. Tsuji,et al.  Direct observation of specific messenger RNA in a single living cell under a fluorescence microscope. , 2000, Biophysical journal.

[10]  T. Misteli,et al.  Nucleocytoplasmic shuttling: a novel in vivo property of antisense phosphorothioate oligodeoxynucleotides. , 2000, Nucleic acids research.

[11]  F. Kramer,et al.  Thermodynamic basis of the enhanced specificity of structured DNA probes. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[12]  R. Singer,et al.  Localization of ASH1 mRNA particles in living yeast. , 1998, Molecular cell.

[13]  A M Gewirtz,et al.  Real time detection of DNA.RNA hybridization in living cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[14]  R. Shoeman,et al.  Active nuclear import of single-stranded oligonucleotides and their complexes with non-karyophilic macromolecules. , 1998, Biology of the cell.

[15]  D. E. Wolf,et al.  Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[16]  R. Singer,et al.  Characterization of a beta-actin mRNA zipcode-binding protein , 1997, Molecular and cellular biology.

[17]  R. Singer,et al.  Actin messenger RNA localization and protein synthesis augment cell motility , 1997 .

[18]  I. Kill.  Localisation of the Ki-67 antigen within the nucleolus. Evidence for a fibrillarin-deficient region of the dense fibrillar component. , 1996, Journal of cell science.

[19]  Sanjay Tyagi,et al.  Molecular Beacons: Probes that Fluoresce upon Hybridization , 1996, Nature Biotechnology.

[20]  T. Mitchison,et al.  Actin-Based Cell Motility and Cell Locomotion , 1996, Cell.

[21]  R. Singer,et al.  Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype , 1994, The Journal of cell biology.

[22]  P. Gunning,et al.  Serum-induced signal transduction determines the peripheral location of beta-actin mRNA within the cell , 1994, The Journal of cell biology.

[23]  F. Szoka,et al.  Intracellular oligonucleotide hybridization detected by fluorescence resonance energy transfer (FRET). , 1994, Nucleic acids research.

[24]  Anne J. Ridley,et al.  The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors , 1992, Cell.

[25]  B. Lebleu,et al.  Intracellular distribution of microinjected antisense oligonucleotides. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[26]  M. Willingham,et al.  O-linked N-acetylglucosamine is attached to proteins of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins. , 1987, The Journal of biological chemistry.

[27]  J. Lawrence,et al.  Intracellular localization of messenger RNAs for cytoskeletal proteins , 1986, Cell.

[28]  M. Zasloff.  tRNA transport from the nucleus in a eukaryotic cell: carrier-mediated translocation process. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[29]  T. Pollard,et al.  Cellular Motility Driven by Assembly and Disassembly of Actin Filaments , 2003, Cell.

[30]  Frederick R Maxfield,et al.  Ratio imaging instrumentation. , 2003, Methods in cell biology.

[31]  H. Tanke,et al.  Methods for visualizing RNA processing and transport pathways in living cells , 2001, Histochemistry and Cell Biology.

[32]  Fred Russell Kramer,et al.  Multicolor molecular beacons for allele discrimination , 1998, Nature Biotechnology.

[33]  M. Dabauvalle,et al.  Inhibition of nuclear accumulation of karyophilic proteins in living cells by microinjection of the lectin wheat germ agglutinin. , 1988, Experimental cell research.