Region-directed phototransfection reveals the functional significance of a dendritically synthesized transcription factor

Multiple nuclear transcription factors including E-26–like protein 1 (Elk-1) have been found in neuronal dendrites, yet the functional significance of such localization has not yet been explained. Here we use a focal transfection procedure, 'phototransfection', to introduce Elk1 mRNA into specific regions of live, intact primary rat neurons. Introduction and translation of Elk1 mRNA in dendrites produced cell death, whereas introduction and translation of Elk1 mRNA in cell bodies did not produce cell death. Elk-1 translated in dendrites was transported to the nucleus, and cell death depended upon transcription, supporting the dendritic imprinting hypothesis and highlighting the importance of the dendritic environment on protein function. Our demonstration of the utility of phototransfection for spatially controlled introduction of mRNAs opens the broader opportunity to use this method to introduce selected quantities of small molecules into discrete regions of live cells to assess their biological functions.

[1]  M. Besson,et al.  In Vivo Expression and Regulation of Elk-1, a Target of the Extracellular-Regulated Kinase Signaling Pathway, in the Adult Rat Brain , 1998, The Journal of Neuroscience.

[2]  J. Eberwine,et al.  Localization and translation of mRNA in dentrites and axons , 2001, Nature Reviews Neuroscience.

[3]  David Baltimore,et al.  Erratum: NF-κB functions in synaptic signaling and behavior (Nature Neuroscince (2003) 6 (1072-1078)) , 2003 .

[4]  O. Steward,et al.  Protein synthesis at synaptic sites on dendrites. , 2001, Annual review of neuroscience.

[5]  Karsten König,et al.  Femtosecond near-infrared laser pulses as a versatile non-invasive tool for intra-tissue nanoprocessing in plants without compromising viability. , 2002, The Plant journal : for cell and molecular biology.

[6]  S. Ochs Rate of fast axoplasmic transport in mammalian nerve fibres , 1972, The Journal of physiology.

[7]  Shuang Liu,et al.  c-Fos oncogene regulator Elk-1 interacts with BRCA1 splice variants BRCA1a/1b and enhances BRCA1a/1b-mediated growth suppression in breast cancer cells , 2001, Oncogene.

[8]  O. Steward,et al.  Inhibition of protein synthesis alters the subcellular distribution of mRNA in neurons but does not prevent dendritic transport of RNA. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[9]  M. Dichter,et al.  Electrophysiological comparison of pyramidal and stellate nonpyramidal neurons in dissociated cell culture of rat hippocampus , 1991, Brain Research Bulletin.

[10]  David Baltimore,et al.  NF-κB functions in synaptic signaling and behavior , 2003, Nature Neuroscience.

[11]  R. Hipskind,et al.  SUMOylation regulates nucleo-cytoplasmic shuttling of Elk-1 , 2004, The Journal of cell biology.

[12]  W. Abraham,et al.  Metabotropic Glutamate Receptors Trigger Homosynaptic Protein Synthesis to Prolong Long-Term Potentiation , 2000, The Journal of Neuroscience.

[13]  Christian T. A. Brown,et al.  Photoporation and cell transfection using a violet diode laser , 2005, 2005 Quantum Electronics and Laser Science Conference.

[14]  J. Eberwine,et al.  Identification of sites for exponential translation in living dendrites , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Alfred Nordheim,et al.  Elk-1 protein domains required for direct and SRF-assisted DNA-binding , 1992, Nucleic Acids Res..

[16]  Takeharu Nagai,et al.  Shift anticipated in DNA microarray market , 2002, Nature Biotechnology.

[17]  J. Eberwine,et al.  Transcription factors in dendrites: dendritic imprinting of the cellular nucleus. , 2001, Results and problems in cell differentiation.

[18]  A. Sharrocks The ETS-domain transcription factor family , 2001, Nature Reviews Molecular Cell Biology.

[19]  O. Steward,et al.  Dendritic transport: quantitative analysis of the time course of somatodendritic transport of recently synthesized RNA , 1990, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[20]  O. Steward,et al.  Selective dendritic transport of RNA in hippocampal neurons in culture , 1987, Nature.

[21]  R. Treisman,et al.  ERK activation induces phosphorylation of Elk-1 at multiple S/T-P motifs to high stoichiometry , 1999, Oncogene.

[22]  P. Crino,et al.  Stimulation of glutamate receptor protein synthesis and membrane insertion within isolated neuronal dendrites. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[23]  J. Eberwine,et al.  Local translation of classes of mRNAs that are targeted to neuronal dendrites , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[24]  K. Becker,et al.  Presence and phosphorylation of transcription factors in developing dendrites. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Erin M. Schuman,et al.  Dynamic Visualization of Local Protein Synthesis in Hippocampal Neurons , 2001, Neuron.

[26]  S. Ochs FAST AXOPLASMIC TRANSPORT OF MATERIALS IN MAMMALIAN NERVE AND ITS INTEGRATIVE ROLE * , 1972, Annals of the New York Academy of Sciences.

[27]  Andrew D Sharrocks Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture. , 2002, Biochemical Society transactions.

[28]  P. Crino,et al.  Molecular Characterization of the Dendritic Growth Cone: Regulated mRNA Transport and Local Protein Synthesis , 1996, Neuron.