Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins

Eric A. Vitriol, Andrea C. Uetrecht, Feimo Shen, Ken Jacobson, James E. Bear

Research output: Contribution to journalArticlepeer-review

56 Scopus citations

Abstract

Chromophore-assisted laser inactivation (CALI) is a light-mediated technique that offers precise spatiotemporal control of protein inactivation, enabling better understanding of the protein's role in cell function. EGFP has been used effectively as a CALI chromophore, and its cotranslational attachment to the target protein avoids having to use exogenously added labeling reagents. A potential drawback to EGFP-CALI is that the CALI phenotype can be obscured by the endogenous, unlabeled protein that is not susceptible to light inactivation. Performing EGFP-CALI experiments in deficient cells rescued with functional EGFP-fusion proteins permits more complete loss of function to be achieved. Here, we present a modified lentiviral system for rapid and efficient generation of knockdown cell lines complemented with physiological levels of EGFP-fusion proteins. We demonstrate that CALI of EGFP-CapZβ increases uncapped actin filaments, resulting in enhanced filament growth and the formation of numerous protrusive structures. We show that these effects are completely dependent upon knocking down the endogenous protein. We also demonstrate that CALI of EGFP-Mena in Mena/VASP-deficient cells stabilizes lamellipodial protrusions.

Original languageEnglish (US)
Pages (from-to)6702-6707
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number16
DOIs
StatePublished - Apr 17 2007
Externally publishedYes

Keywords

  • Capping protein
  • Lentivirus
  • Mena/VASP
  • Spatiotemporal

ASJC Scopus subject areas

  • General

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