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

Abstract

Chromophore-assisted laser inactivation (CALI) is a light-mediatedtechnique 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-CapZ␤ 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.capping protein ͉ Mena/VASP ͉ spatiotemporal ͉ lentivirus

CD2AP Links Cortactin and Capping Protein at the Cell Periphery To Facilitate Formation of Lamellipodia

“…Since CP is important in the formation of actinbased structures at the cell periphery (21,22), our initial experiments examined whether CD2AP plays a role in CP recruitment to the periphery of spreading podocyte cells. Using both confocal and TIRF microscopy, we localized CD2AP and CP during the initial phases of cell spreading.…”

New Gateways to Discovery

“…These novel AFPs, therefore, facilitate tracking experiments in which the movement of a tagged protein from one subcellular locus to another can be monitored following activation. A further innovation is that of chromophore assisted laser inactivation (Vitriol et al, 2007), which is a light-mediated technique that can be used for AFP-tagged protein inactivation. Incorporation of these new AFPs into the vector systems described below is under way, which should be of great benefit to plant biologists seeking precise spatiotemporal control of their AFP fusion.…”

Chromophore-assisted laser inactivation – towards a spatiotemporal–functional analysis of proteins, and the ablation of chromatin, organelle and cell function

“…To address this issue, rescue experiments using EGFP-fusion proteins can be used to evaluate the degree of loss-of-function due to CALI. In fact, they have shown that CALI is comparable with other conventional methods of abrogating protein function (Vitriol et al, 2007).…”

“…1, Table 1). Using genetically encoded photosensitizers, endogenous target proteins cannot be labeled and remain functional (Vitriol et al, 2007). Therefore, in combination with molecular genetic techniques, including RNA interference (RNAi) and genome editing, the pool of endogenous proteins should be eliminated.…”