RESUMEN
Many cellular processes are governed by molecular machineries that involve multiple protein interactions. However, visualizing and identifying multiprotein complexes such as ternary complexes inside cells is always challenging, particularly in the subdiffraction cellular space. Here, we developed a three-fragment fluorescence complementation system (TFFC) based on the splitting of a photoactivatable fluorescent protein, mIrisFP, for the imaging of ternary complexes inside living cells. Using a combination of TFFC and photoactivated localization microscopy (PALM), namely, the TFFC-PALM technique, we are able to identify the multi-interaction of a ternary complex with nanometer-level spatial resolution and single-molecule sensitivity. The TFFC-PALM system has been further applied to the analysis of the Gs ternary complex, which is composed of αs, ß1, and γ2 subunits, providing further insights into the subcellular localization and function of G protein subunits at the single-molecule level. The TFFC-PALM represents a valuable method for the visualization and identification of ternary complexes inside cells at the nanometer scale.
RESUMEN
Actin performs a wide variety of different tasks. This functional diversity may be accomplished either by the formation of different isotypes or by suitable protein decoration that regulates structure and dynamics of actin filaments. To probe for such a potential differential decoration, the actin-binding peptide Lifeact was fused to different photoactivatable fluorescent proteins. These fusions were stably expressed in Nicotiana tabacum L. cv. Bright Yellow 2 cells to follow dynamic reorganization of the actin cytoskeleton during the cell cycle. The Lifeact-monomeric variant of IrisFP fusion protein was observed to indiscriminately label both, central and cortical, actin filaments, whereas the tetrameric Lifeact-photoswitchable red fluorescent protein fusion construct selectively labeled only a specific perinuclear sub-population of actin. By using photoactivated localization microscopy, we acquired super-resolution images with optical sectioning to obtain a 3D model of perinuclear actin. This novel approach revealed that the perinuclear actin basket wraps around the nuclear envelope in a lamellar fashion and repartitions toward the leading edge of the migrating nucleus. Based on these data, we suggest that actin that forms the perinuclear basket differs from other actin assemblies by a reduced decoration with actin binding proteins, which is consistent with the differential decoration model.
