Determination of in Vivo Dissociation Constant, KD, of Cdc42-Effector Complexes in Live Mammalian Cells Using Single Wavelength Fluorescence Cross-correlation Spectroscopy

The RhoGTPase Cdc42 coordinates cell morphogenesis, cell cycle, and cell polarity decisions downstream of membrane-bound receptors through distinct effector pathways. Cdc42-effector protein interactions represent important elements of cell signaling pathways that regulate cell biology in systems as diverse as yeast and humans. To derive mechanistic insights into cell signaling pathways, it is vital that we generate quantitative data from in vivo systems. We need to be able to measure parameters such as protein concentrations, rates of diffusion, and dissociation constants (K_D) of protein-protein interactions in vivo. Here we show how single wavelength fluorescence cross-correlation spectroscopy in combination with Förster resonance energy transfer analysis can be used to determine K_D of Cdc42-effector interactions in live mammalian cells. Constructs encoding green fluorescent protein or monomeric red fluorescent protein fusion proteins of Cdc42, an effector domain (CRIB), and two effectors, neural Wiskott-Aldrich syndrome protein (N-WASP) and insulin receptor substrate protein (IRSp53), were expressed as pairs in Chinese hamster ovary cells, and concentrations of free protein as well as complexed protein were determined. The measured K_D for Cdc42V12-N-WASP, Cdc42V12-CRIB, and Cdc42V12-IRSp53 was 27, 250, and 391 nm, respectively. The determination of K_D for Cdc42-effector interactions opens the way to describe cell signaling pathways quantitatively in vivo in mammalian cells.Over the last 2 decades, we have been successful in describing a myriad of cell signaling pathways that regulate the biology of cells. These pathways are made of elements incorporating protein-protein, protein-lipid and protein-ligand interactions. With the advent of GFP² (1, 2) and its variants (3), it is now possible to genetically encode fluorescent probes into any protein of interest. GFP fusion proteins can be used in live cells giving spatial and temporal resolution to cell signaling pathways (4). To gain mechanistic insights into cellular processes, it is crucial that we measure quantitative parameters to describe cell signaling. In this study, we present an approach based on fluorescence cross-correlation spectroscopy (FCCS) (5, 6) and Förster resonance energy transfer (FRET) to determine quantitative parameters of cell signaling pathways, including the determination of the K_D for Cdc42-effector interactions in live CHO-K-1 (hereafter referred to as CHO) mammalian cells.The RhoGTPase Cdc42 (7, 8) regulates pathways that coordinate cell cycle, morphogenesis, and polarity. Cdc42 is a molecular switch that cycles between an inactive (GDP-bound) and active (GTP-bound) state. The V12 Cdc42 point mutation freezes the protein in an activated GTP-bound form, which binds effectors strongly. In contrast, Cdc42N17 is a dominant negative protein that is GDP-bound and interacts with effectors weakly if at all (9). A major Cdc42 binding site/domain in effector proteins is known as Cdc42- and Rac-interacting binding region (CRIB)³ and was originally found in activated Cdc42 kinase, p21 activated kinase (PAK), and neural Wiskott-Aldrich syndrome protein (N-WASP) (10). The inverse Bin-amphiphysins-Rvs domain adaptor protein IRSp53 is also an effector but binds Cdc42 through a partial CRIB domain (11, 12). Cdc42 interaction with its effectors has two main consequences, which are not mutually exclusive: (i) unfolding of effector to expose the active site and (ii) relocalization of effector to membrane compartments. Thus Cdc42-effector interactions serve as a good model for cell signaling as a whole.Fluorescence correlation spectroscopy and FCCS measure fluctuations in fluorescence of a small number of molecules as they pass through a defined confocal volume, respectively (13, 14, 15). Since the number of molecules in the confocal volume and the confocal volume itself can be determined, concentrations of protein can be measured by fluorescence correlation spectroscopy. Single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS) is an FCCS variant in which excitation of two or more probes is achieved by single wavelength one-photon excitation. To date SW-FCCS has been used successfully to follow receptors and receptor-ligand interactions in vitro and in vivo (6, 16, 17).In the present analysis, we take a two-step approach to determining the K_D of Cdc42 binding to CRIB (domain of PAK), N-WASP, and IRSp53. First, we show that the proteins under investigation are indeed interacting with each other directly in vivo by FRET analysis. Here we use acceptor photobleaching (AP)-FRET as well as changes in lifetime (through fluorescence lifetime imaging microscopy (FLIM)) as indicators of FRET. Second, we use SW-FCCS to determine the K_D of Cdc42 interacting with its effectors by measuring the concentration of free protein versus complexed protein. Thus, the combined use of FRET and FCCS allows quantitative analysis of cell signaling pathways in vivo.