Tilman Rosales

Direct measurement of association and dissociation rates of DNA binding in live cells by fluorescence correlation spectroscopy

Measurement of live-cell binding interactions is vital for understanding the biochemical reactions that drive cellular processes. Here, we develop, characterize, and apply a new procedure to extract information about binding to an immobile substrate from fluorescence correlation spectroscopy (FCS) autocorrelation data. We show that existing methods for analyzing such data by two-component diffusion fits can produce inaccurate estimates of diffusion constants and bound fractions, or even fail altogether to fit FCS binding data. By analyzing live-cell FCS measurements, we show that our new model can satisfactorily account for the binding interactions introduced by attaching a DNA binding domain to the dimerization domain derived from a site-specific transcription factor (the vitellogenin binding protein (VBP)). We find that our FCS estimates are quantitatively consistent with our fluorescence recovery after photobleaching (FRAP) measurements on the same VBP domains. However, due to the fast binding interactions introduced by the DNA binding domain, FCS generates independent estimates for the diffusion constant (6.7 +/- 2.4 microm2/s) and the association (2 +/- 1.2 s(-1)) and dissociation (19 +/- 7 s(-1)) rates, whereas FRAP produces only a single, but a consistent, estimate, the effective-diffusion constant (4.4 +/- 1.4 microm2/s), which depends on all three parameters. We apply this new FCS method to evaluate the efficacy of a potential anticancer drug that inhibits DNA binding of VBP in vitro and find that in vivo the drug inhibits DNA binding in only a subset of cells. In sum, we provide a straightforward approach to directly measure binding rates from FCS data.

Cross-Validating FRAP and FCS to Quantify the Impact of Photobleaching on In Vivo Binding Estimates

Binding can now be quantified in live cells, but the accuracy of such measurements remains uncertain. To address this uncertainty, we compare fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) measurements of the binding kinetics of a transcription factor, the glucocorticoid receptor, in the nuclei of live cells. We find that the binding residence time measured by FRAP is 15 times longer than that obtained by FCS. We show that this discrepancy is not likely due to the significant differences in concentrations typically used for FRAP and FCS, nor is it likely due to spatial heterogeneity of the nucleus, improper calibration of the FCS focal volume, or the intentional FRAP photobleach. Instead, our data indicate that photobleaching of bound molecules in FCS is mainly responsible. When this effect is minimized, FRAP and FCS measurements nearly agree, although cross-validation by other approaches is now required to rule out mutual errors. Our results demonstrate the necessity of a photobleach correction for FCS measurements of GFP-tagged molecules that are bound for >0.25 s, and represent an important step forward in establishing a gold standard for in vivo binding measurements.

Activation of moesin, a protein that links actin cytoskeleton to the plasma membrane, occurs by phosphatidylinositol 4,5-bisphosphate (PIP2) binding sequentially to two sites and releasing an autoinhibitory linker.

Background: Phosphatidylinositol 4,5-bisphosphate (PIP2) activates moesin via two binding sites whose roles are poorly understood. Results: Critical residues are identified in both sites and an inhibitory linker (FLAP) is characterized. Conclusion: Activation of moesin requires PIP2 binding to each site and release of the FLAP. Significance: The results fit a sequential activation model involving conformational change and interfacial release of FLAP. Many cellular processes depend on ERM (ezrin, moesin, and radixin) proteins mediating regulated linkage between plasma membrane and actin cytoskeleton. Although conformational activation of the ERM protein is mediated by the membrane PIP2, the known properties of the two described PIP2-binding sites do not explain activation. To elucidate the structural basis of possible mechanisms, we generated informative moesin mutations and tested three attributes: membrane localization of the expressed moesin, moesin binding to PIP2, and PIP2-induced release of moesin autoinhibition. The results demonstrate for the first time that the POCKET containing inositol 1,4,5-trisphosphate on crystal structure (the “POCKET” Lys-63, Lys-278 residues) mediates all three functions. Furthermore the second described PIP2-binding site (the “PATCH,” Lys-253/Lys-254, Lys-262/Lys-263) is also essential for all three functions. In native autoinhibited ERM proteins, the POCKET is a cavity masked by an acidic linker, which we designate the “FLAP.” Analysis of three mutant moesin constructs predicted to influence FLAP function demonstrated that the FLAP is a functional autoinhibitory region. Moreover, analysis of the cooperativity and stoichiometry demonstrate that the PATCH and POCKET do not bind PIP2 simultaneously. Based on our data and supporting published data, we propose a model of progressive activation of autoinhibited moesin by a single PIP2 molecule in the membrane. Initial transient binding of PIP2 to the PATCH initiates release of the FLAP, which enables transition of the same PIP2 molecule into the newly exposed POCKET where it binds stably and completes the conformational activation.

HIV-1 Nef Binds a Subpopulation of MHC-I throughout Its Trafficking Itinerary and Down-regulates MHC-I by Perturbing Both Anterograde and Retrograde Trafficking

The HIV protein Nef is thought to mediate immune evasion and promote viral persistence in part by down-regulating major histocompatibility complex class I protein (MHC-I or HLA-I) from the cell surface. Two different models have been proposed to explain this phenomenon as follows: 1) stimulation of MHC-I retrograde trafficking from and aberrant recycling to the plasma membrane, and 2) inhibition of anterograde trafficking of newly synthesized HLA-I from the endoplasmic reticulum to the plasma membrane. We show here that Nef simultaneously uses both mechanisms to down-regulate HLA-I in peripheral blood mononuclear cells or HeLa cells. Consistent with this, we found by using fluorescence correlation spectroscopy that a third of diffusing HLA-I at the endoplasmic reticulum, Golgi/trans-Golgi network, and the plasma membrane (PM) was associated with Nef. The binding of Nef was similarly avid for native HLA-I and recombinant HLA-I A2 at the PM. Nef binding to HLA-I at the PM was sensitive to specific inhibition of endocytosis. It was also attenuated by cyclodextrin disruption of PM lipid micro-domain architecture, a change that also retarded lateral diffusion and induced large clusters of HLA-I. In all, our data support a model for Nef down-regulation of HLA-I that involves both major trafficking itineraries and persistent protein-protein interactions throughout the cell.

Quantitative detection of the ligand-dependent interaction between the androgen receptor and the co-activator, Tif2, in live cells using two color, two photon fluorescence cross-correlation spectroscopy

Two-photon, two-color fluorescence cross-correlation spectroscopy (TPTCFCCS) was used to directly detect ligand-dependent interaction between an eCFP-fusion of the androgen receptor (eCFP-AR) and an eYFP fusion of the nuclear receptor co-activator, Tif2 (eYFP-Tif2) in live cells. As expected, these two proteins were co-localized in the nucleus in the presence of ligand. Analysis of the cross-correlation amplitude revealed that AR was on average 81% bound to Tif2 in the presence of agonist, whereas the fractional complex formation decreased to 56% in the presence of antagonist. Residual AR–Tif2 interaction in presence of antagonist is likely mediated by its ligand-independent activation function. These studies demonstrate that using TPTCFCCS it is possible to quantify ligand-dependent interaction of nuclear receptors with co-regulator partners in live cells, making possible a vast array of structure-function studies for these important transcriptional regulators.

A graphical user interface for BIOEQS: a program for simulating and analyzing complex biomolecular interactions.

BIOEQS is a global analysis and simulation program for complex biomolecular interaction data developed during the 1990s. Its continued usefulness derives from the fact that it is based on a numerical solver for complex coupled biological equilibria rather than on closed-form analytical equations for the binding isotherms. Therefore, it is quite versatile, allowing easy testing of multiple binding models and analysis of systems too complex for closed-form solutions. However, a major drawback to a generalized use of this program has been the lack of a graphical user interface (GUI) for setting up the binding models and experimental conditions as well as for visualizing the results. We present here a new GUI for BIOEQS that should be useful in both research and teaching applications.

FRET microscopy autologous tumor lysate processing in mature dendritic cell vaccine therapy

BackgroundAntigen processing by dendritic cells (DC) exposed to specific stimuli has been well characterized in biological studies. Nonetheless, the question of whether autologous whole tumor lysates (as used in clinical trials) are similarly processed by these cells has not yet been resolved.MethodsIn this study, we examined the transfer of peptides from whole tumor lysates to major histocompatibility complex class II molecules (MHC II) in mature dendritic cells (mDC) from a patient with advanced melanoma. Tumor antigenic peptides-MHC II proximity was revealed by Förster Resonance Energy Transfer (FRET) measurements, which effectively extends the application of fluorescence microscopy to the molecular level (<100Å). Tumor lysates were labelled with Alexa-488, as the donor, and mDC MHC II HLA-DR molecules were labelled with Alexa-546-conjugated IgG, as the acceptor.ResultsWe detected significant energy transfer between donor and acceptor-labelled antibodies against HLA-DR at the membrane surface of mDC. FRET data indicated that fluorescent peptide-loaded MHC II molecules start to accumulate on mDC membranes at 16 hr from the maturation stimulus, steeply increasing at 22 hr with sustained higher FRET detected up to 46 hr.ConclusionsThe results obtained imply that the patient mDC correctly processed the tumor specific antigens and their display on the mDC surface may be effective for several days. These observations support the rationale for immunogenic efficacy of autologous tumor lysates.

STAQ: A route toward low power, Multicolor nanoscopy

Nanoscopy has now become a real procedure in fluorescence microscopy of living cells. The STED/RESOLFT family of nanoscopy approaches has the best prospects for delivering high speed imaging, but the history of STED includes a continuing struggle to reduce the deactivation power applied, along with difficulties in achieving simultaneous multicolor images. In this manuscript, we present a concept for a similar real‐time nanoscopy, using a new class of bipartite probes that separate the luminescent and quenching functions into two coupled molecules. In particular, the STAQ (Superresolution via Transiently Activated Quencher) example we show herein employs the excited state absorbance (not ground state) of the partner to accept energy from and quench the luminescent dye. The result is that much less deactivation power is needed for superresolved (∼50 nm) imaging. Moreover, the TAQ partner excited by the “donut” beam is shown to quench several different visible dyes via the same mechanism, opening the door to easier multicolor imaging. We demonstrate three dyes sharing the same deactivation and show examples of superresolved multicolor images. We suggest STAQ will facilitate the growth of real‐time nanoscopy by reducing confounding photodamage within living cells while expanding the nanoscopists palette. Microsc. Res. Tech. 78:343–355, 2015. Published 2015. This article is a U.S. Government work and is in the public domain in the USA