Horst Wallrabe

Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy

Advances in molecular biology provide various methods to define the structure and function of the individual proteins that form the component parts of subcellular structures. The ability to see the dynamic behavior of a specific protein inside the living cell became possible through the application of advanced fluorescence resonance energy transfer (FRET) microscope techniques. The fluorophore molecule used for FRET imaging has a characteristic absorption and emission spectrum that should be considered for characterizing the FRET signal. In this article we describe the system development for the image acquisition for one- and two-photon excitation FRET microscopy. We also describe the precision FRET (PFRET) data analysis algorithm that we developed to remove spectral bleed-through and variation in the fluorophore expression level (or concentration) for the donor and acceptor molecules. The acquired images have been processed using a PFRET algorithm to calculate the energy transfer efficiency and the distance between donor and acceptor molecules. We implemented the software correction to study the organization of the apical endosome in epithelial polarized MDCK cells and dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). For these proteins, the results revealed that the extent of correction affects the conventionally calculated energy transfer efficiency (E) and the distance (r) between donor and acceptor molecules by 38 and 9%, respectively.

IQGAP1 regulates cell motility by linking growth factor signaling to actin assembly

IQGAP1 has been implicated as a regulator of cell motility because its overexpression or underexpression stimulates or inhibits cell migration, respectively, but the underlying mechanisms are not well understood. Here, we present evidence that IQGAP1 stimulates branched actin filament assembly, which provides the force for lamellipodial protrusion, and that this function of IQGAP1 is regulated by binding of type 2 fibroblast growth factor (FGF2) to a cognate receptor, FGFR1. Stimulation of serum-starved MDBK cells with FGF2 promoted IQGAP1-dependent lamellipodial protrusion and cell migration, and intracellular associations of IQGAP1 with FGFR1 – and two other factors – the Arp2/3 complex and its activator N-WASP, that coordinately promote nucleation of branched actin filament networks. FGF2 also induced recruitment of IQGAP1, FGFR1, N-WASP and Arp2/3 complex to lamellipodia. N-WASP was also required for FGF2-stimulated migration of MDBK cells. In vitro, IQGAP1 bound directly to the cytoplasmic tail of FGFR1 and to N-WASP, and stimulated branched actin filament nucleation in the presence of N-WASP and the Arp2/3 complex. Based on these observations, we conclude that IQGAP1 links FGF2 signaling to Arp2/3 complex-dependent actin assembly by serving as a binding partner for FGFR1 and as an activator of N-WASP.

FRET Microscopy in 2010: The Legacy of Theodor Förster on the 100th Anniversary of his Birth

Theodor Förster would have been 100 years old this year, and he would have been astounded to see the impact of his scientific achievement, which is still evolving. Combining his quantitative approach of (Förster) resonance energy transfer (FRET) with state-of-the-art digital imaging techniques allows scientists to breach the resolution limits of light (ca. 200 nm) in light microscopy. The ability to deduce molecular or particle distances within a range of 1-10 nm in real time and to prove or disprove interactions between two or more components is of vital interest to researchers in many branches of science. While Försters groundbreaking theory was published in the 1940s, the availability of suitable fluorophores, instruments, and analytical tools spawned numerous experiments in the last 20 years, as demonstrated by the exponential increase in publications. These cover basic investigation of cellular processes and the ability to investigate them when they go awry in pathological states, the dynamics involved in genetics, and following events in environmental sciences and methods in drug screening. This review covers the essentials of Theodor Försters theory, describes the elements for successful implementation of FRET microscopy, the challenges and how to overcome them, and a leading-edge example of how Försters scientific impact is still evolving in many directions. While this review cannot possibly do justice to the burgeoning field of FRET microscopy, a few interesting applications such as threecolor FRET, which greatly expands the opportunities for investigating interactions of cellular components compared with the traditional two-color method, are described, and an extensive list of references is provided for the interested reader to access.

Confocal FRET Microscopy to Measure Clustering of Ligand-Receptor Complexes in Endocytic Membranes

The dynamics of protein distribution in endocytic membranes are relevant for many cellular processes, such as protein sorting, organelle and membrane microdomain biogenesis, protein-protein interactions, receptor function, and signal transduction. We have developed an assay based on Fluorescence Resonance Energy Microscopy (FRET) and novel mathematical models to differentiate between clustered and random distributions of fluorophore-bound molecules on the basis of the dependence of FRET intensity on donor and acceptor concentrations. The models are tailored to extended clusters, which may be tightly packed, and account for geometric exclusion effects between membrane-bound proteins. Two main criteria are used to show that labeled polymeric IgA-ligand-receptor complexes are organized in clusters within apical endocytic membranes of polarized MDCK cells: 1), energy transfer efficiency (E%) levels are independent of acceptor levels; and 2), with increasing unquenched donor: acceptor ratio, E% decreases. A quantitative analysis of cluster density indicates that a donor-labeled ligand-receptor complex should have 2.5-3 labeled complexes in its immediate neighborhood and that clustering may occur at a limited number of discrete membrane locations and/or require a specific protein that can be saturated. Here, we present a new sensitive FRET-based method to quantify the co-localization and distribution of ligand-receptor complexes in apical endocytic membranes of polarized cells.

Chapter 22: Quantitation of protein-protein interactions: confocal FRET microscopy.

Förster resonance energy transfer (FRET) is an effective and high resolution method to monitor protein-protein interactions in live or fixed specimens. FRET can be used to estimate the distance between interacting protein molecules in vivo or in vitro using laser-scanning confocal FRET microscopy. The spectral overlap of donor and acceptor-essential for FRET-also generates a contamination of the FRET signal, which should be removed in order to carry out quantitative data analysis with confidence. Quantitative FRET data analysis addresses the wealth of information contained in the data set, once optimized FRET imaging has been completed. In this chapter, we describe step-by-step what the issues are in quantitative FRET data analysis, using membrane receptor trafficking and organization as an example. The assays described are applicable to many other biological applications.

Three-Color Spectral FRET Microscopy Localizes Three Interacting Proteins in Living Cells

FRET technologies are now routinely used to establish the spatial relationships between two cellular components (A and B). Adding a third target component (C) increases the complexity of the analysis between interactions AB/BC/AC. Here, we describe a novel method for analyzing a three-color (ABC) FRET system called three-color spectral FRET (3sFRET) microscopy, which is fully corrected for spectral bleedthrough. The approach quantifies FRET signals and calculates the apparent energy transfer efficiencies (Es). The method was validated by measurement of a genetic (FRET standard) construct consisting of three different fluorescent proteins (FPs), mTFP, mVenus, and tdTomato, linked sequentially to one another. In addition, three 2-FP reference constructs, tethered in the same way as the 3-FP construct, were used to characterize the energy transfer pathways. Fluorescence lifetime measurements were employed to compare the relative relationships between the FPs in cells producing the 3-FP and 2-FP fusion proteins. The 3sFRET microscopy method was then applied to study the interactions of the dimeric transcription factor C/EBPalpha (expressing mTFP or mVenus) with the heterochromatin protein 1alpha (HP1alpha, expressing tdTomato) in live-mouse pituitary cells. We show how the 3sFRET microscopy method represents a promising live-cell imaging technique to monitor the interactions between three labeled cellular components.

One- and two-photon fluorescence resonance energy transfer microscopy to establish a clustered distribution of receptor-ligand complexes in endocytic membranes

One- and two-photon fluorescence resonance energy transfer (FRET) microscopy, using different bandwidth emission filters and a novel spectral spillover correction algorithm (PFRET algorithm), provides the basis for a quantitative approach to measure receptor clustering in endocytic membranes. Emission filters with wider bandwidth allow for an increased FRET signal and corresponding spillover. Treatment with the PFRET correction algorithm results in increasing correction levels and comparable energy transfer efficiency (E%) values, thus validating our algorithm-based approach. The relationship between E% and acceptor and donor levels and donor:acceptor (D:A) ratio is used to characterize the distribution of receptor-ligand complexes in endocytic membranes. In addition to the standard test for clustering (E%s independence from acceptor levels), we describe a second parameter: the negative dependence of E% on increasing donor levels and D:A ratio. A donor geometric exclusion hypothesis is proposed to explain this phenomenon. One- and two-photon FRET microscopy assays show that polymeric IgA-receptor-ligand complexes are organized in clusters within apical endocytic membranes of polarized Madin-Darby canine kidney cells.

S-Nitrosoglutathione Reductase in Human Lung Cancer

S-Nitrosoglutathione (GSNO) reductase regulates cell signaling pathways relevant to asthma and protects cells from nitrosative stress. Recent evidence suggests that this enzyme may prevent human hepatocellular carcinoma arising in the setting of chronic hepatitis. We hypothesized that GSNO reductase may also protect the lung against potentially carcinogenic reactions associated with nitrosative stress. We report that wild-type Ras is S-nitrosylated and activated by nitrosative stress and that it is denitrosylated by GSNO reductase. In human lung cancer, the activity and expression of GSNO reductase are decreased. Further, the distribution of the enzyme (including its colocalization with wild-type Ras) is abnormal. We conclude that decreased activity of GSNO reductase could leave the human lung vulnerable to the oncogenic effects of nitrosative stress, as is the case in the liver. This potential should be considered when developing therapies that inhibit pulmonary GSNO reductase to treat asthma and other conditions.

Myosin-Va-Dependent Cell-To-Cell Transfer of RNA from Schwann Cells to Axons

To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

mTOR and neuronal cell cycle reentry: How impaired brain insulin signaling promotes Alzheimer's disease.

A major obstacle to presymptomatic diagnosis and disease‐modifying therapy for Alzheimers disease (AD) is inadequate understanding of molecular mechanisms of AD pathogenesis. For example, impaired brain insulin signaling is an AD hallmark, but whether and how it might contribute to the synaptic dysfunction and neuron death that underlie memory and cognitive impairment has been mysterious. Neuron death in AD is often caused by cell cycle reentry (CCR) mediated by amyloid‐β oligomers (AβOs) and tau, the precursors of plaques and tangles. We now report that CCR results from AβO‐induced activation of the protein kinase complex, mTORC1, at the plasma membrane and mTORC1‐dependent tau phosphorylation, and that CCR can be prevented by insulin‐stimulated activation of lysosomal mTORC1. AβOs were also shown previously to reduce neuronal insulin signaling. Our data therefore indicate that the decreased insulin signaling provoked by AβOs unleashes their toxic potential to cause neuronal CCR, and by extension, neuron death.