Fluorescence Spectroscopy of Low-Level Endogenous ß-Adrenergic Receptor Expression at the Plasma Membrane of Differentiating Human iPSC-Derived Cardiomyocytes.

The potential of human-induced pluripotent stem cells (hiPSCs) to be differentiated into cardiomyocytes (CMs) mimicking adult CMs functional morphology, marker genes and signaling characteristics has been investigated since over a decade. The evolution of the membrane localization of CM-specific G protein-coupled receptors throughout differentiation has received, however, only limited attention to date. We employ here advanced fluorescent spectroscopy, namely linescan Fluorescence Correlation Spectroscopy (FCS), to observe how the plasma membrane abundance of the ß1- and ß2-adrenergic receptors (ß1/2-ARs), labelled using a bright and photostable fluorescent antagonist, evolves during the long-term monolayer culture of hiPSC-derived CMs. We compare it to the kinetics of observed mRNA levels in wildtype (WT) hiPSCs and in two CRISPR/Cas9 knock-in clones. We conduct these observations against the backdrop of our recent report of cell-to-cell expression variability, as well as of the subcellular localization heterogeneity of ß-ARs in adult CMs.

Superresolution microscopy localizes endogenous Dvl2 to Wnt signaling-responsive biomolecular condensates.

During organismal development, homeostasis, and disease, Dishevelled (Dvl) proteins act as key signaling factors in beta-catenin-dependent and beta-catenin-independent Wnt pathways. While their importance for signal transmission has been genetically demonstrated in many organisms, our mechanistic understanding is still limited. Previous studies using overexpressed proteins showed Dvl localization to large, punctate-like cytoplasmic structures that are dependent on its DIX domain. To study Dvl's role in Wnt signaling, we genome engineered an endogenously expressed Dvl2 protein tagged with an mEos3.2 fluorescent protein for superresolution imaging. First, we demonstrate the functionality and specificity of the fusion protein in beta-catenin-dependent and beta-catenin-independent signaling using multiple independent assays. We performed live-cell imaging of Dvl2 to analyze the dynamic formation of the supramolecular cytoplasmic Dvl2_mEos3.2 condensates. While overexpression of Dvl2_mEos3.2 mimics the previously reported formation of abundant large "puncta," supramolecular condensate formation at physiological protein levels is only observed in a subset of cells with approximately one per cell. We show that, in these condensates, Dvl2 colocalizes with Wnt pathway components at gamma-tubulin and CEP164-positive centrosomal structures and that the localization of Dvl2 to these condensates is Wnt dependent. Single-molecule localization microscopy using photoactivated localization microscopy (PALM) of mEos3.2 in combination with DNA-PAINT demonstrates the organization and repetitive patterns of these condensates in a cell cycle-dependent manner. Our results indicate that the localization of Dvl2 in supramolecular condensates is coordinated dynamically and dependent on cell state and Wnt signaling levels. Our study highlights the formation of endogenous and physiologically regulated biomolecular condensates in the Wnt pathways at single-molecule resolution.

Visualization of ß-adrenergic receptor dynamics and differential localization in cardiomyocytes.

A key question in receptor signaling is how specificity is realized, particularly when different receptors trigger the same biochemical pathway(s). A notable case is the two ß-adrenergic receptor (ß-AR) subtypes, ß1 and ß2, in cardiomyocytes. They are both coupled to stimulatory Gs proteins, mediate an increase in cyclic adenosine monophosphate (cAMP), and stimulate cardiac contractility; however, other effects, such as changes in gene transcription leading to cardiac hypertrophy, are prominent only for ß1-AR but not for ß2-AR. Here, we employ highly sensitive fluorescence spectroscopy approaches, in combination with a fluorescent ß-AR antagonist, to determine the presence and dynamics of the endogenous receptors on the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. These techniques allow us to visualize that the ß2-AR is confined to and diffuses within the T-tubular network, as opposed to the ß1-AR, which is found to diffuse both on the outer plasma membrane as well as on the T-tubules. Upon overexpression of the ß2-AR, this compartmentalization is lost, and the receptors are also seen on the cell surface. Such receptor segregation depends on the development of the T-tubular network in adult cardiomyocytes since both the cardiomyoblast cell line H9c2 and the cardiomyocyte-differentiated human-induced pluripotent stem cells express the ß2-AR on the outer plasma membrane. These data support the notion that specific cell surface targeting of receptor subtypes can be the basis for distinct signaling and functional effects.

Linescan microscopy data to extract diffusion coefficient of a fluorescent species using a commercial confocal microscope.

We report here on the measurement of the diffusion coefficient of fluorescent species using a commercial microscope possessing a resonant scanner. Sequential linescans with a rate of up to 12 kHz yield a temporal resolution of 83 µs, making the setup amenable to measure diffusion rates over a range covering at least three orders of magnitude, from 100 µm2/s down to 0.1 µm2/s. We share representative data sets covering (i) the diffusion of a dye molecule, observed in media of different viscosities and (ii) the diffusion of a prototypical membrane receptor. The data can be valuable for researchers interested in the rapid diffusion properties of nuclear, cytosolic or membrane bound proteins fused to fluorescent tags.

Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families.

Signaling pathway modules are often encoded by several closely related paralogous genes that can have redundant roles and are therefore difficult to analyze by loss-of-function analysis. A typical example is the Wnt signaling pathway, which in mammals is mediated by 19 Wnt ligands that can bind to 10 Frizzled (FZD) receptors. Although significant progress in understanding Wnt-FZD receptor interactions has been made in recent years, tools to generate systematic interaction maps have been largely lacking. Here we generated cell lines with multiplex mutant alleles of FZD1, FZD2, and FZD7 and demonstrate that these cells are unresponsive to canonical Wnt ligands. Subsequently, we performed genetic rescue experiments with combinations of FZDs and canonical Wnts to create a functional ligand-receptor interaction map. These experiments showed that whereas several Wnt ligands, such as Wnt3a, induce signaling through a broad spectrum of FZD receptors, others, such as Wnt8a, act through a restricted set of FZD genes. Together, our results map functional interactions of FZDs and 10 Wnt ligands and demonstrate how multiplex targeting by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 can be used to systematically elucidate the functions of multigene families.-Voloshanenko, O., Gmach, P., Winter, J., Kranz, D., Boutros, M. Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families.