multi-color-localization-microscopy-single-membrane-proteins

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

University of Osnabrück

While detailed information on the nanoscale-organization of proteins within intracellular membranes has emerged from electron microcopy, information on their spatiotemporal dynamics is scarce. By use of a photostable rhodamine attached specifically to Halo-tagged proteins in mitochondrial membranes, we were able to track and localize single protein complexes such as Tom20 and ATP synthase in suborganellar structures in live cells. Individual membrane proteins in the inner and outer membrane of mitochondria were imaged over long time periods with localization precisions below 15 nm. Projection of single molecule trajectories revealed diffusion-restricting microcompartments such as individual cristae in mitochondria. At the same time, protein-specific diffusion characteristics within different mitochondrial membranes could be extracted.

Nanoscale organization of mitochondrial microcompartments revealed by combining tracking and localization microscopy.

In living color: efficient intracellular covalent labeling of proteins with a photoswitchable dye using the HaloTag for dSTORM super-resolution imaging in live cells is described. The dynamics of cellular nanostructures at the plasma membrane were monitored with a time resolution of a few seconds. In combination with dual-color FPALM imaging, submicroscopic receptor organization within the context of the membrane skeleton was resolved.

Triple-color super-resolution imaging of live cells: resolving submicroscopic receptor organization in the plasma membrane.

Micropatterned polymer-supported membranes (PSM) are established as a tool for confining the diffusion of transmembrane proteins for single molecule studies. To this end, a photochemical surface modification with hydrophobic tethers on a PEG polymer brush is implemented for capturing of lipid vesicles and subsequent fusion. Formation of contiguous membranes within micropatterns is confirmed by scanning force microscopy, fluorescence recovery after photobleaching (FRAP), and super-resolved single-molecule tracking and localization microscopy. Free diffusion of transmembrane proteins reconstituted into micropatterned PSM is demonstrated by FRAP and by single-molecule tracking. By exploiting the confinement of diffusion within micropatterned PSM, the diffusion and interaction dynamics of individual transmembrane receptors are quantitatively resolved.

Diffusion and interaction dynamics of individual membrane protein complexes confined in micropatterned polymer-supported membranes.

Quantum dots (QD) are powerful labels for probing diffusion and interaction dynamics of proteins on the single molecule level in living cells. Protein cross-linking due to multifunctional QD strongly affects these properties. This becomes particularly critical when labeling interaction partners with QDs for interrogating the dynamics of complexes. We have here implemented a generic method for QD monofunctionalization based on electrostatic repulsion of a highly negatively charged peptide carrier. On the basis of this method, monobiotinylated QDs were prepared with high yield as confirmed by single molecule assays. These QDs were successfully employed for probing the assembly and diffusion dynamics of binary and ternary cytokine-receptor complexes on the surface of living cells by dual color single QD tracking. Thus, sequential and dynamic recruitment of the type I interferon receptor subunits by the ligand could be observed.

Electrostatically controlled quantum dot monofunctionalization for interrogating the dynamics of protein complexes in living cells.

On the basis of a protein cage scaffold, we have systematically explored intracellular application of nanoparticles for single molecule studies and discovered that recognition by the autophagy machinery plays a key role for rapid metabolism in the cytosol. Intracellular stealth nanoparticles were achieved by heavy surface PEGylation. By combination with a generic approach for nanoparticle monofunctionalization, efficient labeling of intracellular proteins with high fidelity was accomplished, allowing unbiased long-term tracking of proteins in the outer mitochondrial membrane. 

Monofunctional stealth nanoparticle for unbiased single molecule tracking inside living cells.

Unraveling the spatiotemporal organization of signaling complexes within the context of plasma membrane nanodomains has remained a highly challenging task. Here, we have applied super-resolution image correlation based on tracking and localization microscopy (TALM) for probing transient confinement as well as ligand binding and intracellular effector recruitment of the type I interferon (IFN) receptor in the plasma membrane of live cells. Ligand and receptor were labeled with monofunctional quantum dots, thus allowing long-term tracking with very high spatial and temporal resolution without an artificial receptor cross-linking at the cell surface. Dual-color TALM was employed for visualizing protein-protein interactions involved in IFN signaling at both sides of the plasma membrane with high spatial and temporal resolution. By pair correlation analyses based on time-lapse TALM images (pcTALM), complex assembly within dynamic submicroscopic zones was identified. Strikingly, recruitment of the IFN effector protein signal transducer and activator of transcription 2 (STAT2) into these dynamic signaling zones could be observed. The results suggest that confined diffusion zones in the plasma membrane are employed as transient platforms for the assembly of signaling complexes. 

Dynamic submicroscopic signaling zones revealed by pair correlation tracking and localization microscopy.

Dimerization of transmembrane receptors is a key regulatory factor in cellular communication, which has remained challenging to study under well-defined conditions in vitro. We developed a novel strategy to explore membrane protein interactions in a controlled lipid environment requiring minute sample quantities. By rapid transfer of transmembrane proteins from mammalian cells into polymer-supported membranes, membrane proteins could be efficiently fluorescence labeled and reconstituted with very low background. Thus, differential ligand-induced dimerization of the type I interferon (IFN) receptor subunits IFNAR1 and IFNAR2 could be probed quantitatively at physiologically relevant concentrations by single molecule imaging. These measurements clearly support a regulatory role of the affinity of IFNs toward IFNAR1 for controlling the level of receptor dimerization. 

Rapid transfer of transmembrane proteins for single molecule dimerization assays in polymer-supported membranes.

Most cell-surface receptors for cytokines and growth factors signal as dimers, but it is unclear whether remodeling receptor dimer topology is a viable strategy to "tune" signaling output. We utilized diabodies (DA) as surrogate ligands in a prototypical dimeric receptor-ligand system, the cytokine Erythropoietin (EPO) and its receptor (EpoR), to dimerize EpoR ectodomains in non-native architectures. Diabody-induced signaling amplitudes varied from full to minimal agonism, and structures of these DA/EpoR complexes differed in EpoR dimer orientation and proximity. Diabodies also elicited biased or differential activation of signaling pathways and gene expression profiles compared to EPO. Non-signaling diabodies inhibited proliferation of erythroid precursors from patients with a myeloproliferative neoplasm due to a constitutively active JAK2V617F mutation. Thus, intracellular oncogenic mutations causing ligand-independent receptor activation can be counteracted by extracellular ligands that re-orient receptors into inactive dimer topologies. This approach has broad applications for tuning signaling output for many dimeric receptor systems.

Tuning Cytokine Receptor Signaling by Re-orienting Dimer Geometry with Surrogate Ligands.

We developed in situ single cell pull-down (SiCPull) of GFP-tagged protein complexes based on micropatterned functionalized surface architectures. Cells cultured on these supports are lysed by mild detergents and protein complexes captured to the surface are probed in situ by total internal reflection fluorescence microscopy. Using SiCPull, we quantitatively mapped the lifetimes of various signal transducer and activator of transcription complexes by monitoring dissociation from the surface and defined their stoichiometry on the single molecule level. 

Single Cell GFP-Trap Reveals Stoichiometry and Dynamics of Cytosolic Protein Complexes.

Type I interferons (IFNs) activate differential cellular responses through a shared cell surface receptor composed of the two subunits, IFNAR1 and IFNAR2. We propose here a mechanistic model for how IFN receptor plasticity is regulated on the level of receptor dimerization. Quantitative single-molecule imaging of receptor assembly in the plasma membrane of living cells clearly identified IFN-induced dimerization of IFNAR1 and IFNAR2. The negative feedback regulator ubiquitin-specific protease 18 (USP18) potently interferes with the recruitment of IFNAR1 into the ternary complex, probably by impeding complex stabilization related to the associated Janus kinases. Thus, the responsiveness to IFNα2 is potently down-regulated after the first wave of gene induction, while IFNβ, due to its ∼100-fold higher binding affinity, is still able to efficiently recruit IFNAR1. Consistent with functional data, this novel regulatory mechanism at the level of receptor assembly explains how signaling by IFNβ is maintained over longer times compared with IFNα2 as a temporally encoded cause of functional receptor plasticity. 

Receptor dimerization dynamics as a regulatory valve for plasticity of type I interferon signaling.

Single-molecule photobleaching has emerged as a powerful non-invasive approach to extract the stoichiometry of multimeric membrane proteins in their native cellular environment. However, this method has mainly been used to determine the subunit composition of ion channels and receptors at the plasma membrane. Here, we applied single-molecule photobleaching to analyze the oligomeric state of an endoplasmic reticulum (ER) resident candidate ceramide sensor protein, SMSr/SAMD8. Co-immunoprecipitation and chemical cross-linking studies previously revealed that the N-terminal sterile alpha motif (or SAM) domain of SMSr drives self-assembly of the protein into oligomers and that SMSr oligomerization is promoted by curcumin, a drug known to perturb ER ceramide and calcium homeostasis. Application of cell spreading surface-active coating materials in combination with total internal reflection fluorescence (TIRF) microscopy allowed us to image GFP-tagged SMSr proteins as single fluorescent spots in the ER of HeLa cells in which expression of endogenous SMSr was abolished. In line with our biochemical analysis, we find that the number of bleaching steps in SMSr-GFP-positive spots displays a substantial drop after removal of the SAM domain. In contrast, treatment of cells with curcumin increased the number of bleaching steps. Our results document the first successful application of single-molecule photobleaching to resolve drug-induced and domain-dependent changes in the oligomeric state of an ER-resident membrane protein, hence establishing a complementary method to unravel the mechanism by which SMSr controls ceramide levels in the ER.

Monitoring Changes in the Oligomeric State of a Candidate Endoplasmic Reticulum (ER) Ceramide Sensor by Single-molecule Photobleaching.

Stress granules (SGs) are cytosolic, nonmembranous RNA-protein complexes. In vitro experiments suggested that they are formed by liquid-liquid phase separation; however, their properties in mammalian cells remain unclear. We analyzed the distribution and dynamics of two paradigmatic RNA-binding proteins (RBPs), Ras GTPase-activating protein SH3-domain-binding protein (G3BP1) and insulin-like growth factor II mRNA-binding protein 1 (IMP1), with single-molecule resolution in living neuronal cells. Both RBPs exhibited different exchange kinetics between SGs. Within SGs, single-molecule localization microscopy revealed distributed hotspots of immobilized G3BP1 and IMP1 that reflect the presence of relatively immobile nanometer-sized nanocores. We demonstrate alternating binding in nanocores and anomalous diffusion in the liquid phase with similar characteristics for both RBPs. Reduction of low-complexity regions in G3BP1 resulted in less detectable mobile molecules in the liquid phase without change in binding in nanocores. The data provide direct support for liquid droplet behavior of SGs in living cells and reveal transient binding of RBPs in nanocores. Our study uncovers a surprising disconnect between SG partitioning and internal diffusion and interactions of RBPs.

Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules.

Synthetically replicating key biological processes requires the ability to puncture lipid bilayer membranes and to remodel their shape. Recently developed artificial DNA nanopores are one possible synthetic route due to their ease of fabrication. However, an unresolved fundamental question is how DNA nanopores bind to and dynamically interact with lipid bilayers. Here we use single-molecule fluorescence microscopy to establish that DNA nanopores carrying cholesterol anchors insert via a two-step mechanism into membranes. Nanopores are furthermore shown to locally cluster and remodel membranes into nanoscale protrusions. Most strikingly, the DNA pores can function as cytoskeletal components by stabilizing autonomously formed lipid nanotubes. The combination of membrane puncturing and remodeling activity can be attributed to the DNA pores' tunable transition between two orientations to either span or co-align with the lipid bilayer. This insight is expected to catalyze the development of future functional nanodevices relevant in synthetic biology and nanobiotechnology.

Article	Year
Nanoscale organization of mitochondrial microcompartments revealed by combining tracking and localization microscopy. Nano letters\| PubMed ID: 22201267	2012
Triple-color super-resolution imaging of live cells: resolving submicroscopic receptor organization in the plasma membrane. Angewandte Chemie (International ed. in English)\| PubMed ID: 22488831	2012
Diffusion and interaction dynamics of individual membrane protein complexes confined in micropatterned polymer-supported membranes. Small (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 23109503	2013
Electrostatically controlled quantum dot monofunctionalization for interrogating the dynamics of protein complexes in living cells. ACS chemical biology\| PubMed ID: 23186299	2013
Monofunctional stealth nanoparticle for unbiased single molecule tracking inside living cells. Nano letters\| PubMed ID: 24655019	2014
Dynamic submicroscopic signaling zones revealed by pair correlation tracking and localization microscopy. Analytical chemistry\| PubMed ID: 25148216	2014
Rapid transfer of transmembrane proteins for single molecule dimerization assays in polymer-supported membranes. ACS chemical biology\| PubMed ID: 25203456	2014
Tuning Cytokine Receptor Signaling by Re-orienting Dimer Geometry with Surrogate Ligands. Cell\| PubMed ID: 25728669	2015
Single Cell GFP-Trap Reveals Stoichiometry and Dynamics of Cytosolic Protein Complexes. Nano letters\| PubMed ID: 25901412	2015
Receptor dimerization dynamics as a regulatory valve for plasticity of type I interferon signaling. The Journal of cell biology\| PubMed ID: 26008745	2015
Monitoring Changes in the Oligomeric State of a Candidate Endoplasmic Reticulum (ER) Ceramide Sensor by Single-molecule Photobleaching. The Journal of biological chemistry\| PubMed ID: 27729449	2016
Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules. The Journal of cell biology\| PubMed ID: 29463567	2018
Multi-functional DNA nanostructures that puncture and remodel lipid membranes into hybrid materials. Nature communications\| PubMed ID: 29670084	2018

Christian P. Richter

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