Single Protein Tracking Research Articles

Super-Resolution Imaging and Single Molecule Tracking of the Nuclear Protein Emerin

Emerin is an integral protein of the nuclear membrane expressed in most somatic cells. When under-expressed or mutated, it leads to Emery-Dreifuss Muscular Dystrophy. This disease is characterized by cardiac abnormalities and progressive muscle wasting. The molecular bases of these phenotypes are not well defined. Previous attempts at characterizing emerin localization and diffusional mobility have relied on ensemble confocal fluorescence microscopy. This approach did not reveal differences between normal and mutated versions of emerin, perhaps due to the limited optical resolution of confocal and FRAP techniques. Here we employ single molecule tracking and super-resolution optical imaging of emerin to further characterize emerin diffusion behaviors and localization at the nanometer scale.We performed single protein tracking of photoactivable-TagRFP-emerin fusions in live cells and study their dynamics in the nuclear membrane. Diffusion analyses indicate the co-existence of four diffusing sub-populations. One emerin sub-population is associated with the endoplasmic reticulum diffusion, two are located exclusively to the nuclear membrane, and a nearly immobile sub-population also locates to the nuclear membrane. Single molecule pulse-chase experiments reveal that the two nuclear-exclusive sub-populations accumulate at different rates, providing further evidence of their mutual uniqueness and their differential distribution between the inner and outer nuclear membrane. Further studies with disease-associated emerin mutants revealed that a single point emerin mutant has a 250% increase in diffusion coefficient for one of the nuclear membrane sub-populations when compared to the wild type.We also map the 3D nanoscale organization of emerin at the nuclear membrane using super-resolution imaging of SNAP-tag-emerin fusions. Analysis of emerin clusters was performed with 3D Ripley's K-function to compare the distribution of wild-type and mutated versions of emerin.

Dendrimer Probes for Enhanced Photostability and Localization in Fluorescence Imaging

Recent advances in fluorescence microscopy have enabled high-resolution imaging and tracking of single proteins and biomolecules in cells. To achieve high spatial resolutions in the nanometer range, bright and photostable fluorescent probes are critically required. From this view, there is a strong need for development of advanced fluorescent probes with molecular-scale dimensions for fluorescence imaging. Polymer-based dendrimer nanoconjugates hold strong potential to serve as versatile fluorescent probes due to an intrinsic capacity for tailored spectral properties such as brightness and emission wavelength. In this work, we report a new, to our knowledge, class of molecular probes based on dye-conjugated dendrimers for fluorescence imaging and single-molecule fluorescence microscopy. We engineered fluorescent dendritic nanoprobes (FDNs) to contain multiple organic dyes and reactive groups for target-specific biomolecule labeling. The photophysical properties of dye-conjugated FDNs (Cy5-FDNs and Cy3-FDNs) were characterized using single-molecule fluorescence microscopy, which revealed greatly enhanced photostability, increased probe brightness, and improved localization precision in high-resolution fluorescence imaging compared to single organic dyes. As proof-of-principle demonstration, Cy5-FDNs were used to assay single-molecule nucleic acid hybridization and for immunofluorescence imaging of microtubules in cytoskeletal networks. In addition, Cy5-FDNs were used as reporter probes in a single-molecule protein pull-down assay to characterize antibody binding and target protein capture. In all cases, the photophysical properties of FDNs resulted in enhanced fluorescence imaging via improved brightness and/or photostability.

High-Speed Atomic Force Microscopy of Protein-Protein Interactions

Protein-protein interactions are of major importance for biological function. Many proteins are oligomers and interact temporaly with other proteins. This is of particular importance in the membrane, where multiprotein assemblies act in important processes like signaling, respiration, photosynthesis, etc. High-speed atomic force microscopy (HS-AFM,[1]) offers unique possibilities to study protein-protein interactions in the membrane. HS-AFM allows not only visualization and tracking of single proteins but also their environment, hence describing the interaction energy between proteins (Fig.1A,[2]), their dynamic supramolecular assemblies (Fig.1B,[3]), and membrane crowding and interaction specificity (Fig.1C, [4]). The interaction profiles can be, though at different time scales, compared to molecular simulations [4]. The perspective of short-cantilever HS-AFM in force spectroscopy will be discussed: the high speed of short cantilevers allows measuring interaction forces at unprecedented loading rates and temporal resolution (Fig.1D).

Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions

Integrins in focal adhesions (FAs) mediate adhesion and force transmission to extracellular matrices essential for cell motility, proliferation and differentiation. Different fibronectin-binding integrins, simultaneously present in FAs, perform distinct functions. Yet, how integrin dynamics control biochemical and biomechanical processes in FAs is still elusive. Using single-protein tracking and super-resolution imaging we revealed the dynamic nano-organizations of integrins and talin inside FAs. Integrins reside in FAs through free-diffusion and immobilization cycles. Integrin activation promotes immobilization, stabilized in FAs by simultaneous connection to fibronectin and actin-binding proteins. Talin is recruited in FAs directly from the cytosol without membrane free-diffusion, restricting integrin immobilization to FAs. Immobilized β3-integrins are enriched and stationary within FAs, whereas immobilized β1-integrins are less enriched and exhibit rearward movements. Talin is enriched and mainly stationary, but also exhibited rearward movements in FAs, consistent with stable connections with both β-integrins. Thus, differential transmission of actin motion to fibronectin occurs through specific integrins within FAs.

Split-GFP Complementation for Targeting and Imaging Single Molecules in Living Cells

Current limitations for studying intracellular dynamics of biomolecules by single molecule fluorescence include the necessity to express a molecule of interest at very low concentration or to target it very specifically with a small number of probes. Inside cells, both conditions are hard to meet. In the first case, very low expression levels of a molecule of interest might influence the cell physiological responses. In the second case, probes with high specificity and very high affinity binding constants for their target are required. To circumvent these issues we introduce the use of split-GFP fusions and their complementation by small synthetic peptides to image the dynamics of single proteins in live cells. Cells expressing the split-GFP fusions are non-fluorescent and a controlled subset of the GFPs can be “lit up” when providing the complementary peptide at different concentrations. With this approach, we imaged the plasma membrane diffusion of single CD4-split GFP and GPI-split GFP proteins in different cell lines. We also demonstrate intracellular single molecule imaging using caveolin-1-split GFP proteins as an example. In all cases, background free tracking of single proteins could be achieved by TIRF microscopy within minutes of complementation. Split-GFP fusions and the development of other split-fluorescent protein variants provide a generic method for multicolor single molecule imaging in living cells even at elevated protein expressions. Split-fluorescent protein fusions also complement the toolbox of intracellular targeting strategies by providing a unique way to verify the specific targeting of molecules introduced in living cells. Toward this aim, we present early efforts at employing split-GFP fusions for addressable targeting of single fluorophores and single quantum dots.

High Affinity scFv−Hapten Pair as a Tool for Quantum Dot Labeling and Tracking of Single Proteins in Live Cells

We describe a general approach to label cell surface proteins using quantum dots (QD) for single-molecule tracking. QDs coated with small-hapten modified peptides are targeted to cell surface fusion proteins containing the corresponding single-chain fragment antibody (scFv). The approach is illustrated with the small hapten fluorescein (FL) and a high-affinity anti-FL scFv fused to two different proteins in yeast and murine neuronal cell line N2a.