Tandem fluorescent protein timers for in vivo analysis of protein dynamics

The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule–mediated protein degradation.     

Inert Pepper aptamer-mediated endogenous mRNA recognition and imaging in living cells

The development of RNA aptamers/fluorophores system is highly desirable for understanding the dynamic molecular biology of RNAs in vivo. Peppers-based imaging systems have been reported and applied for mRNA imaging in living cells. However, the need to insert corresponding RNA aptamer sequences into target RNAs and relatively low fluorescence signal limit its application in endogenous mRNA imaging. Herein, we remolded the original Pepper aptamer and developed a tandem array of inert Pepper (iPepper) fluorescence turn-on system. iPepper allows for efficient and selective imaging of diverse endogenous mRNA species in live cells with minimal agitation of the target mRNAs. We believe iPepper would significantly expand the applications of the aptamer/fluorophore system in endogenous mRNA imaging, and it has the potential to become a powerful tool for real-time studies in living cells and biological processing.     

Actin protein inside DMPC GUVs and its mechanical response to AC electric fields

Cells are dynamic systems with complex mechanical properties, regulated by the presence of different species of proteins capable to assemble (and disassemble) into filamentous forms as required by different cells functions. Giant unilamellar vesicles (GUVs) of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) are systems frequently used as a simplified model of cells because they offer the possibility of assaying separately different stimuli, which is no possible in living cells. Here we present a study of the effect of acting protein on mechanical properties of GUVs, when the protein is inside the vesicles in either monomeric G-actin or filamentous F-actin. For this, rabbit skeletal muscle G-actin is introduced inside GUVs by the electroformation method. Protein polymerization inside the GUVs is promoted by adding to the solution MgCl₂ and the ion carrier A23187 to allow the transport of Mg⁺² ions into the GUVs. To determine how the presence of actin changes the mechanical properties of GUVs, the vesicles are deformed by the application of an AC electric field in both cases with G-actin and with polymerized F-actin. The changes in shape of the vesicles are characterized by optical microscopy and from them the bending stiffness of the membrane are determined. It is found that G-actin has no appreciable effect on the bending stiffness of DMPC GUVs, but the polymerized actin makes the vesicles more rigid and therefore more resistant to deformations. This result is supported by evidence that actin filaments tend to accumulate near the membrane.     

Proteins on the move: insights gained from fluorescent protein technologies

Proteins are always on the move, and this may occur through diffusion or active transport. The realization that the regulation of signal transduction is highly dynamic in space and time has stimulated intense interest in the movement of proteins. Over the past decade, numerous new technologies using fluorescent proteins have been developed, allowing us to observe the spatiotemporal dynamics of proteins in living cells. These technologies have greatly advanced our understanding of protein dynamics, including protein movement and protein interactions.     

Fluorescence protection assay: a novel homogeneous assay platform toward development of aptamer sensors for protein detection

Development of novel aptamer sensor strategies for rapid and selective assays of protein biomarkers plays crucial roles in proteomics and clinical diagnostics. Herein, we have developed a novel aptamer sensor strategy for homogeneous detection of protein targets based on fluorescence protection assay. This strategy is based on our reasoning that interaction of aptamer with its protein target may dramatically increase steric hindrance, which protects the fluorophore, fluorescein isothiocyannate (FITC), labeled at the binding pocket from accessing and quenching by the FITC antibody. The aptamer sensor strategy is demonstrated using a model protein target of immunoglobulin E (IgE), a known biomarker associated with atopic allergic diseases. The results reveal that the aptamer sensor shows substantial (>6-fold) fluorescence enhancement in response to the protein target, thereby verifying the mechanism of fluorescence protection. Moreover, the aptamer sensor displays improved specificity to other co-existing proteins and a desirable dynamic range within the IgE concentration from 0.1 to 50 nM with a readily achieved detection limit of 0.1 nM. Because of great robustness, easy operation and scalability for parallel assays, the developed homogeneous fluorescence protection assay strategy might create a new methodology for developing aptamer sensors in sensitive, selective detection of proteins.     

A genomic integration method for the simultaneous visualization of endogenous mRNAs and their translation products in living yeast

Protein localization within cells can be achieved by the targeting and localized translation of mRNA. Yet, our understanding of the dynamics of mRNA targeting and protein localization, and of how general this phenomenon is, is not clear. Plasmid-based expression systems have been used to visualize exogenously expressed mRNAs and proteins; however, these methods typically produce them at levels greater than endogenous and can result in mislocalization. Hence, a method that allows for the simultaneous visualization of endogenous mRNAs and their translation products in living cells is needed. We previously developed a method (m-TAG) to localize endogenously expressed mRNAs in yeast by chromosomal insertion of the MS2 aptamer sequence between the open-reading frame (ORF) and 3' UTR of any gene. Upon coexpression with the MS2 RNA-binding coat protein (MS2-CP) fused with GFP, the aptamer-tagged mRNAs bearing their 3' UTRs are localized using fluorescence microscopy. Here we describe an advanced method (mp-TAG) that allows for the simultaneous visualization of both endogenously expressed mRNAs and their translation products in living yeast for the first time. Homologous recombination is used to insert the mCherry gene and MS2-CP binding sites downstream from any ORF, in order to localize protein and mRNA, respectively. As proof of the concept, we tagged ATP2 as a representative gene and demonstrated that endogenous ATP2 mRNA and protein localize to mitochondria, as shown previously. In addition, we demonstrate that tagged proteins like Hhf2, Vph1, and Yef3 localize to their expected subcellular location, while the localization of their mRNAs is revealed for the first time.     

A photo-cross-linking approach to monitor protein dynamics in living cells

BackgroundProteins, which comprise one of the major classes of biomolecules that constitute a cell, interact with other cellular factors during both their biogenesis and functional states. Studying not only static but also transient interactions of proteins is important to understand their physiological roles and regulation mechanisms. However, only a limited number of methods are available to analyze the dynamic behaviors of proteins at the molecular level in a living cell. The site-directed in vivo photo-cross-linking approach is an elegant technique to capture protein interactions with high spatial resolution in a living cell.Scope of reviewHere, we review the in vivo photo-cross-linking approach including its recent applications and the potential problems to be considered. We also introduce a new in vivo photo-cross-linking-based technique (PiXie) to study protein dynamics with high spatiotemporal resolution.Major conclusionsIn vivo photo-cross-linking enables us to capture weak/transient protein interactions with high spatial resolution, and allows for identification of interacting factors. Moreover, the PiXie approach can be used to monitor rapid folding/assembly processes of proteins in living cells.General significanceIn vivo photo-cross-linking is a simple method that has been used to analyze the dynamic interactions of many cellular proteins. Originally developed in Escherichia coli, this system has been extended to studies in various organisms, making it a fundamental technique for investigating dynamic protein interactions in many cellular processes. This article is part of a Special issue entitled “Novel major techniques for visualizing ‘live’ protein molecules” edited by Dr. Daisuke Kohda.     

GFP chimeras of E-MAP-115 (ensconsin) domains mimic behavior of the endogenous protein in vitro and in vivo

E-MAP-115 (ensconsin) is a microtubule-associated protein (MAP) abundant in carcinoma and other epithelia-derived cells. We expressed chimeras of green fluorescent protein (GFP) conjugated to ensconsin's N-terminal MT-binding domain (EMTB), to study distribution, dynamics, and function of the MAP in living cells. We tested the hypothesis that behavior of expressed GFP-EMTB accurately matched behavior of endogenous ensconsin. Like endogenous MAP, GFP-EMTB was associated with microtubules in living or fixed cells, and microtubule association of either molecule was impervious to extraction with nonionic detergents. In cell lysates both GFP-EMTB and endogenous ensconsin were dissociated from microtubules by identical salt extraction conditions, and both molecules remained bound to a calcium-stable subset of Taxol-stabilized microtubules. These data show that microtubule association of ensconsin was affected neither by the absence of domains other than its microtubule-binding domain, nor by the presence of appended GFP. We took advantage of this finding to generate constructs in which additional GFP moieties were attached to EMTB, to obtain a more intensely fluorescent reporter of in vivo MAP binding. We show here that expression of chimeric proteins consisting of five GFP molecules attached to a single EMTB molecule produces brightly labeled microtubules without compromising the behavior of the MAP or the microtubules to which it is attached. Thus, we have demonstrated the utility of chimeric proteins containing GFP multimers as authentic reporters of ensconsin distribution and dynamics; expression of these GFP-EMTB chimeric molecules also provides a non-perturbing label of the microtubule system in living cells.     

Structure‐guided design of a reversible fluorogenic reporter of protein‐protein interactions

A reversible green fluorogenic protein‐fragment complementation assay was developed based on the crystal structure of UnaG, a recently discovered fluorescent protein. In living mammalian cells, the nonfluorescent fragments complemented and rapidly became fluorescent upon rapamycin‐induced FKBP and Frb protein interaction, and lost fluorescence when the protein interaction was inhibited. This reversible fluorogenic reporter, named uPPI [UnaG‐based protein‐protein interaction (PPI) reporter], uses bilirubin (BR) as the chromophore and requires no exogenous cofactor. BR is an endogenous molecule in mammalian cells and is not fluorescent by itself. uPPI may have many potential applications in visualizing spatiotemporal dynamics of PPIs.     

Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments

Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.     

A tunable fluorescent timer method for imaging spatial‐temporal protein dynamics using light‐driven photoconvertible protein

Cellular function is largely determined by protein behaviors occurring in both space and time. While regular fluorescent proteins can only report spatial locations of the target inside cells, fluorescent timers have emerged as an invaluable tool for revealing coupled spatial‐temporal protein dynamics. Existing fluorescent timers are all based on chemical maturation. Herein we propose a light‐driven timer concept that could report relative protein ages at specific sub‐cellular locations, by weakly but chronically illuminating photoconvertible fluorescent proteins inside cells. This new method exploits light, instead of oxygen, as the driving force. Therefore its timing speed is optically tunable by adjusting the photoconverting laser intensity. We characterized this light‐driven timer method both in vitro and in vivo and applied it to image spatiotemporal distributions of several proteins with different lifetimes. This novel timer method thus offers a flexible “ruler” for studying temporal hierarchy of spatially ordered processes with exquisite spatial‐temporal resolution. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Detection of altered protein conformations in living cells

The maturation, conformational stability, and the rate of in vivo degradation are specific for each protein and depend on both the intrinsic features of the protein and those of the surrounding cellular environment. While synthesis and degradation can be measured in living cells, stability and maturation of proteins are more difficult to quantify. We developed the split-ubiquitin method into a tool for detecting and analyzing changes in protein conformation. The biophysical parameter that forms the basis of these measurements is the time-averaged distance between the N terminus and C terminus of a protein. Starting from three proteins of known structure, we demonstrate the feasibility of this approach, and employ it to elucidate the effect of a previously described mutation in the protein Sec62p on its conformation in living cells.     

Molecular aptamer beacons for real-time protein recognition

One of the most pressing problems facing those attempting to understand the regulation of gene expression and translation is the necessity to monitor protein production in a variety of metabolic states. Thus far, there is no easy solution that will either identify or quantitate proteins in real time. Here we introduce a novel protein probe, molecular aptamer beacon (MAB), for real time protein recognition and quantitative analysis. The MAB combines the signal transduction mechanism of molecular beacons and the molecular recognition specificity of aptamers. An MAB based on a thrombin-binding aptamer was prepared as a model to demonstrate the feasibility. Significant fluorescent signal change was observed when MAB was bound to thrombin, which is attributed to a significant conformational change in MAB from a loose random coil to a compact unimolecular quadruplex. The MAB recognizes its target protein with high specificity and high sensitivity (112 picomolar thrombin concentration) in homogeneous solutions. Ratiometric imaging has been conducted with MAB labeled with two fluorophores, which makes it feasible for protein quantitation in living specimen. The unique properties of the MAB will enable the development of a class of protein probes for real time protein tracing in living specimen and for efficient biomedical diagnosis in homogeneous solutions.     

Selective control of basolateral membrane protein polarity by cdc42

The rho GTPase cdc42 is implicated in several aspects of cell polarity. A recent study (Kroschewski R, Hall A, Mellman I. Nat Cell Biol 1999;1:8–13) demonstrated that a dominant negative mutant of cdc42 abolishes the polarity of basolateral membrane proteins in MDCK cells, but did not elucidate whether this effect was selective for basolateral proteins or nonselective for all secreted proteins. To answer this question, we analyzed the polarity of newly synthesized membrane and soluble proteins in MDCK cell lines previously induced to overexpress mutant forms of cdc42. GTPase-deficient and dominant negative cdc42 did not affect the apical targeting of a newly synthesized apical membrane protein, but reversed to apical the distribution of two exogenous basolateral membrane proteins. In striking contrast, GTPase-deficient cdc42 did not affect polarized exocytosis of endogenous soluble proteins, either apical or basolateral. The exquisitely selective regulation of polarized protein targeting by cdc42 may allow cells to fine-tune their membrane composition in response to extracellular signals during development, migration and in response to injury.     

Dynamic proteomics in individual human cells uncovers widespread cell-cycle dependence of nuclear proteins

We examined cell cycle-dependent changes in the proteome of human cells by systematically measuring protein dynamics in individual living cells. We used time-lapse microscopy to measure the dynamics of a random subset of 20 nuclear proteins, each tagged with yellow fluorescent protein (YFP) at its endogenous chromosomal location. We synchronized the cells in silico by aligning protein dynamics in each cell between consecutive divisions. We observed widespread (40%) cell-cycle dependence of nuclear protein levels and detected previously unknown cell cycle-dependent localization changes. This approach to dynamic proteomics can aid in discovery and accurate quantification of the extensive regulation of protein concentration and localization in individual living cells.     

Fluorescent probes for imaging endogenous β-actin mRNA in living cells using fluorescent protein-tagged pumilio

Subcellular localization and dynamics of mRNAs control various physiological functions in living cells. A novel technique for visualizing endogenous mRNAs in living cells is necessary for investigation of the spatiotemporal movement of mRNAs. A pumilio homology domain of human pumilio 1 (PUM-HD) is a useful RNA binding protein as a tool for mRNA recognition because the domain can be modified to bind a specific 8-base sequence of target mRNA. In this study, we designed PUM-HD to match the sequence of β-actin mRNA and developed an mRNA probe consisting of two PUM-HD mutants flanking full-length enhanced green fluorescent protein (EGFP). Fluorescence microscopy with the probe in living cells revealed that the probe was labeled precisely with the β-actin mRNA in cytosol. Fluorescent spots from the probe were colocalized with microtubules and moved directionally in living cells. The PUM-HD mutants conjugated with full-length EGFP can enable visualization of β-actin mRNA localization and dynamics in living cells.     

Multiple forms of ubiquitin-protein ligase. Binding of activated ubiquitin to protein substrates

Enzyme activities that catalyzed the covalent attachment of ubiquitin to protein substrates (ubiquitin-protein ligase, UbL) were purified from the extracts of human red blood cells. These activities required the presence of ubiquitin-activating enzyme and ATP for activity. Four fractions (UbL A, B1, B2, and C) were resolved and showed different specificities toward added substrates [carboxymethylated bovine serum albumin (CM-BSA), G-actin, lysozyme, and alpha-lactalbumin]. The enzyme fractions gave different products with a given substrate. UbL A and UbL B1 were exclusively active with CM-BSA and alpha-lactalbumin, respectively. UbL B2 was most active toward CM-BSA with substantial activities to G-actin and alpha-lactalbumin and with no activity to lysozyme. UbL C showed significant activities with all four substrates, having a highest activity toward CM-BSA. There were many endogenous proteins present in the erythrocyte extract which were efficient substrates for ubiquitin conjugation. In particular, a pair of substrates were identified from erythrocyte extracts which were far more efficient substrates than the denatured proteins exogenously added.     

Multiphoton excitation-evoked chromophore-assisted laser inactivation using green fluorescent protein

Noninvasive, straightforward methods to inactivate selected proteins in living cells with high spatiotemporal resolution are needed. Chromophore-assisted laser inactivation (CALI) can be used to photochemically inactivate proteins, but it has several drawbacks, such as procedural complexity and nonspecific photodamage. Here we show that by application of multiphoton excitation to CALI, enhanced green fluorescent protein (EGFP) is an effective chromophore for inactivation of a protein's function without nonspecific photodamage in living mammalian cells.