Visualization Of Protein Interactions Research Articles

Highly Efficient Fluorescent Probe to Visualize Protein Interactions at the Superresolution.

Superresolution microscopy (SR microscopy) of protein-protein interactions (PPIs) occurring in subcellular structures is essential for understanding cellular functions. However, a powerful and useful technology for SR microscopy of PPIs remains elusive. Here, we develop a highly efficient photoconvertible fluorescent probe, named split-Dendra2, for SR microscopy of PPIs in the cell. We found that split-Dendra2 enables a highly efficient detection of PPIs, making it possible to perform SR microscopy of PPIs with high spatial resolution and high image reconstruction fidelity. We demonstrate the utility of split-Dendra2 by visualizing PPIs occurring in small subcellular structures at the superresolution, such as clathrin-coated pits and focal adhesions, which cannot be visualized by the existing tools. Split-Dendra2 offers a powerful and useful tool that greatly expands the possibility of SR microscopy and can contribute to revealing the function of PPIs at the nanoscale resolution.

Acoustofluidic scanning fluorescence nanoscopy with a large field of view

Large-field nanoscale fluorescence imaging is invaluable for many applications, such as imaging subcellular structures, visualizing protein interactions, and high-resolution tissue imaging. Unfortunately, conventional fluorescence microscopy requires a trade-off between resolution and field of view due to the nature of the optics used to form the image. To overcome this barrier, we developed an acoustofluidic scanning fluorescence nanoscope that simultaneously achieves superior resolution, a large field of view, and strong fluorescent signals. The acoustofluidic scanning fluorescence nanoscope utilizes the superresolution capabilities of microspheres that are controlled by a programmable acoustofluidic device for rapid fluorescence enhancement and imaging. The acoustofluidic scanning fluorescence nanoscope resolves structures that cannot be resolved with conventional fluorescence microscopes with the same objective lens and enhances the fluorescent signal by a factor of ~5 without altering the field of view of the image. The improved resolution realized with enhanced fluorescent signals and the large field of view achieved via acoustofluidic scanning fluorescence nanoscopy provides a powerful tool for versatile nanoscale fluorescence imaging for researchers in the fields of medicine, biology, biophysics, and biomedical engineering.

The Molecular Landscape of Lung Metastasis in Primary Head and Neck Squamous Cell Carcinomas.

Background Lung metastasis in head and neck cancer (HNC) patients is a critical concern, often indicating an advanced disease stage and a poor prognosis. This study explores the molecular complexities of such metastases, identifying specific genes and pathways that may serve as valuable targets for diagnosis and treatment. The findings underscore the potential for significantly improved patient outcomes through targeted therapeutic strategies. Methodology In this research, we systematically collected raw gene expression data from head and neck squamous cell carcinoma (HNSCC) and lung squamous cell carcinoma (LSCC). By comparing tumorous and normal gene expression profiles from paired patient samples, we identified differentially expressed genes (DEGs). Network analysis helped visualize protein interactions and pinpoint crucial hub genes. Through validation and comparison across several datasets, we identified common DEGs. Additionally, we employed Kaplan-Meier analysis and log-rank testing to examine the relationship between gene expression patterns and patient survival. Result The study identified 145 overlapping DEGs in both HNSCC and LSCC, which are crucial for cancer progression and linked to lung metastasis, offering vital targets for personalized therapy by identifying key genes affecting disease development and patient survival. Pathway analyses linked these to lung metastasis, while protein-protein interaction network construction and hub gene identification highlighted genes crucial for development and patient survival, offering targets for personalized therapy. Conclusion Identifying key genes and pathways in lung metastasis from HNC, this study highlights potential targets for enhanced diagnosis and therapy. It underscores the crucial role of molecular insights in driving forward personalized treatment approaches and improving patient outcomes.

Using FLIM-FRET for Characterizing Spatial Interactions in the Spindle.

Proper spindle assembly and the attachment of chromosomes to the spindle are key for the accurate segregation of chromosomes to daughter cells. Errors in these processes can lead to aneuploidy, which is a hallmark of cancer. Understanding the mechanisms that drive spindle assembly will provide fundamental insights into how accurate chromosome segregation is achieved. One challenge in elucidating the complexities of spindle assembly is to visualize protein interactions in space and time. The Xenopus egg extract system has been a valuable tool to probe protein function during spindle assembly in vitro. Tagging proteins with fluorescent proteins and utilizing fluorescence-based approaches, such as Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM), have provided visual clues about the mechanics of spindle assembly and its regulators. However, elucidating how spindle assembly factors are spatially regulated is still challenging. Combining the egg extract system and visual FRET approaches provides a powerful tool to probe the processes involved in spindle assembly. Here we describe how a FLIM-FRET biosensor can be used to study protein-protein interactions in spindles assembled in Xenopus egg extracts. This approach should be readily adaptable to a wide variety of proteins to allow for new insights into the regulation of spindle assembly.

Knocksideways in plants: an inducible system for in planta visualization of protein interactions.

Knocksideways in plants: an inducible system for in planta visualization of protein interactions.

The human DEPhOsphorylation Database DEPOD: 2019 update.

The human Dephosphorylation Database (DEPOD) is a manually curated resource that harbors human phosphatases, their protein and non-protein substrates, dephosphorylation sites and the associated signaling pathways. We report here an update to DEPOD by integrating and/or linking to annotations from 69 other open access databases including disease associations, phosphorylating kinases, protein interactions, and also genome browsers. We also provide tools to visualize protein interactions, protein structures, phosphorylation networks, evolutionary conservation of proteins, dephosphorylation sites, and short linear motifs within various proteins. The updated version of DEPOD contains 254 human phosphatases, 336 protein and 83 non-protein substrates, and 1215 manually curated phosphatase-substrate relationships. In addition, we have improved the data access as all the data in DEPOD can now be easily downloaded in a user-friendly format. With multiple significant improvements, DEPOD continues serving as a key resource for research on phosphatase-kinase networks. Database URL: www.depod.org

Comparison of the performance of 1D SDS-PAGE with nondenaturing 2DE on the analysis of proteins from human bronchial smooth muscle cells using quantitative LC-MS/MS

The supernatant and precipitate protein fractions from human bronchial smooth muscle cells (HBSMC) were analyzed using 1D SDS-PAGE-LC-MS/MS and the performance of the method was compared with that of nondenaturing 2DE-LC-MS/MS applied to the supernatant fraction, the methods and the results have been reported previously. The 1D gel lanes were cut into pieces of the same size, in order to enable the reproduction of digital 1D images of the individual MS-assigned proteins. When the obtained information on individual HBSMC proteins was compared between the two methods, SDS-PAGE is advantageous in visualizing the quantity differences between differently treated samples, whereas nondenatuing 2DE is advantageous in visualizing protein interactions. SDS-PAGE-MS of the supernatant fraction provided the assignment of 2552 proteins and their percent abundance ranged from 3.5% to 2 × 10−4%. 2DE-MS of the supernatant fraction provided 4323 proteins with percent abundance ranged from 3.6% to 1 × 10−5%, suggesting that the step of isoelectric focusing served to raise the sensitivity of the method. The proteins in the precipitate fraction, which could not be analyzed by 2DE-MS, were characterized by the abundance of proteins allocated to “membrane” within the category “Cellular component” of UniProtKB database and especially those allocated to “transmembrane” within the subcategory “membrane.” On the other hand, there were about 600 “membrane” proteins which showed more than two-fold higher percent abundance in 2DE-MS than in SDS-PAGE-MS. These results showed that 1D SDS-PAGE and nondenaturing 2DE would provide complementary information on the analysis of proteins and protein interactions in cells.

FRET-FLIM for Visualizing and Quantifying Protein Interactions in Live Plant Cells.

Proteins are the workhorses that control most biological processes in living cells. Although proteins can accomplish their functions independently, the vast majority of functions require proteins to interact with other proteins or biomacromolecules. Protein interactions can be investigated through biochemical assays such as co-immunoprecipitation (co-IP) or Western blot analysis, but such assays lack spatial information. Here we describe a well-developed imaging method, Förster resonance energy transfer (FRET) analyzed by fluorescence lifetime imaging microscopy (FLIM), that can be used to visualize protein interactions with both spatial and temporal resolution in live cells. We demonstrate its use in plant developmental research by visualizing in vivo dimerization of AUXIN RESPONSE FACTOR (ARF) proteins, mediating auxin responses.

Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation.

Death-associated protein kinase 2 (DAPK2) is a Ca(2+)/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-β are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-β was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein's subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions.

Regulation of Ubiquitin Independent Proteasomal Degradation of Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) are labile proteins and undergo degradation by ubiquitin dependent and independent mechanisms. Previously we reported on the 20S proteasomal degradation pathway of the IDPs that is regulated by NQO1, a 20S associated protein, and by Nannies. Nannies are IDP interacting proteins functioning to inhibit IDPs proteasomal degradation. However the question of whether and how the IDPs are targeted to the 20S proteasome remained open. To address this question, we adopted the BiFC cell based technique for visualizing protein interactions with 20S proteasome. We found that IDPs interact with a subset of 20S proteasome alpha subunits. The results suggest that the 20S proteasome has two hub sites for interaction with IDPs in the alpha ring. We focused on the subunit PSMA3 and showed that its C terminus interacts with multiple IDPs. Thus, it implies that IDPs are targeted to the 20S proteasome prior to their degradation. In addition, lately we have identified a new type of 26S proteasomes whereby no ATP but NADH regulates its assembly. We have evidence for this new type of NADH‐26S proteasomes preferentially involved in IDPs degradation. Finally we will report on a new proteomic approach in identifying the selective 20S proteasomes substrates.Asher, G., Reuven, N. and Shaul, Y. (2006). 20S proteasomes and protein degradation by default. BioEssay. 28(8):844‐849.Tsvetkov, P., Reuven, N. and Shaul Y. (2009). The nanny model for IDPs. Nat Chem Biol. 5(11):778‐81Tsvetkov P, Myers N, Eliav R, Adamovich Y, Hagai T, Adler J, Navon A, and Shaul Y. (2014). NADH Binds and Stabilizes the 26S Proteasomes Independent of ATP. J Biol Chem. Apr 18;289(16):11272‐81.

BiFC for protein-protein interactions and protein topology: discussing an integrative approach for an old technique.

BiFC (Bimolecular Fluorescence Complementation) is one of the most widely used techniques to study protein-protein interactions as well as protein topology in living cells. This method allows the visualization of protein interactions or the analysis of their topology in the cell compartments where the proteins belong, without changing their chemical properties, as often occurs after mixing the content of different cellular compartments in cell extracts. Several laboratories use this method because it is relatively easy to perform; however, sometimes a positive protein-protein interaction BiFC signal (i.e., reconstitution of fluorescence of interacting protein pairs) does not necessarily mean that the tested proteins are actually interacting in vivo in a specific way. Here we describe the BiFC approach for assessing protein-protein interactions and for establishing protein topology and we discuss how to best perform this method to avoid false positive results when studying protein interactions in plant cells.

Native protein mapping and visualization of protein interactions in the area of human plasma high‐density lipoprotein by combining nondenaturing micro 2DE and quantitative LC‐MS/MS

A human plasma sample was subjected to nondenaturing micro 2DE and a gel area (5 mm × 18 mm) that includes high-density lipoprotein (HDL) was cut into 1 mm × 1 mm squares, then the proteins in the 90 gel pieces were analyzed by quantitative LC-MS/MS. Grid-cutting of the gel was employed to; (i) ensure the total analysis of the proteins in the area, (ii) standardize the conditions of analysis by LC-MS/MS, (iii) reconstruct the protein distribution patterns from the quantity data. Totally 154 proteins were assigned in the 90 gel pieces and the quantity distribution of each was reconstructed as a color density pattern (a native protein map). The map of apolipoprotein (Apo) A-I showed a wide apparent mass distribution characteristic to HDL and was compared with the maps of the other 153 proteins. Eleven proteins showed maps of wide distribution that overlapped with the map of Apo A-I, and all have been reported to be the components of HDL. Further, seven minor proteins associated with HDL were detected at the gel positions of high Apo A-I quantity. These results for the first time visualized the localization of HDL apolipoproteins on a nondenaturing 2DE gel and strongly suggested their interactions.

The SOL-2/Neto Auxiliary Protein Modulates the Function of AMPA-Subtype Ionotropic Glutamate Receptors

The neurotransmitter glutamate mediates excitatory synaptic transmission by gating ionotropic glutamate receptors (iGluRs). AMPA receptors (AMPARs), a subtype of iGluR, are strongly implicated in synaptic plasticity, learning, and memory. We previously discovered two classes of AMPAR auxiliary proteins in C. elegans that modify receptor kinetics and thus change synaptic transmission. Here, we have identified another auxiliary protein, SOL-2, a CUB-domain protein that associates with both the related auxiliary subunit SOL-1 and with the GLR-1 AMPAR. In sol-2 mutants, behaviors dependent on glutamatergic transmission are disrupted, GLR-1-mediated currents are diminished, and GLR-1 desensitization and pharmacology are modified. Remarkably, a secreted variant of SOL-1 delivered in trans can rescue sol-1 mutants, and this rescue depends on in cis expression of SOL-2. Finally, we demonstrate that SOL-1 and SOL-2 have an ongoing role in the adult nervous system to control AMPAR-mediated currents.

In SituVisualization of Protein Interactions in Sensory Neurons: Glutamic Acid-Rich Proteins (GARPs) Play Differential Roles for Photoreceptor Outer Segment Scaffolding

Vertebrate photoreceptors initiate vision via a G-protein-mediated signaling cascade organized within a specialized cilium, the outer segment (OS). The membranous "stacked pancake" architecture of this organelle must be partially renewed daily to maintain cell function and viability; however, neither its static structure nor renewal process is well described in molecular terms. Glutamic acid-rich proteins (GARPs), including the cyclic nucleotide-gated cation channel (CNGB1) and GARP2 (a CNGB1 splice-variant), are proposed to contribute to OS organization in concert with peripherin/rds (P/rds), a retinal tetraspanin. We developed and applied an in situ fluorescence complementation approach that offers an unprecedented glimpse at the formation, trafficking, and localization of GARP-P/rds interactions in transgenic Xenopus laevis rod photoreceptors. Interactions for these (and other) proteins could be readily visualized using confocal microscopy. Nearly all associations, including CNGB1-P/rds interaction, were initiated within inner segments (ISs) before trafficking to OSs. In contrast, GARP2-P/rds interactions were only observed downstream, at or near sites of disk morphogenesis. These results suggest that GARP2-P/rds interaction participates directly in structuring disk stacks but CNGB1-P/rds interaction does not and instead serves mainly to localize plasma membrane ion channels. Altogether, the results lead us to propose that differential interaction of GARPs with P/rds may contribute to the broad phenotypic heterogeneity produced by inherited defects in P/rds. Analogous experiments applied to the synaptic protein RIBEYE suggest that monomers can oligomerize at the level of the IS before ribbon assembly and demonstrate the general applicability of this strategy for in situ analysis of protein interactions in sensory neurons.

Visualization of protein interactions in living cells

Ligand binding to cell membrane receptors sets off a series of protein interactions that convey the nuances of ligand identity to the cell interior. The information may be encoded in conformational changes, the interaction kinetics and, in the case of multichain immunoreceptors, by chain rearrangements. The signals may be modulated by dynamic compartmentalization of the cell membrane, cellular architecture, motility, and activation-all of which are difficult to reconstitute for studies of receptor signaling in vitro. In this paper, we will discuss how protein interactions in general and receptor signaling in particular can be studied in living cells by different fluorescence imaging techniques. Particularly versatile are methods that exploit Förster resonance energy transfer (FRET), which is exquisitely sensitive to the nanometer-range proximity and orientation between fluorophores. Fluorescence correlation microscopy (FCM) can provide complementary information about the stoichiometry and diffusion kinetics of large complexes, while bimolecular fluorescence complementation (BiFC) and other complementation techniques can capture transient interactions. A continuing challenge is extracting from the imaging data the quantitative information that is necessary to verify different models of signal transduction.

Visualizing protein interactions involved in the formation of the 42S RNP storage particle of Xenopus oocytes

During early phases of Xenopus oogenesis, 5S rRNA and tRNAs are stored in the cytoplasm of young oocytes in the form of a common RNA-protein complex termed the 42S particle. These storage particles comprise two kinds of proteins with different RNA binding specificities. The tRNA-binding protein 42Sp50 belongs to the EF1A (eukaryotic translation elongation factor 1A) family of translation elongation factors, while 42Sp43 is a diverged form of the transcription factor TFIIIA (transcription factor IIIA) and binds 5S rRNA. Little is known about the mode of protein-protein interactions that stabilize the 42S particle. We have determined the intracellular localization of the protein components of the 42S particle by expressing fluorescent protein-tagged fusions in transparent previtellogenic oocytes. 42Sp50 and its isoforms (EF1A-S and EF1A-O) were excluded from the nuclei and distributed uniformly throughout the cytoplasm with no enrichment in the Balbiani bodies, as described earlier by immunocytochemistry. In contrast, 42Sp43 accumulated in the amplified nucleoli. However, when both proteins were simultaneously expressed, 42Sp43 was no longer present in the nucleoli but was retained, together with 42Sp50, in the cytoplasm, the most likely site of 42S particle assembly. In contrast, the somatic-type EF1A isoforms were unable to redirect 42Sp43 from the nucleolar to the cytoplasmic compartment. We also tested for in vivo interactions using transiently transfected mammalian cells (COS-7 cell line). In this heterologous cell system 42Sp43 remained bound to the nucleoli but, on co-expression, induced the redistribution of 42Sp50 from the cytoplasm to the nucleoli. The microscopic approach described allows visualization of protein-protein interactions involved in the assembly of 42S storage particles. In particular, the transfection assay using COS-7 cells provides a rapid screening test that should facilitate identification of critical residues and structural determinants that enable the proteins of the 42S storage particle to interact with each other and to establish distinct higher-order RNP (ribonucleoprotein) complexes.

Visualization of G Protein−Coupled Receptor (GPCR) Interactions in Living Cells Using Bimolecular Fluorescence Complementation (BiFC)

Members of the G protein-coupled receptor (GPCR) superfamily have been shown to homo- and hetero-oligomerize both in vitro and in vivo. Although the functional and pharmacological significance of GPCR oligomerization is far from being completely understood, evidence suggests that, depending on the receptor, oligomerization may influence ligand binding, G protein coupling, and receptor targeting. Bimolecular fluorescence complementation (BiFC) is a technique based on the complementation of fragments from fluorescent proteins that allows the measurement and visualization of protein interactions in living cells. It can be extended to the simultaneous detection of distinct protein-protein interactions using a multicolor setup. This unit describes the application of BiFC and multicolor BiFC to the visualization of GPCR oligomerization in a neuronal cell model. Oligomerization of GPCR fusions to BiFC tags is visualized and measured using fluorescence microscopy and fluorometry. The effect of ligands on the relative formation of distinct oligomeric species is monitored with a ratiometric multicolor BiFC approach.

Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay

The activity of proteins is typically regulated by secondary modifications and by interactions with other partners, resulting in the formation of protein complexes whose functions depend on the participating proteins. Accordingly, it is of central importance to monitor the presence of interaction complexes as well as their localization, thus providing information about the types of cells where the proteins are located and in what sub-cellular compartment these interactions occur. Several methods for visualizing protein interactions in situ have been developed during the last decade. These methods in most cases involve genetic constructs, and they have been successfully used in assays of living cell maintained in tissue culture, but they cannot easily be implemented in studies of clinical specimens. For such samples, affinity reagents like antibodies can be used to target the interacting proteins. In this review we will describe the in situ proximity ligation assays ( in situ PLA), a method that is suitable for visualizing protein interactions in both tissue sections and in vitro cell lines, and we discuss research tasks when this or other method may be selected.

Visualizing protein interactions by bimolecular fluorescence complementation in Xenopus

Bimolecular fluorescence complementation (BiFC) provides a simple and direct way to visualise protein–protein interactions in vivo and in real-time. In this article, we describe methods by which one can implement this approach in embryos of the South African claw-toed frog Xenopus laevis. We have made use of Venus, an improved version of yellow fluorescent protein (YFP), so as to achieve rapid detection of protein interactions. To suppress spontaneous interactions between the N- and C-terminal fragments of Venus, a point mutation (T153M) was introduced into the N-terminal fragment. We have used this reagent to monitor signalling by members of the transforming growth factor type β family in cells of the Xenopus embryo.

Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis

The bimolecular fluorescence complementation (BiFC) assay is a powerful tool for visualizing and identifying protein interactions in living cells. This assay is based on the principle of protein-fragment complementation, using two nonfluorescent fragments derived from fluorescent proteins. When two fragments are brought together in living cells by tethering each to one of a pair of interacting proteins, fluorescence is restored. Here, we provide a protocol for a Venus-based BiFC assay to visualize protein interactions in the living nematode, Caenorhabditis elegans. We discuss how to design appropriate C. elegans BiFC cloning vectors to enable visualization of protein interactions using either inducible heat shock promoters or native promoters; transform the constructs into worms by microinjection; and analyze and interpret the resulting data. When expression of BiFC fusion proteins is induced by heat shock, the fluorescent signals can be visualized as early as 30 min after induction and last for 24 h in transgenic animals. The entire procedure takes 2-3 weeks to complete.