Colocalization Of Molecules Research Articles

KCBC – a correlation-based method for co-localization analysis of super-resolution microscopy images using bivariate Ripley's K functions

Motivated by the high demand for co-localization analysis methods for super-resolution microscopy images which are featured with nanoscale precise locational information of molecules, this paper establishes a novel correlation-based method, KCBC, named after the Coordinated-Based Colocalization (CBC) method proposed by Malkusch et al. in 2012, by using bivariate Ripley's K functions. The local KCBC values are to quantify the local spatial co-localization of molecules between two species by measuring the correlation of bivariate Ripley's K functions over equal-area concentric rings around the base species within a near distance. The mean of local KCBC values is proposed to quantify the co-localization degree of cross-channel to base-channel molecules for the whole image. It could effectively correct the false positives with reduced variance and increased power within the user-defined proximity size. We provide extensive simulation studies under different scenarios to demonstrate the unbiasedness of the KCBC method, and its ability to filter noise signals and random over-counting. Our real data application for super-resolution mitochondria image data illustrates the applicability of our methods with increased effectiveness and power.

Co-Density Distribution Maps for Advanced Molecule Colocalization and Co-Distribution Analysis.

Cellular and subcellular spatial colocalization of structures and molecules in biological specimens is an important indicator of their co-compartmentalization and interaction. Presently, colocalization in biomedical images is addressed with visual inspection and quantified by co-occurrence and correlation coefficients. However, such measures alone cannot capture the complexity of the interactions, which does not limit itself to signal intensity. On top of the previously developed density distribution maps (DDMs), here, we present a method for advancing current colocalization analysis by introducing co-density distribution maps (cDDMs), which, uniquely, provide information about molecules absolute and relative position and local abundance. We exemplify the benefits of our method by developing cDDMs-integrated pipelines for the analysis of molecules pairs co-distribution in three different real-case image datasets. First, cDDMs are shown to be indicators of colocalization and degree, able to increase the reliability of correlation coefficients currently used to detect the presence of colocalization. In addition, they provide a simultaneously visual and quantitative support, which opens for new investigation paths and biomedical considerations. Finally, thanks to the coDDMaker software we developed, cDDMs become an enabling tool for the quasi real time monitoring of experiments and a potential improvement for a large number of biomedical studies.

Revisiting colocalization via optimal transport.

The advent of STED microscopy, which allows observation at a sub-diffraction resolution, raises a challenge in studying spatial proximities of biomolecules’ distributions. In this issue, researchers have attempted to study colocalization of molecules by employing optimal transport.

Stoichiometric analysis of protein complexes by cell fusion and single molecule imaging

The composition, stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit stoichiometry at the single molecule level. However, these typically require artificially low expression levels or detergent isolation to achieve the low fluorophore concentrations required for single molecule imaging, both of which may bias native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this dilution strategy with fluorescence correlation spectroscopy permits quantitative assessment of cytoplasmic oligomerization, while stepwise photobleaching and single molecule colocalization may be used to study the subunit stoichiometry of membrane receptors. Single protein recovery after dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex stoichiometry.

Structure and wettability of heterogeneous monomolecular films of phospholipids with cholesterol or lauryl gallate

The mica supported Langmuir-Blodgett (LB) monolayers composed of 1,2‑dipalmitoyl‐sn‑glycero‑3‑phosphocholine (DPPC) and 1,2‑dioleoyl‑sn‑glycero‑3‑phosphocholine (DOPC) were used to investigate the effect of cholesterol (Chol) or lauryl gallate (LG) on physicochemical properties of the phospholipid DPPC/DOPC monomolecular film treated as a model leaflet of the bilayer structured membrane. First the surface topography studies of these systems were carried out by means of atomic force microscopy (AFM). Then wettability was investigated via the contact angle measurements and the surface free energy determination. Finally, the time-of-flight secondary ion mass spectrometry (TOF-SIMS) was applied for the surface chemical analysis. Wettability of the monolayers was found to be closely related to the surface chemistry, molecular packing and ordering, miscibility and co-localization of molecules rather than nanoscale roughness. The comparison of the surface polarity determined from the values of contact angle and secondary ion signal intensity provided consistent results. The correlation between the changes of the electron-donor parameter and the 184/57 ratio revealed significance of using the latter quantity as a polarity indicator. The carried out studies show preferential association of components providing a framework for understanding the mechanism of heterogeneities formation within the membrane which reflect the specialization of various regions.

Monitoring transcriptional activity by RNA polymerase II in vitro using single molecule co-localization.

RNA polymerase II (Pol II) transcribes eukaryotic mRNA genes. To initiate transcription, pre-initiation complexes (PICs) containing Pol II and general transcription factors (GTFs) form on the core promoters of target genes. In cells this process is regulated by transcriptional activators, co-activators, and chromatin modifying complexes. Reconstituted in vitro transcription systems are important tools for studying the enzymology and fundamental steps in the transcription reaction. In these systems, studying transcription can be complex due to the heterogeneous mixture of transcriptionally active and inactive complexes that assemble at promoters. Accordingly, we developed a technique to use single molecule microscopy to resolve this heterogeneity and distinguish transcriptionally active complexes from inactive complexes. This system uses fluorescently-labeled promoter DNA and a minimal reconstituted transcription system consisting of purified human Pol II and GTFs. Here we describe the materials, methods, and analysis required to study Pol II transcription at the single molecule level. The flexibility of our single molecule method allows for adaptation to answer diverse mechanistic questions about transcription that would otherwise be difficult to study using ensemble assays.

Examining the Coverage of Blood Vessels by Astrocyte Endfeet in an Animal Model of Major Depressive Disorder.

The use of immunofluorescent immunohistochemical methods has been instrumental to characterize the distribution and extents of colocalization of molecules located in various cellular compartments. Such information has been pivotal to formulate hypothesis about their roles in physiological and pathological conditions.In the brain, astrocytes interact with endothelial cells to form the blood-brain barrier (BBB). Astrocytes regulate features of the BBB through the tips of their processes, called astrocyte endfeet, which surround and contact brain microvessels. Any disruption in astrocyte endfeet can have dramatic consequences on the integrity of the BBB, with consequent development of brain disorders.Here we describe a method to characterize the integrity of astrocyte endfeet around blood vessels in an animal model of major depressive disorder (MDD) using confocal microscopy, Adobe Photoshop, and the AutoQuantX3 software.

Single Molecule Fluorescent Colocalization of Split Aptamers for Ultrasensitive Detection of Biomolecules.

Single-molecule fluorescence imaging is a promising strategy for biomolecule detection. However, the accuracy of single-molecule method is often compromised by the false-positive events at the ultralow sample levels that are caused by the nonspecific adsorption of the fluorescent labeled probe and other fluorescent impurities on the imaging surface. Here, we demonstrate an ultrasensitive single molecule detection assay based on dual-color fluorescent colocalization of spilt aptamers that was implemented to the measurement of adenosine triphosphate (ATP). The ATP aptamer was split into two fragments and labeled with green and red dye molecules, respectively. When the two probes of split aptamers were brought together by the target ATP molecule, the two colors of fluorescence of two probes were simultaneously detected through two channels and projected to the correlated locations in the two halves of image. The colocalizaiton imaging of two split apatamer probes greatly excluded the false detection of biomolecules that was usually caused by the fluorescent noise of single nonbound aptamer probes and impurities, and further improved the accuracy of measurement. The assay showed excellent selectivity and high sensitivity for ATP detection with linear range of 1 pM to 5 nM and a detection limit of 100 fM. This versatile protocol of single molecule colocalization of split apatamer can be widely applied to the ultrasensitive and highly accurate detection of many types of biomolecules in basic research and biomedical applications.

Single Molecule Colocalization Analysis of TRPV1 Interactions in Unroofed HEK293T/17 Cells

Single Molecule Colocalization Analysis of TRPV1 Interactions in Unroofed HEK293T/17 Cells

Single Molecule Approaches in RNA-Protein Interactions.

RNA-protein interactions govern every aspect of RNA metabolism, and aberrant RNA-binding proteins are the cause of hundreds of genetic diseases. Quantitative measurements of these interactions are necessary in order to understand mechanisms leading to diseases and to develop efficient therapies. Existing methods of RNA-protein interactome capture can afford a comprehensive snapshot of RNA-protein interaction networks but lack the ability to characterize the dynamics of these interactions. As all ensemble methods, their resolution is also limited by statistical averaging. Here we discuss recent advances in single molecule techniques that have the potential to tackle these challenges. We also provide a thorough overview of single molecule colocalization microscopy and the essential protein and RNA tagging and detection techniques.

Co-Orientation: Quantifying Simultaneous Co-Localization and Orientational Alignment of Filaments in Light Microscopy.

Co-localization analysis is a widely used tool to seek evidence for functional interactions between molecules in different color channels in microscopic images. Here we extend the basic co-localization analysis by including the orientations of the structures on which the molecules reside. We refer to the combination of co-localization of molecules and orientational alignment of the structures on which they reside as co-orientation. Because the orientation varies with the length scale at which it is evaluated, we consider this scale as a separate informative dimension in the analysis. Additionally we introduce a data driven method for testing the statistical significance of the co-orientation and provide a method for visualizing the local co-orientation strength in images. We demonstrate our methods on simulated localization microscopy data of filamentous structures, as well as experimental images of similar structures acquired with localization microscopy in different color channels. We also show that in cultured primary HUVEC endothelial cells, filaments of the intermediate filament vimentin run close to and parallel with microtubuli. In contrast, no co-orientation was found between keratin and actin filaments. Co-orientation between vimentin and tubulin was also observed in an endothelial cell line, albeit to a lesser extent, but not in 3T3 fibroblasts. These data therefore suggest that microtubuli functionally interact with the vimentin network in a cell-type specific manner.

Statistical analysis of molecule colocalization in bioimaging.

The quantitative analysis of molecule interactions in bioimaging is key for understanding the molecular orchestration of cellular processes and is generally achieved through the study of the spatial colocalization between the different populations of molecules. Colocalization methods are traditionally divided into pixel-based methods that measure global correlation coefficients from the overlap between pixel intensities in different color channels, and object-based methods that first segment molecule spots and then analyze their spatial distributions with second-order statistics. Here, we present a review of such colocalization methods and give a quantitative comparison of their relative merits in different types of biological applications and contexts. We show on synthetic and biological images that object-based methods are more robust statistically than pixel-based methods, and allow moreover to quantify accurately the number of colocalized molecules.

Quantitative analysis of single-molecule superresolution images.

This review highlights the quantitative capabilities of single-molecule localization-based superresolution imaging methods. In addition to revealing fine structural details, the molecule coordinate lists generated by these methods provide the critical ability to quantify the number, clustering, and colocalization of molecules with 10-50 nm resolution. Here we describe typical workflows and precautions for quantitative analysis of single-molecule superresolution images. These guidelines include potential pitfalls and essential control experiments, allowing critical assessment and interpretation of superresolution images.

Dual Functioning Genetic Tags for Simultaneous Isolation and Observation of Fluorescent Complexes from Whole Cell Extract

Single molecule colocalization methods provide facile means of detecting and analyzing biomolecular complex formation. However, many of these complexes can not be reconstituted from purified systems and must be studied in cell extracts. We have developed a new approach for tagging these complexes with small molecule fluorophores (e.g., Cy3 and Cy5) and for isolation of these complexes in situ during a single molecule experiment. We are able to isolate complexes using either immunoaffinity techniques or by biotin/streptavidin interaction. We have used this method to isolate a number of complexes involved in pre-mRNA splicing in yeast including both the U1 and U6 snRNPs comprising 17 and 8 proteins respectively. By introducing multiple tags, we have analyzed protein stoichiometry in these isolated complexes. Finally, we have performed functional assays on the immobilized snRNPs to study their interactions with other components of the RNA splicing machinery in yeast. We predict that this approach will be broadly applicable for studies of other macromolecular machines.

Quantifying molecular colocalization in live cell fluorescence microscopy

One of the most challenging tasks in microscopy is the quantitative identification and characterization of molecular interactions. In living cells this task is typically performed by fluorescent labeling of the interaction partners with spectrally distinct fluorophores and imaging in different color channels. Current methods for determining colocalization of molecules result in outcomes that can vary greatly depending on signal-to-noise ratios, threshold and background levels, or differences in intensity between channels. Here, we present a novel and quantitative method for determining the degree of colocalization in live-cell fluorescence microscopy images for two and more data channels. Moreover, our method enables the construction of images that directly classify areas of high colocalization.

281: Assembly of IFNAR1/IFNAR2 is regulated by ubiquitin specific protease 18

Type I Interferons (IFN) are key cytokines in the regulation of the innate immune response. All IFNs bind to a shared cell surface receptor comprised of two subunits, IFNAR1 and IFNAR2. Together with IFN the receptors assemble to hetero-trimeric “ternary” complexes which initiate phosphorylation cascades through the intracellularly associated Janus-Kinases Jak1 and Tyk2. Detailed structure–function analysis of IFNs has established that the dynamics and affinities of IFN interactions with the receptor subunits play a critical role for regulating signalling specificities. Here we have explored the IFN-induced assembly of the ternary signalling complex at physiological expression level by dual colour single molecule imaging. To this end, we employed highly specific and efficient orthogonal posttranslational labelling approaches with photostable organic fluorophores combined with TIRF-microscopy. By single molecule co-localization and co-tracking analysis we have mapped lateral distribution and diffusion of the two receptor subunits with very high spatial and temporal resolution in living cells. Thus, we were able to monitor IFN-induced assembly, co-diffusion and dissociation of individual ternary signalling complexes in the plasma membrane. Systematic modulation of the binding affinities towards the receptor subunits revealed that binding affinity of IFNα2 towards IFNAR1 is optimized for efficient ternary complex formation at physiological receptor surface concentrations. Interestingly, the negative feedback regulator ubiquitin-specific protease 18 (USP18) reduces receptor dimerization by IFNα2, but not IFNβ, which binds to IFNAR1 with higher affinity. Strikingly, this USP18-dependent loss of 2D affinity could be mimicked by truncating the receptor’s intracellular domains. These observations suggest that ternary complexes are stabilized by intracellular interactions of Jak1 and Tyk2 and that these interactions are interfered by USP18, thus selectively abrogating signalling by IFNs with low affinity towards IFNAR1.

Hydrodynamic Co-Localization of Molecules in Supported Lipid Bilayers Detected by Secondary Ion Mass Spectrometry

Here we report on the use of secondary ion mass spectrometry (SIMS) to study the hydrodynamic co-localization of membrane components in supported lipid bilayers formed by the fusion of multi-component giant unilamellar vesicles to oxidized silicon substrates. In these experiments, hydrodynamic drag forces arising from flow above the supported lipid bilayer (SLB) results in the directed motion of molecules protruding from the SLB. In this particular case, protrusion of the cholera toxin B into the aqueous layer serves as a handle for the directed motion of its natural ligand, ganglioside GM1, and any other molecule (i.e. cholesterol) strongly associated with it. Orthogonal isotopic labeling or fluorination of every lipid bilayer component allowed generation of molecule-specific images, using a nanoSIMS, that map the lateral re-distribution of molecules in a lipid bilayer as a result of hydrodynamic flow. Furthermore, simultaneous detection of up to seven different ion species, including secondary electrons, allowed generation of ion ratio images whose signal intensity values could be correlated to composition through the use of calibration curves from standard samples.

Subnanometre single-molecule localization, registration and distance measurements

Remarkable progress in optical microscopy has been made in the measurement of nanometre distances. If diffraction blurs the image of a point object into an Airy disk with a root-mean-squared (r.m.s.) size of s = 0.44lambda/2NA (approximately 90 nm for light with a wavelength of lambda = 600 nm and an objective lens with a numerical aperture of NA = 1.49), limiting the resolution of the far-field microscope in use to d = 2.4s approximately = 200 nm, additional knowledge about the specimen can be used to great advantage. For example, if the source is known to be two spatially resolved fluorescent molecules, the distance between them is given by the separation of the centres of the two fluorescence images. In high-resolution microwave and optical spectroscopy, there are numerous examples where the line centre is determined with a precision of less than 10(-6) of the linewidth. In contrast, in biological applications the brightest single fluorescent emitters can be detected with a signal-to-noise ratio of approximately 100, limiting the centroid localization precision to s(loc) > or = 1% (> or = 1 nm) of the r.m.s. size, s, of the microscope point spread function (PSF). Moreover, the error in co-localizing two or more single emitters is notably worse, remaining greater than 5-10% (5-10 nm) of the PSF size. Here we report a distance resolution of s(reg) = 0.50 nm (1sigma) and an absolute accuracy of s(distance) = 0.77 nm (1sigma) in a measurement of the separation between differently coloured fluorescent molecules using conventional far-field fluorescence imaging in physiological buffer conditions. The statistical uncertainty in the mean for an ensemble of identical single-molecule samples is limited only by the total number of collected photons, to s(loc) approximately 0.3 nm, which is approximately 3 x 10(-3) times the size of the optical PSF. Our method may also be used to improve the resolution of many subwavelength, far-field imaging methods such as those based on co-localization of molecules that are stochastically switched on in space. The improved resolution will allow the structure of large, multisubunit biological complexes in biologically relevant environments to be deciphered at the single-molecule level.

Particle Image Cross Correlation Spectroscopy (PICCS)

In vivo studies of the dynamics of single molecules and particles have produced a wealth of biological insights. The diffusion behavior of a membrane receptor, for example, reveals the structure of the plasma membrane. In recent years we have developed an analysis technique to quantify single molecule translational movement on a nm length and ms time scale (Particle Image Correlation Spectroscopy (PICS), Semrau, Schmidt., Biophys. J., 2007).The insight gained from experiments on a single molecular species is however limited. No biomolecule operates on its own and often it is the very interaction between different types of molecules which is biologically most relevant. To that end we further developed PICS for experiments with two differently labeled molecular species. Particle Image Cross-Correlation Spectroscopy (PICCS) allows us to unambiguously measure molecule colocalization, even at large molecule densities and down to a length scale of 10 nm. To demonstrate the method's power we studied the intracellular transport of the morphogen Dpp enclosed in endosomes. Dpp establishes a gradient in the wing imaginal disk of fruit fly larvae, providing positional information to cells. Using PICCS we found that 52% of apical Dpp is in early endosomes and that early endosomes contain 1.9 times as much Dpp as other endosomes. Our data suggests that Dpp resides shorter in early endosomes compared to late/recycling endosomes.PICCS makes it also possible to push the limits of the time scales on which molecular movement can be measured. By labeling one molecule with two spectrally resolvable fluorophores we can follow the dynamics of the molecule on a 100 μs time scale.To summarize, PICCS opens up a whole new range of in vivo single molecule experiments: Molecular correlations and dynamics can be measured with unprecedented accuracy and temporal resolution.

The origin of protein interactions and allostery in colocalization

Two fundamental principles can account for how regulated networks of interacting proteins originated in cells. These are the law of mass action, which holds that the binding of one molecule to another increases with concentration, and the fact that the colocalization of molecules vastly increases their local concentrations. It follows that colocalization can amplify the effect on one protein of random mutations in another protein and can therefore, through natural selection, lead to interactions between proteins and to a startling variety of complex allosteric controls. It also follows that allostery is common and that homologous proteins can have different allosteric mechanisms. Thus, the regulated protein networks of organisms seem to be the inevitable consequence of natural selection operating under physical laws.