Genome activity and nuclear metabolism clearly depend on accessibility, but it is not known whether and to what extent nuclear structures limit the mobility and access of individual molecules. We used fluorescently labeled streptavidin with a nuclear localization signal as an average-sized, inert protein to probe the nuclear environment. The protein was injected into the cytoplasm of mouse cells, and single molecules were tracked in the nucleus with high-speed fluorescence microscopy. We analyzed and compared the mobility of single streptavidin molecules in structurally and functionally distinct nuclear compartments of living cells. Our results indicated that all nuclear subcompartments were easily and similarly accessible for such an average-sized protein, and even condensed heterochromatin neither excluded single molecules nor impeded their passage. The only significant difference was a higher frequency of transient trappings in heterochromatin, which lasted only tens of milliseconds. The streptavidin molecules, however, did not accumulate in heterochromatin, suggesting comparatively less free volume. Interestingly, the nucleolus seemed to exclude streptavidin, as it did many other nuclear proteins, when visualized by conventional fluorescence microscopy. The tracking of single molecules, nonetheless, showed no evidence for repulsion at the border but relatively unimpeded passage through the nucleolus. These results clearly show that single-molecule tracking can provide novel insights into mobility of proteins in the nucleus that cannot be obtained by conventional fluorescence microscopy. Our results suggest that nuclear processes may not be regulated at the level of physical accessibility but rather by local concentration of reactants and availability of binding sites.
All molecular traffic between nucleus and cytoplasm occurs via the nuclear pore complex (NPC) within the nuclear envelope. In this study we analyzed the interactions of the nuclear transport receptors kapalpha2, kapbeta1, kapbeta1DeltaN44, and kapbeta2, and the model transport substrate, BSA-NLS, with NPCs to determine binding sites and kinetics using single-molecule microscopy in living cells. Recombinant transport receptors and BSA-NLS were fluorescently labeled by AlexaFluor 488, and microinjected into the cytoplasm of living HeLa cells expressing POM121-GFP as a nuclear pore marker. After bleaching the dominant GFP fluorescence the interactions of the microinjected molecules could be studied using video microscopy with a time resolution of 5 ms, achieving a colocalization precision of 30 nm. These measurements allowed defining the interaction sites with the NPCs with an unprecedented precision, and the comparison of the interaction kinetics with previous in vitro measurements revealed new insights into the translocation mechanism.
We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochemical and photochemical analysis indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in Forster resonance energy transfer applications.
Power output of light bulbs changes over time and the total energy delivered will depend on the optical beam path of the microscope, filter sets and objectives used, thus making comparison between experiments performed on different microscopes complicated. Using a thermocoupled power meter, it is possible to measure the exact amount of light applied to a specimen in fluorescence microscopy, regardless of the light source, as the light power measured can be translated into a power density at the sample. This widely used and simple tool forms the basis of a new degree of calibration precision and comparability of results among experiments and setups. Here we describe an easy-to-follow protocol that allows researchers to precisely estimate excitation intensities in the object plane, using commercially available opto-mechanical components. The total duration of this protocol for one objective and six filter cubes is 75 min including start-up time for the lamp.
Messenger RNAs undergo 5' capping, splicing, 3'-end processing, and export before translation in the cytoplasm. It has become clear that these mRNA processing events are tightly coupled and have a profound effect on the fate of the resulting transcript. This processing is represented by modifications of the pre-mRNA and loading of various protein factors. The sum of protein factors that stay with the mRNA as a result of processing is modified over the life of the transcript, conferring significant regulation to its expression.
Export of messenger RNA occurs via nuclear pores, which are large nanomachines with diameters of roughly 120 nm that are the only link between the nucleus and cytoplasm. Hence, mRNA export occurs over distances smaller than the optical resolution of conventional light microscopes. There is extensive knowledge on the physical structure and composition of the nuclear pore complex, but transport selectivity and the dynamics of mRNA export at nuclear pores remain unknown. Here we developed a super-registration approach using fluorescence microscopy that can overcome the current limitations of co-localization by means of measuring intermolecular distances of chromatically different fluorescent molecules with nanometre precision. With this method we achieve 20-ms time-precision and at least 26-nm spatial precision, enabling the capture of highly transient interactions in living cells. Using this approach we were able to spatially resolve the kinetics of mRNA transport in mammalian cells and present a three-step model consisting of docking (80 ms), transport (5-20 ms) and release (80 ms), totalling 180 +/- 10 ms. Notably, the translocation through the channel was not the rate-limiting step, mRNAs can move bi-directionally in the pore complex and not all pores are equally active.
Cellular life can be described as a dynamic equilibrium of a highly complex network of interacting molecules. For this reason, it is no longer sufficient to "only" know the identity of the participants in a cellular process, but questions such as where, when, and for how long also have to be addressed to understand the mechanism being investigated. Additionally, ensemble measurements may not sufficiently describe individual steps of molecular mobility, spatial-temporal resolution, kinetic parameters, and geographical mapping. It is vital to investigate where individual steps exactly occur to enhance our understanding of the living cell. The nucleus, home too many highly complex multi-order processes, such as replication, transcription, splicing, etc., provides a complicated, heterogeneous landscape. Its dynamics were studied to a new level of detail by fluorescence correlation spectroscopy (FCS). Single-molecule tracking, while still in its infancy in cell biology, is becoming a more and more attractive method to deduce key elements of this organelle. Here we discuss the potential of tracking single RNAs and proteins in the nucleus. Their dynamics, localization, and interaction rates will be vital to our understanding of cellular life. To demonstrate this, we provide a review of the HIV life cycle, which is an extremely elegant balance of nuclear and cytoplasmic functions and provides an opportunity to study mechanisms deeply integrated within the structure of the nucleus. In summary, we aim to present a specific, dynamic view of nuclear cellular life based on single molecule and FCS data and provide a prospective for the future.
The internal workings of the nucleus remain a mystery. A list of component parts exists, and in many cases their functional roles are known for events such as transcription, RNA processing, or nuclear export. Some of these components exhibit structural features in the nucleus, regions of concentration or bodies that have given rise to the concept of functional compartmentalization--that there are underlying organizational principles to be described. In contrast, a picture is emerging in which transcription appears to drive the assembly of the functional components required for gene expression, drawing from pools of excess factors. Unifying this seemingly dual nature requires a more rigorous approach, one in which components are tracked in time and space and correlated with onset of specific nuclear functions. In this chapter, we anticipate tools that will address these questions and provide the missing kinetics of nuclear function. These tools are based on analyzing the fluctuations inherent in the weak signals of endogenous nuclear processes and determining values for them. In this way, it will be possible eventually to provide a computational model describing the functional relationships of essential components.
The central dogma of molecular biology - DNA makes RNA makes proteins - is a flow of information that in eukaryotes encounters a physical barrier: the nuclear envelope, which encapsulates, organizes and protects the genome. Nuclear-pore complexes, embedded in the nuclear envelope, regulate the passage of molecules to and from the nucleus, including the poorly understood process of the export of RNAs from the nucleus. Recent imaging approaches focusing on single molecules have provided unexpected insight into this crucial step in the information flow. This review addresses the latest studies of RNA export and presents some models for how this complex process may work.
The nuclear pore complex (NPC) has long been viewed as a point-like entry and exit channel between the nucleus and the cytoplasm. New data support a different view whereby the complex displays distinct spatial dynamics of variable duration ranging from milliseconds to events spanning the entire cell cycle. Discrete interaction sites outside the central channel become apparent, and transport regulation at these sites seems to be of greater importance than currently thought. Nuclear pore components are highly active outside the NPC or impact the fate of cargo transport away from the nuclear pore. The NPC is a highly dynamic, crowded environment-constantly loaded with cargo while providing selectivity based on unfolded proteins. Taken together, this comprises a new paradigm in how we view import/export dynamics and emphasizes the multiscale nature of NPC-mediated cellular transport.
Nuclear pore component Nup98 is a potential tumor suppressor and regulates posttranscriptional expression of select p53 target genes
The p53 tumor suppressor utilizes multiple mechanisms to selectively regulate its myriad target genes, which in turn mediate diverse cellular processes. Here, using conventional and single-molecule mRNA analyses, we demonstrate that the nucleoporin Nup98 is required for full expression of p21, a key effector of the p53 pathway, but not several other p53 target genes. Nup98 regulates p21 mRNA levels by a posttranscriptional mechanism in which a complex containing Nup98 and the p21 mRNA 3'UTR protects p21 mRNA from degradation by the exosome. An in silico approach revealed another p53 target (14-3-3sigma) to be similarly regulated by Nup98. The expression of Nup98 is reduced in murine and human hepatocellular carcinomas (HCCs) and correlates with p21 expression in HCC patients. Our study elucidates a previously unrecognized function of wild-type Nup98 in regulating select p53 target genes that is distinct from the well-characterized oncogenic properties of Nup98 fusion proteins.
Resolution in optical nanoscopy (or super-resolution microscopy) depends on the localization uncertainty and density of single fluorescent labels and on the sample's spatial structure. Currently there is no integral, practical resolution measure that accounts for all factors. We introduce a measure based on Fourier ring correlation (FRC) that can be computed directly from an image. We demonstrate its validity and benefits on two-dimensional (2D) and 3D localization microscopy images of tubulin and actin filaments. Our FRC resolution method makes it possible to compare achieved resolutions in images taken with different nanoscopy methods, to optimize and rank different emitter localization and labeling strategies, to define a stopping criterion for data acquisition, to describe image anisotropy and heterogeneity, and even to estimate the average number of localizations per emitter. Our findings challenge the current focus on obtaining the best localization precision, showing instead how the best image resolution can be achieved as fast as possible.
Obesity, physical activity, and their interaction in incident atrial fibrillation in postmenopausal women
BACKGROUND: Atrial fibrillation (AF) is the most common cardiac arrhythmia and is associated with increased risk of stroke and death. Obesity is an independent risk factor for AF, but modifiers of this risk are not well known. We studied the roles of obesity, physical activity, and their interaction in conferring risk of incident AF.
METHODS AND RESULTS: The Women's Health Initiative (WHI) Observational Study was a prospective observational study of 93 676 postmenopausal women followed for an average of 11.5 years. Incident AF was identified using WHI-ascertained hospitalization records and diagnostic codes from Medicare claims. A multivariate Cox's hazard regression model adjusted for demographic and clinical risk factors was used to evaluate the interaction between obesity and physical activity and its association with incident AF. After exclusion of women with prevalent AF, incomplete data, or underweight body mass index (BMI), 9792 of the remaining 81 317 women developed AF. Women were, on average, 63.4 years old, 7.8% were African American, and 3.6% were Hispanic. Increased BMI (hazard ratio [HR], 1.12 per 5-kg/m(2) increase; 95% confidence interval [CI], 1.10 to 1.14) and reduced physical activity (>9 vs. 0 metabolic equivalent task hours per week; HR, 0.90; 95% CI, 0.85 to 0.96) were independently associated with higher rates of AF after multivariate adjustment. Higher levels of physical activity reduced the AF risk conferred by obesity (interaction P=0.033).
CONCLUSIONS: Greater physical activity is associated with lower rates of incident AF and modifies the association between obesity and incident AF.
Modulating artificial membrane morphology: pH-induced chromatic transition and nanostructural transformation of a bolaamphiphilic conjugated polymer from blue helical ribbons to red nanofibers.
Design and characterization of helical ribbon assemblies of a bolaamphiphilic conjugated polymer and their color-coded transformation into nanofibers are described. An L-glutamic acid modified bolaamphiphilic diacetylene lipid was synthesized and self-assembled into right-handed helical ribbons with micron scale length and nano scale thickness under mild conditions. The ribbon structures were further stabilized by polymerizing well-aligned diacetylene units to form bisfunctional polydiacetylenes (PDAs). Transitions from flat sheets to helical ribbons and tubes were observed by transmission electron microscopy. The helical ribbons appear to originate from the rupture of flat sheets along domain edges and the peeling off between stacked lipid layers. These results point to the applicability of chiral packing theory in bolaamphiphilic supramolecular assemblies. Contact mode atomic force microscopy observations revealed that high order existed in the surface packing arrangement. Hexagonal and pseudorectangular packings were observed in flat and twisted regions of the ribbons, respectively, suggesting a correlation between microscopic morphologies and nanoscopic packing arrangements. The tricarboxylate functionalities of the bolaamphiphilic lipid provide a handle for the manipulation of the bisfunctional PDAs' morphology. Increasing solution pH caused the fraying of helical ribbons into nanofibers accompanied by a sharp blue-to-red chromatic transition. A dramatic change in circular dichroism spectra was observed during this process, suggesting the loss of chirality in packing. A model is proposed to account for the pH-induced morphological change and chromatic transition. The color-coded transition between two distinct microstructures would be useful in the design of sensors and other "smart" nanomaterials requiring defined molecular templates.
Cytocompatible poly(ethylene glycol)-co-polycarbonate hydrogels cross-linked by copper-free, strain-promoted click chemistry
Strategies to encapsulate cells in cytocompatible three-dimensional hydrogels with tunable mechanical properties and degradability without harmful gelling conditions are highly desired for regenerative medicine applications. Here we reported a method for preparing poly(ethylene glycol)-co-polycarbonate hydrogels through copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry. Hydrogels with varying mechanical properties were formed by "clicking" azido-functionalized poly(ethylene glycol)-co-polycarbonate macromers with dibenzocyclooctyne-functionalized poly(ethylene glycol) under physiological conditions within minutes. Bone marrow stromal cells encapsulated in these gels exhibited higher cellular viability than those encapsulated in photo-cross-linked poly(ethylene glycol) dimethacrylate. The precise control over the macromer compositions, cytocompatible SPAAC cross-linking, and the degradability of the polycarbonate segments make these hydrogels promising candidates for scaffold and stem cell assisted tissue repair and regeneration.
(alpha-NaYbF4:Tm(3+))/CaF2 core/shell nanoparticles with efficient near-infrared to near-infrared upconversion for high-contrast deep tissue bioimaging
We describe the development of novel and biocompatible core/shell (alpha-NaYbF(4):Tm(3+))/CaF(2) nanoparticles that exhibit highly efficient NIR(in)-NIR(out) upconversion (UC) for high contrast and deep bioimaging. When excited at ~980 nm, these nanoparticles emit photoluminescence (PL) peaked at ~800 nm. The quantum yield of this UC PL under low power density excitation (~0.3 W/cm(2)) is 0.6 +/- 0.1%. This high UC PL efficiency is realized by suppressing surface quenching effects via heteroepitaxial growth of a biocompatible CaF(2) shell, which results in a 35-fold increase in the intensity of UC PL from the core. Small-animal whole-body UC PL imaging with exceptional contrast (signal-to-background ratio of 310) is shown using BALB/c mice intravenously injected with aqueously dispersed nanoparticles (700 pmol/kg). High-contrast UC PL imaging of deep tissues is also demonstrated, using a nanoparticle-loaded synthetic fibrous mesh wrapped around rat femoral bone and a cuvette with nanoparticle aqueous dispersion covered with a 3.2 cm thick animal tissue (pork).
Amphiphilic degradable polymers for immobilization and sustained delivery of sphingosine 1-phosphate
Controlled delivery of the angiogenic factor sphingosine 1-phosphate (S1P) represents a promising strategy for promoting vascularization during tissue repair and regeneration. In this study, we developed an amphiphilic biodegradable polymer platform for the stable encapsulation and sustained release of S1P. Mimicking the interaction between amphiphilic S1P and its binding proteins, a series of polymers with hydrophilic poly(ethylene glycol) core and lipophilic flanking segments of polylactide and/or poly(alkylated lactide) with different alkyl chain lengths were synthesized. These polymers were electrospun into fibrous meshes, and loaded with S1P in generally high loading efficiencies (>90%). Sustained S1P release from these scaffolds could be tuned by adjusting the alkyl chain length, blockiness and lipophilic block length, achieving 35-55% and 45-80% accumulative releases in the first 8h and by 7 days, respectively. Furthermore, using endothelial cell tube formation assay and chicken chorioallantoic membrane assay, we showed that the different S1P loading doses and release kinetics translated into distinct pro-angiogenic outcomes. These results suggest that these amphiphilic polymers are effective delivery vehicles for S1P and may be explored as tissue engineering scaffolds where the delivery of lipophilic or amphiphilic bioactive factors is desired. reserved.
Bioorthogonally cross-linked hydrogel network with precisely controlled disintegration time over a broad range
Hydrogels with predictable degradation are highly desired for biomedical applications where timely disintegration of the hydrogel (e.g., drug delivery, guided tissue regeneration) is required. However, precisely controlling hydrogel degradation over a broad range in a predictable manner is challenging due to limited intrinsic variability in the degradation rate of liable bonds and difficulties in modeling degradation kinetics for complex polymer networks. More often than not, empirical tuning of the degradation profile results in undesired changes in other properties. Here we report a simple but versatile hydrogel platform that allows us to formulate hydrogels with predictable disintegration time from 2 to >250 days yet comparable macroscopic physical properties. This platform is based on a well-defined network formed by two pairs of four-armed polyethylene glycol macromers terminated with azide and dibenzocyclooctyl groups, respectively, via labile or stable linkages. The high-fidelity bioorthogonal reaction between the symmetric hydrophilic macromers enables robust cross-linking in water, phosphate-buffered saline, and cell culture medium to afford tough hydrogels capable of withstanding >90% compressive strain. Strategic placement of labile ester linkages near the cross-linking site within this superhydrophilic network, accomplished by adjustments of the ratio of the macromers used, enables broad tuning of the disintegration rates precisely matching with the theoretical predictions based on first-order linkage cleavage kinetics. This platform can be exploited for applications where a precise degradation rate is targeted.
Nervous system injury or disease leads to activation of glia, which govern postinjury responses in the nervous system. Axonal injury in Drosophila results in transcriptional up-regulation of the glial engulfment receptor Draper; there is extension of glial membranes to the injury site (termed activation), and then axonal debris is internalized and degraded. Loss of the small GTPase Rac1 from glia completely suppresses glial responses to injury, but upstream activators remain poorly defined. Loss of the Rac guanine nucleotide exchange factor (GEF) Crk/myoblast city (Mbc)/dCed-12has no effect on glial activation, but blocks internalization and degradation of debris. Here we show that the signaling molecules downstream of receptor kinase (DRK) and daughter of sevenless (DOS) (mammalian homologs, Grb2 and Gab2, respectively) and the GEF son of sevenless (SOS) (mammalian homolog, mSOS) are required for efficient activation of glia after axotomy and internalization/degradation of axonal debris. At the earliest steps of glial activation, DRK/DOS/SOS function in a partially redundant manner with Crk/Mbc/dCed-12, with blockade of both complexes strongly suppressing all glial responses, similar to loss of Rac1. This work identifies DRK/DOS/SOS as the upstream Rac GEF complex required for glialresponses to axonal injury, and demonstrates a critical requirement for multiple GEFs in efficient glial activation after injury and internalization/degradation of axonal debris.
Phosphatidic acid phospholipase A1 mediates ER-Golgi transit of a family of G protein-coupled receptors
The coat protein II (COPII)-coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein-coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking.