Superresolution imaging of HIV in infected cells with FlAsH-PALM

Imaging protein assemblies at molecular resolution without affecting biological function is a long-standing goal. The diffraction-limited resolution of conventional light microscopy (∼200–300 nm) has been overcome by recent superresolution (SR) methods including techniques based on accurate localization of molecules exhibiting stochastic fluorescence; however, SR methods still suffer important restrictions inherent to the protein labeling strategies. Antibody labels are encumbered by variable specificity, limited commercial availability and affinity, and are mostly restricted to fixed cells. Fluorescent protein fusions, though compatible with live cell imaging, substantially increase protein size and can interfere with their biological activity. We demonstrate SR imaging of proteins tagged with small tetracysteine motifs and the fluorescein arsenical helix binder (FlAsH-PALM). We applied FlAsH-PALM to image the integrase enzyme (IN) of HIV in fixed and living cells under experimental conditions that fully preserved HIV infectivity. The obtained resolution (∼30 nm) allowed us to characterize the distribution of IN within virions and intracellular complexes and to distinguish different HIV structural populations based on their morphology. We could thus discriminate ∼100 nm long mature conical cores from immature Gag shells and observe that in infected cells cytoplasmic (but not nuclear) IN complexes display a morphology similar to the conical capsid. Together with the presence of capsid proteins, our data suggest that cytoplasmic IN is largely present in intact capsids and that these can be found deep within the cytoplasm. FlAsH-PALM opens the door to in vivo SR studies of microbial complexes within host cells and may help achieve truly molecular resolution.     

Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

Posttranslational modifications (PTMs) are key contributors to chromatin function. The ability to comprehensively link specific histone PTMs with specific chromatin factors would be an important advance in understanding the functions and genomic targeting mechanisms of those factors. We recently introduced a cross-linked affinity technique, BioTAP-XL, to identify chromatin-bound protein interactions that can be difficult to capture with native affinity techniques. However, BioTAP-XL was not strictly compatible with similarly comprehensive analyses of associated histone PTMs. Here we advance BioTAP-XL by demonstrating the ability to quantify histone PTMs linked to specific chromatin factors in parallel with the ability to identify nonhistone binding partners. Furthermore we demonstrate that the initially published quantity of starting material can be scaled down orders of magnitude without loss in proteomic sensitivity. We also integrate hydrophilic interaction chromatography to mitigate detergent carryover and improve liquid chromatography-mass spectrometric performance. In summary, we greatly extend the practicality of BioTAP-XL to enable comprehensive identification of protein complexes and their local chromatin environment.Chromatin encompasses the subset of proteins and RNAs in complex with DNA to form the chromosomes within eukaryotic nuclei. As the primary protein constituents of chromatin, histones organize genomic DNA into nucleosomes and display an extensive array of posttranslational modifications (PTMs) (1). It is increasingly evident that specific histone PTMs are selectively recognized by specific nonhistone proteins that are themselves regulators of many nuclear events such as epigenetic silencing (2, 3). Consequently, defects in these chromatin components often manifest in a number of human diseases (4).As specific interactions between modified histones and nonhistone proteins help distinguish complexes participating in distinct processes, the identification of protein and modification-dependent interactions provides key insights into chromatin biology. Various chromatography and pulldown methods have been developed to identify binding partners. In a typical native pulldown experiment, nuclei from hypotonically lysed cells are digested with micrococcal nuclease (MNase). Extracted chromatin is subjected to immunoprecipitation to isolate the desired complexes. If using a tag such as FLAG, complexes are then competitively eluted and identified. Several drawbacks to native pulldowns as described here are that MNase leaves behind a substantial insoluble nuclear pellet and degrades associating RNAs, that salt extraction of the digested nucleosomes risks dissociation of the tagged bait from its interacting partners, and that most tags do not have sufficient affinity for their respective antibody to permit highly stringent washes. The result is often an incomplete picture of the chromatin-bound complex. BioTAP-XL is a technique that complements native pulldowns. BioTAP-XL uses formaldehyde to inactivate endogenous enzymes and to cross-link labile interactions with the bait, sonication to solubilize the genomic chromatin content, and high affinity tags to capture the bait and its associated factors (K_d = 10⁻⁹ and 10⁻¹⁵ M for the bipartite BioTAP tag compared with 10⁻⁸ M for the FLAG tag). Thus, BioTAP-XL captures interactions that may be inaccessible or disrupted using native pulldowns.BioTAP-XL has successfully identified key interactors of the male-specific lethal (MSL) dosage compensation and heterochromatin protein (HP1a) complexes in Drosophila (5) and revealed novel components of the Polycomb Repressive Complex 2 in human tissue culture cells (6) and fruit flies (7). Despite the aforementioned advantages, BioTAP-XL suffers from several limitations. First, the cell number requirement far exceeds the amounts typically used in standard affinity experiments, namely with 10⁸ cells for native pulldowns (8) compared with 10¹⁰ cells for BioTAP pulldown (9). Second, the high detergent concentrations used during the procedure risks interfering with liquid chromatography mass spectrometry (LC-MS). Third, our analysis of the associated histone PTMs is complicated by both the nearly irreversible binding of the tagged complexes to streptavidin and the inability to obtain histone peptides of comparable ionization efficiencies across modified states using trypsin alone.We sought to address these limitations using Drosophila and human cells expressing BioTAP-tagged factors. To demonstrate scalability, we performed parallel tandem affinity pulldowns of the same bait across various initial chromatin amounts. We recovered the core interactors of MSL3 at even the lowest tested amount of Drosophila cells. To improve detergent removal, we compared a sample processed either with conventional spin columns or hydrophilic interaction chromatography (HILIC). We observed significant improvement in LC-MS quality of the MSL3 elutions prepared using HILIC over spin columns. To facilitate histone modification analysis, we derivatized the associated histones directly on the streptavidin beads. We successfully quantified histone modifications enriched with MSL3 and HP1a from Drosophila cells and with chromobox homolog 4 (CBX4) and bromodomain-containing protein 4 (BRD4) from human cells. In short, this report streamlines BioTAP-XL for biochemically characterizing chromatin complexes and their associated histone modifications.     

Hierarchical looping of zigzag nucleosome chains in metaphase chromosomes

The architecture of higher-order chromatin in eukaryotic cell nuclei is largely unknown. Here, we use electron microscopy-assisted nucleosome interaction capture (EMANIC) cross-linking experiments in combination with mesoscale chromatin modeling of 96-nucleosome arrays to investigate the internal organization of condensed chromatin in interphase cell nuclei and metaphase chromosomes at nucleosomal resolution. The combined data suggest a novel hierarchical looping model for chromatin higher-order folding, similar to rope flaking used in mountain climbing and rappelling. Not only does such packing help to avoid tangling and self-crossing, it also facilitates rope unraveling. Hierarchical looping is characterized by an increased frequency of higher-order internucleosome contacts for metaphase chromosomes compared with chromatin fibers in vitro and interphase chromatin, with preservation of a dominant two-start zigzag organization associated with the 30-nm fiber. Moreover, the strong dependence of looping on linker histone concentration suggests a hierarchical self-association mechanism of relaxed nucleosome zigzag chains rather than longitudinal compaction as seen in 30-nm fibers. Specifically, concentrations lower than one linker histone per nucleosome promote self-associations and formation of these looped networks of zigzag fibers. The combined experimental and modeling evidence for condensed metaphase chromatin as hierarchical loops and bundles of relaxed zigzag nucleosomal chains rather than randomly coiled threads or straight and stiff helical fibers reconciles aspects of other models for higher-order chromatin structure; it constitutes not only an efficient storage form for the genomic material, consistent with other genome-wide chromosome conformation studies that emphasize looping, but also a convenient organization for local DNA unraveling and genome access.The physical packaging of megabase pairs of genomic DNA stored as the chromatin fiber in eukaryotic cell nuclei has been one of the great challenges in biology (1). The limited resolution and disparate levels that can be studied by both experimental and modeling studies of chromatin, which exhibits multiple spatial and temporal scales par excellence, make it challenging to present an integrated structural view, from nucleosomes to chromosomes (2). Because all fundamental template-directed processes of DNA depend on chromatin architecture, advances in our understanding of chromatin higher-order organization are needed to help interpret numerous regulatory events from DNA damage repair to epigenetic control.At the primary structural level, the DNA makes ∼1.7 left-superhelical turns around eight core histones to form a nucleosome core. The nucleosome cores are connected by linker DNA to form nucleosome arrays. An X-ray crystal structure of the nucleosome core has been solved at atomic resolution (3), and a short, four-nucleosome array has also been solved (4). Next, at the secondary structural level, the nucleosome arrays, aided by linker histones (H1 or H5), form a compact chromatin fiber with a diameter of ∼30 nm and longitudinal compaction of 5–7 nucleosomes per 11 nm (5–8). However, evidence for 30-nm fibers in interphase nuclei of living cells has been controversial (reviewed in refs. 9 and 10). For example, whereas a distinct 30-nm fiber architecture is observed in terminally differentiated cells (11, 12), neither continuous nor periodic 30-nm fibers are observed in the nuclei of proliferating cells (13–15). However, zigzag features of the chromatin fibers are well supported by nucleosome interaction mapping in vitro (16) and in vivo (15).For chromatin architecture within metaphase chromosomes, fluorescence studies of mitotic chromosome condensation in vivo (17), cryo-EM observations of unfixed and unstained chromosomes in situ (18), and small-angle X-ray scattering (19) show no structures resembling folded 30-nm fibers and instead suggest random folding of soft polymers. Evidence is also accumulating that during chromosome condensation in mitosis, chromatin higher-order structure is dramatically altered at the global level (20) by significant increase in looping (21). A random type of looping, however, cannot explain sharp chromosomal boundaries separating the translocated genomic regions in metaphase chromosomes (22) as well as formation of highly localized fibers of transgenic DNA, up to 250 nm in diameter, detected by fluorescence imaging in vivo (17). In contrast, a hierarchical or layered looping could explain the above aspects of chromosome organization; in addition, it could help reconcile the experiments in living cells with in vitro data and determine which aspects of the secondary structure are retained in the metaphase chromosome and how these features correlate with the polymer melt model (18, 23).Here we apply the EM-assisted nucleosome interaction capture (EMANIC) technique, which captures nearest-neighbor interactions in combination with mesoscale modeling of chromatin fibers (16) to deduce chromatin architecture in interphase nuclei and metaphase chromosomes. Our results reveal persistence of the zigzag geometry as a dominant architectural motif in these types of chromatin. For metaphase chromosomes, we report a dramatic increase in longer-range interactions, consistent with intrafiber looping, quite different from that seen in compact chromatin fibers in vitro and interphase chromatin in vivo. Modeling also shows hierarchical looping for long fibers, with the loop occurrence strongly modulated by the density of linker histones. Such looping of loosely folded zigzag arrays appears to be an efficient mechanism for both condensing and unraveling the genomic material. Our hierarchical looping mechanism can also explain how distant regulatory DNA sites can be brought together naturally for genic interactions and how linker histone levels and epigenetic histone modifications can further modulate global and local chromatin architecture.     

Multimodal on-chip nanoscopy and quantitative phase imaging reveals the nanoscale morphology of liver sinusoidal endothelial cells

Visualization of three-dimensional (3D) morphological changes in the subcellular structures of a biological specimen is a major challenge in life science. Here, we present an integrated chip-based optical nanoscopy combined with quantitative phase microscopy (QPM) to obtain 3D morphology of liver sinusoidal endothelial cells (LSEC). LSEC have unique morphology with small nanopores (50-300 nm in diameter) in the plasma membrane, called fenestrations. The fenestrations are grouped in discrete clusters, which are around 100 to 200 nm thick. Thus, imaging and quantification of fenestrations and sieve plate thickness require resolution and sensitivity of sub-100 nm along both the lateral and the axial directions, respectively. In chip-based nanoscopy, the optical waveguides are used both for hosting and illuminating the sample. The fluorescence signal is captured by an upright microscope, which is converted into a Linnik-type interferometer to sequentially acquire both superresolved images and phase information of the sample. The multimodal microscope provided an estimate of the fenestration diameter of 119 ± 53 nm and average thickness of the sieve plates of 136.6 ± 42.4 nm, assuming the constant refractive index of cell membrane to be 1.38. Further, LSEC were treated with cytochalasin B to demonstrate the possibility of precise detection in the cell height. The mean phase value of the fenestrated area in normal and treated cells was found to be 161 ± 50 mrad and 109 ± 49 mrad, respectively. The proposed multimodal technique offers nanoscale visualization of both the lateral size and the thickness map, which would be of broader interest in the fields of cell biology and bioimaging.

Far-field optical nanoscopy techniques are frequently used to visualize subcellular structures in biological specimens by surpassing the diffraction limit. Optical nanoscopy encompasses a plethora of techniques, including stimulated emission depletion microscopy (1), structured illumination microscopy (SIM) (2), different variants of single-molecule localization microscopy (SMLM), such as photo-activated localization microscopy (3) and direct stochastic optical reconstruction microscopy (dSTORM) (4), and intensity fluctuation–based techniques such as superresolution optical fluctuation imaging (5). These techniques can help detect subcellular structures (<200 nm) of biological specimens such as lipids, proteins, membrane structures, microtubules, and nucleic acids by specific fluorescence tagging (6). Each technique has respective advantages and disadvantages; for example, SIM has gained popularity for live-cell imaging due to its fast image acquisition time but at limited spatial resolution (7). dSTORM, on the other hand, is slower but offers high resolution for characterization of viral proteins (8) and imaging actin filaments in mammalian cells (9, 10), for example. To reduce the complexity of the typical SMLM setup using a total internal reflection fluorescence (TIRF) configuration, a photonic chip-based optical nanoscopy system was recently proposed (11–13). In the chip-based system, a photonic integrated circuit is used to replace the usual free space optics for excitation. The collection, however, is done through free space optics. The main advantage of this configuration is the decoupling of excitation and collection pathways as well as miniaturization of the excitation light path of the system. In chip-based nanoscopy, the TIRF illumination is generated through the evanescent field of waveguides rather than using conventional high magnification and high numerical aperture (N.A.) TIRF lens. The evanescent field in waveguides can be generated over extraordinarily large areas, as it is only defined by the waveguide geometry. The waveguide geometry makes it possible to use any imaging objective lens to image arbitrarily large areas as compared to the traditional TIRF-based dSTORM (12), which is limited by the field of view (FOV) of the TIRF lens.Quantitative phase microscopy (QPM) is a label-free optical microscopy technique, which facilitates sensitive measurements of the refractive index and thickness of both biological specimens (14). Various QPM methods have been proposed so far for extracting optical phase and dynamics of biological cells (15–17). These techniques offer high phase sensitivity (spatial and temporal), transverse resolution, and high imaging speed (15). The spatial and temporal phase sensitivity of the QPM system is highly dependent on the illumination source and the type of interferometric geometry, respectively (17–19). For example, common path QPM techniques offer better temporal phase sensitivity, which can be used to measure membrane fluctuation of the cells (20). In addition, spatial phase sensitivity of the system can be improved by using low-coherence light sources (halogen lamps and light-emitting diodes [LED]) but requires phase-shifting techniques to utilize the whole FOV of the camera (21). A recent advancement in the QPM technique with superior resolution using structured illumination (22, 23) and three-dimensional (3D) information of the samples has been shown by measuring the phase across multiple angles of illumination. This technique facilitates tomography of various biological specimens such as red blood cells, HT29 cells, and bovine embryos (17, 24). Since the lateral resolution of the QPM technique depends on the N.A. of the objective lens, imaging beyond the diffraction limit (<200 nm) is still challenging and limits the study of subcellular structures. Therefore, it is useful to develop multimodality routes in which different microscopy methods can be utilized to provide complementary information about biological specimens such as liver sinusoidal endothelial cells (LSEC).Fig. 1 depicts LSEC that contain large numbers of fenestrations. These transcellular nanopores vary in diameter from 50 to 300 nm, which is just below the diffraction limit of optical microscopy (25–27). Fenestrations are typically clustered in groups of 5 to 100 within areas called sieve plates (28). The porous morphology of LSEC acts as an ultrafilter between blood and the underlying hepatocytes, facilitating the bidirectional exchange of substrates between the interior of the liver and blood. For example, smaller viruses and drugs can pass this barrier, while blood cells are retained within the sinusoidal vessel lumen (25, 29). The typical thickness of sieve plates is around 100 to 150 nm (30), so fenestrations are consequently nanoscale sized in all three dimensions. As shown in Fig. 1, the fenestrations in sieve plates form openings through the entire LSEC cell body, and therefore TIRF illumination is ideally suited for imaging these structures. Determining the diameter and number of fenestrations, as well as the height of sieve plate regions, is important, as it can be affected by several drugs and conditions (31, 32). The loss of LSEC porous morphology, a process called defenestration, compromises the filtration properties of the liver, which may lead to atherosclerosis (33). Moreover, aging results in “pseudocapillarization,” whereby LSEC simultaneously lose fenestrations and become thicker (34) (Fig. 1). This is believed to be a main factor contributing to the age-related need to increase doses of drugs targeting hepatocytes (e.g., statins) that have to pass through the fenestrations (35). The number of fenestrations in vitro can be increased using actin disrupting agents such as cytochalasin B (27). This treatment decreases the height of LSEC outside of the nuclear area, which contributes to the formation of new fenestrations (36).Open in a separate windowFig. 1.Top view (A) and cross-sectional view (B) of LSEC. LSEC have unique morphology, in which nanoscopic fenestrations are grouped in thin sieve plates. The diameter of fenestrations and thickness of sieve plates are below the diffraction limit of conventional optical microscopes. The number and size of fenestrations, but also LSEC thickness, can be affected by aging and in liver diseases. In vitro, the number of fenestrations can be increased using actin disrupting agents, such as cytochalasin B (27).Here, we have developed a multimodal chip-based optical nanoscopy and highly sensitive QPM system to visualize the 3D morphological changes in LSEC. The proposed system decouples the light illumination path from the collection path and thus enables a straightforward integration of dSTORM and QPM. The nanoscale phase sensitivity of the QPM technique is utilized to extract the optical thickness of sieve plates. Moreover, chip-based dSTORM supports superresolution imaging down to 50 nm over an extraordinarily large FOV up to millimeter scale (12). Therefore, integration of dSTORM and QPM allows superresolution imaging in the lateral dimension (with dSTORM) and nanometric sensitivity in the axial direction (with QPM). In this work, we demonstrate the capabilities of the system by imaging LSEC with both diffraction-limited TIRF microscopy and dSTORM. The fenestrations and sieve plates are observable with dSTORM, and the average optical thickness of the sieve plate region is obtained using diffraction-limited QPM. Furthermore, we investigated the change in the interior morphology of sieve plates by treating the cell with cytochalasin B (10 μg/mL). The deficiency of lateral resolution of QPM was compensated by dSTORM, which enabled us to localize the sieve plate regions containing subdiffraction-sized fenestration. Therefore, in the cell membrane regions distal from the nucleus, the 3D morphology of LSEC can be reconstructed reliably using our multimodal approach. The integrated system offers a combination of simultaneous functional and quantitative imaging of the cells with large FOV, providing a compact imaging platform with a potential for high-throughput morphological and nanometric imaging for specific biological applications.     

Total kinetic analysis reveals how combinatorial methylation patterns are established on lysines 27 and 36 of histone H3

We have developed a targeted method to quantify all combinations of methylation on an H3 peptide containing lysines 27 and 36 (H3K27-K36). By using stable isotopes that separately label the histone backbone and its methylations, we tracked the rates of methylation and demethylation in myeloma cells expressing high vs. low levels of the methyltransferase MMSET/WHSC1/NSD2. Following quantification of 99 labeled H3K27-K36 methylation states across time, a kinetic model converged to yield 44 effective rate constants qualifying each methylation and demethylation step as a function of the methylation state on the neighboring lysine. We call this approach MS-based measurement and modeling of histone methylation kinetics (M4K). M4K revealed that, when dimethylation states are reached on H3K27 or H3K36, rates of further methylation on the other site are reduced as much as 100-fold. Overall, cells with high MMSET have as much as 33-fold increases in the effective rate constants for formation of H3K36 mono- and dimethylation. At H3K27, cells with high MMSET have elevated formation of K27me1, but even higher increases in the effective rate constants for its reversal by demethylation. These quantitative studies lay bare a bidirectional antagonism between H3K27 and H3K36 that controls the writing and erasing of these methylation marks. Additionally, the integrated kinetic model was used to correctly predict observed abundances of H3K27-K36 methylation states within 5% of that actually established in perturbed cells. Such predictive power for how histone methylations are established should have major value as this family of methyltransferases matures as drug targets.     

Differential proteomic profiling of mitochondria from Podospora anserina, rat and human reveals distinct patterns of age-related oxidative changes

According to the 'free radical theory of ageing', the generation and accumulation of reactive oxygen species are key events during ageing of biological systems. Mitochondria are a major source of ROS and prominent targets for ROS-induced damage. Whereas mitochondrial DNA and membranes were shown to be oxidatively modified with ageing, mitochondrial protein oxidation is not well understood. The purpose of this study was an unbiased investigation of age-related changes in mitochondrial proteins and the molecular pathways by which ROS-induced protein oxidation may disturb cellular homeostasis. In a differential comparison of mitochondrial proteins from young and senescent strains of the fungal ageing model Podospora anserina, from brains of young (5 months) vs. older rats (17 and 31 months), and human cells, with normal and chemically accelerated in vitro ageing, we found certain redundant posttranslationally modified isoforms of subunits of ATP synthase affected across all three species. These appear to represent general susceptible hot spot targets for oxidative chemical changes of proteins accumulating during ageing, and potentially initiating various age-related pathologies and processes. This type of modification is discussed using the example of SAM-dependent O-methyltransferase from P. anserina (PaMTH1), which surprisingly was found to be enriched in mitochondrial preparations of senescent cultures.