Bottom-Up Meets Top-Down: The Crossroads of Multiscale Chromatin Modeling

Chromatin can be viewed as a hierarchically structured fiber that regulates gene expression. It consists of a complex network of DNA and proteins whose characteristic dynamical modes facilitate compaction and rearrangement in the cell nucleus. These modes stem from chromatin’s fundamental unit, the nucleosome, and their effects are propagated across length scales. Understanding the effects of nucleosome dynamics on the chromatin fiber, primarily through post-translational modifications that occur on the histones, is of central importance to epigenetics. Within the last decade, imaging and chromosome conformation capture techniques have revealed a number of structural and statistical features of the packaged chromatin fiber at a hitherto unavailable level of resolution. Such experiments have led to increased efforts to develop polymer models that aim to reproduce, explain, and predict the contact probability scaling and density heterogeneity. At nanometer scales, available models have focused on the role of the nucleosome and epigenetic marks on local chromatin structure. At micrometer scales, existing models have sought to explain scaling laws and density heterogeneity. Less work, however, has been done to reconcile these two approaches: bottom-up and top-down models of chromatin. In this perspective, we highlight the multiscale simulation models that are driving toward an understanding of chromatin structure and function, from the nanometer to the micron scale, and we highlight areas of opportunity and some of the prospects for new frameworks that bridge these two scales. Taken together, experimental and modeling advances over the last few years have established a robust platform for the study of chromatin fiber structure and dynamics, which will be of considerable use to the chromatin community in developing an understanding of the interplay between epigenomic regulation and molecular structure.     

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated ^1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated ^4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot ^8-10. We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.     

Localization Microscopy (SPDM) Reveals Clustered Formations of P-Glycoprotein in a Human Blood-Brain Barrier Model

P-glycoprotein (Pgp; also known as MDR1, ABCB1) is the most important and best studied efflux transporter at the blood-brain barrier (BBB); however, the organization of Pgp is unknown. The aim of this study was to employ the recently developed super-resolution fluorescence microscopy method spectral precision distance microscopy/spectral position determination microscopy (SPDM) to investigate the spatial distribution of Pgp in the luminal plasma membrane of brain capillary endothelial cells. Potential disturbing effects of cell membrane curvatures on the distribution analysis are addressed with computer simulations. Immortalized human cerebral microvascular endothelial cells (hCMEC/D3) served as a model of human BBB. hCMEC/D3 cells were transduced with a Pgp-green fluorescent protein (GFP) fusion protein incorporated in a lentivirus-derived vector. The expression and localization of the Pgp-GFP fusion protein was visualized by SPDM. The limited resolution of SPDM in the z-direction leads to a projection during the imaging process affecting the appeared spatial distribution of fluorescence molecules in the super-resolution images. Therefore, simulations of molecule distributions on differently curved cell membranes were performed and their projected spatial distribution was investigated. Function of the fusion protein was confirmed by FACS analysis after incubation of cells with the fluorescent probe eFluxx-ID Gold in absence and presence of verapamil. More than 112,000 single Pgp-GFP molecules (corresponding to approximately 5,600 Pgp-GFP molecules per cell) were detected by SPDM with an averaged spatial resolution of approximately 40 nm in hCMEC/D3 cells. We found that Pgp-GFP is distributed in clustered formations in hCMEC/D3 cells while the influence of present random cell membrane curvatures can be excluded based on the simulation results. Individual formations are distributed randomly over the cell membrane.     

Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins

Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future.     

Correlative Photoactivated Localization and Scanning Electron Microscopy

The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.     

Surface Localization of Escherichia coli 5′-Nucleotidase by Electron Microscopy

The 5'-nucleotidase of Escherichia coli was shown to be located at the cell wall surface by histochemical techniques utilizing the deposition of inorganic phosphate. Penetration of the 5'-nucleotidase in the periplasmic space was seen only in cells treated with ethylenediaminetetraacetic acid (EDTA)-tris(hydroxymethyl)aminomethane (Tris). The 3'-nucleotidase of E. coli was also found to have a surface location, and periplasmic precipitation of inorganic phosphate was seen only after EDTA-Tris-sucrose exposure.     

Impact of Parameter Variation in Fabrication of Nanostructure by Atomic Force Microscopy Nanolithography

In this letter, we investigate the fabrication of Silicon nanostructure patterned on lightly doped (10¹⁵ cm⁻³) p-type silicon-on-insulator by atomic force microscope nanolithography technique. The local anodic oxidation followed by two wet etching steps, potassium hydroxide etching for silicon removal and hydrofluoric etching for oxide removal, are implemented to reach the structures. The impact of contributing parameters in oxidation such as tip materials, applying voltage on the tip, relative humidity and exposure time are studied. The effect of the etchant concentration (10% to 30% wt) of potassium hydroxide and its mixture with isopropyl alcohol (10%vol. IPA ) at different temperatures on silicon surface are expressed. For different KOH concentrations, the effect of etching with the IPA admixture and the effect of the immersing time in the etching process on the structure are investigated. The etching processes are accurately optimized by 30%wt. KOH +10%vol. IPA in appropriate time, temperature, and humidity.     

X-Ray Fluorescence Microscopy Reveals the Role of Selenium in Spermatogenesis

Selenium (Se) is a trace element with important roles in human health. Several selenoproteins have essential functions in development. However, the cellular and tissue distribution of Se remains largely unknown because of the lack of analytical techniques that image this element with sufficient sensitivity and resolution. Herein, we report that X-ray fluorescence microscopy (XFM) can be used to visualize and quantify the tissue, cellular, and subcellular topography of Se. We applied this technique to characterize the role of Se in spermatogenesis and identified a dramatic Se enrichment specifically in late spermatids, a pattern that was not seen in any other elemental maps. This enrichment was due to elevated levels of the mitochondrial form of glutathione peroxidase 4 and was fully dependent on the supplies of Se by selenoprotein P. High-resolution scans revealed that Se concentrated near the lumen side of elongating spermatids, where structural components of sperm are formed. During spermatogenesis, maximal Se associated with decreased phosphorus, whereas Zn did not change. In sperm, Se was primarily in the midpiece and colocalized with Cu and Fe. XFM allowed quantification of Se in the midpiece (0.8 fg) and head (0.2 fg) of individual sperm cells, revealing the ability of sperm cells to handle the amounts of this element well above its toxic levels. Overall, the use of XFM allowed visualization of tissue and cellular Se and provided important insights in the role of this and other trace elements in spermatogenesis.     

Super-Resolution Microscopy Reveals the Native Ultrastructure of the Erythrocyte Cytoskeleton

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Ecological and biogeographical analyses of Mediterranean forests of northern Morocco

Mediterranean forests in northern Morocco have been studied. Tree species composition and abundance were investigated in 84 forest sites, distributed throughout the sandstone formation of the Tangier region. The relative abundance of 15 tree and arborescent shrub species was related, by ordination analysis, to altitude, disturbance by fire, and soil fertility. High-elevation forests were dominated by Cedrus atlantica, Quercus pyrenaica and Pinus pinaster. Sacred forests (protected as holy burial places) had a distinct stand structure, and were considered as refuges, for maintaining biodiversity at landscape scale. The evergreen oak Quercus suber and the semideciduous Quercus canariensis co-dominated the lowland forests. The mean basal area of the studied forests was 34 m² ha^–1. The mean trunk size (dbh) for Q. suber was 24 cm and for Q. canariensis 36.5 cm; in both cases there were indications of declining regeneration at population level. A comparative biogeographical analysis with the equivalent region in southern Spain (separated by the Strait of Gibraltar; 14 km wide), indicated a possible biodiversity loss in the Moroccan forests.     

Quantitative Analysis of Three-Dimensional Fluorescence Localization Microscopy Data

Identifying the three-dimensional molecular organization of subcellular organelles in intact cells has been challenging to date. Here we present an analysis approach for three-dimensional localization microscopy that can not only identify subcellular objects below the diffraction limit but also quantify their shape and volume. This approach is particularly useful to map the topography of the plasma membrane and measure protein distribution within an undulating membrane.Single molecule localization microscopy (SMLM) (1–3) is a superresolution fluorescence microscopy technique that produces coordinate data for single molecule localizations with a precision of tens of nanometers in live and fixed cells. These methods have mainly been performed with total internal reflectance fluorescence microscopy and therefore have generated two-dimensional molecular coordinates. Such two-dimensional data sets have revealed nanosized clusters of membrane proteins at the cell surface (4–7). This was achieved with analysis routines based on pair-correlation analysis (8), Ripley’s K function (9), and related techniques. While three-dimensional localization microscopy techniques such as biplane imaging (10), astigmatic spot analysis (11), and depth-encoding point-spread functions (12) have now been developed, quantitative analysis approaches of three-dimensional coordinate patterns have not.Here, we describe an approach based on Getis and Franklin''s local point pattern analysis to quantitatively analyze three-dimensional subcellular structures and map plasma membrane topography. The latter can also be used to account for topography-induced clustering of membrane proteins in an undulating membrane. To illustrate the approach, we generated three-dimensional SMLM data of the membrane dye DiI and the protein Linker for Activation of T cells (LAT) fused to the photoswitchable fluorescent protein mEos2 in T cells. It has been previously shown that LAT resides within the plasma membrane as well as membrane-proximal vesicles (5,13). The data were acquired using the biplane SMLM technique and highly inclined and laminated optical sheet illumination (14). Three-dimensional molecular coordinates were calculated by fitting a three-dimensional theoretical point-spread-function to the acquired data.As previously described for two-dimensional SMLM data analysis (5), Ripley’s K-function is calculated according to Eq. 1 where V is the analyzed volume, n is the total number of points, and r is the radius of a sphere (a circle for the two-dimensional case) centered on each point. The value K(r) is thus a measure of how many points are encircled within a sphere of radius r:K(r)=V∑i=1n∑j=1n(δij/n2);δij=1ifd(pointi,pointj)<r,0else.(1)For completely spatially random (CSR) data, K(r) scales with the volume of the sphere. We therefore linearize the K-function such that it scales with radius (the L-function) using:L(r)=(3K(r)4π)1/3.(2)The value of L(r)−r is then zero for the CSR case. Values of L(r)−r above zero indicate clustering at the length scale, r.Next we used the related Getis and Franklin''s local point pattern analysis to generate a clustering value (L(r) at r = 50 nm; L(50)) for each point, j, based on the local three-dimensional molecular density. This was calculated using:Lj(50)=((3V4π)∑i=1n(δijn))1/3;δij=1ifd(pointi,pointj)<50,0else.(3)These values can then be interpolated such that every voxel in a volume is assigned a cluster value based on the number of encircled points, relative to the expected CSR case. This allows construction of isosurfaces where all points on the surface have an identical L(50) value. A high threshold imparts a strict criterion for cluster detection compared to a lower one, and this allows users to, for example, determine the efficiency of sequestration into clusters by quantifying the cluster number and size as a function of the threshold.To illustrate the identification of subcellular structures, Lat-mEos2 was imaged by three-dimensional SMLM in activated T cells at the immunological synapse (Fig. 1 A). Three-dimensional projections of isosurfaces (for L(50) = 200) clearly identified intracellular LAT vesicles at varying depths within the synapse (Fig. 1, B and C). Cluster statistics were extracted from this data set to quantify the distribution of clusters in the z direction as well as the volume and sphericity of the LAT objects themselves (Fig. 1, D–F).Open in a separate windowFigure 1Identification of subcellular objects in three dimensions by isosurface rendering of molecular distribution. (A) Schematic of a T cell synapse formed against an activating coverslip where subsynaptic LAT vesicles (red dots) can be imaged with three-dimensional SMLM. (B and C) Isosurfaces, shown in x,z view (B) and as projection (C), identify T cell vesicles as LAT objects with L(50) > 200 (Eq. 3). (D–F) Distribution of LAT objects in z direction (D), volume (E), and sphericity (F) of LAT objects in T cells.Membrane undulations can cause clustering artifacts when the distribution of membrane proteins is recorded as a two-dimensional projection (15) (Fig. 2 A), as is the case in two-dimensional SMLM under total internal reflectance fluorescence illumination. To illustrate a solution to this problem, we obtained three-dimensional SMLM data sets of the membrane dye DiI (16) in resting T cells adhered onto nonactivating coverslips. With appropriately short labeling times to prevent dye internalization, it can be assumed that all DiI molecules reside in the plasma membrane. In this case, as is the case for plasma membrane proteins, neither two-dimensional nor three-dimensional analysis is appropriate, as it is a priori known the points must be derived from a two-dimensional membrane folded in three-dimensional space. To correct for membrane undulations, the plasma membrane topography must first be mapped so that molecular coordinates of membrane molecules can be appropriately corrected in two-dimensional projections. The position of the plasma membrane in three dimensions, i.e., the membrane topography, was determined by averaging the z position of all DiI molecules within a 100-nm radius in x-y at each point. The averaged z-position of DiI molecules was then displayed as a map, which exhibits a smooth, undulating profile (Fig. 2 B). The selection of this radius determines the accuracy of the assigned z position but also causes smoothing of the membrane profile.Open in a separate windowFigure 2Mapping of membrane topography and correction of molecular distributions in undulating membranes. (A) Two-dimensional projections can cause cluster artifacts, for example in membrane ruffles. Molecules (red rectangles) in the upper image are equally spaced along the membrane but appear as clusters in two-dimensional projections in areas with high gradient. (B) Three-dimensional membrane topography of a 2 × 2 μm plasma membrane area of a resting T cell obtained from averaged z positions of DiI molecules. Note that membrane undulation is ∼100 nm. (C) Map of membrane gradient, corresponding to the topography map shown in panel B, with an area of high gradient highlighted (dashed red box). (D) Correction of the circle radii in the Getis and Franklin cluster map calculations to account for projection artifacts. (E and F) Cluster map of data shown in panel C before (E) and after (F) correction for membrane gradient. Boxes in panels C, E, and F highlight the regions with high membrane gradient.Next, the gradient at the position of each DiI molecule was determined and interpolated into a gradient map (Fig. 2 C). Here, blue represents horizontal, i.e., flat membrane areas, whereas red regions indicate areas of high gradient. The information from the gradient map was then used to ensure that the two-dimensional circles in the Getis and Franklin cluster map calculations each correspond to an identical area of membrane, hence accounting for two-dimensional projection artifacts. To do this, the size of the circle (r) used to calculate the L value for each molecule was modified using Eq. 4, where c is calculated for the surface, S, using Eq. 5:r(corr)=r(uncorr)(1+c2)1/4,(4)c=((∂S∂x)2+(∂S∂y)2)1/2.(5)This operation is shown schematically in Fig. 2. The comparison of Getis and Franklin cluster maps before (Fig. 2 E) and after (Fig. 2 F) correction for the gradient shows that cluster values for DiI molecules were substantially reduced by up to 5–10% at sites where the plasma membrane had a high gradient (area highlighted in red box), and where the two-dimensional projection of three-dimensional structures caused an overestimation of clustering.In conclusion, we demonstrated that three-dimensional superresolution localization microscopy data can be used to identify and quantify subcellular structures. The approach has the distinct advantage that subcellular structures are solely identified by the distribution of the fluorescent marker so that no a priori knowledge of the structure is necessary. How precisely the subcellular structures are identified only depends on how efficiently the fluorescent maker is recruited to the structure, and hence does not depend on the resolution limits of optical microscopy. We applied the methods to two very different structures in T cells: small intracellular vesicles and the undulating plasma membrane. Importantly, the topography of plasma membrane can also be used to correct clustering artifacts in two-dimensional projections, which may be useful for distribution analysis within membranes.     

Ion-Abrasion Scanning Electron Microscopy Reveals Surface-Connected Tubular Conduits in HIV-Infected Macrophages

HIV-1-containing internal compartments are readily detected in images of thin sections from infected cells using conventional transmission electron microscopy, but the origin, connectivity, and 3D distribution of these compartments has remained controversial. Here, we report the 3D distribution of viruses in HIV-1-infected primary human macrophages using cryo-electron tomography and ion-abrasion scanning electron microscopy (IA-SEM), a recently developed approach for nanoscale 3D imaging of whole cells. Using IA-SEM, we show the presence of an extensive network of HIV-1-containing tubular compartments in infected macrophages, with diameters of ∼150–200 nm, and lengths of up to ∼5 µm that extend to the cell surface from vesicular compartments that contain assembling HIV-1 virions. These types of surface-connected tubular compartments are not observed in T cells infected with the 29/31 KE Gag-matrix mutant where the virus is targeted to multi-vesicular bodies and released into the extracellular medium. IA-SEM imaging also allows visualization of large sheet-like structures that extend outward from the surfaces of macrophages, which may bend and fold back to allow continual creation of viral compartments and virion-lined channels. This potential mechanism for efficient virus trafficking between the cell surface and interior may represent a subversion of pre-existing vesicular machinery for antigen capture, processing, sequestration, and presentation.     

Model-based Traction Force Microscopy Reveals Differential Tension in Cellular Actin Bundles

Adherent cells use forces at the cell-substrate interface to sense and respond to the physical properties of their environment. These cell forces can be measured with traction force microscopy which inverts the equations of elasticity theory to calculate them from the deformations of soft polymer substrates. We introduce a new type of traction force microscopy that in contrast to traditional methods uses additional image data for cytoskeleton and adhesion structures and a biophysical model to improve the robustness of the inverse procedure and abolishes the need for regularization. We use this method to demonstrate that ventral stress fibers of U2OS-cells are typically under higher mechanical tension than dorsal stress fibers or transverse arcs.     

New material of Ouranopithecus macedoniensis from late Miocene of Macedonia (Greece) and study of its dental attrition

During the last five years our continued excavations in the known late Miocene mammal localities of Macedonia (Greece) provided several new specimens of the hominoid primate Ouranopithecus macedoniensis. This new material includes maxillary and mandibular remains and it is described and compared to the old material of Ouranopithecus in the present article. The material of Ouranopithecus from the three known localities “Ravin de la Pluie” (RPl), “Xirochori 1” (XIR) and “Nikiti 1” (NKT) includes a complete series of tooth rows representing all wearing stages. Thus, the study of the dental wear of Ouranopithecus upper and lower teeth is studied and compared to that of the recent hominoids Gorilla and Pan, as well as to Australopithecus afarensis. The latter species is well known by a series of tooth rows of different wearing stages. The canine’s attrition of Ouranopithecus has a more derived pattern than that of the recent hominoids (Gorilla and Pan) and less derived than A. afarensis. The p3 of Ouranopithecus has similar attrition to that of A. afarensis, the attrition of the molars in Ouranopithecus, A. afarensis and Pan follows a similar pattern, while in Gorilla it is different.