Non-random spatial organization of telomeres varies during the cell cycle and requires LAP2 and BAF.

Spatial genome organization within the nucleus influences major biological processes and is impacted by the configuration of linear chromosomes. Here, we applied 3D spatial statistics and modeling on high-resolution telomere and centromere 3D-structured illumination microscopy images in cancer cells. We found a multi-scale organization of telomeres that dynamically evolved from a mixed clustered-and-regular distribution in early G1 to a purely regular distribution as cells progressed through the cell cycle. In parallel, our analysis revealed two pools of peripheral and internal telomeres, the proportions of which were inverted during the cell cycle. We then conducted a targeted screen using MadID to identify the molecular pathways driving or maintaining telomere anchoring to the nuclear envelope observed in early G1. Lamina-associated polypeptide (LAP) proteins were found transiently localized to telomeres in anaphase, a stage where LAP2α initiates the reformation of the nuclear envelope, and impacted telomere redistribution in the next interphase together with their partner barrier-to-autointegration factor (BAF).

Multi-step atomic mechanism of platinum nanocrystals nucleation and growth revealed by in-situ liquid cell STEM.

The understanding of crystal growth mechanisms has broadened substantially. One significant advancement is based in the conception that the interaction between particles plays an important role in the growth of nanomaterials. This is in contrast to the classical model, which neglects this process. Direct imaging of such processes at atomic-level in liquid-phase is essential for establishing new theoretical models that encompass the full complexity of realistic scenarios and eventually allow for tailoring nanoparticle growth. Here, we investigate at atomic-scale the exact growth mechanisms of platinum nanocrystals from single atom to final crystals by in-situ liquid phase scanning transmission electron microscopy. We show that, after nucleation, the nanocrystals grow via two main stages: atomic attachment in the first stage, where the particles initially grow by attachment of the atoms until depletion of the surrounding zone. Thereafter, follows the second stage of growth, which is based on particle attachment by different atomic pathways to finally form mature nanoparticles. The atomic mechanisms underlying these growth pathways are distinctly different and have different driving forces and kinetics as evidenced by our experimental observations.

Atomic Mechanisms of Nanocrystallization via Cluster-Clouds in Solution Studied by Liquid-Phase Scanning Transmission Electron Microscopy.

The formation of nanocrystals is at the heart of various scientific disciplines, but the atomic mechanisms underlying the early stages of crystallization from supersaturated solutions are still rather unclear. Here, we used in situ liquid-phase scanning transmission electron microscopy to study at the atomic level the very early stages of gold nanocrystal growth, and the evolution of its crystallinity. We found that the nucleation is initiated by the formation of poorly crystalline nanoparticles. These are transformed into monocrystals via nanocrystallization governed by a complex process of multiple out-and-in exchanges of matter between a crystalline-core and a disordered-shell, referred to as the cluster-cloud. Our observations at the crystal/cluster-cloud interface during growth demonstrate that the initially formed nanocrystals expel the poorly crystallized phases as nanoclusters into the cluster-cloud, then readsorb it by two distinct pathways, namely, by (i) monomer attachments and (ii) nanocluster coalescence. This growth process eventually leads to the formation of monocrystalline nanoparticles.

Super-resolved live-cell imaging using random illumination microscopy.

Current super-resolution microscopy (SRM) methods suffer from an intrinsic complexity that might curtail their routine use in cell biology. We describe here random illumination microscopy (RIM) for live-cell imaging at super-resolutions matching that of 3D structured illumination microscopy, in a robust fashion. Based on speckled illumination and statistical image reconstruction, easy to implement and user-friendly, RIM is unaffected by optical aberrations on the excitation side, linear to brightness, and compatible with multicolor live-cell imaging over extended periods of time. We illustrate the potential of RIM on diverse biological applications, from the mobility of proliferating cell nuclear antigen (PCNA) in U2OS cells and kinetochore dynamics in mitotic S. pombe cells to the 3D motion of myosin minifilaments deep inside Drosophila tissues. RIM's inherent simplicity and extended biological applicability, particularly for imaging at increased depths, could help make SRM accessible to biology laboratories.

Atomic mechanisms of gold nanoparticle growth in ionic liquids studied by in situ scanning transmission electron microscopy.

Elementary atomic mechanisms underlying nanoparticle growth in liquids are largely unexplored and mostly a subject of conjectures based on theory and indirect experimental insights. Direct, experimental observation of such processes at an atomic level requires imaging with single-atom sensitivity and control over kinetics. Although conventional liquid-cell (scanning) transmission electron microscopy ((S)TEM) enables nanoscale studies of dynamic processes, the visualization of atomic processes in the liquid phase is inhibited owing to the liquid film thickness and its encapsulation, both limiting the achievable spatial resolution. In contrast, by using thin, free-standing ionic liquid nanoreactors, this work shows that the mechanisms controlling and triggering particle growth can be uncovered at an atom-by-atom level. Our observations of growing particle ensembles reveal that diverse growth pathways proceed simultaneously. We record Ostwald ripening and oriented particle coalescence tracked at the atomic scale, which confirm the mechanisms suggested by theory. However, we also identify unexpected growth phenomena and more intricate coalescence events which show competing mechanisms. The diversity of the observed growth processes thus illustrates that growth reactions in liquids, on the atomic scale, are much more complex than predicted by theory. Furthermore, this work demonstrates that free-standing ionic liquids enable (sub-)Ångström resolution imaging of dynamic processes in liquids with single-atom sensitivity, thus providing a powerful alternative approach to conventional liquid-cell (S)TEM.

Noise2Atom: unsupervised denoising for scanning transmission electron microscopy images.

We propose an effective deep learning model to denoise scanning transmission electron microscopy (STEM) image series, named Noise2Atom, to map images from a source domain [Formula: see text] to a target domain [Formula: see text], where [Formula: see text] is for our noisy experimental dataset, and [Formula: see text] is for the desired clear atomic images. Noise2Atom uses two external networks to apply additional constraints from the domain knowledge. This model requires no signal prior, no noise model estimation, and no paired training images. The only assumption is that the inputs are acquired with identical experimental configurations. To evaluate the restoration performance of our model, as it is impossible to obtain ground truth for our experimental dataset, we propose consecutive structural similarity (CSS) for image quality assessment, based on the fact that the structures remain much the same as the previous frame(s) within small scan intervals. We demonstrate the superiority of our model by providing evaluation in terms of CSS and visual quality on different experimental datasets.

Nanoneedle-Mediated Stimulation of Cell Mechanotransduction Machinery.

Biomaterial substrates can be engineered to present topographical signals to cells which, through interactions between the material and active components of the cell membrane, regulate key cellular processes and guide cell fate decisions. However, targeting mechanoresponsive elements that reside within the intracellular domain is a concept that has only recently emerged. Here, we show that mesoporous silicon nanoneedle arrays interact simultaneously with the cell membrane, cytoskeleton, and nucleus of primary human cells, generating distinct responses at each of these cellular compartments. Specifically, nanoneedles inhibit focal adhesion maturation at the membrane, reduce tension in the cytoskeleton, and lead to remodeling of the nuclear envelope at sites of impingement. The combined changes in actin cytoskeleton assembly, expression and segregation of the nuclear lamina, and localization of Yes-associated protein (YAP) correlate differently from what is canonically observed upon stimulation at the cell membrane, revealing that biophysical cues directed to the intracellular space can generate heretofore unobserved mechanosensory responses. These findings highlight the ability of nanoneedles to study and direct the phenotype of large cell populations simultaneously, through biophysical interactions with multiple mechanoresponsive components.

Formation of gold nanoparticles in a free-standing ionic liquid triggered by heat and electron irradiation.

Ionic liquids (ILs) feature negligibly low vapor pressures and can thus be freely introduced into the high vacuum of a transmission electron microscope. With this extraordinary property, the ILs offer a powerful tool for in situ transmission electron microscopy (TEM) in window-free liquid media at very high resolution. In this work, we use the IL 1-butyl-3-methylimidazolium chloride in order to study nucleation and growth of gold nanoparticles (NPs) in free-standing liquid droplets by scanning TEM (STEM). The results confirm that the used IL allows for generating Au NPs in situ, triggered by electron irradiation and heat. Firstly, the isotropic growth of small, spherical Au NPs was initiated and monitored, whereas different growth mechanisms were observed, i.e. growth by monomer attachment, growth through particle coalescence and possible Ostwald ripening events. After the initial growth phase, a second, anisotropic growth process was induced by a moderate temperature increase and continued electron irradiation. As a result, larger, faceted crystals, such as tetrahedra, octahedra or decahedra, were formed. As all these polymorphs are terminated by {111}-facets, the IL might not only act as liquid medium but in addition as a surfactant which preferentially attaches on the {100}-facets.

Voids and compositional inhomogeneities in Cu(In,Ga)Se2 thin films: evolution during growth and impact on solar cell performance.

Structural defects such as voids and compositional inhomogeneities may affect the performance of Cu(In,Ga)Se2 (CIGS) solar cells. We analyzed the morphology and elemental distributions in co-evaporated CIGS thin films at the different stages of the CIGS growth by energy-dispersive x-ray spectroscopy in a transmission electron microscope. Accumulation of Cu-Se phases was found at crevices and at grain boundaries after the Cu-rich intermediate stage of the CIGS deposition sequence. It was found, that voids are caused by Cu out-diffusion from crevices and GBs during the final deposition stage. The Cu inhomogeneities lead to non-uniform diffusivities of In and Ga, resulting in lateral inhomogeneities of the In and Ga distribution. Two and three-dimensional simulations were used to investigate the impact of the inhomogeneities and voids on the solar cell performance. A significant impact of voids was found, indicating that the unpassivated voids reduce the open-circuit voltage and fill factor due to the introduction of free surfaces with high recombination velocities close to the CIGS/CdS junction. We thus suggest that voids, and possibly inhomogeneities, limit the efficiency of solar cells based on three-stage co-evaporated CIGS thin films. Passivation of the voids' internal surface may reduce their detrimental effects.

Formation of Au Nanoparticles in Liquid Cell Transmission Electron Microscopy: From a Systematic Study to Engineered Nanostructures.

In this work, a systematic study of the effect of electron dose rate, solute concentration, imaging mode (broad beam vs scanning probe mode), and liquid cell setup (static vs flow mode) on the growth mechanism and the ultimate morphology of Au nanoparticles (NPs) was performed in chloroauric acid (HAuCl4) aqueous solutions using in situ liquid-cell TEM (LC-TEM). It was found that a diffusion limited growth dominates at high dose rates, especially for the solution with the lowest concentration (1 mM), resulting in formation of dendritic NPs. Growth of 2D Au plates driven by a reaction limited mechanism was only observed at low dose rates for the 1 mM solution. For the 5 mM and 20 mM solutions, reaction limited growth can still be induced at higher dose rates, due to abundance of the precursor available in the solutions, leading to formation of 2D plates or 3D faceted NPs. As a proof-of-concept, an Au nanostructure with a 3D faceted particle core and a dendritic shell can be in situ produced by simply tuning the electron dose in the 1 mM solution irradiated in a flow cell setup in the STEM mode. This work paves the way to study the growth of complex heteronanostructures composed of multiple elements in LC-TEM.

Enhanced Carrier Collection from CdS Passivated Grains in Solution-Processed Cu2ZnSn(S,Se)4 Solar Cells.

Solution processing of Cu2ZnSn(S,Se)4 (CZTSSe)-kesterite solar cells is attractive because of easy manufacturing using readily available metal salts. The solution-processed CZTSSe absorbers, however, often suffer from poor morphology with a bilayer structure, exhibiting a dense top crust and a porous bottom layer, albeit yielding efficiencies of over 10%. To understand whether the cell performance is limited by this porous layer, a systematic compositional study using (scanning) transmission electron microscopy ((S)TEM) and energy-dispersive X-ray spectroscopy of the dimethyl sulfoxide processed CZTSSe absorbers is presented. TEM investigation revealed a thin layer of CdS that is formed around the small CZTSSe grains in the porous bottom layer during the chemical bath deposition step. This CdS passivation is found to be beneficial for the cell performance as it increases the carrier collection and facilitates the electron transport. Electron-beam-induced current measurements reveal an enhanced carrier collection for this buried region as compared to reference cells with evaporated CdS.

Local band gap measurements by VEELS of thin film solar cells.

This work presents a systematic study that evaluates the feasibility and reliability of local band gap measurements of Cu(In,Ga)Se2 thin films by valence electron energy-loss spectroscopy (VEELS). The compositional gradients across the Cu(In,Ga)Se2 layer cause variations in the band gap energy, which are experimentally determined using a monochromated scanning transmission electron microscope (STEM). The results reveal the expected band gap variation across the Cu(In,Ga)Se2 layer and therefore confirm the feasibility of local band gap measurements of Cu(In,Ga)Se2 by VEELS. The precision and accuracy of the results are discussed based on the analysis of individual error sources, which leads to the conclusion that the precision of our measurements is most limited by the acquisition reproducibility, if the signal-to-noise ratio of the spectrum is high enough. Furthermore, we simulate the impact of radiation losses on the measured band gap value and propose a thickness-dependent correction. In future work, localized band gap variations will be measured on a more localized length scale to investigate, e.g., the influence of chemical inhomogeneities and dopant accumulations at grain boundaries.

Mechanisms of HsSAS-6 assembly promoting centriole formation in human cells.

SAS-6 proteins are thought to impart the ninefold symmetry of centrioles, but the mechanisms by which their assembly occurs within cells remain elusive. In this paper, we provide evidence that the N-terminal, coiled-coil, and C-terminal domains of HsSAS-6 are each required for procentriole formation in human cells. Moreover, the coiled coil is necessary and sufficient to mediate HsSAS-6 centrosomal targeting. High-resolution imaging reveals that GFP-tagged HsSAS-6 variants localize in a torus around the base of the parental centriole before S phase, perhaps indicative of an initial loading platform. Moreover, fluorescence recovery after photobleaching analysis demonstrates that HsSAS-6 is immobilized progressively at centrosomes during cell cycle progression. Using fluorescence correlation spectroscopy and three-dimensional stochastic optical reconstruction microscopy, we uncover that HsSAS-6 is present in the cytoplasm primarily as a homodimer and that its oligomerization into a ninefold symmetrical ring occurs at centrioles. Together, our findings lead us to propose a mechanism whereby HsSAS-6 homodimers are targeted to centrosomes where the local environment and high concentration of HsSAS-6 promote oligomerization, thus initiating procentriole formation.

Potassium-induced surface modification of Cu(In,Ga)Se2 thin films for high-efficiency solar cells.

Thin-film photovoltaic devices based on chalcopyrite Cu(In,Ga)Se2 (CIGS) absorber layers show excellent light-to-power conversion efficiencies exceeding 20%. This high performance level requires a small amount of alkaline metals incorporated into the CIGS layer, naturally provided by soda lime glass substrates used for processing of champion devices. The use of flexible substrates requires distinct incorporation of the alkaline metals, and so far mainly Na was believed to be the most favourable element, whereas other alkaline metals have resulted in significantly inferior device performance. Here we present a new sequential post-deposition treatment of the CIGS layer with sodium and potassium fluoride that enables fabrication of flexible photovoltaic devices with a remarkable conversion efficiency due to modified interface properties and mitigation of optical losses in the CdS buffer layer. The described treatment leads to a significant depletion of Cu and Ga concentrations in the CIGS near-surface region and enables a significant thickness reduction of the CdS buffer layer without the commonly observed losses in photovoltaic parameters. Ion exchange processes, well known in other research areas, are proposed as underlying mechanisms responsible for the changes in chemical composition of the deposited CIGS layer and interface properties of the heterojunction.

Resolution doubling in 3D-STORM imaging through improved buffers.

Super-resolution imaging methods have revolutionized fluorescence microscopy by revealing the nanoscale organization of labeled proteins. In particular, single-molecule methods such as Stochastic Optical Reconstruction Microscopy (STORM) provide resolutions down to a few tens of nanometers by exploiting the cycling of dyes between fluorescent and non-fluorescent states to obtain a sparse population of emitters and precisely localizing them individually. This cycling of dyes is commonly induced by adding different chemicals, which are combined to create a STORM buffer. Despite their importance, the composition of these buffers has scarcely evolved since they were first introduced, fundamentally limiting what can be resolved with STORM. By identifying a new chemical suitable for STORM and optimizing the buffer composition for Alexa-647, we significantly increased the number of photons emitted per cycle by each dye, providing a simple means to enhance the resolution of STORM independently of the optical setup used. Using this buffer to perform 3D-STORM on biological samples, we obtained images with better than 10 nanometer lateral and 30 nanometer axial resolution.

Simple buffers for 3D STORM microscopy.

3D STORM is one of the leading methods for super-resolution imaging, with resolution down to 10 nm in the lateral direction, and 30-50 nm in the axial direction. However, there is one important requirement to perform this type of imaging: making dye molecules blink. This usually relies on the utilization of complex buffers, containing different chemicals and sensitive enzymatic systems, limiting the reproducibility of the method. We report here that the commercial mounting medium Vectashield can be used for STORM of Alexa-647, and yields images comparable or superior to those obtained with more complex buffers, especially for 3D imaging. We expect that this advance will promote the versatile utilization of 3D STORM by removing one of its entry barriers, as well as provide a more reproducible way to compare optical setups and data processing algorithms.

Spindle positioning in human cells relies on proper centriole formation and on the microcephaly proteins CPAP and STIL.

Patients with MCPH (autosomal recessive primary microcephaly) exhibit impaired brain development, presumably due to the compromised function of neuronal progenitors. Seven MCPH loci have been identified, including one that encodes centrosome protein 4.1 associated protein (CPAP; also known as centromere protein J, CENPJ). CPAP is a large coiled-coil protein enriched at the centrosome, a structure that comprises two centrioles and surrounding pericentriolar material (PCM). CPAP depletion impairs centriole formation, whereas CPAP overexpression results in overly long centrioles. The mechanisms by which CPAP MCPH patient mutations affect brain development are not clear. Here, we identify CPAP protein domains crucial for its centriolar localization, as well as for the elongation and the formation of centrioles. Furthermore, we demonstrate that conditions that resemble CPAP MCPH patient mutations compromise centriole formation in tissue culture cells. Using adhesive micropatterns, we reveal that such defects correlate with a randomization of spindle position. Moreover, we demonstrate that the MCPH protein SCL/TAL1 interrupting locus (STIL) is also essential for centriole formation and for proper spindle position. Our findings are compatible with the notion that mutations in CPAP and STIL cause MCPH because of aberrant spindle positioning in progenitor cells during brain development.

The SCF-FBXW5 E3-ubiquitin ligase is regulated by PLK4 and targets HsSAS-6 to control centrosome duplication.

Deregulated centrosome duplication can result in genetic instability and contribute to tumorigenesis. Here, we show that centrosome duplication is regulated by the activity of an E3-ubiquitin ligase that employs the F-box protein FBXW5 (ref. 3) as its targeting subunit. Depletion of endogenous FBXW5 or overexpression of an F-box-deleted mutant version results in centrosome overduplication and formation of multipolar spindles. We identify the centriolar protein HsSAS-6 (refs 4,5) as a critical substrate of the SCF-FBXW5 complex. FBXW5 binds HsSAS-6 and promotes its ubiquitylation in vivo. The activity of SCF-FBXW5 is in turn negatively regulated by Polo-like kinase 4 (PLK4), which phosphorylates FBXW5 at Ser 151 to suppress its ability to ubiquitylate HsSAS-6. FBXW5 is a cell-cycle-regulated protein with expression levels peaking at the G1/S transition. We show that FBXW5 levels are controlled by the anaphase-promoting (APC/C) complex, which targets FBXW5 for degradation during mitosis and G1, thereby helping to reset the centrosome duplication machinery. In summary, we show that a cell-cycle-regulated SCF complex is regulated by the kinase PLK4, and that this in turn restricts centrosome re-duplication through degradation of the centriolar protein HsSAS-6.

ZEB1 limits adenoviral infectability by transcriptionally repressing the coxsackie virus and adenovirus receptor.

BACKGROUND: We have previously reported that RAS-MEK (Cancer Res. 2003 May 1;63(9):2088-95) and TGF-ß (Cancer Res. 2006 Feb 1;66(3):1648-57) signaling negatively regulate coxsackie virus and adenovirus receptor (CAR) cell-surface expression and adenovirus uptake. In the case of TGF-ß, down-regulation of CAR occurred in context of epithelial-to-mesenchymal transition (EMT), a process associated with transcriptional repression of E-cadherin by, for instance, the E2 box-binding factors Snail, Slug, SIP1 or ZEB1. While EMT is crucial in embryonic development, it has been proposed to contribute to the formation of invasive and metastatic carcinomas by reducing cell-cell contacts and increasing cell migration. RESULTS: Here, we show that ZEB1 represses CAR expression in both PANC-1 (pancreatic) and MDA-MB-231 (breast) human cancer cells. We demonstrate that ZEB1 physically associates with at least one of two closely spaced and conserved E2 boxes within the minimal CAR promoter here defined as genomic region -291 to -1 relative to the translational start ATG. In agreement with ZEB1's established role as a negative regulator of the epithelial phenotype, silencing its expression in MDA-MB-231 cells induced a partial Mesenchymal-to-Epithelial Transition (MET) characterized by increased levels of E-cadherin and CAR, and decreased expression of fibronectin. Conversely, knockdown of ZEB1 in PANC-1 cells antagonized both the TGF-ß-induced down-regulation of E-cadherin and CAR and the reduction of adenovirus uptake. Interestingly, even though ZEB1 clearly contributes to the TGF-ß-induced mesenchymal phenotype of PANC-1 cells, TGF-ß did not seem to affect ZEB1's protein levels or subcellular localization. These findings suggest that TGF-ß may inhibit CAR expression by regulating factor(s) that cooperate with ZEB1 to repress the CAR promoter, rather than by regulating ZEB1 expression levels. In addition to the negative E2 box-mediated regulation the minimal CAR promoter is positively regulated through conserved ETS and CRE elements. CONCLUSIONS: This report provides evidence that inhibition of ZEB1 may improve adenovirus uptake of cancer cells that have undergone EMT and for which ZEB1 is necessary to maintain the mesenchymal phenotype. Targeting of ZEB1 may reverse some aspects of EMT including the down-regulation of CAR.