Some tips and tricks for a Correlative Light Electron Microscopy workflow using stable expression of fluorescent proteins.

Correlative Light Electron Microscopy (CLEM) encompasses a wide range of experimental approaches with different degrees of complexity and technical challenges where the attributes of both light and electron microscopy are combined in a single experiment. Although the biological question always determines what technology is the most appropriate, we generally set out to apply the simplest workflow possible. For 2D cell cultures expressing fluorescently tagged molecules, we report on a simple and very powerful CLEM approach by using gridded finder imaging dishes. We first determine the gross localization of the fluorescence using light microscopy and subsequently we retrace the origin/localization of the fluorescence by projecting it onto the ultrastructural reference space obtained by transmission electron microscopy (TEM). Here we describe this workflow and highlight some basic principles of the sample preparation for such a simple CLEM experiment. We will specifically focus on the steps following the resin embedding for TEM and the introduction of the sample in the electron microscope.

Some Guiding Principles for a "Simple" Correlative Light Electron Microscopy Experiment.

In recent years, Correlative Multimodal Imaging (CMI) has become an "en vogue" technique and a bit of a buzzword. It entails combining information from different imaging modalities to extract more information from a sample that would otherwise not be possible from each individual technique. The best established CMI technology is correlative light and electron microscopy (CLEM), which applies light and electron microscopy on the exact same sample/structure. In general, it entails the detection of fluorescently tagged proteins or structures by light microscopy and subsequently their relative intracellular localization is determined with nanometer resolution using transmission electron microscopy (TEM). Here, we describe the different steps involved in a "simple" CLEM approach. We describe the overall workflow, instrumentation, and basic principles of sample preparation for a CLEM experiment exploiting stable expression of fluorescent proteins.

Progression of herpesvirus infection remodels mitochondrial organization and metabolism.

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

Synchronous timing of return to breeding sites in a long-distance migratory seabird with ocean-scale variation in migration schedules.

BACKGROUND: Migratory birds generally have tightly scheduled annual cycles, in which delays can have carry-over effects on the timing of later events, ultimately impacting reproductive output. Whether temporal carry-over effects are more pronounced among migrations over larger distances, with tighter schedules, is a largely unexplored question. METHODS: We tracked individual Arctic Skuas Stercorarius parasiticus, a long-distance migratory seabird, from eight breeding populations between Greenland and Siberia using light-level geolocators. We tested whether migration schedules among breeding populations differ as a function of their use of seven widely divergent wintering areas across the Atlantic Ocean, Mediterranean Sea and Indian Ocean. RESULTS: Breeding at higher latitudes led not only to later reproduction and migration, but also faster spring migration and shorter time between return to the breeding area and clutch initiation. Wintering area was consistent within individuals among years; and more distant areas were associated with more time spent on migration and less time in the wintering areas. Skuas adjusted the period spent in the wintering area, regardless of migration distance, which buffered the variation in timing of autumn migration. Choice of wintering area had only minor effects on timing of return at the breeding area and timing of breeding and these effects were not consistent between breeding populations. CONCLUSION: The lack of a consistent effect of wintering area on timing of return between breeding areas indicates that individuals synchronize their arrival with others in their population despite extensive individual differences in migration strategies.

Light-Induced Nanoscale Deformation in Azobenzene Thin Film Triggers Rapid Intracellular Ca2+ Increase via Mechanosensitive Cation Channels.

Epithelial cells are in continuous dynamic biochemical and physical interaction with their extracellular environment. Ultimately, this interplay guides fundamental physiological processes. In these interactions, cells generate fast local and global transients of Ca2+ ions, which act as key intracellular messengers. However, the mechanical triggers initiating these responses have remained unclear. Light-responsive materials offer intriguing possibilities to dynamically modify the physical niche of the cells. Here, a light-sensitive azobenzene-based glassy material that can be micropatterned with visible light to undergo spatiotemporally controlled deformations is used. Real-time monitoring of consequential rapid intracellular Ca2+ signals reveals that the mechanosensitive cation channel Piezo1 has a major role in generating the Ca2+ transients after nanoscale mechanical deformation of the cell culture substrate. Furthermore, the studies indicate that Piezo1 preferably responds to shear deformation at the cell-material interphase rather than to absolute topographical change of the substrate. Finally, the experimentally verified computational model suggests that Na+ entering alongside Ca2+ through the mechanosensitive cation channels modulates the duration of Ca2+ transients, influencing differently the directly stimulated cells and their neighbors. This highlights the complexity of mechanical signaling in multicellular systems. These results give mechanistic understanding on how cells respond to rapid nanoscale material dynamics and deformations.

Substrate microtopographies induce cellular alignment and affect nuclear force transduction.

Cellular physiology has been mainly studied by using two-dimensional cell culture substrates which lack in vivo-mimicking extracellular environment and interactions. Thus, there is a growing need for more complex model systems in life sciences. Micro-engineered scaffolds have been proven to be a promising tool in understanding the role of physical cues in the co-regulation of cellular functions. These tools allow, for example, probing cell morphology and migration in response to changes in chemo-physical properties of their microenvironment. In order to understand how microtopographical features, what cells encounter in vivo, affect cytoskeletal organization and nuclear mechanics, we used direct laser writing via two-photon polymerization (TPP) to fabricate substrates which contain different surface microtopographies. By combining with advanced high-resolution spectral imaging, we describe how the constructed grid and vertical line microtopographies influence cellular alignment, nuclear morphology and mechanics. Specifically, we found that growing cells on grids larger than 10 × 20 µm2 and on vertical lines increased 3D actin cytoskeleton orientation along the walls of microtopographies and abolished basal actin stress fibers. In concert, the nuclei of these cells were also more aligned, elongated, deformed and less flattened, indicating changes in nuclear force transduction. Importantly, by using fluorescence lifetime imaging microscopy for measuring Förster resonance energy transfer for a genetically encoded nesprin-2 molecular tension sensor, we show that growing cells on these microtopographic substrates induce lower mechanical tension at the nuclear envelope. To conclude, here used substrate microtopographies modulated the cellular mechanics, and affected actin organization and nuclear force transduction.

Nuclear lamina strain states revealed by intermolecular force biosensor.

Nuclear lamins have been considered an important structural element of the nucleus. The nuclear lamina is thought both to shield DNA from excessive mechanical forces and to transmit mechanical forces onto the DNA. However, to date there is not yet a technical approach to directly measure mechanical forces on nuclear lamins at the protein level. To overcome this limitation, we developed a nanobody-based intermolecular tension FRET biosensor capable of measuring the mechanical strain of lamin filaments. Using this sensor, we were able to show that the nuclear lamina is subjected to significant force. These forces are dependent on nuclear volume, actomyosin contractility, functional LINC complex, chromatin condensation state, cell cycle, and EMT. Interestingly, large forces were also present on nucleoplasmic lamins, indicating that these lamins may also have an important mechanical role in the nucleus. Overall, we demonstrate that the nanobody-based approach allows construction of biosensors for complex protein structures for mechanobiology studies.

Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina.

Investigation of nuclear lamina architecture relies on superresolved microscopy. However, epitope accessibility, labeling density, and detection precision of individual molecules pose challenges within the molecularly crowded nucleus. We developed iterative indirect immunofluorescence (IT-IF) staining approach combined with expansion microscopy (ExM) and structured illumination microscopy to improve superresolution microscopy of subnuclear nanostructures like lamins. We prove that ExM is applicable in analyzing highly compacted nuclear multiprotein complexes such as viral capsids and provide technical improvements to ExM method including three-dimensional-printed gel casting equipment. We show that in comparison with conventional immunostaining, IT-IF results in a higher signal-to-background ratio and a mean fluorescence intensity by improving the labeling density. Moreover, we present a signal-processing pipeline for noise estimation, denoising, and deblurring to aid in quantitative image analyses and provide this platform for the microscopy imaging community. Finally, we show the potential of signal-resolved IT-IF in quantitative superresolution ExM imaging of nuclear lamina and reveal nanoscopic details of the lamin network organization-a prerequisite for studying intranuclear structural coregulation of cell function and fate.

G2/M checkpoint regulation and apoptosis facilitate the nuclear egress of parvoviral capsids.

The nuclear export factor CRM1-mediated pathway is known to be important for the nuclear egress of progeny parvovirus capsids in the host cells with virus-mediated cell cycle arrest at G2/M. However, it is still unclear whether this is the only pathway by which capsids exit the nucleus. Our studies show that the nuclear egress of DNA-containing full canine parvovirus. capsids was reduced but not fully inhibited when CRM1-mediated nuclear export was prevented by leptomycin B. This suggests that canine parvovirus capsids might use additional routes for nuclear escape. This hypothesis was further supported by our findings that nuclear envelope (NE) permeability was increased at the late stages of infection. Inhibitors of cell cycle regulatory protein cyclin-dependent kinase 1 (Cdk1) and pro-apoptotic caspase 3 prevented the NE leakage. The change in NE permeability could be explained by the regulation of the G2/M checkpoint which is accompanied by early mitotic and apoptotic events. The model of G2/M checkpoint activation was supported by infection-induced nuclear accumulation of cyclin B1 and Cdk1. Both NE permeability and nuclear egress of capsids were reduced by the inhibition of Cdk1. Additional proof of checkpoint function regulation and promotion of apoptotic events was the nucleocytoplasmic redistribution of nuclear transport factors, importins, and Ran, in late infection. Consistent with our findings, post-translational histone acetylation that promotes the regulation of several genes related to cell cycle transition and arrest was detected. In conclusion, the model we propose implies that parvoviral capsid egress partially depends on infection-induced G2/M checkpoint regulation involving early mitotic and apoptotic events.

Pneumatic equiaxial compression device for mechanical manipulation of epithelial cell packing and physiology.

It is well established that mechanical cues, e.g., tensile- compressive- or shear forces, are important co-regulators of cell and tissue physiology. To understand the mechanistic effects these cues have on cells, technologies allowing precise mechanical manipulation of the studied cells are required. As the significance of cell density i.e., packing on cellular behavior is beginning to unravel, we sought to design an equiaxial cell compression device based on our previously published cell stretching system. We focused on improving the suitability for microscopy and the user-friendliness of the system. By introducing a hinge structure to the substrate stretch generating vacuum chamber, we managed to decrease the z-displacement of the cell culture substrate, thus reducing the focal plane drift. The vacuum battery, the mini-incubator, as well as the custom-made vacuum pressure controller make the experimental setup more flexible and portable. Furthermore, we improved the efficiency and repeatability of manufacture of the device by designing a mold that can be used to cast the body of the device. We also compared several different silicone membranes, and chose SILPURAN® due to its best microscopy imaging properties. Here, we show that the device can produce a maximum 8.5% radial pre-strain which leads to a 15% equiaxial areal compression as the pre-strain is released. When tested with epithelial cells, upon compression, we saw a decrease in cell cross-sectional area and an increase in cell layer height. Additionally, before compression the cells had two distinct cell populations with different cross-sectional areas that merged into a more uniform population due to compression. In addition to these morphological changes, we detected an alteration in the nucleo-cytoplasmic distribution of YAP1, suggesting that the cellular packing is enough to induce mechanical signaling in the epithelium.

Parvovirus nonstructural protein 2 interacts with chromatin-regulating cellular proteins.

Autonomous parvoviruses encode at least two nonstructural proteins, NS1 and NS2. While NS1 is linked to important nuclear processes required for viral replication, much less is known about the role of NS2. Specifically, the function of canine parvovirus (CPV) NS2 has remained undefined. Here we have used proximity-dependent biotin identification (BioID) to screen for nuclear proteins that associate with CPV NS2. Many of these associations were seen both in noninfected and infected cells, however, the major type of interacting proteins shifted from nuclear envelope proteins to chromatin-associated proteins in infected cells. BioID interactions revealed a potential role for NS2 in DNA remodeling and damage response. Studies of mutant viral genomes with truncated forms of the NS2 protein suggested a change in host chromatin accessibility. Moreover, further studies with NS2 mutants indicated that NS2 performs functions that affect the quantity and distribution of proteins linked to DNA damage response. Notably, mutation in the splice donor site of the NS2 led to a preferred formation of small viral replication center foci instead of the large coalescent centers seen in wild-type infection. Collectively, our results provide insights into potential roles of CPV NS2 in controlling chromatin remodeling and DNA damage response during parvoviral replication.

Toward Xeno-Free Differentiation of Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells.

The small intestinal epithelium has an important role in nutrition, but also in drug absorption and metabolism. There are a few two-dimensional (2D) patient-derived induced pluripotent stem cell (iPSC)-based intestinal models enabling easy evaluation of transcellular transport. It is known that animal-derived components induce variation in the experimental outcomes. Therefore, we aimed to refine the differentiation protocol by using animal-free components. More specifically, we compared maturation of 2D-cultured iPCSs toward small intestinal epithelial cells when cultured either with or without serum, and either on Geltrex or on animal-free, recombinant laminin-based substrata. Differentiation status was characterized by qPCR, immunofluorescence imaging, and functionality assays. Our data suggest that differentiation toward definitive endoderm is more efficient without serum. Both collagen- and recombinant laminin-based coating supported differentiation of definitive endoderm, posterior definitive endoderm, and small intestinal epithelial cells from iPS-cells equally well. Small intestinal epithelial cells differentiated on recombinant laminin exhibited slightly more enterocyte specific cellular functionality than cells differentiated on Geltrex. Our data suggest that functional small intestinal epithelial cells can be generated from iPSCs in serum-free method on xeno-free substrata. This method is easily converted to an entirely xeno-free method.

Brick Strex: a robust device built of LEGO bricks for mechanical manipulation of cells.

Cellular forces, mechanics and other physical factors are important co-regulators of normal cell and tissue physiology. These cues are often misregulated in diseases such as cancer, where altered tissue mechanics contribute to the disease progression. Furthermore, intercellular tensile and compressive force-related signaling is highlighted in collective cell behavior during development. However, the mechanistic understanding on the role of physical forces in regulation of cellular physiology, including gene expression and signaling, is still lacking. This is partly because studies on the molecular mechanisms of force transmission require easily controllable experimental designs. These approaches should enable both easy mechanical manipulation of cells and, importantly, readouts ranging from microscopy imaging to biochemical assays. To achieve a robust solution for mechanical manipulation of cells, we developed devices built of LEGO bricks allowing manual, motorized and/or cyclic cell stretching and compression studies. By using these devices, we show that [Formula: see text]-catenin responds differentially to epithelial monolayer stretching and lateral compression, either localizing more to the cell nuclei or cell-cell junctions, respectively. In addition, we show that epithelial compression drives cytoplasmic retention and phosphorylation of transcription coregulator YAP1. We provide a complete part listing and video assembly instructions, allowing other researchers to build and use the devices in cellular mechanics-related studies.

Concepts to Reveal Parvovirus-Nucleus Interactions.

Parvoviruses are small single-stranded (ss) DNA viruses, which replicate in the nucleoplasm and affect both the structure and function of the nucleus. The nuclear stage of the parvovirus life cycle starts at the nuclear entry of incoming capsids and culminates in the successful passage of progeny capsids out of the nucleus. In this review, we will present past, current, and future microscopy and biochemical techniques and demonstrate their potential in revealing the dynamics and molecular interactions in the intranuclear processes of parvovirus infection. In particular, a number of advanced techniques will be presented for the detection of infection-induced changes, such as DNA modification and damage, as well as protein-chromatin interactions.

Longevity record of arctic skua (Stercorarius parasiticus).

The arctic skua (Stercorarius parasiticus) is one of the most long-lived bird species. In 2010, we captured in Finland an adult, female arctic skua which had been ringed as a nestling in 1987. We tagged it also with a color ring. The bird has last been seen in July 2020 at the age of 33 years, making it most likely the oldest known arctic skua of the world. In 2010-2011 the bird carried a light-level measuring geolocator, the data of which revealed that the bird had spent the nonbreeding season in the Canary Current area on the west coast of Africa. Breeding populations of arctic skuas have declined recently especially in British Isles, thus it is useful to get longevity data of this species with a high breeding site fidelity.

Azobenzene-based sinusoidal surface topography drives focal adhesion confinement and guides collective migration of epithelial cells.

Surface topography is a key parameter in regulating the morphology and behavior of single cells. At multicellular level, coordinated cell displacements drive many biological events such as embryonic morphogenesis. However, the effect of surface topography on collective migration of epithelium has not been studied in detail. Mastering the connection between surface features and collective cellular behaviour is highly important for novel approaches in tissue engineering and repair. Herein, we used photopatterned microtopographies on azobenzene-containing materials and showed that smooth topographical cues with proper period and orientation can efficiently orchestrate cell alignment in growing epithelium. Furthermore, the experimental system allowed us to investigate how the orientation of the topographical features can alter the speed of wound closure in vitro. Our findings indicate that the extracellular microenvironment topography coordinates their focal adhesion distribution and alignment. These topographic cues are able to guide the collective migration of multicellular systems, even when cell-cell junctions are disrupted.

Insectivorous birds can see and smell systemically herbivore-induced pines.

Several studies have shown that insectivorous birds are attracted to herbivore-damaged trees even when they cannot see or smell the actual herbivores or their feces. However, it often remained an open question whether birds are attracted by herbivore-induced changes in leaf odor or in leaf light reflectance or by both types of changes. Our study addressed this question by investigating the response of great tits (Parus major) and blue tits (Cyanistes caeruleus) to Scots pine (Pinus sylvestris) damaged by pine sawfly larvae (Diprion pini). We released the birds individually to a study booth, where they were simultaneously offered a systemically herbivore-induced and a noninfested control pine branch. In the first experiment, the birds could see the branches, but could not smell them, because each branch was kept inside a transparent, airtight cylinder. In the second experiment, the birds could smell the branches, but could not see them, because each branch was placed inside a nontransparent cylinder with a mesh lid. The results show that the birds were more attracted to the herbivore-induced branch in both experiments. Hence, either type of the tested cues, the herbivore-induced visual plant cue alone as well as the olfactory cues per se, is attractive to the birds.

Cytoplasmic Parvovirus Capsids Recruit Importin Beta for Nuclear Delivery.

Parvoviruses are an important platform for gene and cancer therapy. Their cell entry and the following steps, including nuclear import, are inefficient, limiting their use in therapeutic applications. Two models exist on parvoviral nuclear entry: the classical import of the viral capsid using nuclear transport receptors of the importin (karyopherin) family or the direct attachment of the capsid to the nuclear pore complex leading to the local disintegration of the nuclear envelope. Here, by laser scanning confocal microscopy and in situ proximity ligation analyses combined with coimmunoprecipitation, we show that infection requires importin ß-mediated access to the nuclear pore complex and nucleoporin 153-mediated interactions on the nuclear side. The importin ß-capsid interaction continued within the nucleoplasm, which suggests a mixed model of nuclear entry in which the classical nuclear import across the nuclear pore complex is accompanied by transient ruptures of the nuclear envelope, also allowing the passive entry of importin ß-capsid complexes into the nucleus.IMPORTANCE Parvoviruses are small DNA viruses that deliver their DNA into the postmitotic nuclei, which is an important step for parvoviral gene and cancer therapies. Limitations in virus-receptor interactions or endocytic entry do not fully explain the low transduction/infection efficiency, indicating a bottleneck after virus entry into the cytoplasm. We thus investigated the transfer of parvovirus capsids from the cytoplasm to the nucleus, showing that the nuclear import of the parvovirus capsid follows a unique strategy, which differs from classical nuclear import and those of other viruses.

Quantitative Microscopy Reveals Stepwise Alteration of Chromatin Structure during Herpesvirus Infection.

During lytic herpes simplex virus 1 (HSV-1) infection, the expansion of the viral replication compartments leads to an enrichment of the host chromatin in the peripheral nucleoplasm. We have shown previously that HSV-1 infection induces the formation of channels through the compacted peripheral chromatin. Here, we used three-dimensional confocal and expansion microscopy, soft X-ray tomography, electron microscopy, and random walk simulations to analyze the kinetics of host chromatin redistribution and capsid localization relative to their egress site at the nuclear envelope. Our data demonstrated a gradual increase in chromatin marginalization, and the kinetics of chromatin smoothening around the viral replication compartments correlated with their expansion. We also observed a gradual transfer of capsids to the nuclear envelope. Later in the infection, random walk modeling indicated a gradually faster transport of capsids to the nuclear envelope that correlated with an increase in the interchromatin channels in the nuclear periphery. Our study reveals a stepwise and time-dependent mechanism of herpesvirus nuclear egress, in which progeny viral capsids approach the egress sites at the nuclear envelope via interchromatin spaces.

Insectivorous Birds Are Attracted by Plant Traits Induced by Insect Egg Deposition.

Insectivorous birds feed upon all developmental stages of herbivorous insects, including insect eggs if larvae and adults are unavailable. Insect egg deposition on plants can induce plant traits that are subsequently exploited by egg parasitoids searching for hosts. However, it is unknown whether avian predators can also use egg-induced plant changes for prey localization. Here, we studied whether great tits (Parus major) and blue tits (Cyanistes caeruleus) are attracted by traits of the Scots pine (Pinus sylvestris) induced by pine sawfly (Diprion pini) egg deposition. We chose this plant - insect system because sawfly egg deposition on pine needles is known to locally and systemically induce a change in pine volatile organic compounds (VOCs), and tits are known to prey upon sawfly eggs. In dual choice laboratory experiments, we simultaneously offered the birds an egg-free control branch and a systemically egg-induced branch. Significantly more birds visited the egg-induced branch first. We confirmed by GC-MS analyses that systemically egg-induced branches released more (E)-ß-farnesene compared to control branches. Spectrophotometric analyses showed that control branches reflected more light than egg-induced branches throughout the avian visual range. Although a discrimination threshold model for blue tits suggests that the birds are poor at discriminating this visual difference, the role of visual stimuli in attracting the birds to egg-induced pines cannot be discounted. Our study shows, for the first time, that egg-induced odorous and/or visual plant traits can help birds to locate insect eggs without smelling or seeing those eggs.