Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.

Lattice light-sheet (LLS) microscopy provides ultrathin light sheets of a two-dimensional optical lattice that allows us imaging three-dimensional (3D) objects for hundreds of time points at sub-second intervals and at or below the diffraction limit. Galectin-3 (Gal3), a carbohydrate-binding protein, triggers glycosphingolipid (GSL)-dependent biogenesis of morphologically distinct endocytic vesicles that are cargo specific and clathrin independent. In this study, we apply LLS microscopy to study the dynamics of Gal3 dependent endocytosis in live T cells. This will allow us to observe Gal3-mediated endocytosis at high temporal and excellent 3D spatial resolution, which may shed light on our understanding of the mechanism and physiological function of Gal3-induced endocytosis.

Mutations affecting the actin regulator WD repeat–containing protein 1 lead to aberrant lymphoid immunity

The immunological synapse is a transient junction that occurs when the plasma membrane of a T cell comes in close contact with an APC after recognizing a peptide from the antigen-MHC. The interaction starts when CRAC channels embedded in the T cell membrane open, flowing calcium ions into the cell. To counterbalance the ion influx and subsequent depolarization, Kv 1.3 and KCa3.1 channels are recruited to the immunological synapse, increasing the extracellular K+ concentration. These processes are crucial as they initiate gene expression that drives T cell activation and proliferation. The T cell-specific function of the K2P channel family member TASK2 channels and their role in autoimmune processes remains unclear. Using mass spectrometry analysis together with epifluorescence and super-resolution single-molecule localization microscopy, we identified TASK2 channels as novel players recruited to the immunological synapse upon stimulation. TASK2 localizes at the immunological synapse, upon stimulation with CD3 antibodies, likely interacting with these molecules. Our findings suggest that, together with Kv 1.3 and KCa3.1 channels, TASK2 channels contribute to the proper functioning of the immunological synapse, and represent an interesting treatment target for T cell-mediated autoimmune disorders.

We introduce and study a novel concept in farfield fluorescence microscopy fundamentally overcoming the classical diffraction resolution limit. This is accomlished by reducing the spatial extent of the effective focus of a scanning fluorescence microscope. The reduction is achieved by depleting the ground-state energy of the molecules located in the outer region of the focus. Our theoretical study shows that ground-state-depletion fluorescence microscopy has the potential of increasing the resolution of far-field fluorescence microscopy by an order of magnitude which is equivalent to a lateral resolution of 15 NM.

Light microscopy has proven to be one of the most versatile analytical tools in cell biology and cytogenetics. The growing spectrum of scientific knowledge demands a continuous improvement of the optical resolution of the instruments. In far-field light microscopy, the attainable resolution is dictated by the limit of diffraction, which, in practice, is about 250 nm for high-numerical-aperture objective lenses. Near-field scanning optical microscopy (NSOM) was the first technique that has overcome this limit up to about one order of magnitude. Typically, the resolution range below 100 nm is accessed for biological applications. Using appropriately designed scanning probes allows for obtaining an extremely small near-field light excitation volume (some tens of nanometers in diameter). Because of the reduction of background illumination, high contrast imaging becomes feasible for light transmission and fluorescence microscopy. The height of the scanning probe is controlled by atomic force interactions between the specimen surface and the probe tip. The control signal can be used for the production of a topographic (nonoptical) image that can be acquired simultaneously. In this chapter, the principle of NSOM is described with respect to biological applications. A brief overview of some requirements in biology and applications described in the literature are given. Practical advice is focused on instruments with aperture-type illumination probes. Preparation protocols focussing on NSOM of cell surfaces and chromosomes are presented.

Background: In anti-tumor immunity, T cells play a central role to eliminate cancer cells. Immune responses of T cells are initiated by communication with antigen presenting cells (APCs). It became clear that an immunological synapse (IS) is formed at the T cell-APC interface and is made up of a supramolecular activation cluster (SMAC) containing the T cell receptors (TCR) microclusters (MCs) and CD28 MCs. TCR MCs consist of TCR, kinases, and adaptor molecules, and are the minimal unit of T cell signal transduction. CD28 MCs also function as the signalosomes of CD28 activation. By using total internal reflection fluorescence microscopy to observe the interaction of T cell and planar bilayer, it was elucidated that TCR activation is regulated by the amount of MCs aggregation, which shows the extent of IS formation. Based on this finding, we hypothesized that ex vivo T cells could be examined IS formation and that exhausted T cells of advanced cancer patients should be low in response to a stimulus. Method: To simplify IS formation of T cells, we used human monoclonal T cells by αCD3 and/or αCD28 antibody and replaced to evaluate the aggregation of MCs by detecting the CD3 or CD28 molecules on the surface of T cells. This phenomenon was defined as IS-like formation. In this method, IS-like formation was detected by confirming an image of each cell with imaging cytometry. After these conditions was established, it was verified whether it is possible to measure IS-like formation rate of T cells. Additionally, the correlation between IS-like formation and interferon-γ cytokine production after this activation was examined. In the same manner as above, IS - like formation rates of T cells derived from PBMCs of healthy donors or esophageal and gastric cancer patients were measured. Result: We have succeeded in observing that CD3 or CD28 molecules on a T cell aggregated in one area after stimulation as IS-like formation by imaging cytometry. In dot plot study, positive area of CD3 or CD28 decreased after the stimulation, which showed the aggregation of these molecules. Accordingly, we could measure IS-like formation rate of a population of T cell clones. In addition, the rate of IS-like formation showed a positive correlation with interferon-γ production. Similar results were obtained T cell from PBMC in healthy donors. Moreover, IS-like formation rate of T cell from cancer patients decreased compared with healthy donors. Conclusion: IS-like formation of T cell succeeded to observe by CD3 and/or CD28 antibody stimulation. We established the evaluation method of IS-like formation degree by imaging cytometry. Moreover, IS-like formation rate correlated with cytokine production of IFN-g. We established new method to evaluate the strength of T cell after activation. Citation Format: Tomoko Tanaka, Kimihiro Yamashita, Yutaka Sugita, Eiji Fukuoka, Akira Arimoto, Yoshihiro Kakeji. A new method for evaluating T cell response after stimulation on gastrointestinal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5699.

The formation of stars is a key process in astrophysics. Detailed knowledge of the physical mechanisms that govern stellar birth is a prerequisite for understanding the formation and evolution of our galactic home, the Milky Way. A theory of star formation is an essential part of any model for the origin of our solar system and of planets around other stars. Despite this pivotal importance, and despite many decades of research, our understanding of the processes that initiate and regulate star formation is still limited. Stars are born in cold interstellar clouds of molecular hydrogen gas. Star formation in these clouds is governed by the complex interplay between the gravitational attraction in the gas and agents such as turbulence, magnetic fields, radiation and thermal pressure that resist compression. The competition between these processes determines both the locations at which young stars form and how much mass they ultimately accrete. It plays out over many orders of magnitude in space and time, ranging from galactic to stellar scales. In addition, star formation is a highly stochastic process in which rare and hard-to-predict events, such as the formation of very massive stars and the resulting feedback, can play a dominant role in determining the evolution of a star-forming cloud. As a consequence of the wide range of scales and processes that control star formation, analytic models are usually restricted to highly idealized cases. These can yield insight, but the complexity of the problem means that they must be used in concert with large-scale numerical simulations. Here we summarize the state of modern star formation theory and review the recent advances in numerical simulation techniques.

The defi nition of a volcano is discussed, and a new encompassing version is provided. The discussion focuses on the observations that volcanism is a self-similar process that ranges many orders of magnitude in space and time scales, and that all kinds of geologic processes act on volcanoes. Former defi nitions of volcano, such as that from the Glossary of Geology (1997, p. 690)—“a vent in the surface of the Earth through which magma and associated gases and ash erupt” or “the form or structure, usually conical, that is produced by the ejected material” are clearly insuffi cient. All defi nitions that we encountered tend to consider volcanoes from the point of view of a single discipline, each of them neglecting relevant aspects belonging to other disciplines. For the two cases mentioned above a volcano is seen only from the point of view of eruptive activity or of morphology. We attempt to look at volcano holistically to provide a more comprehensive defi nition. We defi ne a volcano as a geologic environment that, at any scale, is characterized by three elements: magma, eruption, and edifi ce. It is suffi cient that only one of these elements is proven, as long as the others can be inferred to exist, to have existed, or to have the potential to exist in the future.

Drosophila melanogaster has become an indispensable model organism in toxicology research due to its rapid life cycle, robust genetic toolkit, and significant genetic overlap with humans (≈75% of disease-related genes). Drosophila melanogaster serves as a valuable model for evaluating neurotoxicity, oxidative stress, and metabolic disturbances. This study employed a systematic methodology to identify relevant literature on Drosophila melanogaster in toxicity testing, regulatory frameworks, and environmental health. The selection process prioritized toxicological research, regulatory relevance, and recent advancements from 2022 to 2025. Drosophila melanogaster models cellular and molecular responses to toxicants, leveraging its life cycle and genetic features despite limitations such as the size and structural differences and certain metabolic differences. Drosophila melanogaster offers unique advantages in genetic manipulation and rapid result generation compared to other models like Caenorhabditis elegans, zebrafish, and mice, which provide complementary insights. Additionally, established regulatory and ethical guidelines support its widespread use in toxicology. Real-life case studies underscore its predictive value in environmental and pharmaceutical toxicology, bridging the gap between in vitro assays and more complex mammalian models. Overall, Drosophila melanogaster stands as a pivotal organism for advancing our understanding of toxic mechanisms and improving risk assessment protocols in toxicology research.

We examined the potential value of the natural killer (NK) cell line; NK-92, as immunotherapy tool for breast cancer (BC) treatment and searched for biomarker(s) of sensitivity to NK-92-mediated cytotoxicity. The cytotoxic activity of NK-92 cells towards one breast precancerous and nine BC cell lines was analyzed using calcein-AM and degranulation assays. The molecules associated with NK-92-responsiveness were determined by differential gene expression analysis using RNA-sequencing and validated by RT-PCR, immunostaining and flow cytometry. NK-target interactions and immunological synapse formation were assessed by fluorescence microscopy. Potential biomarker expression was determined by IHC in 99 patient-derived BC tissues and 10 normal mammary epithelial tissues. Most (8/9) BC cell lines were resistant while only one BC and the precancerous cell lines were effectively killed by NK-92 lymphocytes. NK-92-sensitive target cells specifically expressed CD56, which ectopic expression in CD56-negative BC cells induced their sensitivity to NK-92-mediated killing, suggesting that CD56 is not only a biomarker of responsiveness but actively regulates NK function. CD56 adhesion molecules which are also expressed on NK cells accumulate at the immunological synapse enhancing NK-target interactions, cytotoxic granzyme B transfer from NK-92 to CD56-expressing target cells and induction of caspase 3 activation in targets. Interestingly, CD56 expression was found to be reduced in breast tumor tissues (36%) with strong inter- and intratumoral heterogeneity in comparison to normal breast tissues (80%). CD56 is a potential predictive biomarker for BC responsiveness to NK-92-cell based immunotherapy and loss of CD56 expression might be a mechanism of escape from NK-immunity.

Research into aging has been galvanized by the discovery of mutations in single genes that extend life span and evolutionary conservation of their effects. An environmental intervention—dietary restriction—also extends life span in evolutionarily diverse animals. These discoveries have opened the way to using laboratory model organisms to understand human aging. Invertebrate species, budding yeast Saccharomyces cerevisiae , the nematode worm Caenorhabditis elegans , and the fruit fly Drosophila melanogaster , have a vital role in this process of discovery. Their ease of culture and handling in the laboratory and short life spans (~3 days in yeast, ~3 weeks in the worm, and ~3 months in Drosophila ) mean that much more rapid progress can be made than in the mouse, whose life spans are about 3 years. Completion of the genome sequences for the invertebrates and their closely related species, together with the development of many genetic and other resources, also make them powerful experimental systems. Each of these organisms has strengths and weaknesses for research into aging. We highlight here some of the particular strengths of Drosophila and uses to which they have been put and could be put in the future. The jaw-dropping genetics applied to a complex tissue structure approaching that of vertebrates leaves Drosophila flying at the front edge of aging research. The time has long passed when a full review of the biology of aging in the fruit fly D. melanogaster could be usefully accommodated in a single chapter. Drosophila has been an established model organism for...

Five polypeptides in the soluble fraction of chloroplasts and three polypeptides in the thylakoid membranes in chloroplasts from green spores of the fern Osmunda japonica decreased during spore germination. The 22-kDa protein in thylakoid membranes almost completely disappeared during germination over 48 h. The 22-kDa protein had been purified (Physiologia Plantarum 95: 465. 1995). A protease for the 22-kDa protein also had been separated from chloroplasts. Several fragments appeared after proteolysis of the 22-kDa protein by the protease (Physiologia Plantarum 109: 129. 2000). We have attempted to determine a gene of the 22-kDa protein by PCR using the information of amino-terminal sequences of proteolytic products. The cDNA obtained from the mRNA in the spores shows that the precursor protein is composed of the signal peptide having 16 amino acid residues and the mature protein having 180 amino acid residues. A computer search of the protein database revealed similar features between the 22-kDa protein and LEA 3 homologs of Deinococcus radiodurans, Drosophila melanogaster, Caenorhabditis elegans, and cDNA corresponding to cold hardening-induced Chlorella genes. It seems possible that the 22-kDa protein is a homolog of ABA-inducible proteins, desiccation-related proteins, or a LEA protein. The presence of a chloroplast transit peptide in a sequence of the cDNA supports that the 22-kDa protein is one of the thylakoid membranes. The 22-kDa protein may be a desiccation-tolerant protein in quiescent spores, and a role of the protease is a scavenger for the protein during germination.

The first isoform of phosphoinositide 3-kinases (PI3K) had been found in Saccharomyces cerevisiae when screening for mutants not exhibiting normal vacuolar protein sorting (Vps), Vps34. Class III PI3K/Vps34 has long been worked on in regards to its role in endosomal sorting and autophagy, a process allowing cells to survive nutrient-deprived conditions. Most research groups have investigated the functions of Vps34 in the yeast model system Saccharomyces cerevisiae. Newer publications now use mammalian cell lines, Caenorhabditis elegans or Drosophila melanogaster, deciphering interesting differences between the various species in regards to Vps34 characteristics. Our cancer model system, melanoma tumors, are known to be very aggressive and their treatment difficult, due to mutations leading to drug resistance. Autophagy and whether its induction would be beneficial or not for cancer patients, has been the topic of discussions in the field lately. In this work, we investigated the role of class III PI3K by two different methods, a pharmacological and a genetic approach. We started with natural compound screenings on hVps34 in genetically modified yeast systems. The pure fraction of Citrus medica extracts giving best results turned out to be limettin. Limettin inhibited hVps34 in both our in vivo yeast system and in in vitro kinase assays using the immunoprecipitated enzyme from HEK293 cells. Our candidate inhibitor seemed very specific for the human isoform, but still required quite high concentrations in the assays performed. Further chemical designing and eventual fitting to the hVps34 ATP binding pocket would be necessary to render this molecule into one of the first specific class III PI3K inhibitors. In addition to pharmacological approaches, class III PI3K state-of-the-art genetic knockdown experiments were done in melanoma cell lines in order to characterize this isoform’s role in melanoma more specifically. Vps34 is not essential in yeast, but leads to serious temperature sensitivity phenotypes. In one melanoma cell line (A375) tested, knockdown had similar as but milder effects than known in other cancer types. Two others though (A2058 and 1205lu), did not tolerate the longterm loss of class III PI3K. We suppose that the importance of hVps34 depends on the genetic background of cell types. Further studies are required to define precisely which effectors determine the intolerance described i.e. which melanoma types could be targeted by inhibition of class III PI3K.

The immunological synapse (IS) regulates immune responses by integrating extracellular stimuli into intracellular signalling networks, which causes leukocyte differentiation and effector functions. The dynamic spatial organisation of molecules at the IS was initially characterised by wide-field fluorescence microscopy of cell conjugates and cells interacting with planar lipid bilayers. These methods showed stable supramolecular clusters of several microns in size, which were proposed to be responsible for sustained signalling and cell-cell adhesion. The recent emergence of microspectroscopy techniques with higher spatial and temporal resolution nonetheless reveals the complex dynamics of molecular reactions that mediate IS assembly and function. This review describes microspectroscopy-based in vitro experimental approaches for imaging the molecular dynamics at the IS, as well as their contributions and open questions in the field. It also describes experimental methods to obtain quantitative parameters of dynamic biochemical reactions in living cells, and discusses about the important role of quantitative imaging and theoretical science in our understanding of molecular mechanisms underlying lymphocyte activation.

When killing through the granule exocytosis pathway, cytotoxic lymphocytes release key effector molecules into the immune synapse, perforin and granzymes, to initiate target cell killing. The pore-forming perforin is essential for the function of cytotoxic lymphocytes, as its pores disrupt the target cell membrane and allow diffusion of pro-apoptotic serine proteases, granzyme, into the target cell, where they initiate various cell death cascades. Unlike human perforin, the detection of its murine counterpart in a live cell system has been problematic due its relatively low expression level and the lack of sensitive antibodies. The lack of a suitable methodology to visualise murine perforin secretion into the synapse hinders the study of the cytotoxic lymphocyte secretory machinery in murine models of human disease. Here, we describe a novel recombinant technology, whereby a short ALFA-tag sequence has been fused with the amino-terminus of a mature murine perforin, and this allowed its detection by the highly specific FluoTag®-X2 anti-ALFA nanobodies using both Total Internal Reflection Fluorescence (TIRF) microscopy of an artificial synapse, and confocal microscopy of the physiological immune synapse with a target cell. This methodology can have broad application in the field of cytotoxic lymphocyte biology and for the many models of human disease.