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Staphylococcus epidermidis is a major causative agent of prosthetic joint infections (PJI). The ability to form biofilms supports this highly selective pathogenic potential. In vitro studies essentially relying on phenotypic assays and genetic approaches have provided a detailed picture of the molecular events contributing to biofilm assembly. A major limitation in these studies is the use of synthetic growth media, which significantly differs from the environmental conditions S. epidermidis encounters during host invasion. Building on evidence showing that growth in serum substantially affects S. epidermidis gene expression profiles and phenotypes, the major aim of this study was to develop and characterize a growth medium mimicking synovial fluid, thereby facilitating research addressing specific aspects related to PJI. Using fresh human plasma, a protocol was established allowing for the large-scale production of a medium that by biochemical analysis matches key characteristics of synovial fluid and therefore is referred to as artificial synovial fluid (ASF). By analysis of biofilm-positive, polysaccharide intercellular adhesion (PIA)-producing S. epidermidis 1457 and its isogenic, PIA- and biofilm-negative mutant 1457-M10, evidence is provided that the presence of ASF induces cluster formation in S. epidermidis 1457 and mutant 1457-M10. Consistent with the aggregative properties, both strains formed multilayered biofilms when analyzed by confocal laser scanning microscopy. In parallel to the phenotypic findings, expression analysis after growth in ASF found upregulation of genes encoding for intercellular adhesins (icaA, aap, and embp) as well as atlE, encoding for the major cell wall autolysin being responsible for eDNA release. In contrast, growth in ASF was associated with reduced expression of the master regulator agr. Collectively, these results indicate that ASF induces expression profiles that are able to support intercellular adhesion in both PIA-positive and PIA-negative S. epidermidis. Given the observation that ASF overall induced biofilm formation in a collection of S. epidermidis isolates from PJI, the results strongly support the idea of using growth media mimicking host environments. ASF may play an important role in future studies related to the pathogenesis of S. epidermidis PJI.

Stress granules (SGs) are membrane-less condensates composed of RNA and protein that assemble in response to stress stimuli and disassemble when stress is lifted. Both assembly and disassembly are tightly controlled processes, yet, it remains elusive whether mRNAs in SGs completely recover for translation following stress relief. Using RNA-seq of translating fractions in human cell line, we found that higher fraction of the m

Introductory ParagraphIn many species, including Arabidopsis, heterochromatin often comprises repetitive DNA elements, such as arrays of ribosomal DNA (rDNA). Repetitive regions pose a risk in meiosis since recombination between them can lead to gross genomic rearrangements. However, meiotic recombination at rDNA arrays and other heterochromatic repeat regions is blocked by not well understood mechanisms. Here, we have identified RAD21.2, an α-kleisin subunit of cohesin, as a repressor of meiotic recombination at the rDNA regions in Arabidopsis. We show that RAD21.2 co-localizes with heterochromatic factors and is specifically enriched at rDNA repeats, which are devoid of the meiosis specific α-kleisin REC8, needed for recombination. Knocking down RAD21.2, we find that REC8 moves into the nucleolus organizing regions (NORs), where we see an increase of RAD51 recombinase foci numbers. Concomitantly, we find extensive rearrangements of the NORs and the offspring of these plants have large variation in rDNA copy numbers demonstrating that RAD21.2 is necessary for transgenerational genome stability.One-Sentence SummaryThe cohesin component RAD21.2 represses meiotic recombination and by that contributes to genome stability over generations.

Molecular psychiatry xx, xx (2022). doi:10.1038/s41380-022-01882-3, Postsynaptic scaffold proteins such as Shank, PSD-95, Homer and SAPAP/GKAP family members establish the postsynaptic density of glutamatergic synapses through a dense network of molecular interactions. Mutations in SHANK genes are associated with neurodevelopmental disorders including autism and intellectual disability. However, no SHANK missense mutations have been described which interfere with the key functions of Shank proteins believed to be central for synapse formation, such as GKAP binding via the PDZ domain, or Zn2+-dependent multimerization of the SAM domain. We identify two individuals with a neurodevelopmental disorder carrying de novo missense mutations in SHANK2. The p.G643R variant distorts the binding pocket for GKAP in the Shank2 PDZ domain and prevents interaction with Thr(−2) in the canonical PDZ ligand motif of GKAP. The p.L1800W variant severely delays the kinetics of Zn2+-dependent polymerization of the Shank2-SAM domain. Structural analysis shows that Trp1800 dislodges one histidine crucial for Zn2+ binding. The resulting conformational changes block the stacking of helical polymers of SAM domains into sheets through side-by-side contacts, which is a hallmark of Shank proteins, thereby disrupting the highly cooperative assembly process induced by Zn2+. Both variants reduce the postsynaptic targeting of Shank2 in primary cultured neurons and alter glutamatergic synaptic transmission. Super-resolution microscopy shows that both mutants interfere with the formation of postsynaptic nanoclusters. Our data indicate that both the PDZ- and the SAM-mediated interactions of Shank2 contribute to the compaction of postsynaptic protein complexes into nanoclusters, and that deficiencies in this process interfere with normal brain development in humans., Published by Macmillan, London

The use of disease-specific signatures of microRNAs (miRNAs) in exosomes has become promising for clinical applications, either as biomarkers or direct therapeutic targets. However, a new approach for exosome enrichment and quantification of miRNAs is urgently needed for its clinical application, since the commercial techniques have shortcomings in quantity and quality. To overcome these deficiencies, we developed a new method for purification of exosomes with subsequent miRNA extraction, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR), and compared our assays with commercial techniques. For the establishment of these methods, numerous reagents, parameters, and combinations thereof were examined. Our new technique for exosome extraction is based on a mannuronate-guluronate polymer (MGP) which avoids co-precipitating plasma proteins. Quality, concentration and biological activity of the isolated exosomes were examined by Western blot, Nanoparticle Tracking Analysis (NTA), and confocal microscopy. A combination of chaotropic and non-chaotropic salts was used to extract miRNAs from plasma, serum, and exosomes, allowing the exclusion of hazardous components, such as phenol/chloroform. The performance of the miRNAs extraction was verified by RT-qPCR. The chemistry and TaqMan probe were also optimized for RT-qPCR. Sensitivity, efficiency, and linearity of RT-qPCR were tested on serial dilutions of synthetic miR-16 and miR-142. Our established procedure covers all steps of miRNA analyses, and measures the levels of either cell-free and exosomal miRNAs in plasma, serum and other body fluids with high performance.

Background About 3%–5% of adults with membranous nephropathy have autoantibodies directed against thrombospondin type 1 domain–containing 7A (THSD7A), a podocyte-expressed transmembrane protein. However, the temporal and spatial expression of THSD7A and its biologic function for podocytes are unknown, information that is needed to understand the effects of THSD7A autoantibodies in this disease. Methods Using a variety of microscopic techniques, we analyzed THSD7A localization in postnatal, adult, and autoantibody-injected mice as well as in human podocytes. We also analyzed THSD7A function in human podocytes using confocal microscopy; Western blotting; and adhesion and migration assays. Results We found that THSD7A expression begins on glomerular vascularization with slit diaphragm formation in development. THSD7A localizes to the basal aspect of foot processes, closely following the meanders of the slit diaphragm in human and mice. Autoantibodies binding to THSD7A localize to the slit diaphragm. In human podocytes, THSD7A expression is accentuated at filopodia and thin arborized protrusions, an expression pattern associated with decreased membrane activity of cytoskeletal regulators. We also found that, phenotypically, THSD7A expression in human podocytes is associated not only with increases in cell size, enhanced adhesion, and reduced detachment from collagen type IV–coated plates but also, with decreased ability to migrate. Conclusions Our findings suggest that THSD7A functions as a foot process protein involved in the stabilization of the slit diaphragm of mature podocytes and that autoantibodies to THSD7A, on the basis of their localization, might structurally and functionally alter the slit diaphragm’s permeability to protein.

Type 3 secretion systems (T3SS) are essential virulence factors of numerous bacterial pathogens and inject effector proteins into host cells. The needle-like T3SS machinery consists of more than 20 components, has a length of around 100 nm and a diameter of up to 30 nm according to EM studies. Its intrabacterial components are highly dynamic and in permanent exchange with other bacterial structures. Therefore, a temporally and spatially resolved visualization of the T3SS using fluorescence microscopy techniques has been challenging. In the present study, novel labeling strategies were combined with super-resolution microscopy such as STED, STORM and MINFLUX. MINFLUX nanoscopy allowed to visualize components of the T3SS machinery such as the dynamic sorting platform component YscL and the extrabacterial pore protein YopD at unprecedented resolutions. The presented results represent the basis for an in depth investigation of T3SS structure and function and therefore gain new insights into the infection process of human pathogens in order to develop novel treatment and prevention strategies.

Dissociation of hyper-phosphorylated Tau from neuronal microtubules and its pathological aggregates, are hallmarks in the etiology of tauopathies. The Tau-microtubule interface is subject to polyglutamylation, a reversible posttranslational modification, increasing negative charge at tubulin C-terminal tails. Here, we asked whether tubulin polyglutamylation may contribute to Tau pathology in vivo. Since polyglutamylases modify various proteins other than tubulin, we generated a knock-in mouse carrying gene mutations to abolish Tuba4a polyglutamylation in a substrate-specific manner. We found that Tuba4a lacking C-terminal polyglutamylation prevents the binding of Tau and GSK3 kinase to neuronal microtubules, thereby strongly reducing phospho-Tau levels. Notably, crossbreeding of the Tuba4a knock-in mouse with the hTau tauopathy model, expressing a human Tau transgene, reversed hyper-phosphorylation and oligomerization of Tau and normalized microglia activation in brain. Our data highlight tubulin polyglutamylation as a potential therapeutic strategy in fighting tauopathies.

A new method is proposed to perform Mueller matrix polarimetry using a Full Poincaré beam (i.e., a non-uniformly polarized beam presenting all polarization states across its section) as a parallel polarization state generator and a charge-coupled device (CCD) camera as a detector of the polarization state analyzer. In this way, the polarization change is measured for all possible input states simultaneously. To obtain the Mueller matrix of the sample, the overdetermined system of equations that relates the input and output states of polarization is solved by means of the Moore–Penrose pseudo-inverse. Preliminary numerical simulations are performed to identify and exhaustively analyze the main sources of error. In order to test the method, experimental measurements are presented for several known samples, showing an excellent agreement between the experimentally obtained Mueller matrices and the theoretically expected ones.

NAADP-evoked Ca2+ release through type 1 ryanodine receptors (RYR1) is a major mechanism underlying the earliest signals in T cell activation, which are the formation of Ca2+ microdomains. In our characterization of the molecular machinery underlying NAADP action, we identified an NAADP-binding protein, called hematological and neurological expressed 1-like protein (HN1L) [also known as Jupiter microtubule-associated homolog 2 (JPT2)]. Gene deletion of Hn1l/Jpt2 in human Jurkat and primary rat T cells resulted in decreased numbers of initial Ca2+ microdomains and delayed the onset and decreased the amplitude of global Ca2+ signaling. Photoaffinity labeling demonstrated direct binding of NAADP to recombinant HN1L/JPT2. T cell receptor/CD3-dependent coprecipitation of HN1L/JPT2 with RYRs and colocalization of these proteins suggest that HN1L/JPT2 connects NAADP formation with the activation of RYR channels within the first seconds of T cell activation. Thus, HN1L/JPT2 enables NAADP to activate Ca2+ release from the endoplasmic reticulum through RYR.

Extracellular vesicles (EVs) are important means of intercellular communication and a potent tool for regenerative therapy. In ischaemic stroke, transient blockage of a brain artery leads to a lack of glucose and oxygen in the affected brain tissue, provoking neuronal death by necrosis in the core of the ischaemic region. The fate of neurons in the surrounding penumbra region depends on the stimuli, including EVs, received during the following hours. A detailed characterization of such stimuli is crucial not only for understanding stroke pathophysiology but also for new therapeutic interventions. In the present study, we characterize the EVs in mouse brain under physiological conditions and 24 h after induction of transient ischaemia in mice. We show that, in steady-state conditions, microglia are the main source of small EVs (sEVs), whereas after ischaemia the main sEV population originates from astrocytes. Brain sEVs presented high amounts of the prion protein (PrP), which were further increased after stroke. Moreover, EVs were enriched in a proteolytically truncated PrP fragment (PrP-C1). Because of similarities between PrP-C1 and certain viral surface proteins, we studied the cellular uptake of brain-derived sEVs from mice lacking (PrP-KO) or expressing PrP (WT). We show that PrP-KO-sEVs are taken up significantly faster and more efficiently than WT-EVs by primary neurons. Furthermore, microglia and astrocytes engulf PrP-KO-sEVs more readily than WT-sEVs. Our results provide novel information on the relative contribution of brain cell types to the sEV pool in murine brain and indicate that increased release of sEVs by astrocytes together with elevated levels of PrP in sEVs may play a role in intercellular communication at early stages after stroke. In addition, amounts of PrP (and probably PrP-C1) in brain sEVs seem to contribute to regulating their cellular uptake.

Immunotherapeutic strategies are increasingly important in neuro-oncology, and the elucidation of escape mechanisms that lead to treatment resistance is crucial. We investigated the impact of immune pressure on the clonal dynamics and immune escape signature by comparing glioma growth in immunocompetent versus immunodeficient mice. Glioma-bearing WT and Pd-1(–/–) mice survived significantly longer than immunodeficient Pfp(–/–) Rag2(–/–) mice. While tumors in Pfp(–/–) Rag2(–/–) mice were highly polyclonal, immunoedited tumors in WT and Pd-1(–/–) mice displayed reduced clonality with emergence of immune escape clones. Tumor cells in WT mice were distinguished by an IFN-γ–mediated response signature with upregulation of genes involved in immunosuppression. Tumor-infiltrating stromal cells, which include macrophages/microglia, contributed even more strongly to the immunosuppressive signature than the actual tumor cells. The identified murine immune escape signature was reflected in human patients and correlated with poor survival. In conclusion, immune pressure profoundly shapes the clonal composition and gene regulation in malignant gliomas.

Understanding the cellular processes that occur between the cytosol and the plasma membrane is an important task for biological research. Till now, however, it was not possible to combine fast and high-resolution imaging of both the isolated plasma membrane and the surrounding intracellular volume. Here, we demonstrate the combination of fast high-resolution spinning disk (SD) and total internal reflection fluorescence (TIRF) microscopy for specific imaging of the plasma membrane. A customised SD-TIRF microscope was used with specific design of the light paths that allowed, for the first time, live SD-TIRF experiments at high acquisition rates. A series of experiments is shown to demonstrate the feasibility and performance of our setup.

Extracellular vesicles (EVs) are important means of intercellular communication and a potent tool for regenerative therapy. In ischemic stroke, transient blockage of a brain artery leads to a lack of glucose and oxygen in the affected brain tissue, provoking neuronal death by necrosis in the core of the ischemic region. The fate of neurons in the surrounding penumbra depends on the stimuli, including EVs, received during the following hours. A detailed characterization of such stimuli is crucial not only for understanding stroke pathophysiology but also for new therapeutic interventions.In the present study, we characterize the EVs in mouse brain under physiological conditions and 24h after induction of transient ischemia in mice. We show that, in steady-state conditions, microglia are the main source of small EVs (sEVs) whereas after ischemia, the main EV population originates from astrocytes. Moreover, sEVs presented high amounts of the prion protein (PrP) which were increased after stroke. Conspicuously, sEVs were particularly enriched in a truncated PrP fragment (PrP-C1). Because of similarities between PrP-C1 and certain viral surface proteins, we studied the cellular uptake of brain-derived sEVs from mice lacking (PrP-KO) or containing PrP (WT). We show that PrP-KO-EVs are rapidly taken up by neurons and colocalize with lysosomes. Although eventually WT-EVs are also found in lysosomes, the amount taken up by neurons is significantly higher for PrP-KO-EVs. Likewise, microglia and astrocytes were also engulfing PrP-KO-sEVs more efficiently than WT-sEVs.Our results provide information on the relative contribution of brain cell types to the sEV pool in mice and indicate that increased release of sEVs by astrocytes together with elevated levels of PrP in sEVs may play a role in intercellular communication at early stages after stroke. In addition, amounts of PrP (and probably PrP-C1) in brain sEVs seem to contribute to their cellular uptake.

Keratins are the main identification markers of circulating tumor cells (CTCs), however, whether their deregulation is associated with the metastatic process is largely unknown. Previously we have shown by in silico analysis that keratin 16 (KRT16) mRNA upregulation might be associated with more aggressive cancer. Therefore, in this study, we investigated the biological role and the clinical relevance of K16 in metastatic breast cancer. By performing RT-qPCR, western blot, and immunocytochemistry, we investigated the expression patterns of K16 in metastatic breast cancer cell lines and evaluated the clinical relevance of K16 expression in CTCs of 20 metastatic breast cancer patients. High K16 protein expression was associated with an intermediate mesenchymal phenotype. Functional studies showed that K16 has a regulatory effect on EMT and overexpression of K16 significantly enhanced cell motility (p &lt, 0.001). In metastatic breast cancer patients, 64.7% of the detected CTCs expressed K16, which was associated with shorter relapse-free survival (p = 0.0042). Our findings imply that K16 is a metastasis-associated protein that promotes EMT and acts as a positive regulator of cellular motility. Furthermore, determining K16 status in CTCs provides prognostic information that helps to identify patients whose tumors are more prone to metastasize.

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

The earliest intracellular signals that occur after T cell activation are local, subsecond Ca2+ microdomains. Here, we identified a Ca2+ entry component involved in Ca2+ microdomain formation in both unstimulated and stimulated T cells. In unstimulated T cells, spontaneously generated small Ca2+ microdomains required ORAI1, STIM1, and STIM2. Super-resolution microscopy of unstimulated T cells identified a circular subplasmalemmal region with a diameter of about 300 nm with preformed patches of colocalized ORAI1, ryanodine receptors (RYRs), and STIM1. Preformed complexes of STIM1 and ORAI1 in unstimulated cells were confirmed by coimmunoprecipitation and Forster resonance energy transfer studies. Furthermore, within the first second after T cell receptor (TCR) stimulation, the number of Ca2+ microdomains increased in the subplasmalemmal space, an effect that required ORAI1, STIM2, RYR1, and the Ca2+ mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). These results indicate that preformed clusters of STIM and ORAI1 enable local Ca2+ entry events in unstimulated cells. Upon TCR activation, NAADP-evoked Ca2+ release through RYR1, in coordination with Ca2+ entry through ORAI1 and STIM, rapidly increases the number of Ca2+ microdomains, thereby initiating spread of Ca2+ signals deeper into the cytoplasm to promote full T cell activation.

Inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) has been considered as an actin bundling protein because its N-terminal actin binding domain (ABD) induces formation of linear actin bundles. Since in many cancer cell lines ITPKA is essential for formation of lamellipodia, which consist of cross-linked actin filaments, here we analyzed if full length-ITPKA may induce formation of more complex actin structures. Indeed, we found that incubation of F-actin with ITPKA resulted in formation of dense, branched actin networks. Based on our result that ITPKA does not exhibit an additional C-terminal ABD, we exclude that ITPKA cross-links actin filaments by simultaneous F-actin binding with two different ABDs. Instead, stimulated-emission-depletion-microscopy and measurement of InsP3Kinase activity give evidence that that N-terminal ABD-homodimers of ITPKA bind to F-actin while the monomeric C-termini insert between adjacent actin filaments. Thereby, they prevent formation of thick actin bundles but induce formation of thin branched actin structures. Interestingly, when embedded in this dense actin network, InsP3Kinase activity is doubled and the product of InsP3Kinase activity, Ins(1,3,4,5)P4, inhibits spontaneous actin polymerization which may reflect a local negative feedback regulation of InsP3Kinase activity. In conclusion, we demonstrate that not only the ABD of ITPKA modulates actin dynamics but reveal that the InsP3Kinase domain substantially contributes to this process. © 2015 Wiley Periodicals, Inc.

Although inactivating mutations of PLS3, encoding the actin-bundling protein plastin-3, have been identified to cause X-linked osteoporosis, the cellular and molecular influence of PLS3 on bone remodeling is poorly defined. Moreover, although a previous study has demonstrated moderate osteopenia in 12 week-old Pls3-deficient mice based on μCT scanning, there is no reported analysis of such a model on the basis of undecalcified histology and bone-specific histomorphometry. To fill this knowledge gap we applied a deep phenotyping approach and studied Pls3-deficient mice at different ages. Surprisingly, we did not detect significant differences between wildtype and Pls3-deficient littermates with respect to trabecular bone mass, and the same was the case for all histomorphometric parameters determined at 12 weeks of age. Remarkably however, the cortical thickness in both, tibia and femur, was significantly reduced in Pls3-deficient mice in all age groups. We additionally studied the ex vivo behavior of Pls3-deficient primary osteoblasts, which displayed moderately impaired mineralization capacity. Of note, while most osteoblastogenesis markers were not differentially expressed between wildtype and Pls3-deficient cultures, the expression of Sfrp4 was significantly reduced in the latter, a potentially relevant finding, since Sfrp4 inactivation, in mice and humans, specifically causes cortical thinning. We finally addressed the question, if Pls3-deficiency would impair the osteoanabolic influence of parathyroid hormone (PTH). For this purpose we applied daily injection of PTH into wildtype and Pls3-deficient mice and found a similar response regardless of the genotype. Taken together, our data reveal that Pls3-deficiency in mice only recapitulates the cortical bone phenotype of individuals with X-linked osteoporosis by negatively affecting the early stage of cortical bone acquisition.

The capture of circulating tumor cells (CTCs) is still a challenging application for microfluidic chips, as these cells are rare and hidden in a huge background of blood cells. Here, different microfluidic ceiling designs in regard to their capture efficiency for CTCs in model experiments and more realistic conditions of blood samples spiked with a clinically relevant amount of tumor cells are evaluated. An optimized design for the capture platform that allows highly efficient recovery of CTCs from size-based pre-enriched samples under realistic conditions is obtained. Furthermore, the viability of captured tumor cells as well as single cell recovery for downstream genomic analysis is demonstrated. Additionally, the authors' findings underline the importance of evaluating rational design rules for microfluidic devices based on theoretical models by application-specific experiments.

While single-molecule fluorescence from emitters with high quantum efficiencies such as organic dye molecules can easily be detected by modern apparatus, many less efficient emission processes such as Raman scattering and metal luminescence require dramatic enhancement to exceed the single-particle detection limit. This enhancement can be achieved using resonant optical systems such as plasmonic particles or nanoantennas, the study of which has led to substantial progress in understanding the interaction of quantum emitters with their electromagnetic environment. This review is focused on the advances in measurement techniques and potential applications enabled by a deeper understanding of fundamental optical interaction processes occurring between single quantum systems on the nanoscale. While the affected phenomena are numerous, including molecular fluorescence and also exciton luminescence and Raman scattering, the interaction itself can often be described from a unified point of view. Starting from a single underlying model, this work elucidates the dramatic enhancement potential of plasmonic tips and nanoparticles and also the more deterministic influence of a Fabry-Pérot microresonator. With the extensive knowledge of the radiative behavior of a quantum system, insight can be gained into nonradiative factors as well, such as energy transfer phenomena or spatial and chemical configurations in single molecules.

Mucolipidosis type II (MLII) is a severe multi-systemic genetic disorder caused by missorting of lysosomal proteins and the subsequent lysosomal storage of undegraded macromolecules. Although affected children develop disabling skeletal abnormalities, their pathogenesis is not understood. Here we report that MLII knock-in mice, recapitulating the human storage disease, are runted with accompanying growth plate widening, low trabecular bone mass and cortical porosity. Intralysosomal deficiency of numerous acid hydrolases results in accumulation of storage material in chondrocytes and osteoblasts, and impaired bone formation. In osteoclasts, no morphological or functional abnormalities are detected whereas osteoclastogenesis is dramatically increased in MLII mice. The high number of osteoclasts in MLII is associated with enhanced osteoblastic expression of the pro-osteoclastogenic cytokine interleukin-6, and pharmacological inhibition of bone resorption prevented the osteoporotic phenotype of MLII mice. Our findings show that progressive bone loss in MLII is due to the presence of dysfunctional osteoblasts combined with excessive osteoclastogenesis. They further underscore the importance of a deep skeletal phenotyping approach for other lysosomal diseases in which bone loss is a prominent feature.

This work demonstrates for the first time that one-photon luminescence in gold nanorods is an entirely plasmon-induced process. To achieve this goal, we used confocal microscopy in combination with...

Understanding the cellular processes that occur between the cytosol and the plasma membrane is an important task for biological research. Till now, however, it was not possible to combine fast and high-resolution imaging of both the isolated plasma membrane and the surrounding intracellular volume. Here, we demonstrate the combination of fast high-resolution spinning disk (SD) and total internal reflection fluorescence (TIRF) microscopy for specific imaging of the plasma membrane. A customised SD-TIRF microscope was used with specific design of the light paths that allowed, for the first time, live SD-TIRF experiments at high acquisition rates. A series of experiments is shown to demonstrate the feasibility and performance of our setup.

Higher order laser modes, mainly called doughnut modes (DMs) have use in many different branches of research, such as, bio-imaging, material science, single-molecule microscopy, and spectroscopy. The main reason of their increasing importance is that recently, the techniques to generate well-defined DMs have been refined or rediscovered. Although their potential is still not fully utilized, their specifically polarized field distribution gives rise to a wide field of applications. They are contributing to complete our fundamental knowledge of the optical properties of single emitting species, such as molecules, nanoparticles, or quantum dots, offering insight into the three-dimensional dipole or particle orientation in space. The perfect zero intensity in the focus center qualifies some DMs for stimulated emission depletion (STED) microscopy. For the same reason, they have been suggested for trapping and tweezing applications.

Lasermoden mit ringformigem Strahlprofil, die zumeist als Doughnut-Moden (DMs) bezeichnet werden, finden Anwendung in unterschiedlichen Forschungsbereichen wie den Bio- und Materialwissenschaften oder der Einzelmolekulmikroskopie und -spektroskopie. Der Hauptgrund fur die wachsende Bedeutung der DMs liegt darin, dass in letzter Zeit die Techniken zur Herstellung dieser Moden mit hoher Prazision verfeinert oder wiederentdeckt wurden. Obwohl ihr Potential bisher noch nicht vollkommen ausgeschopft wird, ermoglichen die speziellen Polarisationseigenschaften der DMs eine Vielzahl von Anwendungen. So tragen sie dazu bei, unser grundlegendes Verstandnis der optischen Eigenschaften einzelner emittierender Spezies wie Molekule, Nanopartikel oder Quantenpunkte zu vervollstandigen und erlauben Einblick in die dreidimensionale Dipol- oder Partikelorientierung im Raum. Durch ihre vollstandig verschwindende Intensitat im Zentrum des Fokus eignen sich einige DMs auch fur die stimulierte Emissionsloschung in der Fluoreszenzmikroskopie. Aus demselben Grund hat man sie auch fur die Verwendung in optischen Fallen und Pinzetten vorgeschlagen.

Genome instability is considered as an enabling hallmark of cancer formation and progression as well as a source of intratumoral heterogeneity. Retinoid acid-induced protein 2 (RAI2) was initially identified as a new metastasis-associated protein especially related with the presence of disseminated tumor cells in the bone marrow of breast cancer patients. In primary breast tumors low RAI2 gene expression correlates with mutant TP53 status and moreover, RAI2 depleted luminal breast cancer cell lines show decreased expression of key regulators of G2/M transition. We hypothesize that RAI2 has an influence on mitotic fidelity and is involved in maintaining genomic stability in breast cancer cell lines. To analyze consequences of RAI2 depletion on mitotic fidelity in luminal breast cancer cells, we followed mitotic cells by live cell imaging and analyzed chromosomal segregation by immune fluorescence staining and karyotyped RAI2 depleted cells. For analysis of cell cycle dependent RAI2 protein expression we executed Western blot analysis after cell synchronization and treatment with genotoxic agents. Localization of ectopically expressed HA-tagged RAI2 protein was analyzed in breast cancer cells treated with genotoxic agents using immune fluorescence staining. Finally, we performed in silico validation in different breast cancer data sets. Our findings show that RAI2 depletion in luminal breast cancer cell lines leads to increased chromosomal instability, associated with loss of acentric chromosomal fragments during ana- and metaphase as well as a prolonged mitosis. Furthermore, karyotyping revealed a significant reduction of chromosome number from 78 to 73 in RAI2 depleted cells. In synchronized parental breast cancer cells we could show an increased RAI2 expression during S/G2 phase compared to unsynchronized cells. Interestingly, genotoxic treatment resulted in a significant upregulation of RAI2 protein, which can also be seen as an increased formation of nuclear speckles (p0,001). Our findings show that RAI2 depletion in luminal breast cancer cell lines leads to increased chromosomal instability. Our results show that RAI2 depletion causes pre-mitotic DNA lesions. Moreover, in breast cancer patients a low RAI2 expression was significantly associated with chromosomal instability. We conclude that the RAI2 protein preserves mitotic fidelity and chromosomal integrity in luminal breast cancer cell lines by affecting either the DNA damage response or DNA replication during S- and/or G2-phase. Citation Format: Lena Boettcher, Bernd Zobiak, Kerstin Borgmann, Alexandra Zielinski, Antonio Virgilio Failla, Klaus Pantel, Harriet Wikman, Stefan Werner. RAI2 maintains genome stability of breast cancer cells [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 3364.

We analyzed the scattering patterns of individual Au nanorods detected by means of confocal interference scattering microscopy in combination with a higher order laser mode. We placed the Au nanorods at the interface between two dielectric media and examined the influence of different interfaces on the signal amplitude, the signal-to-noise ratio as well as on the precision of topological measurements. Approaching the index matching regime allows for topological measurements with high accuracy minimizing the acquisition time. We were also able to track the position and the orientation of particles embedded in water even when they were not thoroughly sticking to the glass surface. These results underscore the potential of the presented technique for applications in life sciences.

We have investigated the influence of the plasmon resonances of individual, spatially isolated gold nanoparticle aggregates on their emission properties using combined optical confocal and dark field scattering microscopy and spectroscopy. The emission intensity of the same aggregate is enhanced by up to 1 order of magnitude if the emission wavelengths overlap with the plasmon resonance in the corresponding white light scattering spectrum. Regardless of the specific geometry of an individual aggregate, the in situ measurement of the plasmon characteristics delivers unique information about its potential as a substrate for surface-enhanced spectroscopy and allows its characterization as a nanoscatterer, nanoemitter, or local heater.

Summary We demonstrate a novel optical method for characterizing single Au nanoparticles by acquiring their scattering patterns. This technique combines confocal microscopy and higher-order laser modes for detecting the light scattered by sub-wavelength–sized nanoobjects. The optical patterns are generated by the coherent superposition of the field scattered by individual metallic particles and the excitation field reflected at the cover slide–air interface and provide information about the particles' position, orientation, size and shape. Detectable changes in the full width at half maximum (FWHM) of the signal intensity permit to distinguish between 20- and 60-nm diameter Au spheres. The confocal images are also very sensitive to the particle's geometry and polarizability, that is, Au nanospheres, Au nanorods and triangular Au nanoplates give different characteristic patterns if the excitation wavelength is varied.

In reeler mutant mice, which are deficient in reelin (Reln), the lamination of the cerebral cortex is disrupted. Reelin signaling induces phosphorylation of LIM kinase 1, which phosphorylates the actin-depolymerizing protein cofilin in migrating neurons. Conditional cofilin mutants show neuronal migration defects. Thus, both reelin and cofilin are indispensable during cortical development. To analyze the effects of cofilin phosphorylation on neuronal migration we used in utero electroporation to transfect E14.5 wild-type cortical neurons with pCAG-EGFP plasmids encoding either a nonphosphorylatable form of cofilin 1 (cofilin(S3A)), a pseudophosphorylated form (cofilin(S3E)) or wild-type cofilin 1 (cofilin(WT)). Wild-type controls and reeler neurons were transfected with pCAG-EGFP. Real-time microscopy and histological analyses revealed that overexpression of cofilin(WT) and both phosphomutants induced migration defects and morphological abnormalities of cortical neurons. Of note, reeler neurons and cofilin(S3A)- and cofilin(S3E)-transfected neurons showed aberrant backward migration towards the ventricular zone. Overexpression of cofilin(S3E), the pseudophosphorylated form, partially rescued the migration defect of reeler neurons, as did overexpression of Limk1. Collectively, the results indicate that reelin and cofilin cooperate in controlling cytoskeletal dynamics during neuronal migration.

Follicular penetration has gained increasing interest regarding (i) safety concerns about (environmentally born) xenobiotics available to the hair follicle (HF), e.g. nanomaterials or allergens which should not enter the skin, and (ii) the possibility for non-invasive follicular drug and antigen delivery. However, not much is known about barriers in the HF which have to be surpassed upon uptake and/or penetration into surrounding tissue. Thus, aim of this work was a detailed investigation of this follicular barrier function, as well as particle uptake into the HF of porcine skin which is often used as a model system for human skin for such purposes. We show that follicular tight junctions (TJs) form a continuous barrier from the infundibulum down to the suprabulbar region, complementary to the stratum corneum in the most exposed upper follicular region, but remaining as the only barrier in the less accessible lower follicular regions. In the bulbar region of the HF no TJ barrier was found, demonstrating the importance of freely supplying this hair-forming part with e.g. nutrients or hormones from the dermal microenvironment. Moreover, the dynamic character of the follicular TJ barrier was shown by modulating its permeability using EDTA. After applying polymeric model-nanoparticles (154 nm) to the skin, transmission electron microscopy revealed that the majority of the particles were localized in the upper part of the HF where the double-barrier is present. Only few penetrated deeper, reaching regions where TJs act as the only barrier, and no particles were observed in the bulbar, barrier-less region. Lastly, the equivalent expression and distribution of TJ proteins in human and porcine HF further supports the suitability of porcine skin as a predictive model to study the follicular penetration and further biological effects of dermally applied nanomaterials in humans.

We present a new method for the imaging of single metallic nanoparticles that provides information about their shape and orientation. Using confocal microscopy in combination with higher order laser modes, scattering images of individual particles are recorded. Gold nanospheres and nonorods render characteristic patterns reflecting the different particle geometries. In the case of nanorods, the scattering patterns also reveal the orientation of the particles. This novel technique provides a promising tool for the visualization of nonbleaching labels in the biosciences.

Recently developed far field light optical methods are a powerful tool to analyze biological nanostructures and their dynamics, in particular including the interior of three-dimensionally conserved cells. In this article, the recently described method of spatially modulated illumination (SMI) microscopy has been further extended to the online determination of the extension of small, subwavelength sized, fluorescent objects (nanosizing). Using fluorescence excitation with 488 nm, the determination of fluorescent labeled object diameters down to 40 nm corresponding to about 1/12th of the wavelength used for one-photon excitation could be shown. The results of the SMI nanosizing procedure for a detailed, systematic variation of the object diameter are presented together with a fast algorithm for online size evaluation. In addition, we show a direct comparison of the diameter of “colocalization volumes” between SMI nanosizing and conventional confocal laser scanning microscopy.

For an improved understanding of cellular processes, it is highly desirable to develop light optical methods for the analysis of biological nanostructures and their dynamics in the interior of three-dimensionally (3D) conserved cells. Here, important structural parameters to be considered are the topology, i.e. the mutual positions and distances, as well as the sizes of the constituting subunits. This has become possible by the development of a novel method of far-field light fluorescence microscopy, spatially modulated illumination (SMI) microscopy. Using this approach, axial distances between fluorescence-labeled targets can be measured with an accuracy close to 1 nm; their sizes can be determined down to a few tens of nanometers. This approach can be extended to the determination of 3D positions and mutual 3D distances and sizes of any number of small objects/subunits that can be discriminated due to their spectral signatures. Consequently, the new approach allows an ‘in situ nanostructure elucidation, until now regarded to be beyond the possibilities of far-field light microscopy. Application examples discussed are: colocalization/nanosizing and topological analysis of large protein-protein complexes, of nucleic acid-protein complexes (such as transcription factories), or of the highly complex DNA-protein nanostructures of which active/ inactive gene regions in the eukaryotic cell nucleus are constituted.

In this paper, we explain in detail the wavelength dependence of the elastic scattering pattern of individual, optically isolated gold nanorods by using confocal microscopy in combination with higher order laser modes, i.e., radially/azimuthally polarized laser modes. We demonstrate that the spectral dependence of the scattering pattern is mostly caused by the relative strength of the gold nanorods’ plasmonic modes at different wavelengths. Since the gold nanorods’ plasmonic modes are determined by the particles’ geometrical parameter, e.g., size and aspect ratio, as well as the refractive index of the surrounding medium, we show that the spectral dependence of the scattering pattern is a simple, not invasive way to determine, e.g., the gold nanorod aspect ratio or physical variation of the local environment. Thus, a further development of spectral imaging of gold nanorods can lead to the employment of this technique in biomedical assays involving also living samples.

Given the increasing importance of immunotherapeutic treatment strategies in neuro-oncology, the investigation of immune escape mechanisms, which lead to treatment resistance, is crucial. Therefore, studying tumor heterogeneity to understand the clonal dynamics and emergence of immune escape variants during cancer immunoediting can yield valuable insights into the adaptive processes of treatment resistance. We applied a novel multicolor labeling strategy, termed optical barcoding, which makes use of flow-cytometric read-out to compare in-vivo clonality in an immunocompetent versus -deficient environment. The optically barcoded syngeneic GL261 glioma was administered to wildtype and immunodeficient C57BL/6 pfp-/- rag2-/- mice, and tumor growth was monitored. We also analyzed the phenotype of tumor-infiltrating lymphocytes and the gene-expression signatures of explanted tumors using RNAseq. Ex-vivo analysis of individual tumor clones demonstrated that tumors grown in immunocompetent C57BL/6 mice were composed of a significantly different clonal architecture compared to pfp-/- rag2-/- C57BL/6. Whereas pfp-/- rag2-/- mice displayed a homogeneous clonal distribution in all tumors, immunoedited tumors in wildtype mice exhibited reduced clonality with emergence of immune escape clones, independent of proliferation rates. Beyond the expected prolonged survival of wildtype mice, immunoediting was further associated with differences in macroscopic growth patterns, vascularity and invasiveness. Tissue staining, time-course microarray analyses and flow-cytometric phenotyping of tumor-infiltrating immune cells highlighted the exemplary role of PD-L1 as mechanism to adapt to interferon-ɣ and infiltration of T-cells. Furthermore, RNAseq profiling of GL261 tumors grown in wt vs immunodeficient mice allowed the identification of an interferon-ɣ-mediated T-cell-shaped gene expression signature and new pathways potentially involved in the immune escape of glioma. Taken together, using optical barcoding we have demonstrated that the process of cancer immunoediting during tumor evolution not only shapes the gene expression profile and growth pattern, but also has a profound impact on the intratumoral heterogeneity of glioma in the syngeneic GL261 model.

Most of the genomic and epigenetic alterations in GBM have been described thanks to the effort of the Tumor Cancer Genome Atlas and others, highlighting the highly heterogeneous and complex genomic landscape of GBM. Our understanding of how such heterogeneity is maintained is however still limited, and tumor growth driven by micro-environmental selective pressure follows a clonal-evolution model that leads to the emergence of new subpopulation and regional heterogeneity. We established GBM-patient derived cell lines (PDCL) and mouse xenografts from tissue resections and followed the growth progression of multiple clones in vivo by using optical barcoding to track and quantify the clonal evolution. We isolated single cells out of GBM-PDCL and tagged them with specific combinations of two out of six different fluorescent proteins, thus generating 21 optically barcoded (OBC) clones. These clones were then grown either separately or as a mix in vitro and in vivo after intracranial injection. Using this innovative technique, we were able to track the fate of individual clones and analyzed their growth dynamics in different microenvironments. We discovered that fast growing clones in vitro are not necessarily the ones that are responsible for most of the tumor growth in vivo. Interestingly, the clones that do not have the capacity to overgrow other clones when injected in the brain as a mix, are still tumorigenic when injected alone. Moreover, we discovered that distinct clones were more resistant to radiotherapy than others, opening the possibility to identify specific clonal subpopulations in the tumor that are resistant to treatment and will probably develop into the major resistant clone when the tumor will recur. Understanding how tumor heterogeneity is shaped by the microenvironment and how the different clonal population react to treatment will help predicting drug treatment efficiency and potential resistance.

The vascular endothelium as well as subendothelium are objects of many researches as it is directly involved in a multiplicity of physiological and pathological settings. Detailed study of endothelial function became feasible with the development of techniques to culture endothelial cells (EC) in vitro. Limitations of this approach have become apparent with the realization that cell culture dedifferentiate with time and do not exhibit properties of intact tissue. Here we describe the development of a novel ex vivo tissue model to study cell–vascular wall interactions by using isolated mouse aorta patches. Validation of this model was performed by demonstrating cell attachment and changes in cell shape typical for cell spreading during adhesion. A major advantage of this model is that cell–endothelium interaction and its molecular backgrounds can now be studied more feasibly on an intact and native tissue.

Inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) has been considered as an actin bundling protein because its N-terminal actin binding domain (ABD) induces formation of linear actin bundles. Since in many cancer cell lines ITPKA is essential for formation of lamellipodia, which consist of cross-linked actin filaments, here we analyzed if full length-ITPKA may induce formation of more complex actin structures. Indeed, we found that incubation of F-actin with ITPKA resulted in formation of dense, branched actin networks. Based on our result that ITPKA does not exhibit an additional C-terminal ABD, we exclude that ITPKA cross-links actin filaments by simultaneous F-actin binding with two different ABDs. Instead, stimulated-emission-depletion-microscopy and measurement of InsP3 Kinase activity give evidence that that N-terminal ABD-homodimers of ITPKA bind to F-actin while the monomeric C-termini insert between adjacent actin filaments. Thereby, they prevent formation of thick actin bundles but induce formation of thin branched actin structures. Interestingly, when embedded in this dense actin network, InsP3 Kinase activity is doubled and the product of InsP3 Kinase activity, Ins(1,3,4,5)P4 , inhibits spontaneous actin polymerization which may reflect a local negative feedback regulation of InsP3 Kinase activity. In conclusion, we demonstrate that not only the ABD of ITPKA modulates actin dynamics but reveal that the InsP3 Kinase domain substantially contributes to this process.

Merkel cells, the neurosecretory cells of skin, are essential for light-touch responses and may probably fulfill additional functions. Whether these cells derive from an epidermal or a neural lineage has been a matter of dispute for a long time. In mice, recent studies have clearly demonstrated an epidermal origin of Merkel cells. Given the differences in Merkel cell distribution between human and murine skin, it is, however, unclear whether the same holds true for human Merkel cells. We therefore attempted to gain insight into the human Merkel cell lineage by co-immunodetection of the Merkel cell marker protein cytokeratin 20 (CK20) with various proteins known to be expressed either in epidermal or in neural stem cells of the skin. Neither Sox10 nor Pax3, both established markers of the neural crest lineage, exhibited any cell co-labeling with CK20. By contrast, β1 integrin, known to be enriched in epidermal stem cells, was found in nearly 70 % of interfollicular epidermal and 25 % of follicular Merkel cells. Moreover, LRIG1, also enriched in epidermal stem cells, displayed significant co-immunolabeling with CK20 as well (approximately 20 % in the interfollicular epidermis and 7 % in the hair follicle, respectively). Further epidermal markers were detected in sporadic Merkel cells. Cells co-expressing CK20 with epidermal markers may represent a transitory state between stem cells and differentiated cells. β1 integrin is probably also synthesized by a large subset of mature Merkel cells. Summarizing, our data suggest that human Merkel cells may originate from epidermal rather than neural progenitors.

In this work we demonstrate that freely oriented individual gold nanorods (GNRs) can be used for sensing variations of the refractive index at the interface between two dielectric media. Both the elastic scattering and the luminescence signal of individual GNRs have been used to characterize the dielectric medium surrounding the particles. The scattering signal depends strongly on the distance from the focusing interface and the refractive index mismatch at the focusing interface, while the luminescence signal is only influenced by the last parameter. We used radially and azimuthally polarized light as an excitation source to directly determine the orientation of individual gold nanorods embedded within a dielectric medium.

We investigated the influence of the orientation of individual gold nanorods on the polarization-dependent single-particle surface-enhanced Raman spectroscopy (SERS) of adenine. Higher-order laser beams (radial and azimuthal polarizations) have been used in combination with a parabolic mirror-assisted confocal optical microscope. Based on the photoluminescence (PL) patterns of the single gold nanorods and the simulated electric-field distribution in the focus, we distinguished between isolated gold rods and clusters as well as single nanorods with different orientations. We found that for single gold nanorods lying flat on the substrate, the longitudinal particle plasmon resonance (PPR) mode can be excited more efficiently with the in-plane field component in the focus of an azimuthally polarized laser beam, which enables the observation of stronger enhanced adenine Raman spectra from the single gold nanorods compared to the case of a radially polarized beam.

We present a powerful technique to fully characterize individual gold nanorods by using confocal microscopy in combination with higher order laser modes. We obtain topological information far beyond the optical resolution limit, although the used method is diffraction limited. We perform, for the first time, the imaging of gold nanorods by recording simultaneously their scattering and luminescence signal. In the future, this might permit to extent the results achieved already in [1] to a 3D system. Moreover, the scattering pattern is strongly dependent on the phase relation between the light scattered and reflected at the sample interface, while the luminescence pattern does not depend on this phase relation. By exploiting an index matched sample geometry, we were able to omit the phase relation and therefore detect the pure scattering signal and qualitatively compare it with the luminescence one. Furthermore, as previously shown [2, 3], our technique is capable to track the rotation of single noble metal nanorods. We show that measuring the rotation rate of gold nanorods might be useful to estimate the local viscosity of the surrounding medium.

We present a novel scattering microscopy method to detect the orientation of individual silver nanorods and to measure their relative distances. Using confocal microscopy in combination with either the fundamental or higher order laser modes, scattering images of silver nanorods were recorded. The distance between two individual nanorods was measured with an accuracy in the order of 1 nm. We detected the orientation of isolated silver nanorods with a precision of 0.5 degree that corresponds to a rotational arch of about 1 nm. The results demonstrate the potential of the technique for the visualization of non-bleaching labels in biosciences.

We used confocal interference scattering microscopy combined with higher order laser modes to determine the position, the shape, the orientation and the effect of the environment on individual metal nanoparticles by studying their scattering patterns.