Progerin forms an abnormal meshwork and has a dominant-negative effect on the nuclear lamina.

Progerin, the protein that causes Hutchinson-Gilford progeria syndrome, triggers nuclear membrane (NM) ruptures and blebs, but the mechanisms are unclear. We suspected that the expression of progerin changes the overall structure of the nuclear lamina. High-resolution microscopy of smooth muscle cells (SMCs) revealed that lamin A and lamin B1 form independent meshworks with uniformly spaced openings (~0.085 µm2). The expression of progerin in SMCs resulted in the formation of an irregular meshwork with clusters of large openings (up to 1.4 µm2). The expression of progerin acted in a dominant-negative fashion to disrupt the morphology of the endogenous lamin B1 meshwork, triggering irregularities and large openings that closely resembled the irregularities and openings in the progerin meshwork. These abnormal meshworks were strongly associated with NM ruptures and blebs. Of note, the progerin meshwork was markedly abnormal in nuclear blebs that were deficient in lamin B1 (~50% of all blebs). That observation suggested that higher levels of lamin B1 expression might normalize the progerin meshwork and prevent NM ruptures and blebs. Indeed, increased lamin B1 expression reversed the morphological abnormalities in the progerin meshwork and markedly reduced the frequency of NM ruptures and blebs. Thus, progerin expression disrupts the overall structure of the nuclear lamina, but that effect-along with NM ruptures and blebs-can be abrogated by increased lamin B1 expression.

Imaging innate immunity.

Innate immunity is the first line of defense against infectious intruders and also plays a major role in the development of sterile inflammation. Direct microscopic imaging of the involved immune cells, especially neutrophil granulocytes, monocytes, and macrophages, has been performed since more than 150 years, and we still obtain novel insights on a frequent basis. Initially, intravital microscopy was limited to small-sized animal species, which were often invertebrates. In this review, we will discuss recent results on the biology of neutrophils and macrophages that have been obtained using confocal and two-photon microscopy of individual cells or subcellular structures as well as light-sheet microscopy of entire organs. This includes the role of these cells in infection defense and sterile inflammation in mammalian disease models relevant for human patients. We discuss their protective but also disease-enhancing activities during tumor growth and ischemia-reperfusion damage of the heart and brain. Finally, we provide two visions, one experimental and one applied, how our knowledge on the function of innate immune cells might be further enhanced and also be used in novel ways for disease diagnostics in the future.

Specific protein interactions between rice members of the GT43 and GT47 families form various central cores of putative xylan synthase complexes.

Members of the glycosyltransferase (GT)43 and GT47 families have been associated with heteroxylan synthesis in both dicots and monocots and are thought to assemble into central cores of putative xylan synthase complexes (XSCs). Currently, it is unknown whether protein-protein interactions within these central cores are specific, how many such complexes exist, and whether these complexes are functionally redundant. Here, we used gene association network and co-expression approaches in rice to identify four OsGT43s and four OsGT47s that assemble into different GT43/GT47 complexes. Using two independent methods, we showed that (i) these GTs assemble into at least six unique complexes through specific protein-protein interactions and (ii) the proteins interact directly in vitro. Confocal microscopy showed that, when alone, all OsGT43s were retained in the endoplasmic reticulum (ER), while all OsGT47s were localized in the Golgi. co-expression of OsGT43s and OsGT47s displayed complexes that form in the ER but accumulate in Golgi. ER-to-Golgi trafficking appears to require interactions between OsGT43s and OsGT47s. Comparison of the central cores of the three putative rice OsXSCs to wheat, asparagus, and Arabidopsis XSCs, showed great variation in GT43/GT47 combinations, which makes the identification of orthologous central cores between grasses and dicots challenging. However, the emerging picture is that all central cores from these species seem to have at least one member of the IRX10/IRX10-L clade in the GT47 family in common, suggesting greater functional importance for this family in xylan synthesis. Our findings provide a new framework for future investigation of heteroxylan biosynthesis and function in monocots.

Probing the in situ volumes of Arabidopsis leaf plastids using three-dimensional confocal and scanning electron microscopy.

Leaf plastids harbor a plethora of biochemical reactions including photosynthesis, one of the most important metabolic pathways on Earth. Scientists are eager to unveil the physiological processes within the organelle but also their interconnection with the rest of the plant cell. An increasingly important feature of this venture is to use experimental data in the design of metabolic models. A remaining obstacle has been the limited in situ volume information of plastids and other cell organelles. To fill this gap for chloroplasts, we established three microscopy protocols delivering in situ volumes based on: (i) chlorophyll fluorescence emerging from the thylakoid membrane, (ii) a CFP marker embedded in the envelope, and (iii) calculations from serial block-face scanning electron microscopy (SBFSEM). The obtained data were corroborated by comparing wild-type data with two mutant lines affected in the plastid division machinery known to produce small and large mesophyll chloroplasts, respectively. Furthermore, we also determined the volume of the much smaller guard cell plastids. Interestingly, their volume is not governed by the same components of the division machinery which defines mesophyll plastid size. Based on our three approaches, the average volume of a mature Col-0 wild-type mesophyll chloroplasts is 93 µm3 . Wild-type guard cell plastids are approximately 18 µm3 . Lastly, our comparative analysis shows that the chlorophyll fluorescence analysis can accurately determine chloroplast volumes, providing an important tool to research groups without access to transgenic marker lines expressing genetically encoded fluorescence proteins or costly SBFSEM equipment.

Dot Scanner: open-source software for quantitative live-cell imaging in planta.

Confocal microscopy has greatly aided our understanding of the major cellular processes and trafficking pathways responsible for plant growth and development. However, a drawback of these studies is that they often rely on the manual analysis of a vast number of images, which is time-consuming, error-prone, and subject to bias. To overcome these limitations, we developed Dot Scanner, a Python program for analyzing the densities, lifetimes, and displacements of fluorescently tagged particles in an unbiased, automated, and efficient manner. Dot Scanner was validated by performing side-by-side analysis in Fiji-ImageJ of particles involved in cellulose biosynthesis. We found that the particle densities and lifetimes were comparable in both Dot Scanner and Fiji-ImageJ, verifying the accuracy of Dot Scanner. Dot Scanner largely outperforms Fiji-ImageJ, since it suffers far less selection bias when calculating particle lifetimes and is much more efficient at distinguishing between weak signals and background signal caused by bleaching. Not only does Dot Scanner obtain much more robust results, but it is a highly efficient program, since it automates much of the analyses, shortening workflow durations from weeks to minutes. This free and accessible program will be a highly advantageous tool for analyzing live-cell imaging in plants.

Multi-view confocal microscopy enables multiple organ and whole organism live-imaging.

Understanding how development is coordinated in multiple tissues and gives rise to fully functional organs or whole organisms necessitates microscopy tools. Over the last decade numerous advances have been made in live-imaging, enabling high resolution imaging of whole organisms at cellular resolution. Yet, these advances mainly rely on mounting the specimen in agarose or aqueous solutions, precluding imaging of organisms whose oxygen uptake depends on ventilation. Here, we implemented a multi-view multi-scale microscopy strategy based on confocal spinning disk microscopy, called Multi-View confocal microScopy (MuViScopy). MuViScopy enables live-imaging of multiple organs with cellular resolution using sample rotation and confocal imaging without the need of sample embedding. We illustrate the capacity of MuViScopy by live-imaging Drosophila melanogaster pupal development throughout metamorphosis, highlighting how internal organs are formed and multiple organ development is coordinated. We foresee that MuViScopy will open the path to better understand developmental processes at the whole organism scale in living systems that require gas exchange by ventilation.

The molecular and phenotypic makeup of fetal human skin T lymphocytes.

The adult human skin contains a vast number of T cells that are essential for skin homeostasis and pathogen defense. T cells are first observed in the skin at the early stages of gestation; however, our understanding of their contribution to early immunity has been limited by their low abundance and lack of comprehensive methodologies for their assessment. Here, we describe a new workflow for isolating and expanding significant amounts of T cells from fetal human skin. Using multiparametric flow cytometry and in situ immunofluorescence, we found a large population with a naive phenotype and small populations with a memory and regulatory phenotype. Their molecular state was characterized using single-cell transcriptomics and TCR repertoire profiling. Importantly, culture of total fetal skin biopsies facilitated T cell expansion without a substantial impact on their phenotype, a major prerequisite for subsequent functional assays. Collectively, our experimental approaches and data advance the understanding of fetal skin immunity and potential use in future therapeutic interventions.

Quantitation of F-actin in cytoskeletal reorganization: Context, methodology and implications.

The actin cytoskeleton is involved in a large number of cellular signaling events in addition to providing structural integrity to the cell. Actin polymerization is a key event during cellular signaling. Although the role of actin cytoskeleton in cellular processes such as trafficking and motility has been extensively studied, the reorganization of the actin cytoskeleton upon signaling has been rarely explored due to lack of suitable assays. Keeping in mind this lacuna, we developed a confocal microscopy based approach that relies on high magnification imaging of cellular F-actin, followed by image reconstruction using commercially available software. In this review, we discuss the context and relevance of actin quantitation, followed by a detailed hands-on approach of the methodology involved with specific points on troubleshooting and useful precautions. In the latter part of the review, we elucidate the method by discussing applications of actin quantitation from our work in several important problems in contemporary membrane biology ranging from pathogen entry into host cells, to GPCR signaling and membrane-cytoskeleton interaction. We envision that future discovery of cell-permeable novel fluorescent probes, in combination with genetically encoded actin-binding reporters, would allow real-time visualization of actin cytoskeleton dynamics to gain deeper insights into active cellular processes in health and disease.

Three-Dimensional Optical Imaging of Internal Deformations in Polymeric Microscale Mechanical Metamaterials.

Recent advances in two-photon polymerization fabrication processes are paving the way to creating macroscopic metamaterials with microscale architectures, which exhibit mechanical properties superior to their bulk material counterparts. These metamaterials typically feature lightweight, complex patterns such as lattice or minimal surface structures. Conventional tools for investigating these microscale structures, such as scanning electron microscopy, cannot easily probe the internal features of these structures, which are critical for a comprehensive assessment of their mechanical behavior. In turn, we demonstrate an optical confocal microscopy-based approach that allows for high-resolution optical imaging of internal deformations and fracture processes in microscale metamaterials under mechanical load. We validate this technique by investigating an exemplary metamaterial lattice structure of 80 × 80 × 80 µm3 in size. This technique can be extended to other metamaterial systems and holds significant promise to enhance our understanding of their real-world performance under loading conditions.

Tetrahedral serial multiview microscopy and image fusion for improved resolution and extent in stained zebrafish embryos.

BACKGROUND: Spatial mapping on the single-cell level over the whole organism can uncover roles of molecular players involved in vertebrate development. Custom microscopes have been developed that use multiple objectives to view a sample from multiple views at the same time. Such multiview imaging approaches can improve resolution and uniformity of image quality as well as allow whole embryos to be imaged (Swoger et al., Opt Express, 2007;15(13):8029). However, multiview imaging is highly restricted to specialized equipment requiring multiple objectives or sample rotation with automated hardware. RESULTS: Our approach uses a standard single-objective confocal microscope to perform serial multiview imaging. Multiple views are imaged sequentially by mounting the fixed sample in an agarose tetrahedron that is manually rotated in between imaging each face. Computational image fusion allows for a joint 3D image to be created from multiple tiled Z-stacks acquired from different angles. The resulting fused image has improved resolution and imaging extent. CONCLUSION: With this technique, multiview imaging can be performed on a variety of common single-objective microscopes to allow for whole-embryo, high-resolution imaging.

The dendritic ERGIC: Microtubule and actin cytoskeletons participate in stop-and-go movement of mobile carriers between stable structures.

The endoplasmic reticulum (ER)-to-Golgi intermediate compartment (ERGIC) is a membranous organelle that mediates protein transport between the ER and the Golgi apparatus. In neurons, clusters of these vesiculotubular structures are situated throughout the cell in proximity to the ER, passing cargo to the cis-Golgi cisternae, located mainly in the perinuclear region. Although ERGIC markers have been identified in neurons, the distribution and dynamics of neuronal ERGIC structures have not been characterized yet. Here, we show that long-distance ERGIC transport occurs via an intermittent mechanism in dendrites, with mobile elements moving between stationary structures. Slow and fast live-cell imaging have captured stable ERGIC structures remaining in place over long periods of time, as well as mobile ERGIC structures advancing very short distances along dendrites. These short distances have been consistent with the lengths between the stationary ERGIC structures. Kymography revealed ERGIC elements that moved intermittently, emerging from and fusing with stationary ERGIC structures. Interestingly, this movement apparently depends not only on the integrity of the microtubule cytoskeleton, as previously reported, but on the actin cytoskeleton as well. Our results indicate that the dendritic ERGIC has a dual nature, with both stationary and mobile structures. The neural ERGIC network transports proteins via a stop-and-go movement in which both the microtubule and the actin cytoskeletons participate.

Human Fis1 directly interacts with Drp1 in an evolutionarily conserved manner to promote mitochondrial fission.

Mitochondrial fission protein 1 (Fis1) and dynamin-related protein 1 (Drp1) are the only two proteins evolutionarily conserved for mitochondrial fission, and directly interact in Saccharomyces cerevisiae to facilitate membrane scission. However, it remains unclear if a direct interaction is conserved in higher eukaryotes as other Drp1 recruiters, not present in yeast, are known. Using NMR, differential scanning fluorimetry, and microscale thermophoresis, we determined that human Fis1 directly interacts with human Drp1 (KD = 12-68 µM), and appears to prevent Drp1 assembly, but not GTP hydrolysis. Similar to yeast, the Fis1-Drp1 interaction appears governed by two structural features of Fis1: its N-terminal arm and a conserved surface. Alanine scanning mutagenesis of the arm identified both loss-of-function and gain-of-function alleles with mitochondrial morphologies ranging from highly elongated (N6A) to highly fragmented (E7A), demonstrating a profound ability of Fis1 to govern morphology in human cells. An integrated analysis identified a conserved Fis1 residue, Y76, that upon substitution to alanine, but not phenylalanine, also caused highly fragmented mitochondria. The similar phenotypic effects of the E7A and Y76A substitutions, along with NMR data, support that intramolecular interactions occur between the arm and a conserved surface on Fis1 to promote Drp1-mediated fission as in S. cerevisiae. These findings indicate that some aspects of Drp1-mediated fission in humans derive from direct Fis1-Drp1 interactions that are conserved across eukaryotes.

Spatial organization and dynamics of chromosomes and microtubules during barley mitosis.

Mitosis and cytokinesis are fundamental processes through which somatic cells increase their numbers and allow plant growth and development. Here, we analyzed the organization and dynamics of mitotic chromosomes, nucleoli, and microtubules in living cells of barley root primary meristems using a series of newly developed stable fluorescent protein translational fusion lines and time-lapse confocal microscopy. The median duration of mitosis from prophase until the end of telophase was 65.2 and 78.2 min until the end of cytokinesis. We showed that barley chromosomes frequently start condensation before mitotic pre-prophase as defined by the organization of microtubules and maintain it even after entering into the new interphase. Furthermore, we found that the process of chromosome condensation does not finish at metaphase, but gradually continues until the end of mitosis. In summary, our study features resources for in vivo analysis of barley nuclei and chromosomes and their dynamics during mitotic cell cycle.

Adenosine A2B receptors differently modulate oligodendrogliogenesis and myelination depending on their cellular localization.

Differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs) is a key event for axonal myelination in the brain; this process fails during demyelinating pathologies. Adenosine is emerging as an important player in oligodendrogliogenesis, by activating its metabotropic receptors (A1R, A2AR, A2BR, and A3R). We previously demonstrated that the Gs-coupled A2BR reduced differentiation of primary OPC cultures by inhibiting delayed rectifier (IK) as well as transient (IA) outward K+ currents. To deepen the unclear role of this receptor subtype in neuron-OL interplay and in myelination process, we tested the effects of different A2BR ligands in a dorsal root ganglion neuron (DRGN)/OPC cocultures, a corroborated in vitro myelination assay. The A2BR agonist, BAY60-6583, significantly reduced myelin basic protein levels but simultaneously increased myelination index in DRGN/OPC cocultures analyzed by confocal microscopy. The last effect was prevented by the selective A2BR antagonists, PSB-603 and MRS1706. To clarify this unexpected data, we wondered whether A2BRs could play a functional role on DRGNs. We first demonstrated, by immunocytochemistry, that primary DRGN monoculture expressed A2BRs. Their selective activation by BAY60-6583 enhanced DRGN excitability, as demonstrated by increased action potential firing, decreased rheobase and depolarized resting membrane potential and were prevented by PSB-603. Throughout this A2BR-dependent enhancement of neuronal activity, DRGNs could release factors to facilitate myelination processes. Finally, silencing A2BR in DRGNs alone prevents the increased myelination induced by BAY60-6583 in cocultures. In conclusion, our data suggest a different role of A2BR during oligodendrogliogenesis and myelination, depending on their activation on neurons or oligodendroglial cells.

Enhanced extracellular vesicles mediated uttroside B (Utt-B) delivery to Hepatocellular carcinoma cell: Pharmacokinetics based on PBPK modelling.

Our prior investigation has confirmed that the anti-hepatocellular carcinoma activity of the plant saponin, specifically Uttroside B (Utt-B), derived from the leaves of Solanum nigrum Linn. This study concentrated on formulating a novel biocompatible nanocarrier utilizing Extracellular vesicles (EVs) to enhance the delivery of plant saponin into cells. The physicochemical attributes of Extracellular Vesicles/UttrosideB (EVs/Utt-B) were comprehensively characterized through techniques such as Transmission Electron Microscopy (TEM) and Fourier-transform infrared spectroscopy (FTIR). Despite the promising therapeutic potential of this uttroside B, mechanistic know-how about its entry into cells is still in its infancy. Our research sheds light on the extracellular vesicle-mediated mechanism facilitating the entry of the saponin into cells, a phenomenon confirmed through the use of by confocal microscopy. We further analysed drug-releasing kinetics and simulated the Pharmacokinetics by PBPK modelling. The simulated pharmacokinetics revealed the bioavailability of Uttroside-B in oral administration against intravenous administration.

Neural Tracing Protein-Functionalized Nanoparticles Capable of Fast Retrograde Axonal Transport in Live Neurons.

Neural tracing proteins like horseradish peroxidase-conjugated wheat germ agglutinin (WGA-HRP) can target the central nervous system (CNS) through anatomic retrograde transport without crossing the blood-brain barrier (BBB). Conjugating WGA-HRP to nanoparticles may enable the creation of BBB-bypassing nanomedicine. Microfluidics and two-photon confocal microscopy is applied to screen nanocarriers for transport efficacy and gain mechanistic insights into their interactions with neurons. Protein modification of gold nanoparticles alters their cellular uptake at the axonal terminal and activates fast retrograde transport. Trajectory analysis of individual endosomes carrying the nanoparticles reveals a run-and-pause pattern along the axon with endosomes carrying WGA-HRP-conjugated gold nanoparticles exhibiting longer run duration and faster instantaneous velocity than those carrying nonconjugated nanoparticles. The results offer a mechanistic explanation of the different axonal transport dynamics as well as a cell-based functional assay of neuron-targeted nanoparticles with the goal of developing BBB-bypassing nanomedicine for the treatment of nervous system disorders.

Functionalizing Yeast Lipid Droplets as Versatile Biomaterials.

Lipid droplets (LD) are dynamic cellular organelles of ≈1 µm diameter in yeast where a neutral lipid core is surrounded by a phospholipid monolayer and attendant proteins. Beyond the storage of lipids, opportunities for LD engineering remain underdeveloped but they show excellent potential as new biomaterials. In this research, LD from yeast Saccharomyces cerevisiae is engineered to display mCherry fluorescent protein, Halotag ligand binding protein, plasma membrane binding v-SNARE protein, and carbonic anhydrase enzyme via linkage to oleosin, an LD anchoring protein. Each protein-oleosin fusion is coded via a single gene construct. The expressed fusion proteins are specifically displayed on LD and their functions can be assessed within cells by fluorescence confocal microscopy, TEM, and as isolated materials via AFM, flow cytometry, spectrophotometry, and by enzyme activity assay. LD isolated from the cell are shown to be robust and stabilize proteins anchored into them. These engineered LD function as reporters, bind specific ligands, guide LD and their attendant proteins into union with the plasma membrane, and catalyze reactions. Here, engineered LD functions are extended well beyond traditional lipid storage toward new material applications aided by a versatile oleosin platform anchored into LD and displaying linked proteins.

Shedding Light on the D1 -Like Receptors: A Fluorescence-Based Toolbox for Visualization of the D1 and D5 Receptors.

Dopamine D1 -like receptors are the most abundant type of dopamine receptors in the central nervous system and, even after decades of discovery, still highly interesting for the study of neurological diseases. We herein describe the synthesis of a new set of fluorescent ligands, structurally derived from D1 R antagonist SCH-23390 and labeled with two different fluorescent dyes, as tool compounds for the visualization of D1 -like receptors. Pharmacological characterization in radioligand binding studies identified UR-NR435 (25) as a high-affinity ligand for D1 -like receptors (pKi (D1 R)=8.34, pKi (D5 R)=7.62) with excellent selectivity towards D2 -like receptors. Compound 25 proved to be a neutral antagonist at the D1 R and D5 R in a Gs heterotrimer dissociation assay, an important feature to avoid receptor internalization and degradation when working with whole cells. The neutral antagonist 25 displayed rapid association and complete dissociation to the D1 R in kinetic binding studies using confocal microscopy verifying its applicability for fluorescence microscopy. Moreover, molecular brightness studies determined a single-digit nanomolar binding affinity of the ligand, which was in good agreement with radioligand binding data. For this reason, this fluorescent ligand is a useful tool for a sophisticated characterization of native D1 receptors in a variety of experimental setups.

Fluorescent Tools for the Imaging of Dopamine D2 -Like Receptors.

The family of dopamine D2 -like receptors represents an interesting target for a variety of neurological diseases, e. g. Parkinson's disease (PD), addiction, or schizophrenia. In this study we describe the synthesis of a new set of fluorescent ligands as tools for visualization of dopamine D2 -like receptors. Pharmacological characterization in radioligand binding studies identified UR-MN212 (20) as a high-affinity ligand for D2 -like receptors (pKi (D2long R)=8.24, pKi (D3 R)=8.58, pKi (D4 R)=7.78) with decent selectivity towards D1 -like receptors. Compound 20 is a neutral antagonist in a Go1 activation assay at the D2long R, D3 R, and D4 R, which is an important feature for studies using whole cells. The neutral antagonist 20, equipped with a 5-TAMRA dye, displayed rapid association to the D2long R in binding studies using confocal microscopy demonstrating its suitability for fluorescence microscopy. Furthermore, in molecular brightness studies, the ligand's binding affinity could be determined in a single-digit nanomolar range that was in good agreement with radioligand binding data. Therefore, the fluorescent compound can be used for quantitative characterization of native D2 -like receptors in a broad variety of experimental setups.

Fluorescence Confocal Microscopy Can Accelerate Diagnosis of Cervical Lymphadenopathy.

Fluorescence confocal microscopy (FCM) is an optical technique that uses laser light sources of different wavelengths to generate real-time images of fresh, unfixed tissue specimens. Unlike conventional histologic evaluation methods, FCM is able to assess fresh tissue samples without the associated cryo artifacts typically observed after frozen sectioning. The purpose of this study was to evaluate the utility of FCM imaging in the differential diagnosis of cervical lymphadenopathy. Twenty-two cervical lymph node specimens from patients with lymphadenopathy of unknown origin were imaged by FCM. Two pathologists independently evaluated the scans for suspicion of malignancy and preliminary diagnosis. Malignancy was reliably excluded or confirmed by both pathologists with a sensitivity of 90.9% for pathologist 1 and 100% for pathologist 2. The specificity was 100% for both pathologists. For the preliminary diagnosis, almost perfect agreement with the final diagnosis was observed for both pathologists (κ = 0.94 for pathologist 1 and κ = 1.00 for pathologist 2). This is the first study to investigate lymph node specimens with different diagnoses, including lymphoma, using FCM. Our results indicate that differential diagnosis of lymph node specimens is feasible in FCM images, thus encouraging further exploration of FCM imaging in lymph node specimens to accelerate diagnosis and open the possibility of digitizing diagnosis on fresh, unfixed tissue.