Membrane Tension Regulation is Required for Wound Repair.

Disruptions of the eukaryotic plasma membrane due to chemical and mechanical challenges are frequent and detrimental and thus need to be repaired to maintain proper cell function and avoid cell death. However, the cellular mechanisms involved in wound resealing and restoration of homeostasis are diverse and contended. Here, it is shown that clathrin-mediated endocytosis is induced at later stages of plasma membrane wound repair following the actual resealing of the wound. This compensatory endocytosis occurs near the wound, predominantly at sites of previous early endosome exocytosis which is required in the initial stage of membrane resealing, suggesting a spatio-temporal co-ordination of exo- and endocytosis during wound repair. Using cytoskeletal alterations and modulations of membrane tension and membrane area, membrane tension is identified as a major regulator of the wounding-associated exo- and endocytic events that mediate efficient wound repair. Thus, membrane tension changes are a universal trigger for plasma membrane wound repair modulating the exocytosis of early endosomes required for resealing and subsequent clathrin-mediated endocytosis acting at later stages to restore cell homeostasis and function.

Introducing FISCAS, a Tool for the Effective Generation of Single Cell MALDI-MSI Data.

We introduce Fluorescence Integrated Single-Cell Analysis Script (FISCAS), which combines fluorescence microscopy with MALDI-MSI to streamline single-cell analysis. FISCAS enables automated selection of tight measurement regions, thereby reducing the acquisition of off-target pixels, and makes use of established algorithms for cell segmentation and coregistration to rapidly compile single-cell spectra. MALDI-compatible staining of membranes, nuclei, and lipid droplets allows the collection of fluorescence data prior to the MALDI-MSI measurement on a timsTOF fleX MALDI-2. Usefulness of the software is demonstrated by the example of THP-1 cells during stimulated differentiation into macrophages at different time points. In this proof-of-principle study, FISCAS was used to automatically generate single-cell mass spectra along with a wide range of morphometric parameters for a total number of roughly 1300 cells collected at 24, 48, and 72 h after the onset of stimulation. Data analysis of the combined morphometric and single-cell mass spectrometry data shows significant molecular heterogeneity within the cell population at each time point, indicating an independent differentiation of each individual cell rather than a synchronized mechanism. Here, the grouping of cells based on their molecular phenotype revealed an overall clearer distinction of the different phases of differentiation into macrophages and delivered an increased number of lipid signals as possible markers compared with traditional bulk analysis. Utilizing the linkage between mass spectrometric data and fluorescence microscopy confirmed the expected positive correlation between lipid droplet staining and the overall signal for triacylglyceride (TG), demonstrating the usefulness of this multimodal approach.

Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner.

Multiple factors are required to form functional lymphatic vessels. Here, we uncover an essential role for the secreted protein Svep1 and the transmembrane receptor Tie1 during the development of subpopulations of the zebrafish facial lymphatic network. This specific aspect of the facial network forms independently of Vascular endothelial growth factor C (Vegfc) signalling, which otherwise is the most prominent signalling axis in all other lymphatic beds. Additionally, we find that multiple specific and newly uncovered phenotypic hallmarks of svep1 mutants are also present in tie1, but not in tie2 or vegfc mutants. These phenotypes are observed in the lymphatic vasculature of both head and trunk, as well as in the development of the dorsal longitudinal anastomotic vessel under reduced flow conditions. Therefore, our study demonstrates an important function for Tie1 signalling during lymphangiogenesis as well as blood vessel development in zebrafish. Furthermore, we show genetic interaction between svep1 and tie1 in vivo, during early steps of lymphangiogenesis, and demonstrate that zebrafish as well as human Svep1/SVEP1 protein bind to the respective Tie1/TIE1 receptors in vitro. Since compound heterozygous mutations for SVEP1 and TIE2 have recently been reported in human glaucoma patients, our data have clinical relevance in demonstrating a role for SVEP1 in TIE signalling in an in vivo setting.

Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy.

Three-photon excitation has recently been demonstrated as an effective method to perform intravital microscopy in deep, previously inaccessible regions of the mouse brain. The applicability of 3-photon excitation for deep imaging of other, more heterogeneous tissue types has been much less explored. In this work, we analyze the benefit of high-pulse-energy 1 MHz pulse-repetition-rate infrared excitation near 1300 and 1700 nm for in-depth imaging of tumorous and bone tissue. We show that this excitation regime provides a more than 2-fold increased imaging depth in tumor and bone tissue compared to the illumination conditions commonly used in 2-photon excitation, due to improved excitation confinement and reduced scattering. We also show that simultaneous 3- and 4-photon processes can be effectively induced with a single laser line, enabling the combined detection of blue to far-red fluorescence together with second and third harmonic generation without chromatic aberration, at excitation intensities compatible with live tissue imaging. Finally, we analyze photoperturbation thresholds in this excitation regime and derive setpoints for safe cell imaging. Together, these results indicate that infrared high-pulse-energy low-repetition-rate excitation opens novel perspectives for intravital deep-tissue microscopy of multiple parameters in strongly scattering tissues and organs.

Sialic Acid Blockade Suppresses Tumor Growth by Enhancing T-cell-Mediated Tumor Immunity.

Sialic acid sugars on the surface of cancer cells have emerged as potent immune modulators that contribute to the immunosuppressive microenvironment and tumor immune evasion. However, the mechanisms by which these sugars modulate antitumor immunity as well as therapeutic strategies directed against them are limited. Here we report that intratumoral injections with a sialic acid mimetic Ac53FaxNeu5Ac block tumor sialic acid expression in vivo and suppress tumor growth in multiple tumor models. Sialic acid blockade had a major impact on the immune cell composition of the tumor, enhancing tumor-infiltrating natural killer cell and CD8+ T-cell numbers while reducing regulatory T-cell and myeloid regulatory cell numbers. Sialic acid blockade enhanced cytotoxic CD8+ T-cell-mediated killing of tumor cells in part by facilitating antigen-specific T-cell-tumor cell clustering. Sialic acid blockade also synergized with adoptive transfer of tumor-specific CD8+ T cells in vivo and enhanced CpG immune adjuvant therapy by increasing dendritic cell activation and subsequent CD8+ T-cell responses. Collectively, these data emphasize the crucial role of sialic acids in tumor immune evasion and provide proof of concept that sialic acid blockade creates an immune-permissive tumor microenvironment for CD8+ T-cell-mediated tumor immunity, either as single treatment or in combination with other immune-based intervention strategies.Significance: Sialic acid sugars function as important modulators of the immunosuppressive tumor microenvironment that limit potent antitumor immunity.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3574/F1.large.jpg Cancer Res; 78(13); 3574-88. ©2018 AACR.