VISION - an open-source software for automated multi-dimensional image analysis of cellular biophysics.

Environment-sensitive probes are frequently used in spectral and multi-channel microscopy to study alterations in cell homeostasis. However, the few open-source packages available for processing of spectral images are limited in scope. Here, we present VISION, a stand-alone software based on Python for spectral analysis with improved applicability. In addition to classical intensity-based analysis, our software can batch-process multidimensional images with an advanced single-cell segmentation capability and apply user-defined mathematical operations on spectra to calculate biophysical and metabolic parameters of single cells. VISION allows for 3D and temporal mapping of properties such as membrane fluidity and mitochondrial potential. We demonstrate the broad applicability of VISION by applying it to study the effect of various drugs on cellular biophysical properties. the correlation between membrane fluidity and mitochondrial potential, protein distribution in cell-cell contacts and properties of nanodomains in cell-derived vesicles. Together with the code, we provide a graphical user interface for easy adoption.

Superresolution microscopy localizes endogenous Dvl2 to Wnt signaling-responsive biomolecular condensates.

During organismal development, homeostasis, and disease, Dishevelled (Dvl) proteins act as key signaling factors in beta-catenin-dependent and beta-catenin-independent Wnt pathways. While their importance for signal transmission has been genetically demonstrated in many organisms, our mechanistic understanding is still limited. Previous studies using overexpressed proteins showed Dvl localization to large, punctate-like cytoplasmic structures that are dependent on its DIX domain. To study Dvl's role in Wnt signaling, we genome engineered an endogenously expressed Dvl2 protein tagged with an mEos3.2 fluorescent protein for superresolution imaging. First, we demonstrate the functionality and specificity of the fusion protein in beta-catenin-dependent and beta-catenin-independent signaling using multiple independent assays. We performed live-cell imaging of Dvl2 to analyze the dynamic formation of the supramolecular cytoplasmic Dvl2_mEos3.2 condensates. While overexpression of Dvl2_mEos3.2 mimics the previously reported formation of abundant large "puncta," supramolecular condensate formation at physiological protein levels is only observed in a subset of cells with approximately one per cell. We show that, in these condensates, Dvl2 colocalizes with Wnt pathway components at gamma-tubulin and CEP164-positive centrosomal structures and that the localization of Dvl2 to these condensates is Wnt dependent. Single-molecule localization microscopy using photoactivated localization microscopy (PALM) of mEos3.2 in combination with DNA-PAINT demonstrates the organization and repetitive patterns of these condensates in a cell cycle-dependent manner. Our results indicate that the localization of Dvl2 in supramolecular condensates is coordinated dynamically and dependent on cell state and Wnt signaling levels. Our study highlights the formation of endogenous and physiologically regulated biomolecular condensates in the Wnt pathways at single-molecule resolution.

Quantifying Fluorescence Lifetime Responsiveness of Environment-Sensitive Probes for Membrane Fluidity Measurements.

The structural diversity of different lipid species within the membrane defines its biophysical properties such as membrane fluidity, phase transition, curvature, charge distribution, and tension. Environment-sensitive probes, which change their spectral properties in response to their surrounding milieu, have greatly contributed to our understanding of such biophysical properties. To realize the full potential of these probes and avoid misinterpretation of their spectral responses, a detailed investigation of their fluorescence characteristics in different environments is necessary. Here, we examined the fluorescence lifetime of two newly developed membrane order probes, NR12S and NR12A, in response to alterations in their environments such as the degree of lipid saturation, cholesterol content, double bond position and configuration, and phospholipid headgroup. As a comparison, we investigated the lifetime sensitivity of the membrane tension probe Flipper in these environments. Applying fluorescence lifetime imaging microscopy (FLIM) in both model membranes and biological membranes, all probes distinguished membrane phases by lifetime but exhibited different lifetime sensitivities to varying membrane biophysical properties (e.g., cholesterol). While the lifetime of Flipper is particularly sensitive to the membrane cholesterol content, the NR12S and NR12A lifetimes are moderately sensitive to both the cholesterol content and lipid acyl chains. Moreover, all of the probes exhibit longer lifetimes at longer emission wavelengths in membranes of any complexity. This emission wavelength dependency results in varying lifetime resolutions at different spectral regions, which are highly relevant for FLIM data acquisition. Our data provide valuable insights on how to perform FLIM with these probes and highlight both their potential and limitations.

Apoptosis-mediated ADAM10 activation removes a mucin barrier promoting T cell efferocytosis.

Efferocytic clearance of apoptotic cells in general, and T cells in particular, is required for tissue and immune homeostasis. Transmembrane mucins are extended glycoproteins highly expressed in the cell glycocalyx that function as a barrier to phagocytosis. Whether and how mucins may be regulated during cell death to facilitate efferocytic corpse clearance is not well understood. Here we show that normal and transformed human T cells express a subset of mucins which are rapidly and selectively removed from the cell surface during apoptosis. This process is mediated by the ADAM10 sheddase, the activity of which is associated with XKR8-catalyzed flipping of phosphatidylserine to the outer leaflet of the plasma membrane. Mucin clearance enhances uptake of apoptotic T cells by macrophages, confirming mucins as an enzymatically-modulatable barrier to efferocytosis. Together these findings demonstrate a glycocalyx regulatory pathway with implications for therapeutic intervention in the clearance of normal and transformed apoptotic T cells.

Dissecting the mechanisms of environment sensitivity of smart probes for quantitative assessment of membrane properties.

The plasma membrane, as a highly complex cell organelle, serves as a crucial platform for a multitude of cellular processes. Its collective biophysical properties are largely determined by the structural diversity of the different lipid species it accommodates. Therefore, a detailed investigation of biophysical properties of the plasma membrane is of utmost importance for a comprehensive understanding of biological processes occurring therein. During the past two decades, several environment-sensitive probes have been developed and become popular tools to investigate membrane properties. Although these probes are assumed to report on membrane order in similar ways, their individual mechanisms remain to be elucidated. In this study, using model membrane systems, we characterized the probes Pro12A, NR12S and NR12A in depth and examined their sensitivity to parameters with potential biological implications, such as the degree of lipid saturation, double bond position and configuration (cis versus trans), phospholipid headgroup and cholesterol content. Applying spectral imaging together with atomistic molecular dynamics simulations and time-dependent fluorescent shift analyses, we unravelled individual sensitivities of these probes to different biophysical properties, their distinct localizations and specific relaxation processes in membranes. Overall, Pro12A, NR12S and NR12A serve together as a toolbox with a wide range of applications allowing to select the most appropriate probe for each specific research question.

Nuclear IGF1R interact with PCNA to preserve DNA replication after DNA-damage in a variety of human cancers.

Nuclear IGF1R has been linked to poor outcome in cancer. We recently showed that nuclear IGF1R phosphorylates PCNA and increases DNA damage tolerance. In this paper we aimed to describe this mechanism in cancer tissue as well as in cancer cell lines. In situ proximity ligation assay identified frequent IGF1R and PCNA colocalization in many cancer types. IGF1R/PCNA colocalization was more frequently increased in tumor cells than in adjacent normal, and more prominent in areas with dysplasia and invasion. However, the interaction was often lost in tumors with poor response to neoadjuvant treatment and most metastatic lesions. In two independent cohorts of serous ovarian carcinomas and oropharyngeal squamous cell carcinomas, stronger IGF1R/PCNA colocalization was significantly associated with a higher overall survival. Ex vivo irradiation of ovarian cancer tissue acutely induced IGF1R/PCNA colocalization together with γH2AX-foci formations. In vitro, RAD18 mediated mono-ubiquitination of PCNA during replication stress was dependent on IGF1R kinase activity. DNA fiber analysis revealed that IGF1R activation could rescue stalled DNA replication forks, but only in cancer cells with baseline IGF1R/PCNA interaction. We believe that the IGF1R/PCNA interaction is a basic cellular mechanism to increase DNA stress tolerance during proliferation, but that this mechanism is lost with tumor progression in conjunction with accumulated DNA damage and aberrant strategies to tolerate genomic instability. To exploit this mechanism in IGF1R targeted therapy, IGF1R inhibitors should be explored in the context of concomitant induction of DNA replication stress as well as in earlier clinical stages than previously tried.