Rapamycin-dependent delocalization as a novel tool to reveal protein-protein interactions in plants

Identifying protein-protein interactions (PPI) is crucial to understand any type of biological process. Many PPI tools are available, yet only some function within the context of a plant cell. Narrowing down even further, only few PPI tools allow visualizing higher order interactions. Here, we present a novel and conditional in vivo PPI tool for plant research. Knocksideways in plants (KSP) uses the ability of rapamycin to alter the localization of a bait protein and its interactors via the heterodimerization of FKBP and FRB domains. KSP is inherently free from many limitations, which other PPI systems hold. It is an in vivo tool, it is flexible concerning the orientation of protein tagging as long as this does not interfere with the interaction and it is compatible with a broad range of fluorophores. KSP is also a conditional tool and therefore does not require additional controls. The interactions can be quantified and in high throughput by the scripts that we provide. Finally, we demonstrate that KSP can visualize higher-order interactions. It is therefore a versatile tool, complementing the PPI methods field with unique characteristics and applications

The ORMDL3 asthma susceptibility gene regulates systemic ceramide levels without altering key asthma features in mice

Background: Genome-wide association studies in asthma have repeatedly identified single nucleotide polymorphisms in the ORM (yeast)-like protein isoform 3 (ORMDL3) gene across different populations. Although the ORM homologues in yeast are well-known inhibitors of sphingolipid synthesis, it is still unclear whether and how mammalian ORMDL3 regulates sphingolipid metabolism and whether altered sphingolipid synthesis would be causally related to asthma risk. Objective: We sought to examine the in vivo role of ORMDL3 in sphingolipid metabolism and allergic asthma. Methods: Ormdl3-LacZ reporter mice, gene-deficient Ormdl3(-/-) mice, and overexpressing Ormdl3(Tg/wt) mice were exposed to physiologically relevant aeroallergens, such as house dust mite (HDM) or Alternaria alternata, to induce experimental asthma. Mass spectrometry-based sphingolipidomics were performed, and airway eosinophilia, T(H)2 cytokine production, immunoglobulin synthesis, airway remodeling, and bronchial hyperreactivity were measured. Results: HDM challenge significantly increased levels of total sphingolipids in the lungs of HDM-sensitized mice compared with those in control mice. In Ormdl3(Tg/wt) mice the allergen-induced increase in lung ceramide levels was significantly reduced, whereas total sphingolipid levels were not affected. Conversely, in liver and serum, levels of total sphingolipids, including ceramides, were increased in Ormdl3(-/-) mice, whereas they were decreased in Ormdl3(Tg/wt) mice. This difference was independent of allergen exposure. Despite these changes, all features of asthma were identical between wildtype, Ormdl3(Tg/wt), and Ormdl3(-/-) mice across several models of experimental asthma. Conclusion: ORMDL3 regulates systemic ceramide levels, but genetically interfering with Ormdl3 expression does not result in altered experimental asthma

Osteopontin expression identifies a subset of recruited macrophages distinct from Kupffer cells in the fatty liver

Metabolic-associated fatty liver disease (MAFLD) represents a spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs), are suggested to play important roles in the pathogenesis of MAFLD through their activation, although the exact roles played by these cells remain unclear. Here, we demonstrated that KCs were reduced in MAFLD being replaced by macrophages originating from the bone marrow. Recruited macrophages existed in two subsets with distinct activation states, either closely resembling homeostatic KCs or lipid-associated macrophages (LAMs) from obese adipose tissue. Hepatic LAMs expressed Osteopontin, a biomarker for patients with NASH, linked with the development of fibrosis. Fitting with this, LAMs were found in regions of the liver with reduced numbers of KCs, characterized by increased Desmin expression. Together, our data highlight considerable heterogeneity within the macrophage pool and suggest a need for more specific macrophage targeting strategies in MAFLD

Analyzing protein clusters on the plasma membrane: application of spatial statistical analyses on super-resolution microscopy images

The spatial distribution of proteins within the cell affects their capability to interact with other molecules and directly influences cellular processes and signaling. At the plasma membrane, multiple factors drive protein compartmentalization into specialized functional domains, leading to the formation of clusters in which intermolecule interactions are facilitated. Therefore, quantifying protein distributions is a necessity for understanding their regulation and function. The recent advent of super-resolution microscopy has opened up the possibility of imaging protein distributions at the nanometer scale. In parallel, new spatial analysis methods have been developed to quantify distribution patterns in super-resolution images. In this chapter, we provide an overview of super-resolution microscopy and summarize the factors influencing protein arrangements on the plasma membrane. Finally, we highlight methods for analyzing clusterization of plasma membrane proteins, including examples of their applications.status: publishe

Excess LINC complexes impair brain morphogenesis in a mouse model of recessive TOR1A disease

Heterozygosity for the TOR1A-Δgag mutation causes semi-penetrant childhood-onset dystonia (OMIM #128100). More recently, homozygous TOR1A mutations were shown to cause severe neurological dysfunction in infants. However, there is little known about the recessive cases, including whether existing reports define the full spectrum of recessive TOR1A disease. Here we describe abnormal brain morphogenesis in ∼30% of Tor1a-/- mouse embryos while, in contrast, this is not found in Tor1aΔgag/Δgag mice. The abnormal Tor1a-/- brains contain excess neural tissue, as well as proliferative zone cytoarchitectural defects related to radial glial cell polarity and cytoskeletal organization. In cultured cells torsinA effects the linker of nucleoskeleton and cytoskeleton (LINC) complex that couples the nucleus and cytoskeleton. Here we identify that torsinA loss elevates LINC complex levels in the proliferative zone, and that genetic reduction of LINC complexes prevents abnormal brain morphogenesis in Tor1a-/- embryos. These data show that Tor1a affects radial glial cells via a LINC complex mediated mechanism. They also predict human TOR1A disease will include incompletely penetrant defects in embryonic brain morphogenesis in cases where mutations ablate TOR1A function.status: publishe

3-D reconstructions of intercellular junctions of the intercalated disc and their connection to complexes of the excitation-contraction coupling unit of cardiomyocytes

C

A Label-free Multicolor Optical Surface Tomography (ALMOST) imaging method for nontransparent 3D samples

Abstract Background Current mesoscale 3D imaging techniques are limited to transparent or cleared samples or require the use of X-rays. This is a severe limitation for many research areas, as the 3D color surface morphology of opaque samples—for example, intact adult Drosophila, Xenopus embryos, and other non-transparent samples—cannot be assessed. We have developed “ALMOST,” a novel optical method for 3D surface imaging of reflective opaque objects utilizing an optical projection tomography device in combination with oblique illumination and optical filters. Results As well as demonstrating image formation, we provide background information and explain the reconstruction—and consequent rendering—using a standard filtered back projection algorithm and 3D software. We expanded our approach to fluorescence and multi-channel spectral imaging, validating our results with micro-computed tomography. Different biological and inorganic test samples were used to highlight the versatility of our approach. To further demonstrate the applicability of ALMOST, we explored the muscle-induced form change of the Drosophila larva, imaged adult Drosophila, dynamically visualized the closure of neural folds during neurulation of live Xenopus embryos, and showed the complementarity of our approach by comparison with transmitted light and fluorescence OPT imaging of a Xenopus tadpole. Conclusion Thus, our new modality for spectral/color, macro/mesoscopic 3D imaging can be applied to a variety of model organisms and enables the longitudinal surface dynamics during development to be revealed

A Label-free Multicolor Optical Surface Tomography (ALMOST) imaging method for nontransparent 3D samples

Background Current mesoscale 3D imaging techniques are limited to transparent or cleared samples or require the use of X-rays. This is a severe limitation for many research areas, as the 3D color surface morphology of opaque samples—for example, intact adult Drosophila, Xenopus embryos, and other non-transparent samples—cannot be assessed. We have developed “ALMOST,” a novel optical method for 3D surface imaging of reflective opaque objects utilizing an optical projection tomography device in combination with oblique illumination and optical filters. Results As well as demonstrating image formation, we provide background information and explain the reconstruction—and consequent rendering—using a standard filtered back projection algorithm and 3D software. We expanded our approach to fluorescence and multi-channel spectral imaging, validating our results with micro-computed tomography. Different biological and inorganic test samples were used to highlight the versatility of our approach. To further demonstrate the applicability of ALMOST, we explored the muscle-induced form change of the Drosophila larva, imaged adult Drosophila, dynamically visualized the closure of neural folds during neurulation of live Xenopus embryos, and showed the complementarity of our approach by comparison with transmitted light and fluorescence OPT imaging of a Xenopus tadpole. Conclusion Thus, our new modality for spectral/color, macro/mesoscopic 3D imaging can be applied to a variety of model organisms and enables the longitudinal surface dynamics during development to be revealed.status: Published onlin