SAMM50 acts with p62 in piecemeal basal- and OXPHOS-induced mitophagy of SAM and MICOS components.

Mitophagy is the degradation of surplus or damaged mitochondria by autophagy. In addition to programmed and stress-induced mitophagy, basal mitophagy processes exert organelle quality control. Here, we show that the sorting and assembly machinery (SAM) complex protein SAMM50 interacts directly with ATG8 family proteins and p62/SQSTM1 to act as a receptor for a basal mitophagy of components of the SAM and mitochondrial contact site and cristae organizing system (MICOS) complexes. SAMM50 regulates mitochondrial architecture by controlling formation and assembly of the MICOS complex decisive for normal cristae morphology and exerts quality control of MICOS components. To this end, SAMM50 recruits ATG8 family proteins through a canonical LIR motif and interacts with p62/SQSTM1 to mediate basal mitophagy of SAM and MICOS components. Upon metabolic switch to oxidative phosphorylation, SAMM50 and p62 cooperate to mediate efficient mitophagy.

Multiscale and Multimodal Optical Imaging of the Ultrastructure of Human Liver Biopsies.

The liver as the largest organ in the human body is composed of a complex macroscopic and microscopic architecture that supports its indispensable function to maintain physiological homeostasis. Optical imaging of the human liver is particularly challenging because of the need to cover length scales across 7 orders of magnitude (from the centimeter scale to the nanometer scale) in order to fully assess the ultrastructure of the entire organ down to the subcellular scale and probe its physiological function. This task becomes even more challenging the deeper within the organ one hopes to image, because of the strong absorption and scattering of visible light by the liver. Here, we demonstrate how optical imaging methods utilizing highly specific fluorescent labels, as well as label-free optical methods can seamlessly cover this entire size range in excised, fixed human liver tissue and we exemplify this by reconstructing the biliary tree in three-dimensional space. Imaging of tissue beyond approximately 0.5 mm length requires optical clearing of the human liver. We present the successful use of optical projection tomography and light-sheet fluorescence microscopy to derive information about the liver architecture on the millimeter scale. The intermediate size range is covered using label-free structural and chemically sensitive methods, such as second harmonic generation and coherent anti-Stokes Raman scattering microscopy. Laser-scanning confocal microscopy extends the resolution to the nanoscale, allowing us to ultimately image individual liver sinusoidal endothelial cells and their fenestrations by super-resolution structured illumination microscopy. This allowed us to visualize the human hepatobiliary system in 3D down to the cellular level, which indicates that reticular biliary networks communicate with portal bile ducts via single or a few ductuli. Non-linear optical microscopy enabled us to identify fibrotic regions extending from the portal field to the parenchyma, along with microvesicular steatosis in liver biopsies from an older patient. Lastly, super-resolution microscopy allowed us to visualize and determine the size distribution of fenestrations in human liver sinusoidal endothelial cells for the first time under aqueous conditions. Thus, this proof-of-concept study allows us to demonstrate, how, in combination, these techniques open up a new chapter in liver biopsy analysis.

Photonic-chip assisted correlative light and electron microscopy.

Correlative light and electron microscopy (CLEM) unifies the versatility of light microscopy (LM) with the high resolution of electron microscopy (EM), allowing one to zoom into the complex organization of cells. Here, we introduce photonic chip assisted CLEM, enabling multi-modal total internal reflection fluorescence (TIRF) microscopy over large field of view and high precision localization of the target area of interest within EM. The photonic chips are used as a substrate to hold, to illuminate and to provide landmarking of the sample through specially designed grid-like numbering systems. Using this approach, we demonstrate its applicability for tracking the area of interest, imaging the three-dimensional (3D) structural organization of nano-sized morphological features on liver sinusoidal endothelial cells such as fenestrations (trans-cytoplasmic nanopores), and correlating specific endo-lysosomal compartments with its cargo protein upon endocytosis.

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip.

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 µm x 500 µm region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Quantitative phase microscopy of red blood cells during planar trapping and propulsion.

Red blood cells (RBCs) have the ability to undergo morphological deformations during microcirculation, such as changes in surface area, volume and sphericity. Optical waveguide trapping is suitable for trapping, propelling and deforming large cell populations along the length of the waveguide. Bright field microscopy employed with waveguide trapping does not provide quantitative information about structural changes. Here, we have combined quantitative phase microscopy and waveguide trapping techniques to study changes in RBC morphology during planar trapping and transportation. By using interference microscopy, time-lapsed interferometric images of trapped RBCs were recorded in real-time and subsequently utilized to reconstruct optical phase maps. Quantification of the phase differences before and after trapping enabled study of the mechanical effects during planar trapping. During planar trapping, a decrease in the maximum phase values, an increase in the surface area and a decrease in the volume and sphericity of RBCs were observed. QPM was used to analyze the phase values for two specific regions within RBCs: the annular rim and the central donut. The phase value of the annular rim decreases whereas it increases for the central donut during planar trapping. These changes correspond to a redistribution of cytosol inside the RBC during planar trapping and transportation.

FITC Conjugation Markedly Enhances Hepatic Clearance of N-Formyl Peptides.

In both septic and aseptic inflammation, N-formyl peptides may enter the circulation and induce a systemic inflammatory response syndrome similar to that observed during septic shock. The inflammatory response is brought about by the binding of N-formyl peptide to formyl peptide receptors (FPRs), specific signaling receptors expressed on myeloid as well as non-myeloid cells involved in the inflammatory process. N-formyl peptides conjugated with fluorochromes, such as fluorescein isothiocyanate (FITC) are increasingly experimentally used to identify tissues involved in inflammation. Hypothesizing that the process of FITC-conjugation may transfer formyl peptide to a ligand that is efficiently cleared from the circulation by the natural powerful hepatic scavenging regime we studied the biodistribution of intravenously administered FITC-fNLPNTL (Fluorescein-isothiocyanate- N-Formyl-Nle-Leu-Phe-Nle-Tyr-Lys) in mice. Our findings can be summarized as follows: i) In contrast to unconjugated fNLPNTL, FITC-fNLPNTL was rapidly taken up in the liver; ii) Mouse and human liver sinusoidal endothelial cells (LSECs) and hepatocytes express formyl peptide receptor 1 (FRP1) on both mRNA (PCR) and protein (Western blot) levels; iii) Immunohistochemistry showed that mouse and human liver sections expressed FRP1 in LSECs and hepatocytes; and iv) Uptake of FITC-fNLPNTL could be largely blocked in mouse and human hepatocytes by surplus-unconjugated fNLPNTL, thereby suggesting that the hepatocytes in both species recognized FITC-fNLPNTL and fNLPNTL as indistinguishable ligands. This was in contrast to the mouse and human LSECs, in which the uptake of FITC-fNLPNTL was mediated by both FRP1 and a scavenger receptor, specifically expressed on LSECs. Based on these results we conclude that a significant proportion of FITC-fNLPNTL is taken up in LSECs via a scavenger receptor naturally expressed in these cells. This calls for great caution when using FITC-fNLPNTL and other chromogen-conjugated formyl peptides as a probe to identify cells in a liver engaged in inflammation. Moreover, our finding emphasizes the role of the liver as an important neutralizer of otherwise strong inflammatory signals such as formyl peptides.

In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.

In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

Rat liver sinusoidal endothelial cells (LSECs) express functional low density lipoprotein receptor-related protein-1 (LRP-1).

BACKGROUND & AIMS: The low density lipoprotein receptor-related protein-1 (LRP-1) is a large, multifunctional endocytic receptor from the LDL receptor family, highly expressed in liver parenchymal cells (PCs), neurons, activated astrocytes, and fibroblasts. The aim of the study was to investigate if liver sinusoidal endothelial cells (LSECs), highly specialized scavenger cells, express LRP-1. METHODS: To address this question, experiments were performed in vivo and in vitro to determine if receptor associated protein (RAP) and trypsin-activated α(2)-macroglobulin (α(2)M∗) were endocytosed in LSECs. RESULTS: Both ligands were cleared from the circulation mainly by the liver. Hepatocellular distribution of intravenously administered ligands, assessed after magnetic bead cell separation using LSEC- and KC-specific antibodies, showed that PCs contained 93% and 82% of liver-associated (125)I-RAP and (125)I-α(2)M∗, whereas 5% and 11% were associated with LSECs. Uptake of RAP and α(2)M∗ in the different liver cell population in vitro was specific and followed by degradation. The uptake of (125)I-RAP was not inhibited by ligands to known endocytosis receptors in LSECs, while uptake of (125)I-α(2)M∗ was significantly inhibited by RAP, suggesting the involvement of LRP-1. Immunofluorescence using LRP-1 antibody showed positive staining in LSECs. Ligand blot analyses using total cell proteins and (125)I-RAP followed by mass spectrometry further confirmed and identified LRP-1 in LSECs. CONCLUSIONS: LSECs express functional LRP-1. An important implication of our findings is that LSECs contribute to the rapid removal of blood borne ligands for LRP-1 and may thus play a role in lipid homeostasis.

Liver sinusoidal endothelial cells are the principal site for elimination of unfractionated heparin from the circulation.

The mechanism of elimination of blood borne heparin was studied. To this end unfractionated heparin (UFH) was tagged with FITC, which served as both a visual marker and a site of labeling with (125)I-iodine. UFH labeled in this manner did not alter the anticoagulant activity or binding specificity of the glycosaminoglycan. Labeled heparin administered intravenously to rats (0.1 IU/kg) had a circulatory t(1/2) of 1.7 min, which was increased to 16 min upon coinjection with unlabeled UFH (100 IU/kg). At 15 min after injection, 71% of recovered radioactivity was found in liver. Liver cell separation revealed the following relative uptake capacity, expressed per cell: liver sinusoidal endothelial cell (LSEC)-parenchymal cell-Kupffer cell = 15:3.6:1. Fluorescence microscopy on liver sections showed accumulation of FITC-UFH only in cells lining the liver sinusoids. No fluorescence was detected in parenchymal cells or endothelial cells lining the central vein. Fluorescence microscopy of cultured LSECs following binding of FITC-UFH at 4 degrees C and chasing at 37 degrees C, showed accumulation of the probe in vesicles located at the periphery of the cells after 10 min, with transfer to large, evenly stained vesicles in the perinuclear region after 2 h. Immunogold electron microscopy of LSECs to probe the presence of FITC following injection of FITC-UFH along with BSA-gold to mark lysosomes demonstrated colocalization of the probe with the gold particles in the lysosomal compartment. Receptor-ligand competition experiments in primary cultures of LSECs indicated the presence of a specific heparin receptor, functionally distinct from the hyaluronan/scavenger receptor (Stabilin2). The results suggest a major role for LSECs in heparin elimination.