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Elucidating the 3D nanoscale structure of tissues and cells is essential for understanding the complexity of biological processes. Electron microscopy (EM) offers the resolution needed for reliable interpretation, but the limited throughput of electron microscopes has hindered its ability to effectively image large volumes. We report a workflow for volume EM with FAST-EM, a novel multibeam scanning transmission electron microscope that speeds up acquisition by scanning the sample in parallel with 64 electron beams. FAST-EM makes use of optical detection to separate the signals of the individual beams. The acquisition and 3D reconstruction of ultrastructural data from multiple biological samples is demonstrated. The results show that the workflow is capable of producing large reconstructed volumes with high resolution and contrast to address biological research questions within feasible acquisition time frames.

We report the evolution of mScarlet3, a cysteine-free monomeric red fluorescent protein with fast and complete maturation, as well as record brightness, quantum yield (75%) and fluorescence lifetime (4.0 ns). The mScarlet3 crystal structure reveals a barrel rigidified at one of its heads by a large hydrophobic patch of internal residues. mScarlet3 behaves well as a fusion tag, displays no apparent cytotoxicity and it surpasses existing red fluorescent proteins as a Förster resonance energy transfer acceptor and as a reporter in transient expression systems.

Biogenesis of nuclear pore complexes (NPCs) includes the formation of the permeability barrier composed of phenylalanine-glycine-rich nucleoporins (FG-Nups) that regulate the selective passage of biomolecules across the nuclear envelope. The FG-Nups are intrinsically disordered and prone to liquid–liquid phase separation and aggregation when isolated. How FG-Nups are protected from making inappropriate interactions during NPC biogenesis is not fully understood. Here we find that DNAJB6, a molecular chaperone of the heat shock protein network, forms foci in close proximity to NPCs. The number of these foci decreases upon removal of proteins involved in the early steps of interphase NPC biogenesis. Conversely, when this process is stalled in the last steps, the number of DNAJB6-containing foci increases and these foci are identified as herniations at the nuclear envelope. Immunoelectron tomography shows that DNAJB6 localizes inside the lumen of the herniations arising at NPC biogenesis intermediates. Loss of DNAJB6 results in the accumulation of cytosolic annulate lamellae, which are structures containing partly assembled NPCs, a feature associated with disturbances in NPC biogenesis. We find that DNAJB6 binds to FG-Nups and can prevent the aggregation of the FG region of several FG-Nups in cells and in vitro. Together, our data show that the molecular chaperone DNAJB6 provides quality control during NPC biogenesis and is involved in the surveillance of native intrinsically disordered FG-Nups.

Extracellular vesicles (EVs), such as exosomes, can mediate long-distance communication between cells by delivering biomolecular cargo. It is speculated that EVs undergo back-fusion at multivesicular bodies (MVBs) in recipient cells to release their functional cargo. However, direct evidence is lacking. Tracing the cellular uptake of EVs with high resolution as well as acquiring direct evidence for the release of EV cargo is challenging mainly because of technical limitations. Here, we developed an analytical methodology, combining state-of-the-art molecular tools and correlative light and electron microscopy, to identify the intracellular site for EV cargo release. GFP was loaded inside EVs through the expression of GFP-CD63, a fusion of GFP to the cytosolic tail of CD63, in EV producer cells. In addition, we genetically engineered a cell line which expresses anti-GFP fluobody that specifically recognizes the EV cargo (GFP). Incubation of anti-GFP fluobody-expressing cells with GFP-CD63 EVs resulted in the formation of fluobody punctae, designating cytosolic exposure of GFP. Endosomal damage was not observed in EV acceptor cells. Ultrastructural analysis of the underlying structures at GFP/fluobody double-positive punctae demonstrated that EV cargo release occurs from endosomes/lysosomes. Finally, we show that neutralization of endosomal pH and cholesterol accumulation in endosomes leads to blockage of EV cargo exposure. In conclusion, we report that a fraction of internalized EVs fuse with the limiting membrane of endosomes/lysosomes in an acidification-dependent manner, which results in EV cargo exposure to the cell cytosol.

Congenital diarrheal disorders are rare, often fatal, diseases that are difficult to diagnose (often requiring biopsies) and that manifest in the first few weeks of life as chronic diarrhea and the malabsorption of nutrients. The etiology of congenital diarrheal disorders is diverse, but several are associated with defects in the predominant intestinal epithelial cell type, enterocytes. These particular congenital diarrheal disorders (CDDENT) include microvillus inclusion disease and congenital tufting enteropathy, and can feature in other diseases, such as hemophagocytic lymphohistiocytosis type 5 and trichohepatoenteric syndrome. Treatment options for most of these disorders are limited and an improved understanding of their molecular bases could help to drive the development of better therapies. Recently, mutations in genes that are involved in normal intestinal epithelial physiology have been associated with different CDDENT. Here, we review recent progress in understanding the cellular mechanisms of CDDENT. We highlight the potential of animal models and patient-specific stem-cell-based organoid cultures, as well as patient registries, to integrate basic and clinical research, with the aim of clarifying the pathogenesis of CDDENT and expediting the discovery of novel therapeutic strategies.

Liver regeneration is stimulated by blood platelets, but the molecular mechanisms involved are largely unexplored. Although platelets are anucleate, they do contain coding or regulatory RNAs that can be functional within the platelet or, after transfer, in other cell types. Here, we show that platelets and platelet-like particles (PLPs) derived from the megakaryoblastic cell line MEG-01 stimulate proliferation of HepG2 cells. Platelets or PLPs were internalized within 1 hour by HepG2 cells and accumulated in the perinuclear region of the hepatocyte. Platelet internalization also occurred following a partial hepatectomy in mice. Annexin A5 blocked platelet internalization and HepG2 proliferation. We labeled total RNA of MEG-01 cells by incorporation of 5-ethynyluridine (EU) and added EU-labeled PLPs to HepG2 cells. PLP-derived RNA was detected in the cytoplasm of the HepG2 cell. We next generated PLPs containing green fluorescent protein (GFP)-tagged actin messenger RNA. PLPs did not synthesize GFP, but in coculture with HepG2 cells, significant GFP protein synthesis was demonstrated. RNA-degrading enzymes partly blocked the stimulating effect of platelets on hepatocyte proliferation. Thus, platelets stimulate hepatocyte proliferation via a mechanism that is dependent on platelet internalization by hepatocytes followed by functional transfer of RNA stored in the anucleate platelet. This mechanism may contribute to platelet-mediated liver regeneration.

Many brain diseases involve activation of resident and peripheral immune cells to clear damaged and dying neurons. Which immune cells respond in what way to cues related to brain disease, however, remains poorly understood. To elucidate these in vivo immunological events in response to brain cell death we used genetically targeted cell ablation in zebrafish. Using intravital microscopy and large-scale electron microscopy, we defined the kinetics and nature of immune responses immediately following injury. Initially, clearance of dead cells occurs by mononuclear phagocytes, including resident microglia and macrophages of peripheral origin, whereas amoeboid microglia are exclusively involved at a later stage. Granulocytes, on the other hand, do not migrate towards the injury. Remarkably, following clearance, phagocyte numbers decrease, partly by phagocyte cell death and subsequent engulfment of phagocyte corpses by microglia. Here, we identify differential temporal involvement of microglia and peripheral macrophages in clearance of dead cells in the brain, revealing the chronological sequence of events in neuroinflammatory resolution. Remarkably, recruited phagocytes undergo cell death and are engulfed by microglia. Because adult zebrafish treated at the larval stage lack signs of pathology, it is likely that this mode of resolving immune responses in brain contributes to full tissue recovery. Therefore, these findings suggest that control of such immune cell behavior could benefit recovery from neuronal damage.

To evaluate the effect of soluble CD14 (sCD14) on human neutrophil response to lipopolysaccharide (LPS), we developed an LPS‐priming assay that measures the chemiluminescence response to N‐formyl‐methionyl‐leucyl‐phenylalanine stimulation. Priming by 1 ng/mL rough LPS occurred in the presence of either serum or recombinant LPS‐binding protein (LBP) only. Priming was completely CD14‐dependent because preincubation of the neutrophils with an anti‐CD14 monoclonal antibody prevented priming. We hypothesize that sCD14 enhances LPS response in neutrophils, but this response is not as effective as LPS response via membrane CD14 (mCD14). In our experiments sCD14 is present in an excess compared with mCD14. Priming of neutrophils occurs with low LBP, supposedly via sCD14‐LPS complexes. With high LBP, addition of sCD14 inhibited LPS‐priming of neutrophils. In that case, LPS may be transported to sCD14, preventing a more effective response via mCD14. In this study we demonstrate that the effect of sCD14 on neutrophil response to LPS is a delicate balance between activation and inhibition depending on concentration of serum or LBP. J. Leukoc. Biol. 61: 173–178; 1997.