Molecular chaperones aid protein folding in the cell, but their effects on the conformation of the substrate protein have largely eluded experimental investigation. Single‐molecule fluorescence spectroscopy was used to extract structural and dynamic information from a protein–chaperone complex (see figure; yellow: rhodanase, blue: GroEL). This approach will aid in a more physical understanding of the role of cellular factors in protein folding.

The main limiting factor in spatial resolution of localization microscopy is the number of detected photons. Recently we showed that cryogenic measurements improve the photostability of fluorophores, giving access to Angstrom precision in localization of single molecules. Here, we extend this method to colocalize two fluorophores attached to well-defined positions of a double-stranded DNA. By measuring the separations of the fluorophore pairs prepared at different design positions, we verify the feasibility of cryogenic distance measurement with sub-nanometer accuracy. We discuss the important challenges of our method as well as its potential for further improvement and various applications.

Molekulare Chaperone unterstützen die Proteinfaltung in der Zelle, ihr Einfluss auf die Konformation ihrer Substratproteine war bislang aber wenig verstanden. Einzelmolekülfluoreszenzspektroskopie wurde nun eingesetzt, um Informationen über Struktur und Dynamik von Protein‐Chaperon‐Komplexen (siehe Abbildung; gelb: Rhodanese, blau: GroEL) zu erhalten. Dieser Ansatz ebnet den Weg für ein physikalisches Verständnis der Rolle zellulärer Faktoren in der Proteinfaltung.

Blood low molecular weight proteins (LMWPs) are freely filtered in the kidney glomeruli and subsequently reabsorbed via receptor‐mediated endocytosis (RME) by epithelial cells lining the proximal convoluted tubule (PCT). After RME, the destiny of LMWPs that enter the endo‐lysosomal system (ELS) is to either be degraded in lysosomes or recycled to the blood stream via transcytosis. The PCT is classically divided into early and late segments (S1 and S2, respectively), based on differences in cellular ultrastructure observed with electron microscopy (EM). We recently found that RME of LMWPs takes place almost entirely in S1 (JASN 2018). The aim of this study was to investigate how the structure of the ELS in S1 cells is adapted to this purpose.To further investigate the reasons for axial differences in LMWP reabsorption capacity, we first performed transmission‐EM of C57Bl/6J‐Rj mouse PCTs using a fixation strategy optimized to preserve the structure of the ELS. We found electron‐transparent small apical vesicles densely packed beneath the apical brush border, which formed a much thicker layer in S1 than in S2. Moreover, S1 cells contained numerous large apical vacuoles (LAVs) heterogeneous in electron density, shape and dimension. Lysosomes were observed as rounded structures that could be easily distinguished from the irregular LAVs in S1. Lysosomes in S1 had a different appearance to those in S2, being much more electron‐dense in the latter.To gain deeper insights into the micro‐anatomical features responsible for the greater capacity of LMWP reabsorption in S1, we performed high resolution 3‐D imaging using focused ion beam‐scanning electron microscopy (FIB‐SEM). Sections were acquired every 5nm through the entire thickness of a single S1 cell. Afterwards, using a machine‐learning based approach (ilastik‐1.3.2), we segmented the different components of the ELS and reconstructed their 3‐D appearance. Using this approach, we found that the vast majority of the electron‐transparent vesicles found underneath the apical brush border actually have direct openings into the tubular lumen, and thus represent apical membrane invaginations. LAVs, which are about 1.5μm in diameter, exhibited complex and irregular 3‐D structures, suggesting they are highly dynamic, In contrast, lysosomes were spherical in shape and ranged from 500nm to 1μm in diameter. Finally, we observed a few electron‐transparent small vesicles in the basal third of the cell, which may represent organelles responsible for transcytosis of internalized cargoes to the basolateral membrane.In summary, 2‐D electron microscopy in C57Bl/6J‐Rj mice revealed micro‐anatomical differences in ELS structure between S1 and S2 PCT cells, which may help to explain differences in endocytic capacity. Furthermore, using 3‐D EM we were able to reconstruct the complex organization of the endocytic machinery of S1 cells and to visualize the connections between its components. Taken together, these findings have important implications for understanding reabsorption and handling of LMWPs in the PCT in vivo.Support or Funding InformationNCCR Kidney. CH and S NSF grant 31003A166507This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.