Glycation Interferes with the Activity of the Bi-Functional UDP-N-Acetylglucosamine 2-Epimerase/N-Acetyl-mannosamine Kinase (GNE)

Mutations in the gene coding for the bi-functional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of the sialic acid biosynthesis, are responsible for autosomal-recessive GNE myopathy (GNEM). GNEM is an adult-onset disease with a yet unknown exact pathophysiology. Since the protein appears to work adequately for a certain period of time even though the mutation is already present, other effects appear to influence the onset and progression of the disease. In this study, we want to investigate whether the late onset of GNEM is based on an age-related effect, e.g., the accumulation of post-translational modifications (PTMs). Furthermore, we also want to investigate what effect on the enzyme activity such an accumulation would have. We will particularly focus on glycation, which is a PTM through non-enzymatic reactions between the carbonyl groups (e.g., of methylglyoxal (MGO) or glyoxal (GO)) with amino groups of proteins or other biomolecules. It is already known that the levels of both MGO and GO increase with age. For our investigations, we express each domain of the GNE separately, treat them with one of the glycation agents, and determine their activity. We demonstrate that the enzymatic activity of the N-acetylmannosamine kinase (GNE-kinase domain) decreases dramatically after glycation with MGO or GO—with a remaining activity of 13% ± 5% (5 mM MGO) and 22% ± 4% (5 mM GO). Whereas the activity of the UDP-N-acetylglucosamine 2-epimerase (GNE-epimerase domain) is only slightly reduced after glycation—with a remaining activity of 60% ± 8% (5 mM MGO) and 63% ± 5% (5 mM GO).Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)Deutsche ForschungsgemeinschaftPeer Reviewe

Analyse der Plasmamembran-Rauigkeit unterschiedlicher Zelltypen mittels Raster-Ionenleitfähigkeitsmikroskopie (SICM)

In dieser Arbeit wurde ein neues hochauflösendes Raster-Ionenleitfähigkeitsmikroskop (SICMic) charakterisiert. Im Anschluss wurde das SICMic zur Untersuchung der örtlichen Verteilung von Membranstrukturen auf der Zelloberfläche von lebenden HT29-Zellen verwendet. Zur Detektion von Veränderungen in der Nanostruktur wurde eine Rauigkeits-Analyse entwickelt. Dabei konnten signifikante Unterschiede zwischen einzelnen Bereichen der Zelle festgestellt werden. Um den Einfluss der Topographie auf die Diffusion von Molekülen über die Plasmamembran zu untersuchen, wurden im Anschluss Fluoreszenzkorrelationsspektroskopie (FCS)-Messungen in unterschiedlichen Bereichen der Zelle modelliert. Die Rauigkeits-Analyse wurde ebenfalls für die Untersuchung des Einflusses von Wachstumsfaktoren und Nanopartikeln auf die Nanostruktur von Astrozyten und HeLa-Zellen verwendet. Des Weiteren wurde in einem $\it proof-of-principle$-Versuch die Topographie mit der Verteilung eines Zytoskelett-Proteins korreliert

Correlative Stimulated Emission Depletion and Scanning Ion Conductance Microscopy

Correlation microscopy combining fluorescence and scanning probe or electron microscopy is limited to fixed samples due to the sample preparation and nonphysiological imaging conditions required by most probe or electron microscopy techniques. Among the few scanning probe techniques that allow imaging of living cells under physiological conditions, scanning ion conductance microscopy (SICM) has been shown to be the technique that minimizes the impact on the investigated sample. However, combinations of SICM and fluorescence microscopy suffered from the mismatch in resolution due to the limited resolution of conventional light microscopy. In the last years, the diffraction limit of light microscopy has been circumvented by various techniques, one of which is stimulated emission depletion (STED) microscopy. Here, we aimed at demonstrating the combination of STED and SICM. We show that both methods allow recording a living cellular specimen and provide a SICM and STED image of the same sample, which allowed us to correlate the membrane surface topography and the distribution of the cytoskeletal protein actin. Our proof-of-concept study exemplifies the benefit of correlating SICM with a subdiffraction fluorescence method and might form the basis for the development of a combined instrument that would allow the simultaneous recording of subdiffraction fluorescence and topography information

Variations in Plasma Membrane Topography Can Explain Heterogenous Diffusion Coefficients Obtained by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is frequently used to study diffusion in cell membranes, primarily the plasma membrane. The diffusion coefficients reported in the plasma membrane of the same cell type and even within single cells typically display a large spread. We have investigated whether this spread can be explained by variations in membrane topography throughout the cell surface, that changes the amount of membrane in the FCS focal volume at different locations. Using FCS, we found that diffusion of the membrane dye DiI in the apical plasma membrane was consistently faster above the nucleus than above the cytoplasm. Using live cell scanning ion conductance microscopy (SICM) to obtain a topography map of the cell surface, we demonstrate that cell surface roughness is unevenly distributed with the plasma membrane above the nucleus being the smoothest, suggesting that the difference in diffusion observed in FCS is related to membrane topography. FCS modeled on simulated diffusion in cell surfaces obtained by SICM was consistent with the FCS data from live cells and demonstrated that topography variations can cause the appearance of anomalous diffusion in FCS measurements. Furthermore, we found that variations in the amount of the membrane marker DiD, a proxy for the membrane, but not the transmembrane protein TCRζ or the lipid-anchored protein Lck, in the FCS focal volume were related to variations in diffusion times at different positions in the plasma membrane. This relationship was seen at different positions both at the apical cell and basal cell sides. We conclude that it is crucial to consider variations in topography in the interpretation of FCS results from membranes.QC 20201013</p