Single molecule fluorescence: from physical fascination to biological relevance

Confocal fluorescence microscopy is particularly well-known from the beautiful\ud images that have been obtained with this technique from cells. Several cellular\ud components could be nicely visualized simultaneously by staining them with\ud different fluorophores. Not only for ensemble applications but also in single\ud molecule research confocal fluorescence microscopy became a popular technique.\ud In this thesis the possibilities are shown to study a complicated biological process, which is Nucleotide Excision Repair (NER), on the single molecule level with confocal fluorescence microscopy. In chapter one an introduction is given in single molecule fluorescence, properties of fluorescent labels and immobilization methods. Also the NER process is described and the configuration of the confocal fluorescence microscope used

Evidence for Hoogsteen GC Base Pairs in the Proton-Induced Transition from Right-Handed to Left-Handed Poly(dG-dC) . Poly(dG-dC)

The structure of double-helical poly(dG-dC)·poly(dG-dC) is investigated at various pH values with Raman spectroscopy, absorption spectroscopy, and circular dichroism. A comparison is made between the B-form with Watson−Crick base pairing at 1 mM [Na+] and pH 7.2, the Z-form with Watson−Crick base pairing at 4 M [Na+] and pH 7.2, and a different structure at 1 mM [Na+] and pH 4.5 as well as at 150 mM [Na+] and pH 3.1. The CD spectrum of poly(dG-dC)·poly(dG-dC) under the latter conditions does not show a negative band at 290 nm. The structure is a double-helical structure different from the B-form and the Z-form according to circular dichroism, Raman, and absorption spectroscopic studies. The Raman spectra evidence that the structure contains Hoogsteen base pairing. This can be accommodated in the double helix when the cytosine group is protonated and the sugar−guanine conformer has adopted a C2‘-endo/syn conformation. It is shown that this antiparallel-stranded Hoogsteen base paired structure can be maintained under varying conditions, balancing the decrease in pH with an increased salt concentration. It is further concluded that the proton-induced transition from a Watson−Crick to a Hoogsteen base pair is aided by a decrease of [Na+] at pH 4.5 and occurs prior to a conversion from a right-handed helix to a left-handed heli

Occurrence and a possible mechanism of penetration of natural killer cells into k562 target cells during the cytotoxic interaction

The cytotoxic interaction between cloned human Natural Killer (NK) cells and K562 target cells was studied using confocal laser scanning microscopy (CLSM) and conventional fluorescence microscopy. We observed, using fixed as well as living cells, the occurrence of (pseudo)emperipolesis during the interaction. About 30% of conjugated NK cells penetrated, partly or completely, into the target cells (in-conjugation). Virtually all in-conjugated target cells exhibited polymerized actin. Killer cells of in-conjugates were frequently seen approaching the target cell nucleus or aligning along it. If the cytotoxic process was inhibited by the absence of calcium neither actin polymerization nor in-conjugation were observed. A kinetic study showed that in-conjugation starts somewhat later than actin polymerization but still within a few minutes after addition of calcium to conjugates previously formed in the absence of calcium. The presence of cytochalasin D (an inhibitor of actin polymerization) completely inhibited in-conjugation and partly reduced the cytotoxic activity. Zinc ions (endonuclease inhibition) inhibited in-conjugation and decreased the total number of target cells with polymerized actin in a concentration dependent manner. Cytotoxic activity was also reduced but not as efficiently as in-conjugation. \ud Our study demonstrates that in-conjugation represents a significant fraction of the cytotoxic interaction. The results indicate that it may be a consequence of an actin polymerization and endonuclease activity dependent part of a cytotoxic mechanism. \u

SARS-CoV-2 N-protein induces the formation of composite α-synuclein/N-protein fibrils that transform into a strain of α-synuclein fibrils

The presence of deposits of alpha-synuclein (αS) fibrils in the cells of the brain is a hallmark of several α-synucleinopathies, including Parkinson's disease. As most disease cases are not familial, it is likely that external factors play a role in the disease onset. One of the external factors that may influence the disease onset is viral infection. It has recently been shown in in vitro assays that in the presence of SARS-Cov-2 N-protein, αS fibril formation is faster and proceeds in an unusual two-step aggregation process. Here, we show that faster fibril formation is not due to the SARS-CoV-2 N-protein-catalysed formation of an aggregation-prone nucleus. Instead, aggregation starts with the formation of a population of mixed αS/N-protein fibrils with low affinity for αS. Mixed amyloid fibrils, composed of two different proteins, have not been observed before. After the depletion of N-protein, fibril formation comes to a halt, until a slow transformation into fibrils with characteristics of a pure αS fibril strain occurs. This transformation into a strain of αS fibrils subsequently results in a second phase of fibril growth until a new equilibrium is reached. We hypothesize that this fibril strain transformation may be of relevance in the cell-to-cell spread of the αS pathology and disease onset

Elimination of adsorptive behaviour of biomolecules at the glass-solution interface in fluorescence correlation spectroscopy

We study adsorptive behaviour of biomolecules at the glass-solution interface in fluorescence correlation spectroscopy (FCS), and propose a negatively charged coating to eliminate the adsorption of molecules. In this article, we demonstrate confocal microscopic measurements on Cy3.5-90-mer-ssDNA and Cy3.5-90-bp-dsDNA in different solutions, and use two polymers – poly (acrylic acid, sodium salt) and poly (sodium 4-styrenesulfonate) to produce the negatively charged coating on glass coverslips. This technology enables more stable FCS measurements in extremely low concentration samples and reveals that the adsorptive behaviour of biomolecules is responsible for sudden disappearance of many iomolecules in low concentration solutions