• Journal of Microbiology
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• 제40권4호
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• pp.319-326
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• 2002

Cellular DNA in prokaryotes is organized in nucleic acid-protein self-assemblies referred to as the nucleoid. The physical forces responsible for its stability inside the poor solvent properties of the cytoplasm and their functional implications are not understood. Studies on the organisation and functioning of the cytosol of cells largely rely on experimental protocols performed in highly dilute solutions using biochemically purified molecules, which is not a reliable substitute for the situation existing in vivo. Our current research interest is focused on the characterization of biological and physical forces determining the compaction and phase separation of DNA in Escherichia coli cytoplasm. We have emphasized the effect of excluded volume in solutions with high macromolecular concentrations (macromolecular crowding) upon self-association patterns of reactions. The prokaryotic cytosol was simulated by addition of inert polymer polyethylene glycol (PEG) (average molecular weight 20000), as an agent which afterwards facilitates the self-association of macromolecules. Fluorescence microscopy was used for direct visualization of nucleoids in intact cells, after staining with DAPI (4',6-diamidino-2-phenylindole dihydrochloride). Addition of the crowding agent PEG 20,000, in increasing concentrations generated progressively enhanced nucleoid compaction, the effect being stronger in the presence of 0.2 M NaCl and 5 mM MgCl$\_$2/. Under these conditions, the nucleoids were compacted to volumes of around 2 ㎛$\^$3/ or comparable sizes with that of living cells.

• 대한의생명과학회지
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• 제18권4호
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• pp.329-337
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• 2012

${\alpha}$-synuclein (${\alpha}$-syn) is known to be implicated in the pathogenesis of Parkinson's disease and transiently bind to biological vesicles. In this study, we examined the effect of molecular crowding on the interaction of ${\alpha}$-syn with biological vesicles by using inert polymers since the environment of proteins in cells are crowded with other macromolecules. The addition of different polymers including polyethylene glycol, dextran, and ficoll enhanced the binding of ${\alpha}$-syn to vesicles in a concentration-dependent manner and the association of ${\alpha}$-syn with vesicle was proportionally augmented by increased expression of ${\alpha}$-syn. However, molecular crowding had a neglectable effect on the vesicle binding of ${\alpha}$-syn mutants (A30P, TG6), which has been reported to show impaired vesicle binding capacity. These results suggest that transient interaction of ${\alpha}$-syn with vesicles occurs more commonly in cells than expected implying interaction with vesicles may be one of the physiological processes in which ${\alpha}$-syn is involved.