title: Excluded-volume effects in living cells.
abstract: Biomolecules evolve and function in densely crowded and highly heterogeneous cellular environments. Such conditions are often mimicked in the test tube by the addition of artificial macromolecular crowding agents. Still, it is unclear if such cosolutes indeed reflect the physicochemical properties of the cellular environment as the in-cell crowding effect has not yet been quantified. We have developed a macromolecular crowding sensor based on a FRET-labeled polymer to probe the macromolecular crowding effect inside single living cells. Surprisingly, we find that excluded-volume effects, although observed in the presence of artificial crowding agents, do not lead to a compression of the sensor in the cell. The average conformation of the sensor is similar to that in aqueous buffer solution and cell lysate. However, the in-cell crowding effect is distributed heterogeneously and changes significantly upon cell stress. We present a tool to systematically study the in-cell crowding effect as a modulator of biomolecular reactions.
authors.affiliation: Department of Physical Chemistry II, Ruhr University Bochum, Universitätsstrasse 150, 44801 Bochum (Germany).
authors.forename: David
authors.initials: D
authors.lastname: Gnutt
authors.forename: Mimi
authors.initials: M
authors.lastname: Gao
authors.forename: Oliver
authors.initials: O
authors.lastname: Brylski
authors.forename: Matthias
authors.initials: M
authors.lastname: Heyden
authors.forename: Simon
authors.initials: S
authors.lastname: Ebbinghaus
mesh_terms: D015374:Biosensing Techniques; D005452:Fluoresceins / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D011092:Polyethylene Glycols / Q000737:chemistry; D011108:Polymers / Q000737:chemistry*
chemical_list: C585059:Atto 488 maleimide; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011108:Polymers; D011092:Polyethylene Glycols
keywords: FRET; biophysics; biosensors; excluded-volume effect; macromolecular crowding