Self-Assembly of Pseudo-Isocyanine Chloride as a Sensor for Macromolecular Crowding In Vitro and In Vivo.

ABSTRACT

Pseudo-isocyanine chloride (PIC) is a cationic dyestuff that exhibits self-assembly in aqueous solution, promoted either by increasing the PIC concentration or by decreasing the temperature. PIC-aggregates exhibit a characteristic and sharp absorption band as well as a fluorescence band at a wavelength of 573 nm making PIC an interesting candidate to analyze the self-assembly process in various environments. The present work developed PIC-based, synthetic model systems, suitable to investigate how macromolecular crowding influences self-assembly processes. Four synthetic additives were used as potential crowders Triethylene glycol (TEG), polyethylene glycol (PEG), Ficoll 400 as a highly branched polysaccharide, and sucrose corresponding to the monomeric unit of Ficoll. Combined UV/Vis spectroscopy and time-resolved light scattering revealed a strong impact of crowding based on excluded volume effects only for Ficoll 400. Sucrose had hardly any influence on the self-assembly of PIC and PEG and TEG impeded the PIC self-assembly. Development of such a PIC based model system led over to in-cell experiments. HeLa cells were infiltrated with PIC solutions well below the aggregation threshold in the infiltrating solution. In the cellular environment, PIC was exposed to a significant crowding and immediately started to aggregate. As was demonstrated by fluorescence imaging, the extent of aggregation can be modulated by exposing the cells to salt-induced osmotic stress. The results suggest future use of such a system as a sensor for the analysis of in vitro and in vivo crowding effects on self-assembly processes.