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

Abstract

Pseudo-isocyanine chloride (PIC) is ac ationic dyestuff that exhibits self-assembly in aqueous solution, promoted either by increasing the PIC concentration or by decreasing the temperature. PIC-aggregates exhibit ac haracteristic and sharp absorption band as wella saf luorescence band at aw avelength of 573 nm making PIC an interesting candidate to analyze the self-assembly process in variousenvironments. The presentw ork developed PIC-based, synthetic model systems, suitablet oi nvestigate how macromolecular crowding influences self-assembly processes. Four synthetic additives were used as potentialc rowders:T riethylene glycol (TEG), polyethylene glycol (PEG), Ficoll 400 as ah ighly branched polysaccharide, and sucrose corresponding to the monomericu nit of Ficoll. Combined UV/Vis spectroscopy and time-resolved light scattering revealed as trong impact of crowding based on excludedv olume 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.D evelopment of such aP IC based models ystem led over to in-cell experiments.H eLa cells were infiltrated with PIC solutionswell below the aggregation threshold in the infiltrating solution. In the cellular environment, PIC was exposed to as ignificant crowding andi mmediatelys tarted to aggregate. As was demonstrated by fluorescencei maging, the extent of aggregation can be modulated by exposing the cells to salt-induced osmotic stress. The resultss uggest future use of such as ystem as as ensor for the analysis of in vitro and in vivo crowding effects on self-assembly processes. Sample preparationAqueous solutions of crowding agents with contents varying from 2wt% to 25 wt %s erved as solvents. Solvents containing Ficoll 400 were slightly acidic. As Ficoll would undergo chemical decomposition, especially at elevated temperatures, the pH of the solvent thus had to be adjusted to 7t oe nsure chemical stability of Ficoll 400. Stock solutions of PIC in the presence of crowding agents had

Thermodynamic Analysis of the Self‐Assembly of Pseudo Isocyanine Chloride in the Presence of Crowding Agents

“…The self-assembly of PIC depends on the PIC concentration and temperature. With a preceding work [12] we were able to verify that the self-assembly of PIC is prone to macromolecular crowding, visualised by shifted aggregation boundaries in in vitro experiments and supported by a strong aggregation in cellular environment exposed to a supernatant with a PIC content well below the respective aggregation boundary during in vivo experiments. This further illustrates close analogies of self-assembly of PIC to self-assembly of proteins in biological systems and identifies PIC to be a suitable model system for mimicking fibril formation of proteins.…”

“…The chemical potential derivatives μ 23 for all investigated systems from the solubility approach based on Equation (3), from data of the ceiling temperature analogy based on Equation (6) and from the ASA-model [13] based on Equation (4). The present analysis together with the preceding work [12] identifies PIC as a suitable candidate for further studies regarding the elucidation of fibril formation in biological systems. The striking change of the absorption pattern of PIC during aggregation gives access to a straightforward data analysis and similarities of self-assembly of PIC and of proteins may allow to transfer established principles of crowding effects on PIC to amyloidogenic proteins.…”

“…The present analysis together with the preceding work [12] identifies PIC as a suitable candidate for further studies regarding the elucidation of fibril formation in biological systems. The striking change of the absorption pattern of PIC during aggregation gives access to a straightforward data analysis and similarities of self‐assembly of PIC and of proteins may allow to transfer established principles of crowding effects on PIC to amyloidogenic proteins.…”

“…[8] A previous study has already addressed the impact of various crowding agents on the fibrillization of PIC. [12] In vitro experiments verified that the presence of triethylene glycol (TEG) and polyethylene glycol (PEG) both suppressed the formation of PIC fibrils. Ficoll 400 on the other hand promoted the formation of PIC fibrils, whereas sucrose, the corresponding monomeric unit of Ficoll, did not show any effects on fibrillization.…”

“…A previous study has already addressed the impact of various crowding agents on the fibrillization of PIC [12] . In vitro experiments verified that the presence of triethylene glycol (TEG) and polyethylene glycol (PEG) both suppressed the formation of PIC fibrils.…”