Self-assembly of biomacromolecules into fibrillar nanostructures is a fundamental process in both prokaryotic and eukaryotic cells. In contrast to the well-defined (dys)functions of endogenous protein filaments, the fate of intracellular assembly of small molecules remains largely unknown.  Besides being driven by non-covalent bonds to form ordered filamentous assemblies, the nanofibers of molecular hydrogelators have shown certain unexpected bioactivities, e.g., they promote activation of procaspase-3, disrupt the elongation of microtubules, and serve as a mimic of cytoskeleton in a model protocell.  For a better understanding of small molecular self-assembly inside living bio-entities, we have proposed a general imaging method, correlative light and electron microscopy (CLEM), for the investigation of self-assembly of small molecules without fluorescence labeling (native form) inside mammalian cells after a necessary incubation time.  The idea is to incorporate dansyl labeled molecules into the nanofibers formed from the native molecules.  This method allows us to determine the formation, localization, and progression of molecular assemblies generated from non-fluorescent small molecular hydrogelators by an enzyme-triggered hydrogelation mechanism inside live mammalian cells. We demonstrated that (i) the precursors passively diffuse inside the cells; (ii) molecular assembly occurs on the endoplasmic reticulum (ER); and (iii) CLEM further indicates that the molecular assemblies are localized near or inside the ER and are likely processed via the cellular secretory pathway (e.g., ER-Golgi-lysosomes/secretion) by cells, albeit less efficiently. This work not only establishes a general strategy to provide the spatiotemporal profile of molecular assemblies inside cells, but indicates that molecular assemblies of small molecules may provide a new model system to mimic and to understand the cellular mechanisms and processes related to endogenous-normal and aberrant-protein nanofibers.

Probing self-assembly of non-fluorescent small molecules inside live mammalian cells

247th National Meeting (2014)

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