Highlights Method enables investigation of amyloid-β oligomer stoichiometry without requiring extrinsic fluorescent probes. Uses single-molecule stepwise photobleaching in vitro . Unveils heterogeneity within populations of oligomers. Assays oligomer-induced dysregulation of intracellular Ca 2+ homeostasis in living cells.

Membrane solubilization by sodium dodecyl sulfate (SDS) is indispensable for many established biotechnological applications, including viral inactivation and protein extraction. Although the ensemble thermodynamics have been thoroughly explored, the underlying molecular dynamics have remained inaccessible, owing to major limitations of traditional measurement tools. Here, we integrate multiple advanced biophysical approaches to gain multiangle insight into the time-dependence and fundamental kinetic steps associated with the solubilization of single submicron sized vesicles in response to SDS. We find that the accumulation of SDS molecules on intact vesicles triggers biphasic solubilization kinetics comprising an initial vesicle expansion event followed by rapid lipid loss and micellization. Our findings support a general mechanism of detergent-induced membrane solubilization, and we expect that the framework of correlative biophysical technologies presented here will form a general platform for elucidating the complex kinetics of membrane perturbation induced by a wide variety of surfactants and disrupting agents.

In eukaryotes, intracellular physico-chemical properties like macromolecular crowding and cytoplasmic viscoelasticity influence key processes such as metabolic activities, molecular diffusion and protein folding. However, mapping crowding and viscoelasticity in living cells remains challenging. One approach uses passive rheology in which diffusion of exogenous fluorescent particles internalized in cells is tracked and physico-chemical properties inferred from derived mean square displacement relations. Recently, the crGE2.3 Förster resonance energy transfer biosensor was developed to quantify crowding in cells, though it is unclear how this readout depends on viscoelasticity and the molecular weight of the crowder. Here, we present correlative, multi-dimensional data to explore diffusion and molecular crowding characteristics of molecular crowding agents using super-resolved fluorescence microscopy and ensemble time-resolved spectroscopy. We firstly characterize in vitro and then apply these insights to live cells of budding yeast Saccharomyces cerevisiae . It is to our knowledge the first time this has been attempted. We demonstrate that these are usable both in vitro and in the case of endogenously expressed sensors in live cells. Finally, we present a method to internalize fluorescent beads as in situ viscoelasticity markers in the cytoplasm of live yeast cells and discuss limitations of this approach including impairment of cellular function.

Membrane solubilization by sodium dodecyl sulfate (SDS) is indispensable for many established biotechnological applications, including viral inactivation and protein extraction. Although the ensemble thermodynamics have been thoroughly explored, the underlying molecular dynamics have remained inaccessible, owing to major limitations of traditional measurement tools. Here, we integrate multiple advanced biophysical approaches to gain multi-angle insight into the time-dependence and fundamental kinetic steps associated with the solubilization of single sub-micron sized vesicles in response to SDS. We find that the accumulation of SDS molecules on in-tact vesicles triggers biphasic solubilization kinetics comprising an initial vesicle expansion event followed by rapid lipid loss and micellization. Our findings support a general mechanism of detergent-induced membrane solubilization and we expect the framework of correlative biophysical technologies presented here will form a general platform for elucidating the complex kinetics of membrane perturbation induced by a wide variety of surfactants and disrupting agents.

The solubilization of lipid membranes by Tween-20 is crucial for a number of biotechnological applications, but the mechanistic details remain elusive. Evidence from ensemble assays supports a solubilization model that encompasses surfactant association with the membrane and the release of mixed micelles to solution, but whether this process also involves intermediate transitions between regimes is unanswered. In search of mechanistic origins, increasing focus is placed on identifying Tween-20 interactions with controllable membrane mimetics. Here, we employed ultrasensitive biosensing approaches, including single-vesicle spectroscopy based on fluorescence and energy transfer from membrane-encapsulated molecules, to interrogate interactions between Tween-20 and submicrometer-sized vesicles below the optical diffraction limit. We discovered that Tween-20, even at concentrations below the critical micellar concentration, triggers stepwise and phase-dependent structural remodeling events, including permeabilization and swelling, in both freely diffusing and surface-tethered vesicles, highlighting the substantial impact the surfactant has on vesicle conformation and stability prior to lysis.