The E. coli MinCDE system has become a paradigmatic reaction–diffusion system in biology. The membrane-bound ATPase MinD and ATPase-activating protein MinE oscillate between the cell poles followed by MinC, thus positioning the main division protein FtsZ at midcell. Here we report that these energy-consuming MinDE oscillations may play a role beyond constraining MinC/FtsZ localization. Using an in vitro reconstitution assay, we show that MinDE self-organization can spatially regulate a variety of functionally completely unrelated membrane proteins into patterns and gradients. By concentration waves sweeping over the membrane, they induce a direct net transport of tightly membrane-attached molecules. That the MinDE system can spatiotemporally control a much larger set of proteins than previously known, may constitute a MinC-independent pathway to division site selection and chromosome segregation. Moreover, the here described phenomenon of active transport through a traveling diffusion barrier may point to a general mechanism of spatiotemporal regulation in cells.
The healthy growth and maintenance of a biological system depends on the precise spatial organization of molecules within the cell through the dissipation of energy. Reaction–diffusion mechanisms can facilitate this organization, as can directional cargo transport orchestrated by motor proteins, by relying on specific protein interactions. However, transport of material through the cell can also be achieved by active processes based on non-specific, purely physical mechanisms, a phenomenon that remains poorly explored. Here, using a combined experimental and theoretical approach, we discover and describe a hidden function of the Escherichia coli MinDE protein system: in addition to forming dynamic patterns, this system accomplishes the directional active transport of functionally unrelated cargo on membranes. Remarkably, this mechanism enables the sorting of diffusive objects according to their effective size, as evidenced using modular DNA origami–streptavidin nanostructures. We show that the diffusive fluxes of MinDE and non-specific cargo couple via density-dependent friction. This non-specific process constitutes a diffusiophoretic mechanism, as yet unknown in a cell biology setting. This nonlinear coupling between diffusive fluxes could represent a generic physical mechanism for establishing intracellular organization.
The limited photon budget of fluorescent dyes is the main limitation for localization precision in localization-based super-resolution microscopy. Points accumulation for imaging in nanoscale topography (PAINT)-based techniques use the reversible binding of fluorophores and can sample a single binding site multiple times, thus elegantly circumventing the photon budget limitation. With DNA-based PAINT (DNA-PAINT), resolutions down to a few nanometers have been reached on DNA-origami nanostructures. However, for long acquisition times, we find a photo-induced depletion of binding sites in DNA-PAINT microscopy that ultimately limits the quality of the rendered images. Here we systematically investigate the loss of binding sites in DNA-PAINT imaging and support the observations with measurements of DNA hybridization kinetics via surface-integrated fluorescence correlation spectroscopy (SI-FCS). We do not only show that the depletion of binding sites is clearly photo-induced, but also provide evidence that it is mainly caused by dye-induced generation of reactive oxygen species (ROS). We evaluate two possible strategies to reduce the depletion of binding sites: By addition of oxygen scavenging reagents, and by the positioning of the fluorescent dye at a larger distance from the binding site.
Single-molecule localization microscopy (SMLM) has revolutionized optical microscopy, extending resolution down to the level of individual molecules. However, the actual counting of molecules relies on preliminary knowledge of the blinking behavior of individual targets or on a calibration to a reference. In particular for biological applications, great care has to be taken because a plethora of factors influence the quality and applicability of calibration-dependent approaches to count targets in localization clusters particularly in SMLM data obtained from heterogeneous samples. Here, we present localization-based fluorescence correlation spectroscopy (lbFCS) as the first absolute molecular counting approach for DNA-points accumulation for imaging in nanoscale topography (PAINT) microscopy and, to our knowledge, for SMLM in general. We demonstrate that lbFCS overcomes the limitation of previous DNA-PAINT counting and allows the quantification of target molecules independent of the localization cluster density. In accordance with the promising results of our systematic proof-of-principle study on DNA origami structures as idealized targets, lbFCS could potentially also provide quantitative access to more challenging biological targets featuring heterogeneous cluster sizes in the future.
We present a simple and versatile single-molecule-based method for the accurate determination of binding rates to surfaces or surface bound receptors. To quantify the reversible surface attachment of fluorescently labeled molecules, we have modified previous schemes for fluorescence correlation spectroscopy with total internal reflection illumination (TIR-FCS) and camera-based detection. In contrast to most modern applications of TIR-FCS, we completely disregard spatial information in the lateral direction. Instead, we perform correlation analysis on a spatially integrated signal, effectively converting the illuminated surface area into the measurement volume. In addition to providing a high surface selectivity, our new approach resolves association and dissociation rates in equilibrium over a wide range of time scales. We chose the transient hybridization of fluorescently labeled single-stranded DNA to the complementary handles of surface-immobilized DNA origami structures as a reliable and well-characterized test system. We varied the number of base pairs in the duplex, yielding different binding times in the range of hundreds of milliseconds to tens of seconds, allowing us to quantify the respective surface affinities and binding rates.
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