Tau, an intrinsically disordered protein confined to neuronal axons, binds to and regulates microtubule dynamics. Although there have been observations of string-like microtubule fascicles in the axon initial segment (AIS) and hexagonal bundles in neurite-like processes in non-neuronal cells overexpressing Tau, cell-free reconstitutions have not replicated either geometry. Here we map out the energy landscape of Tau-mediated, GTP-dependent ‘active' microtubule bundles at 37 °C, as revealed by synchrotron SAXS and TEM. Widely spaced bundles (wall-to-wall distance Dw–w≈25–41 nm) with hexagonal and string-like symmetry are observed, the latter mimicking bundles found in the AIS. A second energy minimum (Dw–w≈16–23 nm) is revealed under osmotic pressure. The wide spacing results from a balance between repulsive forces, due to Tau's projection domain (PD), and a stabilizing sum of transient sub-kBT cationic/anionic charge–charge attractions mediated by weakly penetrating opposing PDs. This landscape would be significantly affected by charge-altering modifications of Tau associated with neurodegeneration.

Despite the recent development of several super-resolution fluorescence microscopic techniques, there are still few techniques that can be readily employed in conventional imaging systems. We present a very simple, rapid, general and cost-efficient super-resolution imaging method, which can be directly employed in a simple fluorescent imaging system with general fluorophores. Based on diffusion-assisted Förster resonance energy transfer (FRET), fluorescent donor molecules that label specific target structures can be stochastically quenched by diffusing acceptor molecules, thereby temporally separating otherwise spatially overlapped fluorescence signals and allowing super-resolution imaging. The proposed method provides two- to three-fold-enhancement in spatial resolution, a significant optical sectioning property, and favorable temporal resolution in live-cell imaging. We demonstrate super-resolution live-cell dynamic imaging using general fluorophores in a standard epi-fluorescence microscope with light-emitting diode (LED) illumination. Due to the simplicity of this approach, we expect that the proposed method will prove an attractive option for super-resolution imaging.

Microtubules (MTs) are nanometer scale hollow cylindrical biological polyelectrolytes. They are assembled from α/β-tubulin dimers, which stack to form protofilaments (PFs) with lateral interactions between PFs resulting in the curved MT. In cells, MTs and their assemblies are critical components in a range of functions from providing tracks for the transport of cargo to forming the spindle structure during mitosis. Previous studies have shown that while cations with valence equal to or larger than 3+ tend to assemble tight 3D bundles of taxol-stabilized MTs, certain divalent cations induce relatively loose 2D bundles of different symmetry [D. J. Needleman, et al., PNAS, 2004, 101, 16099]. Similarly, divalent cations form 2D bundles of DNA adsorbed on cationic membranes [I. Koltover, et al., PNAS, 2000, 97, 14046]. The bundling behavior for these biological polyelectrolyte systems is qualitatively in agreement with current theory. Here, we present results, which show that unlike the case for DNA adsorbed on cationic membranes, bundling of taxol-stabilized MTs occurs only for certain divalent cations above a critical ion concentration (e.g. Ca2+, Sr2+, Ba2+). Instead many divalent cations preempt the bundling transition and depolymerize taxol-stabilized MTs at a lower counterion concentration. Although previous cryogenic TEM has shown that, in the absence of taxol, Ca2+ depolymerizes MTs assembling in buffers containing GTP (guanosine triphosphate), our finding is surprising given the known stabilizing effects of taxol on GDP (guanosine diphosphate)-MTs. The ion concentration required for MT depolymerization decreases with increasing atomic number for the divalents Mg2+, Mn2+, Co2+, and Zn2+. GdCl3 (3+) is found to be extremely efficient at MT depolymerization requiring about 1 mM, while oligolysine (2+), is observed not to depolymerize MTs at concentrations as high as 144 mM. The surprising MT depolymerization results are discussed in the context of divalents disrupting either lateral interactions between PFs (which are strengthened for taxol containing β-tubulin) or interfering with taxol’s ability to induce flexibility at the interface between two tubulin dimers in the same PF (which has been recently suggested as a mechanism by which taxol stabilizes MTs post-hydrolysis with the induced flexibility counteracting the kink between GDP-tubulin dimers in a PF).

The manipulation of droplets with sizes on the millimetre scale and below has attracted considerable attention over the past few decades for applications in microfluidics, biology, and chemistry. In this paper, we report the response of an oil droplet floating in an aqueous solution to local laser heating. Depending on the laser power, distinct dynamic transitions of the shape and motion of the droplet are observed, namely, breathing, crawling, budding, and splitting. We found that the selection of the dynamic modes is determined by dynamic instabilities due to the interplay between the convection flows and capillary effects. Our findings can be useful for constructing microfluidic devices to control the motion and shape of a small droplet by simply altering the laser power, and for understanding thermal convective systems with fully soft boundaries.

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