Microtubule-crosslinking motor proteins, which slide antiparallel microtubules, are required for remodeling of microtubule networks. Hitherto, all microtubule-crosslinking motors have been shown to slide microtubules at constant velocity until no overlap between the microtubules remains, leading to breakdown of the initial microtubule geometry. Here, we show in vitro that the sliding velocity of microtubules, driven by human kinesin-14, HSET, decreases when microtubules start to slide apart, resulting in the maintenance of finite-length microtubule overlaps. We quantitatively explain this feedback by the local interaction kinetics of HSET with overlapping microtubules, causing retention of HSET in shortening overlaps. Consequently, the increased HSET density in the overlaps leads to a density-dependent decrease in sliding velocity and the generation of an entropic force antagonizing the force exerted by the motors. Our results demonstrate that a spatial arrangement of microtubules can regulate the collective action of molecular motors through local alteration of their individual interaction kinetics.

Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The mechanisms of their interactions with the plasma membrane remain poorly understood. Here we show by cell-free reconstitution that membrane binding requires electrostatic interactions of septins with anionic lipids and promotes the ordered self-assembly of fly septins into filamentous meshworks. Transmission electron microscopy reveals that both fly and mammalian septins form arrays of single and paired filaments. Atomic force microscopy and quartz crystal microbalance demonstrate that the fly filaments form mechanically rigid, 12 to 18 nm thick, double layers of septins. By contrast, C-terminally truncated septin mutants form 4 nm thin monolayers, indicating that stacking requires the C-terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is required for fly septins to form ordered arrays of single and paired filaments and provides new insights into the mechanisms by which septins may regulate cell surface mechanics.

Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The mechanisms of their interactions with the plasma membrane remain poorly understood. Here we show by cell-free reconstitution that binding to flat lipid membranes requires electrostatic interactions of septins with anionic lipids and promotes the ordered self-assembly of fly septins into filamentous meshworks. Transmission electron microscopy reveals that both fly and mammalian septin hexamers form arrays of single and paired filaments. Atomic force microscopy and quartz crystal microbalance demonstrate that the fly filaments form mechanically rigid, 12 to 18 nm thick, double layers of septins. By contrast, C-terminally truncated septin mutants form 4 nm thin monolayers, indicating that stacking requires the C-terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is required for fly septins to form ordered arrays of single and paired filaments and provides new insights into the mechanisms by which septins may regulate cell surface mechanics.

We propose two-focus fluorescence correlation spectroscopy (2fFCS) on basis of plasmonic nanoantennas that provide distinct hot spots that are individually addressable through polarization, yet lie within a single diffraction limited microscope focus. The importance of two-focus FCS is that a calibrated distance between foci provides an intrinsic calibration to derive diffusion constants from measured correlation times. Through electromagnetic modelling we analyze a geometry of perpendicular nanorods, and their inverse, i.e., nanoslits. While we find that nanorods are not suited for nano-antenna enhanced 2fFCS due to substantial background signal, a nanoslit geometry is expected to provide a di tinct cross-correlation between orthogonally polarized detection channels. Furthermore, by utilizing a periodic array of nanoslits instead of a single pair, the amplitude of the cross-correlation can be enhanced. To demonstrate this technique, we present a proof of principle experiment on the basis of a periodic array of nanoslits, applied to lipid diffusion in a supported lipid bilayer.