Fast Leaps between Millisecond Confinements Govern Ase1 Diffusion along Microtubules

Abstract

Diffusion is the most fundamental mode of protein translocation within cells. Confined diffusion of proteins along the electrostatic potential constituted by the surface of microtubules, although modeled meticulously in molecular dynamics simulations, has not been experimentally observed in real‐time. Here, interferometric scattering microscopy is used to directly visualize the movement of the microtubule‐associated protein Ase1 along the microtubule surface at nanometer and microsecond resolution. Millisecond confinements of Ase1 and fast leaps between these positions of dwelling preferentially occurring along the microtubule protofilaments are resolved, revealing Ase1's mode of diffusive translocation along the microtubule's periodic surface. The derived interaction potential closely matches the tubulin‐dimer periodicity and the distribution of the electrostatic potential on the microtubule lattice. It is anticipated that mapping the interaction landscapes for different proteins on microtubules, finding plausible energetic barriers of different positioning and heights, can provide valuable insights into regulating the dynamics of essential cytoskeletal processes, such as intracellular cargo trafficking, cell division, and morphogenesis, all of which rely on diffusive translocation of proteins along microtubules.

Electro-detachment of kinesin motor domain from microtubule in silico

“…Overall, our computational results have a profound importance for the guidance of the experimental design: not only EF strength, but also EF direction, with respect to the microtubule and kinesin orientation, significantly affect the detachment process. To experimentally verify the predictions, one would ideally need to collect the data from single-molecule imaging techniques, such as TIRF microscopy [60] , but with a high temporal resolution to resolve EF effects within the time step, such as iSCAT [61] , [62] , [63] , [64] . Furthermore, suitable chip technology is required that integrates microfluidics for the sample position and manipulation, electrodes system capable of delivering high EF strengths at sufficiently broad frequency bandwidth (required for ns and shorter electric pulses) and compatibility with the advanced microscopy techniques mentioned above.…”

Photothermal Spectro-Microscopy as Benchmark for Optoplasmonic Bio-Detection Assays

“… 4 − 12 Sensitive refractometric sensors such as optical microresonators also provide powerful platforms for molecular recognition, 13 , 14 especially in combination with plasmonic particles. 15 − 21 At the same time novel microscopic methods make use of plasmonic particles as photostable labels 22 , 23 and combine them with optical, electromagnetic, or electric devices for trapping and manipulation of sensor particles or even of the molecules themselves. 24 − 29 In the following we will exclusively focus on optoplasmonic assays that facilitate analyte recognition via observation of a plasmonic nanostructure’s response to (single) analytes perturbing its dielectric environment.…”

“…Optoplasmonic methods, which harness strong near fields around plasmonic metal nanostructures to enhance the sensitivity and selectivity of optical detection, have evolved over the past decade into powerful tools for biomolecular recognition. Dedicated versions of these methods now enable the detection of a wide range of molecules and nanoparticles, on a single-object basis. − Sensitive refractometric sensors such as optical microresonators also provide powerful platforms for molecular recognition, , especially in combination with plasmonic particles. − At the same time novel microscopic methods make use of plasmonic particles as photostable labels , and combine them with optical, electromagnetic, or electric devices for trapping and manipulation of sensor particles or even of the molecules themselves. − In the following we will exclusively focus on optoplasmonic assays that facilitate analyte recognition via observation of a plasmonic nanostructure’s response to (single) analytes perturbing its dielectric environment. The volume in which such perturbations are recognizable is defined by the extent of the structures’ enhanced near-field and is limited to distances on the order of 10 nm away from the structures’ surface.…”