Single microtubules and small networks become significantly stiffer on short time-scales upon mechanical stimulation

Abstract

The transfer of mechanical signals through cells is a complex phenomenon. To uncover a new mechanotransduction pathway, we study the frequency-dependent transport of mechanical stimuli by single microtubules and small networks in a bottom-up approach using optically trapped beads as anchor points. We interconnected microtubules to linear and triangular geometries to perform micro-rheology by defined oscillations of the beads relative to each other. We found a substantial stiffening of single filaments above a characteristic transition frequency of 1–30 Hz depending on the filament’s molecular composition. Below this frequency, filament elasticity only depends on its contour and persistence length. Interestingly, this elastic behavior is transferable to small networks, where we found the surprising effect that linear two filament connections act as transistor-like, angle dependent momentum filters, whereas triangular networks act as stabilizing elements. These observations implicate that cells can tune mechanical signals by temporal and spatial filtering stronger and more flexibly than expected.

Label-free Imaging and Bending Analysis of Microtubules by ROCS Microscopy and Optical Trapping

“…We use established active microrheology techniques and analyzed the viscoelastic response of single MTs in Fourier space (4,(33)(34)(35), expressed by the frequency-dependent viscoelastic shear modulus G(u) ¼ G 0 (u) þ iG 00 (u) as the complex sum of the elastic and viscous moduli G' and G 00 . between the active driving force DF D generated by the oscillating optical trap and the resulting superposition of deformationsũ qn ðuÞ of the MT is assumed.…”

“…In addition, fluorophores do not emit enough photons for fast imaging at high resolution and contrast. Therefore, label-free techniques producing strong photon signals from small structures have become of utmost importance (3,4).…”

“…Therefore, it is desirable to combine such a label-free imaging technique with optical tweezers and sensitive, high-bandwidth particle-tracking ability such as with back-focal-plane (BFP) interferometry (7,8) to measure mechanical interactions through time-varying forces and energy landscapes of single proteins, cellular structures, and whole cells (9)(10)(11). Common approaches rely on fluorescence, e.g., to investigate entropic forces in microtubule networks by total internal reflection (TIR) fluorescence microscopy (12,13), in single molecule experiments such as DNAbinding protein assays using the STED principle (14), or to probe their frequency-dependent elastic properties using epifluorescence (4). As a label-free technique, differential interference contrast microscopy is widely used in combination with optical traps but suffers a 1D image contrast that completely disappears in the direction perpendicular to the fast axis of the differential interference contrast prism (15).…”

“…the constant low-frequency elastic modulus G 0 ðu ¼ 0Þ ¼ 1=2:16p 2 ' p ðu ¼ 0Þðp=LÞ4 kT in Eq. 4, we derived the persistence length ' p ðu ¼ 0; LÞ ¼ G 0 ðu ¼ 0; LÞ,2:16=kTp 2 L 4 as a function of the contour Viscoelastic properties of single microtubules.…”

The power of time—how to set up a rhythm

“…In other words, rather than to rely upon the obvious, but somewhat metaphysic notion that the cell needs to sense holistic properties such as symmetry or axis, when it comes to lay down the PPB, the reach model can derive PPB orientation based on local constraints (distance between nuclear envelope and cell wall). The preference for the closest site might be of mechanic nature-microtubules are rigid structures that are able to convey compression forces (Gittes et al 1993;Koch et al 2017) and undergo dynamic decay upon pressure (Akhmanova and Steinmetz, 2008).…”