High-Resolution Imaging of a Single Gliding Protofilament of Tubulins by HS-AFM

Abstract

In vitro gliding assay of microtubules (MTs) on kinesins has provided us with valuable biophysical and chemo-mechanical insights of this biomolecular motor system. Visualization of MTs in an in vitro gliding assay has been mainly dependent on optical microscopes, limited resolution of which often render them insufficient sources of desired information. In this work, using high speed atomic force microscopy (HS-AFM), which allows imaging with higher resolution, we monitored MTs and protofilaments (PFs) of tubulins while gliding on kinesins. Moreover, under the HS-AFM, we also observed splitting of gliding MTs into single PFs at their leading ends. The split single PFs interacted with kinesins and exhibited translational motion, but with a slower velocity than the MTs. Our investigation at the molecular level, using the HS-AFM, would provide new insights to the mechanics of MTs in dynamic systems and their interaction with motor proteins.

A bio-pen for direct writing of single molecules on user-functionalized surfaces

“…Considering that the orientation of both APTES and antikinesin antibodies leads to anisotropic geometries, 37 we further hypothesized that any kinesin dispensed from the microtubule pen as a pattern to be written onto the anti-kinesin antibodies would not only show an additional increase in local surface roughness at its place of binding, but also a surface dotlike geometry. Our hypothesis is supported by previous reports that showed that single molecules could be imaged using AFM, [17][18][19][20][21] with kinesin molecules in particular being resolved by AFM as individual visible blobs. 17 Indeed, kinesin writing led to an additional increase in surface roughness, i.e., from 1.12 to 4.00 nm for R q and from 1.54 to 5.02 nm for R a for the APTES-glutaraldehyde-antikinesin antibody-kinesin surface relative to the APTESglutaraldehyde-anti-kinesin antibody functionalized surface respectively ( Fig.…”

“…Moreover, our analysis is supported by Kacher et al and Schaap et al, who noted that AFM can resolve single kinesin motors, with kinesins being visible as blobs when using AFM. 17,18 Upon demonstrating kinesin's ability to recognize and ink a microtubule pen, we created a "holder" to affix such a bio-pen. The holder consisted of a biologically inert AFM tip functionalized with anti-tubulin antibodies (Fig.…”

“…At least 6 experiments for each one of the samples were performed. 17,18 For microtubules, mica sheets washed with DI water, ethanol and again with DI water, and subsequently dried overnight under vacuum and at room temperature, were used as substrates. Mica was functionalized with 20 mL APTES through incubation at room temperature for 15 min.…”

“…Contact mode AFM in the respective buffer of each sample was performed to evaluate changes in surface roughness. [17][18][19][20][21] The obtained images were attened to remove curvature and slope effects. The root mean square average of height deviation (as taken from the mean image data plane) and the arithmetic average of absolute values of the surface height deviations (i.e., R q and R a ) were calculated from the obtained attened images.…”

Radial alignment of microtubules through tubulin polymerization in an evaporating droplet

“…The higher-ordered alignments may give rise to these sharp peaks in the layer lines of detected d-spacing values which are similar to the previously reported values [39]. The d values may also indicate the size of the tubulin monomers [40] which were closely packed resulting radial array of MTs. Upon cooling, the sharp peaks from the near-meridional reflections disappeared which confirms the spherulite was formed by the MTs (Fig 4C (iii)).…”