Sudhakar, S. | Search bioRxiv

bioRxiv2020

Single depolymerizing and transport kinesins stabilize microtubule ends

Microtubules are highly dynamic cellular filaments and many intracellular processes like cell division depend on an accurate control of their length. Among other factors, microtubule length is actively modulated by motors from the kinesin superfamily. For example, yeast kinesin-8, Kip3, depolymerizes microtubules in a collective manner by a force- and length-dependent mechanism. However, whether single motors depolymerize or stabilize microtubule ends is unclear. Here, using interference reflection microscopy, we measured the influence of single kinesin motors on the stability of microtubules in an in vitro assay. Surprisingly, using unlabeled, stabilized microtubules, we found that both single kinesin-8 and non-depolymerizing kinesin-1 transport motors stabilized microtubule ends further by reducing the spontaneous microtubule depolymerization rate. Since we observed this effect for two very different kinesins, it implies a more general stabilization mechanism. For Kip3, this behavior is contrary to the collective force-dependent depolymerization activity of multiple motors. The complex, concentration-dependent interaction with microtubule ends provides new insights into the molecular mechanism of kinesin-8 and its regulatory function of microtubule length.

biophysics↗

bioRxiv2020

Polycationic gold nanorods as multipurpose in vitro microtubule markers

Gold nanoparticles are intriguing because of their unique size- and shape-dependent chemical, electronic and optical properties. Various microscopy and biomedical applications are based on the particles biocompatibility, surface functionalizability, light absorption, and plasmon resonances. Gold nanorods (AuNRs) are particularly promising for various sensor applications due to their tip-enhanced plasmonic fields. For biomolecule attachment, AuNRs are often stabilized with amphiphilic molecules and functionalized with antibodies or biotin-binding proteins. However, by their intrinsic size such molecules block the most sensitive near-field region of the AuNRs. Here, we used short cationic thiols to covalently functionalize the gold surface. We show that the functionalization layer is thin and that these polycationic AuNRs bind in vitro to negatively charged microtubule filaments. Furthermore, we can plasmonically stimulate light emission from the AuNRs and, therefore, use them as bleach- and blinkfree microtubule markers. We confirmed colocalization by transmission electron microscopy or the combination of interference reflection and single-molecule fluorescence microscopy of fluorescently-labeled or plasmonic photoluminescent versions of the AuNRs. We expect that polycationic AuNRs may be applicable to in vivo systems and other negatively charged molecules like DNA. In the long-term, microtubule-bound AuNRs can be used as ultrasensitive single-molecule sensors for molecular machines that interact with microtubules.

biophysics↗