Abstract
SSNA1 (Sjögren's Syndrome Nuclear Autoantigen 1) is a microtubule-associated protein involved in key cellular processes, including cell division, intraflagellar transport, and axonal branching. SSNA1 specifically localizes to sites of damage along the microtubule lattice, thus acting as a microtubule damage sensor. However, the effects of SSNA1 on microtubule mechanics or on the process of microtubule self-repair, which involves the incorporation of soluble tubulin dimers into lattice damage sites, are not known. Here, we use in vitro reconstitution with purified proteins and total internal reflection fluorescence (TIRF) microscopy to probe SSNA1's effects on microtubule mechanics and self-repair. We apply two distinct sources of force to investigate microtubule mechanics: kinesin-driven gliding assays and microfluidic flow. We find that SSNA1 binding increases microtubule rigidity and resistance to breakage under the physiological and controlled forces in our assays. Interestingly, SSNA1's localization to microtubule damage sites prevents the incorporation of new tubulin dimers and thus inhibits lattice self-repair. Conversely, we find that SSNA1 does not recognize damage sites that have been repaired by tubulin incorporation. Together, our findings demonstrate that SSNA1 reinforces the mechanical strength of microtubules without promoting self-repair, suggesting an alternative mechanism for restoring microtubule integrity in the absence of tubulin-mediated repair and providing new insights into SSNA1's mechanism of microtubule stabilization.