Biology subjects

Lawrence, E. J.

Publications and source records attributed to Lawrence, E. J..

4 recordsLinked to original sources

bioRxiv2026

SSNA1 mechanically reinforces the damaged microtubule lattice

SSNA1 (Sjogrens 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 SSNA1s 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, SSNA1s 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 SSNA1s mechanism of microtubule stabilization. Significance StatementMicrotubules are cytoskeletal polymers that experience mechanical stress during essential cellular processes such as cargo transport, cell division, and ciliary beating. To maintain their integrity, microtubules rely on both stabilizing proteins and repair mechanisms. Here, we show that microtubule-associated protein SSNA1 strengthens microtubules by increasing their rigidity and resistance to force-induced breakage, while simultaneously blocking tubulin-mediated lattice repair at sites of damage. By distinguishing between damaged and repaired microtubule lattices, SSNA1 enforces a stabilization strategy that favors mechanical reinforcement over self-repair. These findings reveal a new mode of microtubule regulation that decouples mechanical stability from lattice repair and provide insight into how cells preserve cytoskeletal integrity under force.

biophysics↗

bioRxiv2022

CLASPs stabilize the intermediate state between microtubule growth and catastrophe

CLASPs regulate microtubules in many fundamental cellular processes. CLASPs stabilize dynamic microtubules by suppressing catastrophe and promoting rescue, the switch-like transitions between microtubule growth and shrinkage. However, the molecular mechanisms underlying CLASPs activity are not understood. Here, we investigate the effects of CLASPs on distinct microtubule substrates in the absence of tubulin to gain insight into how CLASPs regulate microtubule dynamics. Surprisingly, we find that human CLASP1 depolymerizes stable microtubules in the presence of GTP, but not in the absence of nucleotide. Conversely, CLASP1 stabilizes dynamic microtubules upon tubulin dilution in the presence of GTP. Our results demonstrate that CLASP1 drives microtubule substrates with different inherent stabilities into the same slowly-depolymerizing state in the absence of tubulin in a nucleotide-dependent manner. We interpret this state as the pre-catastrophe intermediate state between microtubule growth and shrinkage. Thus, we conclude that CLASPs stabilize the intermediate state between microtubule growth and shrinkage to suppress microtubule catastrophe and promote rescue.

cell biology↗

bioRxiv2022

CLASP2 facilitates dynamic actin filament organization along the microtubule lattice

Coordination between the microtubule and actin networks is essential for cell motility, neuronal growth cone guidance, and wound healing. Members of the CLASP (Cytoplasmic Linker-Associated Protein) family of proteins have been implicated in the cytoskeletal crosstalk between microtubules and actin networks, however, the molecular mechanisms underlying CLASPs role in cytoskeletal coordination are unclear. Here, we investigate CLASP2s crosslinking function with microtubules and F-actin. Our results demonstrate that CLASP2 crosslinks F-actin to the microtubule lattice in vitro. We find that the crosslinking ability is retained by L-TOG2-S, a minimal construct containing the TOG2 domain and serine-arginine rich region of CLASP2. Furthermore, CLASP2 promotes the accumulation of multiple actin filaments along the microtubule, supporting up to 11 F-actin landing events on a single microtubule lattice region. CLASP2 also facilitates dynamic organization of polymerizing actin filaments templated by the microtubule network, with F-actin forming bridges between individual microtubules. Finally, we find that depletion of CLASPs in vascular smooth muscle cells results in disorganized actin fibers and reduced co-alignment of actin fibers with microtubules, suggesting that CLASP and microtubules contribute to higher-order actin structures. Taken together, our results indicate that CLASP2 can directly crosslink F-actin to microtubules, and that this microtubule-CLASP-actin interaction may influence overall cytoskeletal organization in cells.

cell biology↗

bioRxiv2019

Stutter: a Transient Dynamic Instability Phase that is Strongly Associated with Catastrophe

Microtubules (MTs) are cytoskeletal fibers that undergo dynamic instability (DI), a remarkable process involving phases of growth and shortening separated by stochastic transitions called catastrophe and rescue. Dissecting dynamic instability mechanism(s) requires first characterizing and quantifying these dynamics, a subjective process that often ignores complexity in MT behavior. We present a Statistical Tool for Automated Dynamic Instability Analysis (STADIA), which identifies and quantifies not only growth and shortening, but also a category of intermediate behaviors that we term stutters. During stutters, the rate of MT length change tends to be smaller in magnitude than during typical growth or shortening phases. Quantifying stutters and other behaviors with STADIA demonstrates that stutters precede most catastrophes in our dimer-scale MT simulations and in vitro experiments, suggesting that stutters are mechanistically involved in catastrophes. Related to this idea, we show that the anti-catastrophe factor CLASP2{gamma} works by promoting the return of stuttering MTs to growth. STADIA enables more comprehensive and data-driven analysis of MT dynamics compared to previous methods. The treatment of stutters as distinct and quantifiable DI behaviors provides new opportunities for analyzing mechanisms of MT dynamics and their regulation by binding proteins.

cell biology↗