Microtubule stiffening by doublecortin-domain protein ZYG-8 contributes to spindle orientation during C. elegans zygote division

Abstract

In the Caenorhabditis elegans zygote, mutations in zyg-8DCLK1, the sole Doublecortin-family member, disrupt mitotic spindle positioning, as seen by immunofluorescence. Doublecortin proteins bind microtubules and are thought to stabilise or rigidify them. In the zygote, ZYG-8 only modestly affects microtubule growth and nucleation. We thus investigated whether these moderate dynamic perturbations alone could explain the spindle mispositioning observed in zyg-8 mutants. Using three complementary genetic perturbations--RNAi-mediated depletion of ZYG-8, its overexpression, and the thermosensitive zyg-8(or484ts) mutant (that disrupts microtubule binding)--we observed altered spindle pole oscillations and changes in microtubule cortical-contact behaviour, indicative of impaired cortical forces. Importantly, these phenotypes could not be fully explained by previously reported alterations in microtubule dynamics, suggesting an additional mechanism. Our findings indicate that ZYG-8 increases microtubule rigidity: ZYG-8 depletion or mutation led to more frequent microtubule bending and higher curvature and tortuosity. Simulations confirmed that reduced rigidity prolongs cortical contact lifetimes, an effect we experimentally observed in zyg-8(RNAi) embryos. Using custom biophysical assays, we showed that microtubule softening in zyg-8(RNAi) embryos and zyg-8 mutants reduced the efficiency of centring forces, leading to exaggerated spindle-pole oscillations. In mutants, the largest oscillations caused spindle poles to move closer to the cell periphery, preventing re-centring and resulting in spindle mispositioning and misorientation during late anaphase. Importantly, reducing cortical pulling forces rescued orientation defects, highlighting the importance of balanced pulling-pushing forces for proper spindle positioning. We propose that sufficient microtubule rigidity is essential for generating effective cortical pushing forces, potentially in synergy with other microtubule properties, which contribute to centring mechanisms that ensure accurate spindle orientation in late mitosis. Given that DCLK1 is frequently deregulated in human cancers and that accurate spindle positioning is essential for maintaining cell proliferation-differentiation balance, these findings may have implications for understanding how disruptions in microtubule mechanics contribute to carcinogenesis. Author summaryDuring cell division, the mitotic spindle--a structure made of microtubules that separates chromosomes in daughter cells--must position itself precisely to ensure healthy cell growth and differentiation. Microtubules are tiny, dynamic, semi-flexible fibers that notably generate forces at the cell periphery, cortical pulling and pushing. These two opposite forces contribute to position the spindle. In the worm C. elegans, the protein ZYG-8 (similar to human DCLK1) stabilizes the microtubules. Our study reveals that ZYG-8 also makes microtubules stiffer, a property critical for generating strong enough pushing forces. When zyg-8 was impaired (mutation or RNAi), microtubules bent excessively, weakening the pushing forces that keep the spindle correctly centered and oriented. As a result, softer microtubules in zyg-8 mutants caused the spindle to drift toward the cells periphery, resulting in mispositioning and misorientation when mitosis ends. By reducing the opposing cortical pulling forces in zyg-8 mutants, we restored normal spindle orientation, highlighting that microtubule rigidity helps balance the forces that position the spindle correctly. Our findings suggest that microtubule stiffness is essential for accurate cell division--a process vital for both normal development and disease prevention, including cancers where homologous proteins are often disrupted.