Kinetochore microtubules flux poleward along fixed centrosome-anchored microtubules during the metaphase ofC. elegansone-cell embryo

Abstract

AbstractThe microtubule array, assembled into the mitotic spindle, polymerises from the centrosomes and the chromosomes in many organisms. Their plus ends alternate between growing and shrinking. This dynamic instability plays a key role in pulling on the kinetochores to check the spindle assembly and correct the errors in chromosome attachments. In addition, the minus ends at centrosomes can undergo depolymerisation coordinated with the polymerisation of the plus ends at the kinetochores. Such a mechanism, among others, creates treadmilling,id esta net poleward movement of microtubules called poleward flux. This flux is involved in many roles, chromosome congression in prometaphase, chromosome misattachment detection and correction, spindle length maintenance in metaphase, and synchronous segregation of sister chromatids in anaphase. Interestingly, no poleward flux was measured in theCaenorhabditis eleganssingle-cell embryo, despite it is equipped with all homologous proteins involved in this mechanism in other organisms. To investigate this peculiarity, we labelled the microtubules and photobleached them in a rectangular region. Surprisingly, we observed that both edges of the bleached zone (fronts) move inwards, closing the dark area. However, the middle of the bleached zone does not move clearly, confirming the absence of a global poleward flow. The dynamics of the microtubules emanating from the centrosomes combined with the diffraction due to microscopy imaging account for the apparent movement of the front on the centrosome side. Therefore, we suggest no flux of the centrosome-anchored (spindle) microtubules. In contrast, on the chromosome side, we observed a front moving poleward, faster than the one on the other side, and dependent on proteins ensuring the attachment and growth of microtubules at kinetochores, NDC-80, CLS-2CLASP, and ZYG-9XMAP215. Besides, we found that the depletion of the depolymerase KLP-7MCAKdoes not impair this poleward recovery. Finally, the faster recovery is restricted to the spindle region close to the chromosomes. Therefore, we suggest that the kinetochore microtubules undergo a poleward flux, moving with respect to spindle microtubules. Because the kinetochore microtubules are shorter than the half-spindle, this flux is localised close to the chromosomes. Furthermore, it may not rely on treadmilling as KLP-7MCAKis dispensable. This spatially restricted flux found in the nematode may be related to the slow elongation of the spindle during metaphase and may buffer the strong pulling forces exerted by the cortical force generators at the spindle poles.