Making Microtubules and Mitotic Spindles in Cells without Functional Centrosomes

SummaryCentrosomes are considered to be the major sites of microtubule nucleation in mitotic cells (reviewed in 1, 2 and 3), yet mitotic spindles can still form after laser ablation or disruption of centrosome function 4, 5, 6 and 7. Although kinetochores have been shown to nucleate microtubules [3], mechanisms for acentrosomal spindle formation remain unclear. Here, we performed live-cell microscopy of GFP-tubulin to examine spindle formation in Drosophila S2 cells after RNAi depletion of either γ-tubulin, a microtubule nucleating protein [8], or centrosomin, a protein that recruits γ-tubulin to the centrosome 7 and 9. In these RNAi-treated cells, we show that poorly focused bipolar spindles form through the self-organization of microtubules nucleated from chromosomes (a process involving γ-tubulin), as well as from other potential sites, and through the incorporation of microtubules from the preceding interphase network. By tracking EB1-GFP (a microtubule-plus-end binding protein) in acentrosomal spindles, we also demonstrate that the spindle itself represents a source of new microtubule formation, as suggested by observations of numerous microtubule plus ends growing from acentrosomal poles toward the metaphase plate. We propose that the bipolar spindle propagates its own architecture by stimulating microtubule growth, thereby augmenting the well-described microtubule nucleation pathways that take place at centrosomes and chromosomes.