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Female meiotic spindles in many organisms form in the absence of centrosomes, the organelle typically associated with microtubule (MT) nucleation. Previous studies have proposed that these meiotic spindles arise from RanGTP-mediated MT nucleation in the vicinity of chromatin; however, whether this process is sufficient for spindle formation is unknown. Here, we investigated whether a recently proposed spindle-based MT nucleation pathway that involves augmin, an 8-subunit protein complex, also contributes to spindle morphogenesis. We used an assay system in which hundreds of meiotic spindles can be observed forming around chromatin-coated beads after introduction of Xenopus egg extracts. Spindles forming in augmin-depleted extracts showed reduced rates of MT formation and were predominantly multipolar, revealing a function of augmin in stabilizing the bipolar shape of the acentrosomal meiotic spindle. Our studies also have uncovered an apparent augmin-independent MT nucleation process from acentrosomal poles, which becomes increasingly active over time and appears to partially rescue the spindle defects that arise from augmin depletion. Our studies reveal that spatially and temporally distinct MT generation pathways from chromatin, spindle MTs, and acentrosomal poles all contribute to robust bipolar spindle formation in meiotic extracts.
Fig. 4. Visualization of growing MT plus ends via GFP-EB1 in extracts with control antibody (Left) or anti-Dgt4 antibody (Right). (A) Representative images of tubulin (Cy3-channel) and GFP–EB1 (140 nM) are shown for extract incubated with control or Dgt4 antibody (see Fig. 3G and Fig. S3 C–E) at three assembly stages (see Fig. 1D). (B and C) Augmin-inhibited spindles show a reduction of EB1–GFP comets from the middle of the spindles. Note the prominent density of EB1–GFP comets at the acentrosomal poles in C (both control and Dgt4 AB). (Scale bars, 10 μm.) See also Movie S3 (control AB) and Movie S4 (Dgt4 AB). (D) Quantitation of the ratio between the EB1 intensity in the midzone and near the poles (see circles for zone of measurement). Mean and SEM are derived from three independent experiments (2–7 spindles measured for each time point in each independent experiment).
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