Figure 3.

Plk4 Is Required for MT Dynamics and Growth

(A) Schematic representation of MT regrowth assay. A single two-cell stage blastomere was injected with mRNA for EGFP-EB3 either with or without (control) Plk4 dsRNA. EGFP-EB3 mRNA expression begins within 2 hr and at least 6 additional hours are required for blastomeres to enter mitosis during which period, Plk4 is depleted. Upon entry into mitosis, embryos were subjected to cold treatment for 26 min, after which they were either fixed for immunostaining or imaged alive to follow dynamics of MT regrowth.

(B and C) Two-cell embryos fixed at indicated times following release from cold treatment and stained for DNA (blue) and α-tubulin (green). The cold treatment completely depolymerized MTs (0 min) that regrew into a bipolar mitotic spindle within 10 min. In contrast, MT repolymerization was severely delayed in Plk4 dsRNAi-depleted blastomeres.

(D and E) In vivo time-lapse microscopy following release from cold treatment. In control two-cell stage embryos (D) in which one blastomere was injected with mRNA for EGFP-EB3 and Plk4-mcherry, regrowth of MTs begins around sites of major MTOCs, in the vicinity of Plk4-mcherry fluorescence; bipolar spindle formation is seen by 48 min. In Plk4-RNAi-treated embryos (E), MT regrowth after cold exposure is impaired. See also Movie S3.

(F and G) Time projection of metaphase spindles depicted by EGFP-EB3 expression. MT growth at plus tips shown on kymographs of EGFP-EB3 signals in control (F′) and Plk4-depleted (G′) blastomeres within the indicated areas of the spindles. The average pixel intensity projection of a metaphase spindle is analyzed for control (n = 12) and Plk4 RNAi blastomeres (n = 8). MT growth was determined from the slopes of EGFP-EB3 (yellow lines on kymographs).

(H) MT growth rate distribution in control (average = 18.2 ± 3.5 μm/min; n = 56) and Plk4 RNAi cells (5.9 ± 4.04 μm/min; n = 35).