Validation of a ZYG-9-AID strain.

(A) Schematic of the degron::GFP::ZYG-9 AID strain and description of auxin treatment lengths. ZYG-9 is tagged at the endogenous locus and the TIR1 transgene is expressed from a germline-specific promoter. Types of auxin treatment: (1) Long-term: worms are incubated on auxin-containing plates overnight, (2) Short-term: worms are soaked in auxin-containing media for 25–30 minutes prior to dissecting oocytes for immunofluorescence, (3) Acute: embryos are dissected into auxin-containing media and filmed immediately. (B) Linescan profiles of degron::GFP::ZYG-9 (green) and mCherry::histone (magenta) fluorescence intensities from metaphase spindles in live oocytes. The plot shows that ZYG-9 is enriched at the poles of the bipolar metaphase spindle, but ZYG-9 is localized in the midspindle region as well. n represents the number of spindles analyzed. Bar = 2.5 μm. (C) Linescan profiles of fixed metaphase spindles stained for DNA (blue), tubulin (green), degron::GFP::ZYG-9 (using the GFP antibody), and ASPM-1. The plot shows that ZYG-9 is enriched at the spindle poles but also has more signal in the midspindle region than ASPM-1. n represents the number of spindles analyzed. Bar = 2.5μm. (D) Meiotic oocyte spindles from ZYG-9 AID worms plated overnight on control plates (- auxin) and 1 mM auxin-containing plates (+ auxin) stained for DNA (blue), tubulin (green), ZYG-9 (not shown in merge), and ASPM-1 (red). Long-term ZYG-9 depletion results in the formation of multipolar spindles (44/45 spindles). Bars = 2.5μm. (E) Mitotic spindles in 1-cell stage embryos from ZYG-9 AID worms plated overnight on control plates (- auxin) and 1 mM auxin-containing plates (+ auxin) stained for DNA (blue), tubulin (green), ZYG-9 (red), and ASPM-1 (not shown in merge). Long-term ZYG-9 depletion recapitulates published mitotic phenotypes (8/8 embryos) [14,33]. Bars = 10μm.