Volume 16, Issue 2, Pages 214-220 (January 2006) Cdc42 Activation Couples Spindle Positioning to First Polar Body Formation in Oocyte Maturation  Chunqi Ma, Héléne A. Benink, Daye Cheng, Véronique Montplaisir, Ling Wang, Yanwei Xi, Pei-Pei Zheng, William M. Bement, X. Johné Liu  Current Biology  Volume 16, Issue 2, Pages 214-220 (January 2006) DOI: 10.1016/j.cub.2005.11.067 Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 1 Cdc42T17N Inhibited First Polar Body Formation (A) Typical images of oocytes after different treatment. All except for control (GV) were treated with progesterone overnight. When cytochalasin B (CB, 5 μg/ml) was used, it was added 2 hr before the addition of progesterone. Oocytes injected with Cdc42T17N or Rac1T17N mRNA were incubated for at least 6 hr before the addition of progesterone. Oocytes injected with C3 mRNA were immediately placed in medium containing progesterone. (B) Oocytes treated as described in (A) were fixed, stained with Sytox green, and viewed from the animal pole under a dissecting fluorescence microscope. First polar body (PB) is indicated. (C) Control oocytes and oocytes injected with Cdc42T17N or Rac1T17N mRNA were coinjected with tubulin-Oregon green 514 conjugate (Molecular Probes, 5 nl per oocyte) before the addition of progesterone. Following overnight incubation, the oocytes were fixed, stained with propidium iodide (red), and viewed following lateral bisection of the oocytes. Pictures are representative confocal images viewed from the cutting surface. C3 mRNA-injected oocytes and CB-treated oocytes were fixed, bisected, and costained with anti-tubulin antibodies (red) and Sytox green. A dashed line indicates the oocyte cortex in each image. (D) Uninjected (control) oocytes and oocytes injected with mRNA for HA-Cdc42T17N or HA-Rac1T17N were incubated overnight. Extracts were prepared and analyzed by SDS-PAGE followed by immunoblotting with antibodies against HA. (E) Uninjected oocytes (control, lanes 1 to 4) and oocytes injected with HA-Cdc42T17N mRNA (lanes 5 to 8) were incubated overnight before the addition of progesterone. Individual oocytes were lysed at GVBD or at the indicated times following GVBD. GV oocytes (without progesterone) were lysed at the same time as the GVBD 3 hr oocytes. Extracts were subjected to MPF assays (top panel) or immunoblotting with antibodies against cyclin B2 (middle panel) or xFZY (lower panel). Shown is a representative of three independent experiments. Current Biology 2006 16, 214-220DOI: (10.1016/j.cub.2005.11.067) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 2 Cdc42T17N Inhibits Contractile Ring Formation (A) En face series from time-lapse experiments showing the typical chromosomal changes in control oocytes (top) and oocytes injected with Cdc42T17N (bottom). Time lapses (in minutes) are from the first appearance of a maturation spot (i.e, GVBD = 0). PB, first polar body. (B) Time-lapse experiments showing the F-actin (green) contractile ring in relationship with the spindle (red) in control oocytes (first row, en face series; second row, Z series). Similar experiments (third row, en face series; fourth row, Z series) reveal the lack of contractile ring in oocytes injected with Cdc42T17N, despite the correct spindle positioning and anaphase initiation. Time lapses (minutes) are from the first appearance of a maturation spot (i.e., GVBD = 0). Current Biology 2006 16, 214-220DOI: (10.1016/j.cub.2005.11.067) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 3 Spindle-Associated Activation of Cdc42 during Meiosis I in Frog Oocytes (A) En face series from 4D movie showing metaphase I spindle (red) and GFP-wGBD (green). An increase in wGBD signal is apparent (arrowheads, 51:20) coincident with metaphase I spindle assuming perpendicular orientation relative to the cortex. (B) Double-label Z series of wGBD (green) and metaphase I spindle (red) during the formation of the first polar body. Active Cdc42 is initially apparent as a patch over the metaphase I spindle (00:00; equivalent to 51:20 in [A]) and then spreads downward from its concentration above the spindle (arrow, 02:00). Active Cdc42 ultimately surrounds first polar body (16:40). All times are in min:sec from the start of imaging. Current Biology 2006 16, 214-220DOI: (10.1016/j.cub.2005.11.067) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 4 Coordination of RhoA and Cdc42 in Polar Body Formation (A) Double-label 4D movie series showing eGFP-rGBD (indicating active RhoA, red) and GFP-wGBD (indicating active Cdc42, green). RhoA activity first appears as a zone around a patch of Cdc42 activity (3:40). As RhoA zone contracts to form the first polar body, Cdc42 activity spreads to cover the polar body (more evident in the Z series, 11:20). Note the lack of colocalization of rGBD and wGBD in any of the pictures. All times in min:sec from the start of imaging. (B) The schematic (top, en face view; bottom, lateral view) depicts the relationship between RhoA activity zone (red) and Cdc42 activity zone (green) and the possible microtubule-mediated activation of the two small GTPases. Brown circles represent spindle poles (microtubule organizing centers). Black lines are microtubules, with their plus ends (+) and minus ends (−) indicated. Purple bars are separating chromosomes in late anaphase or telophase. Current Biology 2006 16, 214-220DOI: (10.1016/j.cub.2005.11.067) Copyright © 2006 Elsevier Ltd Terms and Conditions