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. 2009 Dec 15;20(24):5195–5210. doi: 10.1091/mbc.E09-05-0428

Figure 3.

Figure 3.

Cdc12p nodes nucleate and elongate actin filaments in random directions during contractile-ring formation. Cdc12p (red) and actin (green) in merged images. (A–E) Cdc12p-tdTomato GFP-CHD (strain JW1452). (A) A time-lapse series of a meshwork of actin filaments condensing into a ring. Only the GFP signal is shown. The meshwork forms at 1–2 min relative to time zero (white arrow), whereas structures similar to the actin aster and leading cable are seen at 5–6 min (red arrowhead). (B) Radial projections (see Materials and Methods) of Cdc12p nodes and GFP-CHD marked actin filaments of Z-sections spaced at 0.2 μm showing random actin-filament orientation (see Video 6). The long axis of the cell is vertical, but only the medial region is shown as indicated in the schematic to the left (red box). Right, enlarged region (white box) with arrows pointing to two nodes connected by a single actin filament. (C) Montage showing elongation of an actin filament/bundle (green) from a Cdc12p node (red, arrowhead) at high temporal resolution (see Video 7). The node signal from the first frame was smudged (averaging the intensity values over a box of 3 × 3 pixels in the x and y directions) and repeated in all frames for better visualization. (D and E) Histograms of orientations and lengths of actin filaments, n = 317 filaments from 14 cells. (D) Filaments from the top (dark) and bottom half (light) of the cells exhibit a random filament angle distribution. (F) Synchronized cdc25-22 cells (JW1465) after releasing to 25°C at the same time that Lat-A was washed out. These cells have nodes and actin filaments at the division site. (G) Histogram of orientations of >500 filaments binned every 10° including all data from asynchronous and synchronized cultures including data from (D). (H) Time course of new actin filaments growing from Cdc12p nodes after Lat-A was washed out in six for3Δ cells with the long axis of the cell positioned vertically (JW1488). The region of the cells that is shown corresponds to the schematic in B, but these are much longer cells after synchronization. Times given are from Lat-A washout. Top indicates maximum intensity projections of slices 3–9 of 26 sections spaced at 0.2 μm (72 min) or slices 1–5 of 11 sections spaced at 0.5 μm (87, 90 min). Bottom, maximum intensity projections of slices 15–21 (72 min) or slices 6–10 (87, 90 min). (I) Kymograph constructed along the length of an actin filament in JW1488 cells from H, showing elongation of an actin filament (green) from a Cdc12p node (red, arrowhead). The node signal from the first frame was smudged and repeated in all frames for better visualization. Bars, 5 μm; except in B enlargement and C, bars, 1 μm.