Figure 2.

Cdc12p nodes appear similar to known nodes and are dynamically associated with Rlc1p nodes. (A) Cdc12p-3YFP (KV346) and mYFP-Cdc15p (JW1054) localize to a broad band of nodes (arrowheads) at the equator of cells. Left, wild-type cells (JW81) with low autofluorescence when excited at 514 nm. Note Cdc15p also localizes to patches concentrated at the cell tips. (B) Time-lapse series of lateral condensation of Cdc12p and Cdc15p nodes into contractile rings (see Videos 3 and 4). Time zero is an arbitrary time with nodes present in both cells. (C) Cdc12p colocalizes with Rlc1p in nodes. Cells expressing Cdc12p-3YFP (middle, JW1404), Rlc1p-mCherry (bottom, JW1340), or both (top, JW1413) were imaged using exactly the same settings. The boxed regions in the double-tagged strain are enlarged, and nodes are circled to show the colocalization of nodes. Cdc12p-3YFP was imaged with 488-nm excitation and an emission filter for YFP, resulting in less obvious speckles. (D) Time course of condensation of Rlc1p and Cdc12p nodes (strain JW1411) during contractile-ring assembly. (E and F) Dynamics of Cdc12p nodes and ring revealed by FRAP of Cdc12p-3YFP (JW1404). (E) Montages are from time-lapse microscopy during FRAP (see Materials and Methods). Cells were bleached within the white box at time zero. (i) Middle section of Cdc12p-3YFP ring. (ii) The slice with nodes chosen for FRAP (see Video 5). (iii) Maximum intensity projection (left) of the cell in ii and the chosen slice for bleaching (right). (F) Recovery of the Cdc12p ring (n = 14 cells, red) and nodes (n = 11 cells, gray). Relative intensity is normalized to the average prebleach intensity, and the immobile fraction for both is ∼30%. Bars, 5 μm, except in C enlarged regions, bar, 1 μm.