Assembly Mechanism of the Contractile Ring for Cytokinesis by Fission Yeast
Dimitrios Vavylonis, Jian-Qiu Wu, Steven Hao, Ben O'Shaughnessy, Thomas D. Pollard

Supporting Online Material

This supplement contains:
Materials and Methods
Figs. S1 to S18
Table S1
References

This file is in Adobe Acrobat PDF format.

Other Supporting Online Material for this manuscript includes the following:
(available at www.sciencemag.org/cgi/content/full/1151086/DC1)
Movies S1 to S19

Movie S1
Rotated 3D reconstruction of 81 z-sections of 0.1 µm of the broad band of a cell expressing Rlc1p-3GFP (strain JW1258) showing the distribution of nodes near the plasma membrane.

Movie S2
Time-lapse series of a cell expressing Rlc1p-3GFP showing the condensation of nodes into a ring through stochastic node motions. Movie shows a single focal plane at the top of a cell. The time interval between frames is 1 s (display rate: 8X) and the movie is corrected for photobleaching. The long axis of the cell runs from left to right. To increase the signal to noise ratio at the expense of time resolution, each frame shown is the average of six frames of the original movie (frame 1 is the average of original frames 1-6, frame 2 the average of 2-7, etc).

Movie S3
Movie showing the results of a simulation. The time interval between frames is 1 s. Display rate: 8X. The parameter values are the same as those of Fig. 1J of the main text. Nodes in red, actin filaments in green (broadened to optical resolution).

Movie S4
Enlarged region of a portion of Movie S3. Right part shows nodes in red and actin in green. Left part shows only the nodes, whose motions are similar to those in experiment (Movie S2). Display rate: 8X.

Movie S5
Time-lapse series of a cell expressing Rlc1p-3GFP showing the appearance of nodes at a single focal plane at the top of a cell. The time interval between frames is 1 s (display rate: 8X) and the movie is corrected for photobleaching. The long axis of the cell runs from top left to bottom right. To increase the signal to noise ratio at the expense of time resolution, each frame shown is the average of six frames of the original movie, as for Movie S2. Note that a few nodes at the top of the movie apparently form by a splitting event of a node already present at t = 0. This particular event could have been triggered by movement of material within the cell unrelated to node formation.

Movie S6
Time-lapse series of a cell expressing Rlc1p-3GFP showing stationary nodes at a single focal plane at top of cells. Nodes transition to a mobile phase near frame 300 and start to condense into a contractile ring. The time interval between frames is 1 s (display rate: 8X) and the movie was corrected for photobleaching. The long axis of the cell runs from top to bottom. Each frame shown is the average of six frames of the original movie, as for Movie S2.

Movie S7
A time-lapse series of two representative cdc25-22 cells (strain JW1329) expressing GFP-CHD (green) and Sad1p-mRFP1 (red) showing the timing of actin filament accumulation at the division site relative to the duplication and separation of the spindle pole body labeled with Sad1p-mRFP1. The time zero is the start of imaging. The real time is shown as h:min:s.

Movie S8
A time-lapse series of a cell expressing GFP-CHD showing that the contractile actin ring assembles from a meshwork of actin filaments. A stack of 12 z-sections spaced at 0.6 μm was collected every minute for 41 minutes and projected into a 2D image using maximum intensity projection. The bright spots are actin patches. Display rate: 190 X.

Movie S9
Movie showing that actin filaments connect nodes together to form a network in cdc25-22 cells expressing GFP-CHD and Rlc1p-mRFP1. Stacks of 24 z-sections of 0.3 µm were collected. 3D projections of the middle portions of cells b to d in Fig. 3G are shown. Note: The Fig. 3B shows the maximum intensity projections of the bottom half of the cells a to f.

Movie S10
Time series showing dynamic actin network associating with Rlc1p nodes. The movie shows a single focal plane at the top of a cell expressing GFP-CHD and Rlc1p-tdTomato (strain JW1349). Time interval between frames is 0.3 s. The Rlc1p signal (red) is static and is the average of all the Rlc1p frames from the first 6 sec (the Rlc1p signal per frame is otherwise very weak). The GFP-CHD signal (green) is a moving average over 3 successive frames. The transient bright spots are actin patches. Display rate: 3.6X.

Movie S11
Time series of an actin filament associating with a node before the condensation of nodes into a contractile ring, consistent with a search and capture mechanism. The movie shows a single focal plane at the top of cdc25-22 cells expressing GFP-CHD to mark actin filaments and Rlc1p-tdTomato to mark nodes (strain JW1351). The long axis of the cell is vertical. An actin filament/bundle (green) starts at a node, moves laterally by ~ 0.2 µm and becomes captured by a neighboring node (marked by an arrowhead). The filament/bundle appears to break 10 s after capture. Time interval between frames is 0.2 s. Display rate: 2X.

Movie S12
Time series showing actin filaments dynamically associated with nodes as they condense into a contractile ring in for3 Δ cells expressing GFP-CHD and Rlc1ptdTomato (strain JW1353). The long axis of the cell is vertical. Two nodes near the ring (arrowheads) move closer to one another, then split and move in separate paths. The nodes move after associating with linear elements marked by GFP-CHD, consistent with a search, capture, pull and release mechanism. A filament/bundle appears between the nodes as they merge, while a new filament/bundle originates from the center of the forming ring and seems to pull the nodes apart and towards the contractile ring. A stack of two z-sections spaced at 0.1 µm were collected with an exposure time of 0.2 s for each section in each channel and projected into a 2D image using average intensity projection. The transient bright spots are actin patches. Time interval between frames is 1.8 s. Display rate: 16X.

Movie S13
Time series showing actin filaments marked by GFP-CHD growing from a node, associating with other actin structures and causing the node to move towards the contractile ring. The movie shows a single focal plane at the top of for3 Δ cells expressing GFP-CHD and Rlc1p-tdTomato (strain JW1353). The long axis of the cell is vertical. An actin filament/bundle elongates from a lagging node (marked by arrowhead). The filaments/bundles appear to move laterally as they connect to other filaments before the movement of the node towards the contractile ring. Time interval between frames is 0.8 s. The transient bright spots are actin patches. Display rate: 4X.

Movie S14
Evidence for search, capture, pull and release mechanism. A time series shows actin filaments associating with a lagging node as it moves towards the contractile ring during the late stages of condensation of nodes into a contractile ring in cells expressing GFP-CHD and Rlc1p-tdTomato (strain JW1349). The long axis of the cell is vertical. The movie is consistent with actin filaments (green) growing to capture a lagging node (marked by an arrowhead), and pulling it towards the contractile ring until the filaments break (sudden decrease in local GFP-CHD intensity marked by an arrow). Some actin filaments associated with the node at the beginning of the movie are lost before new filaments establish connections with the node. A stack of two z-sections spaced at 0.2 µm were collected with an exposure time of 0.3 s for each section in each channel and projected into a 2D image using average intensity projection. Fig. 3D shows a montage and kymograph of the cell from a region below the contractile ring. Time interval between frames is 2.5 s. The transient bright spots are actin patches. Display rate: 12.5X.

Movie S15
Time series showing actin filaments associated with and growing from Rlc1p nodes. The movie shows actin filaments transiently connecting two nodes and regrowing from one of these nodes. Single focal plane at the top of a cell expressing GFP-CHD and Rlc1p-tdTomato (strain JW1349). Time interval between frames is 0.3 s. The Rlc1p signal (red) is static and is the average of all the Rlc1p frames from the first 6 sec (the Rlc1p signal per frame is otherwise very weak). The GFP-CHD signal (green) is a moving average over 3 successive frames. The transient bright spots are actin patches. Display rate: 2.1X.

Movie S16
Time series showing actin filaments growing and turning over on the lateral margins of a fully formed contractile ring. The movie shows a single focal plane at the top of for3 Δ cells expressing GFP-CHD and Rlc1p-tdTomato (strain JW1353). The long axis of the cell is vertical. GFP-CHD: left panel and green in the merged panel. Rlc1- tdTomato: middle panel and red in the merged panel. An actin filament/bundle (marked by arrowhead) elongates from the contractile ring. Some filaments (marked by ?) detach (i.e. turnover) from the contractile ring. Time interval between frames is 0.8 s. The bright spots are actin patches. Display rate: 5X.

Movie S17
Movie showing a linear actin element buckling in the broad band. Average of two z slices spaced at 0.3 µm at the top of a cdc25-22 cell expressing GFP-CHD (strain JW1311). The transient bright spots are actin patches. Time interval between frames is 0.8 s. Display rate: 8X.

Movie S18
Times series showing a linear actin filament/bundle breaking at the division site. Movie shows a single focal plane at the top of a cell expressing GFP-CHD (strain FC1218). Only the left side of the cell is shown. Time interval between frames is 0.4 s. The transient bright spots are actin patches. A linear actin filament/bundle (marked with a green arrowhead) grows from the middle portion of the contractile ring to the left. It then detaches from the ring and moves rapidly towards the left side of the cell. Each frame shown is the average of three frames of the original movie (frame 1 is the average of original frames 1-3, frame 2 the average of 2-4, etc). Display rate: 2X.

Movie S19
Time-lapse series of cells expressing Rlc1p-3GFP showing the transient alignment of nodes into linear structures during the late stages of contractile ring formation. The movie shows a single focal plane at the top of a cell. The time interval between frames is 1 s (display rate: 8X). The long axis of the cell runs from top left to bottom right. To increase the signal to noise ratio at the expense of time resolution, each frame shown is the average of six frames of the original movie.

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