Supplementary information

From the following article

Role of actin polymerization and actin cables in actin-patch movement in Schizosaccharomyces pombe

Robert J. Pelham, Jr & Fred Chang

Nature Cell Biology 3, 235 - 244 (2001) Published online: 1 February 2001

doi:10.1038/35060020

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Movie 1

Three-dimensional images of the S. pombe actin cytoskeleton.
Rotating image of a three-dimensional reconstruction of wild-type cells stained with phalloidin-conjugated AlexaFluor 488 to visualize the actin cytoskeleton. Cells are slightly flattened because of pressure from the coverslip. Individual frames are pre-sented sequentially at projections of 2° angles, rotating about the y-axis.

Movie 2

Three-dimensional images of the S. pombe actin cytoskeleton.
Rotating image of a three-dimensional reconstruction of wild-type cells as in Movie 1, rotating about the x-axis.

Movie 3

Actin patches are highly dynamic.
Time-lapse recording of actin-patch dynamics in S. pombe cells expressing Crn1p–GFP, which labels actin patches. Images are two-dimensional projections of 5 optical sections (1 mum). The time inter-val between frames is 5 s. Scale bar represents 5 mum.

Movie 4

Actin patches are located both in the cytoplasm and in association with the cell cortex. Series of optical sections of a wild-type cell expressing Crn1p–GFP. Eighteen optical sections are spaced 0.2 mum apart and go from the upper cell surface to the lower. Scale bar represents 5 mum.

Movie 5

Actin patches exhibit directed and non-directed movements.
Time-lapse recording of actin patches exhibiting directed movement along linear tracks, and non-directed movement at cell tips. The time interval between frames is 0.5 s. The first frame is represented by Fig. 2a, 0 s. Scale bar represents 5 mum.

Movie 6

Time-lapse recording of an actin patch exhibiting directed movement along a Crn1p–GFP cable.
The time interval between frames is 0.5 s. The first frame is rep-resented by Fig. 2a, 0 s. Scale bar represents 5 mum.

Movie 7

Movement of actin patches requires actin polymerization.
Time-lapse recording showing inhibition of actin-patch movement after attenuation of actin polymerization. Cells were treated with 50 mum LatA for 2 min and movement of Crn1p–GFP patches in live cells was then recorded. Inhibition of actin polymerization with LatA abolishes actin-patch movement. The time interval between frames is 0.5 s. The first frame is represented by Fig. 3a, left panel. Scale bar represents 5mum.

Movie 8

Wild-type control treated with 1% dimethylsulphoxide (DMSO).
Control time-lapse recording of actin patch-movement after treatment with 1% DMSO for 2 min. This treatment does not affect the pattern or rate of actin-patch movement. The time interval between frames is 0.5 s. The first frame is represent-ed by Fig. 3a, right panel. Scale bar represents 5 mum.

Movie 9

Arp3 is required for movement of actin patches.
Time-lapse recording showing reduction of actin-patch movement in the cold-sensitive arp3 mutant. Cells were shifted to 19 °C for 100 min before imaging Crn1p–GFP fluorescence. The time interval between frames is 0.5 s. The first frame is represented by Fig. 4a, arp3. Scale bar represents 5 microm.

Movie 10

Profilin is required for movement of actin patches.
Time-lapse recording showing reduction of actin-patch movement in the temperature-sensitive cdc3 (profilin) mutant. Cells were shifted to 36 °C for 4 h before imaging Crn1p–GFP fluorescence. The time interval between frames is 0.5 s. The first frame is represented by Fig. 4a, cdc3. Scale bar represents 5 mum.

Movie 11

Microtubules are not required for movement of actin patches.
Time-lapse recording showing that actin-patch movement is unaffected in cells treated with 25 mg ml–1 MBC for 10 min to depolymerize microtubules. The time interval between frames is 0.5 s. The first frame is represented by Fig. 4a, +MBC. Scale bar represents 5 mum.

Movie 12

Wild-type control treated with 1% DMSO.
Control time-lapse record-ing of actin-patch movement after treatment with 1% DMSO for 10 min. This treat-ment does not effect the pattern or rate of actin-patch movement. The time interval between frames is 0.5 s. The first frame is represented by Fig. 4a, + 1% DMSO. Scale bar represents 5 mum.

Movie 13

Wild-type 36 °C control.
Control time-lapse recording of actin-patch movement after a shift to 36 °C for 4 h. Incubation at 36 °C does not effect the pattern or rate of actin-patch movement. The time interval between frames is 0.5 s. The first frame is represented by Fig. 4a, 36 °C. Scale bar represents 5 mum.

Movie 14

Wild-type 19 °C control.
Control time-lapse recording of actin-patch movement after a shift to 19 °C for 100 min. Incubation at 19 °C does not effect the pattern or rate of actin-patch movement. The time interval between frames is 0.5 s. The first frame is represented by Fig. 4a, 19 °C. Scale bar represents 5 mum.

Movie 15

Directional movement of actin patches requires tropomyosin.
Time-lapse recording showing loss of directed actin-patch movement in the temperature- sensitive cdc8 (tropomyosin) mutant, which lacks actin cables. Cells were shifted to 35.5 °C for 25 min before imaging Crn1p–GFP fluorescence. The time interval between frames is 0.5 s. The first frame is represented by Fig. 5a, cdc8. Scale bar represents 5 mum.

Movie 16

Wild-type 35.5 °C control after 25 min.
Control time-lapse recording of actin-patch movement after a shift to 35.5 °C for 25 min. Incubation at 35.5 °C does not effect the pattern or rate of actin-patch movement. The time interval between frames is 0.5 s. The first frame is represented by Fig. 5a, wild type. Scale bar represents 5 mum.

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