Abstract
Factors that are involved in actin polymerization, such as the Arp2/3 complex, have been found to be packaged into discrete, motile, actin-rich foci. Here we investigate the mechanism of actin-patch motility in S. pombe using a fusion of green fluorescent protein (GFP) to a coronin homologue, Crn1p. Actin patches are associated with cables and move with rates of 0.32 μm s−1 primarily in an undirected manner at cell tips and also in a directed manner along actin cables, often away from cell tips. Patches move more slowly or stop when actin polymerization is attenuated by Latrunculin A or in arp3 and cdc3 (profilin) mutants. In a cdc8 (tropomyosin) mutant, actin cables are absent, and patches move with similar speed but in a non-directed manner. Patches are sites of Arp3-dependent F-actin polymerization in vitro. Rapid F-actin turnover rates in vivo indicate that patches and cables are maintained continuously by actin polymerization. Our studies give rise to a model in which actin patches are centres for actin polymerization that drive their own movement on actin cables using Arp2/3-based actin polymerization.
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Acknowledgements
We thank B. Goode for showing us the S. pombe coronin homologue sequence in the database, and L. Pon and members of her laboratory for discussions and insights. We also thank K. Gould for the arp3 strain, D. Drubin for LatA, J. Q. Wu and J. Bahler for kanMX plasmids, S. Randall (Improvision), and B. Semon (Morrell Instruments, New Jersey) for microscopy support, and R. Lustig for technical assistance. We are grateful to B. Feierbach for assistance with molecular biology and for support and suggestions throughout this study. We thank L. Pon, B. Feierbach and D. Drubin and his laboratory for comments on the manuscript. R.J.P. was supported by an NIH predoctoral training grant; FC was supported by the NIH, the American Cancer Society, a March of Dimes Basil O'Connor Scholar award and the Irma T. Hirschl Charitable Trust.
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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. (MOV 396 kb)
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. (MOV 403 kb)
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 μm). The time inter-val between frames is 5 s. Scale bar represents 5 μm. (MOV 176 kb)
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 μm apart and go from the upper cell surface to the lower. Scale bar represents 5 μm. (MOV 188 kb)
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 μm. (MOV 164 kb)
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 μm. (MOV 245 kb)
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 μm 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 5μm. (MOV 769 kb)
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 μm. (MOV 198 kb)
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 µm. (MOV 742 kb)
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 μm. (MOV 209 kb)
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 μm. (MOV 1441 kb)
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 μm. (MOV 1853 kb)
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 μm. (MOV 1897 kb)
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 μm. (MOV 1756 kb)
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 μm. (MOV 251 kb)
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 μm. (MOV 472 kb)
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Pelham, R., Chang, F. Role of actin polymerization and actin cables in actin-patch movement in Schizosaccharomyces pombe. Nat Cell Biol 3, 235–244 (2001). https://doi.org/10.1038/35060020
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DOI: https://doi.org/10.1038/35060020