Movie S1 Recruitment of BUD-6-GFP to the incipient site of CAR assembly occurs seconds before plasma membrane invagi- nation becomes visible by FM4–64 staining. BUD-6-GFP remains associated to the CAR during septum formation. (MP4) Movie S2 BUD-6-GFP accumulates at homing CAT tips and remains present at the fusion site until cytoplasmic continuity is established. Note the bright clusters of BUD-6 at other locations along the cell cortex, and the disappearing ring of BUD-6 at the fusion site. (MP4) Movie S3 SPA-2-GFP accumulates at homing CAT tips and remains present at the fusion site until cytoplasmic continuity is established. During CAT homing and fusion germ tube growth is arrested, but resumes shortly after fusion pore opening. (MP4) Movie S4 BUD-6-GFP forms a highly dynamic subapical cloud surrounding the Spitzenko¨ rper (stained with FM4–64). The merged seqeunce shows that BUD-6-GFP does not colocalize with the Spitzenko¨ rper. (MP4) Movie S5 Time course of concatenated z-stacks showing BUD-6- GFP recruitment to the septal plug prior to tip repolarization from the sealed septum. BUD-6 and membranous material (FM4–64 staining) accumulate around the Woronin body (phase contrast). (MP4) Movie S6 Time course of concatenated z-stacks showing BUD-6- GFP recruitment during tip repolarization from the sealed septum. BUD-6 accumulations are present left and right to the septal plug even before the new tip emerges. An apical BUD-6 crescent accumulates as soon as elongation growth of the tip commences. (MP4) Movie S7 Time course of concatenated z-stacks showing SPA-2- GFP recruitment during tip repolarization from the sealed septum. SPA-2-GFP becomes recruited to the septal plug and permanently remains associated to the emerging and elongating tip as apical cap. (MP4) References 1. Piel M, Tran PT (2009) Cell shape and cell division in fission yeast. Current Biology 19: R823–827. 2. Pollard TD, Wu JQ (2010) Understanding cytokinesis: lessons from fission yeast. Nature Reviews Molecular Cell Biology 11: 149–155. 3. Bornens M (2008) Organelle positioning and cell polarity. Nature Reviews Molecular and Cellular Biolology 9: 874–886. 4. Li R, Gundersen GG (2008) Beyond polymer polarity: how the cytoskeleton builds a polarized cell. Nature Reviews Molecular Cell Biology 9: 860–873. 5. Perez P, Rincon SA (2010) Rho GTPases: regulation of cell polarity and growth in yeasts. Biochemical Journal 426: 243–253. 6. Slaughter BD, Smith SE, Li R (2009) Symmetry breaking in the life cycle of the budding yeast. Cold Spring Harbor Perspectives in Biology 1: a003384. 7. Wedlich-So¨ldner R, Li R (2008) Yeast and fungal morphogenesis from an evolutionary perspective. Seminars in Cell and Developmental Biology 19: 224–233. 8. Harris SD (2006) Cell polarity in filamentous fungi: shaping the mold. International Review of Cytology 251: 41–77. 9. Harris SD (2008) Branching of fungal hyphae: regulation, mechanisms and comparison with other branching systems. Mycologia 100: 823–832. 10. Harris SD, Momany M (2004) Polarity in filamentous fungi: moving beyond the yeast paradigm. Fungal Genetics and Biology 41: 391–400. 11. Roca GM, Arlt J, Jeffree CE, Read ND (2005) Cell biology of conidial anastomosis tubes in Neurospora crassa. Eukaryotic Cell 4: 911–919. 12. Roca GM, Read ND, Wheals AE (2005) Conidial anastomosis tubes in filamentous fungi. FEMS Microbiological Letters 249: 191–198. 13. Read ND, Lichius A, Shoji J, Goryachev AB (2009) Self-signalling and self- fusion in filamentous fungi. Current Opinion in Microbiology 12: 608–615. Neurospora Crassa Polarisome Architecture Movie S8 BUD-6-GFP recruitment to tips of vegetative fusion hyphae and the rim of the opening fusion pore. Shortly after cytoplasmic continuity between the two hyphae is established, BUD-6-GFP fluorescence disappears from the fusion site. (MP4) Movie S9 Vegetative hyphal fusion at the leading colony edge of Dbni-1. Due to the lack of septa, huge vacuoles accumulate at the edge of the mutant colony, which can be seen passing through fusion pores. (MP4) Movie S10 A group of germlings demonstrating dynamics of BN-1-GFP recruitment during different stages of cell fusion. Taken together, this sequence demonstrates that BNI-1 is present at homing CAT tips, focuses at the attachment site, forms an opening ring of fluorescence during fusion pore formation, and finally disappears from the fusion site. Note, the cell in the middle becomes lifted upwards due to the torque forces transmitted upon cell-cell attachment, and thus disappears from the focal plane. This ‘‘spore-torque’’ is commonly observed when imaging germling fusion of N. crassa in liquid medium. (MP4) Movie S11 Time course of concatenated z-stacks showing BNI- 1-GFP recruitment during tip repolarization from the sealed septum. 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Cellular and Molecular Biology of Filamentous Fungi. Washington DC.: American Society of Microbiology. 17. Jones LA, Sudbery PE (2010) Spitzenko¨ rper, exocyst, and polarisome components in Candida albicans hyphae show different patterns of localization and have distinct dynamic properties. Eukaryotic Cell 9: 1455–1465. 18. Ko¨hli M, Galati V, Boudier K, Roberson RW, Philippsen P (2008) Growth- speed-correlated localization of exocyst and polarisome components in growth zones of Ashbya gossypii hyphal tips. Journal of Cell Science 121: 3878–3889. 19. Harris SD (2009) The Spitzenko¨rper: a signalling hub for the control of fungal development? Molecular Microbiology 73: 733–736. 20. Sudbery P (2011) Fluorescent proteins illuminate the structure and function of the hyphal tip apparatus. Fungal Genetics and Biology 48: 849–857. 21. Lichius A, Berepiki A, Read ND (2011) Form follows function: the versatile fungal cytoskeleton. Fungal Biology 115: 518–540. 22. Berepiki A, Lichius A, Read ND (2011) Actin organization and dynamics in filamentous fungi. Nature Reviews Microbiology (in press). 23. Taheri-Talesh N, Horio T, Araujo-Baza´n L, Dou X, Espeso EA, et al. (2008) The tip growth apparatus of Aspergillus nidulans. Molecular Biology of the Cell 19: 1439–1449. 24. Upadhyay S, Shaw BD (2008) The role of actin, fimbrin and endocytosis in growth of hyphae in Aspergillus nidulans. Molecular Microbiology 68: 690–705. PLoS ONE | www.plosone.org 21 January 2012 | Volume 7 | Issue 1 | e30372