Two Woronin body interacting proteins localize in rings around the septal pore. (A) Isolation of Woronin body (WB)-associated proteins. The indicated fractions were separated by SDS/PAGE. The Woronin body-specific membrane marker Woronin sorting complex (WSC) is GFP-tagged (WSC-GFP), and it allowed assessment of Woronin body enrichment during the purification process (Lower). HEX, WSC-GFP, SPA1, and SPA2 are indicated. (Scale bar: 2 μm.) (B) SPA1 localizes at the septal pore and rearranges in stressed hyphae (stress) and during wound-induced (wounding) membrane resealing. (Bottom) Expanded views of the indicated regions are presented. (Scale bars: Upper and Middle, 5 μm; Bottom, 1 μm.) (C) SPA2-GFP is localized to the pore, and its fluorescence coincides with refractive structures that can be observed by differential interference contrast microscopy (DIC) (arrow). (Scale bar: 1 μm.) (D) SPA proteins associate with Woronin bodies in cellular extracts. (Scale bar: 1 μm.) (E) Detergent-treated cell wall ghosts retain SPA1-GFP rings. Arrows point to septa. (Scale bars: 10 μm; Inset, 1 μm.) (F) SPA1 and SPA2 interactions are shown by yeast two-hybrid assay. The indicated versions of the activation domain (AD) and binding domain (BD) were expressed in yeast and assayed on the indicated media. his, histidine; ade, adenine, αGal, 5-Bromo-4-chloro-3-indoxyl-α-D-Galactopyranoside. (G) Septal pore-associated electron-dense aggregates as seen by TEM. White arrows indicate peripheral material, and the white arrowhead points to central pore-occluding material. The black arrow points to plasma membrane traversing the pore. An asterisk identifies artifact of the staining process. (Inset) Another septum from the same experiment, which is open and through which a mitochondrion (m) is trafficking. (Scale bar: 200 nm.)

SPA proteins localize to the septal pore and possess features of intrinsically disordered proteins. (A) Images show the localization of the indicated SPA proteins at the septal pore. Type I localization produces a fine ring around the septal pore rim. Type II signal emanates from the center of the septal pore, and type III localization occurs in a broad disk centered at the septal pore. DIC, differential interfering contrast microscopy. (Scale bar: 2 μm.) (B) Charge-hydropathy analysis of SPA proteins (red □) and folded (○), FG-repeat (△), and SR-repeat splicing (◇) factors. The order/disorder boundary is indicated with a solid line. (C) Amino acid composition of disordered domains from SPA, FG-repeat (FG), SR-repeat (SR), and disordered proteins (Disprot) is shown relative to folded globular domains.

Disorder, coiled-coil domains, and sequence conservation of SPA proteins. The y axis indicates the predicted probability of disorder [red line; IUPred ()] and predicted coiled-coil domains (blue line). The x axis corresponds to amino acid sequence, and green bars indicate approximate regions of primary sequence conservation with homologous sequences from other filamentous Ascomycetes. Regions with homology to known domains [Shg1 homologous sequence in SPA5 and annexin domain in SPA14] are identified with a blue bar. DO, disordered; OR, ordered.

Biochemical analysis of SPA aggregates and in vitro assembly of SPA domains. (A) Cytosolic extract from WT and hex mutant hyphae was fractionated by centrifugation into supernatant (S) and pellet (P) fractions in the absence and presence of Triton X-100 (+TX-100), and SPA1 distribution was determined by Western blotting. T, total extract. (B) Stability of SPA1 aggregates. A 10,000 × g pellet fraction was resuspended in the indicated chemicals, followed by centrifugation to produce supernatant and pellet fractions. SPA1 distribution was determined by Western blotting. (C) His-tagged versions of SPA1^OR, SPA1^DO, and SPA5^DO domains were purified under denaturing conditions and dialyzed into Tris-buffered saline, followed by microscopic examination and fractionation into pellet and supernatant fractions by centrifugation. Lysozyme, BSA, SOFT^DO, and Nsp-1 subjected to the same conditions produced no visible aggregates and remained in the S fraction. DO, disordered; OR, ordered. (D) SPA1^OR aggregates were collected by centrifugation (arrow) and examined by SEM. Insets show enlarged SPA sphere (Lower Left) and disassembly of concentrated SPA1^OR aggregates in 2 mM SDS (Lower Right) are shown. (Scale bars: 10 μm; Inset, 2 μm.) (E) SPA5^DO aggregates have gel-like properties. Individual images correspond to frames extracted from Movie S1. (Scale bar: 50 μm.) (F) Both SPA1^OR and SPA1^DO domains are capable of recognizing the septal pore. (Left) Cartoon shows the genomic structure produced by marker fusion tagging. (Right) Localization is shown. DIC, differential interfering contrast microscopy. (Scale bar: 2 μm.)

SPA proteins regulate Woronin body function, septation, and septal pore stability. (A) SPA9 controls Woronin body release. (Top) spa9 deletion strain exhibits impaired colony growth and intrahyphal hyphae. (Middle) Deletion of HEX eliminates Woronin bodies and suppresses the growth defect (graph). DIC, differential interfering contrast microscopy. (Bottom) Localization of GFP-SPA9 to cortical Woronin bodies during the systemic response to injury. (Scale bar: 1 μm.) (B) SPA10 regulates septation. The spa10 deletion mutants develop normal numbers of septa in apical compartments but elaborate abnormally high numbers of septa as the hypha ages. Septa were counted in the apical and subapical compartments ∼1 cm behind the colony growth front. Septal numbers are quantified in the graph (Upper), and representative hyphae are shown (Lower). (Scale bar: 10 μm.) (C) SPA13 is required for septal pore maintenance. SPA13 deletion mutants “bleed” protoplasm exclusively from subapical regions of the colony (arrowheads), and reconstruction of septal pores using confocal microscopy shows that this phenotype is due to septal pore degeneration in old regions of the colony. A, apical; SA, subapical; +, point of colony initiation. (Scale bar: 2 μm.)