(C) PLOS One This story was originally published by PLOS One and is unaltered. . . . . . . . . . . The Ync13–Rga7–Rng10 complex selectively coordinates secretory vesicle trafficking and secondary septum formation during cytokinesis [1] ['Sha Zhang', 'Department Of Molecular Genetics', 'The Ohio State University', 'Columbus', 'Ohio', 'United States Of America', 'Davinder Singh', 'Yi-Hua Zhu', 'Katherine J. Zhang', 'Alejandro Melero'] Date: 2025-11 Cytokinesis requires precise coordination of contractile-ring constriction, vesicle trafficking and fusion to the plasma membrane, and extracellular matrix assembly/remodeling at the cleavage furrow to ensure faithful cell division and maintain cell integrity. These processes and proteins involved are broadly conserved across eukaryotes, yet molecular mechanisms controlling the spatiotemporal pathways of membrane trafficking remain poorly understood. Here, using fission yeast genetics, microscopy, and in vitro binding assays, we identify a conserved module including the Munc13 protein Ync13, F-BAR protein Rga7, and coiled-coil protein Rng10 to be critical for precise and selective vesicle targeting to the plasma membrane during cytokinesis. The module specifically recruits the TRAPP-II but not the exocyst complex to tether vesicles containing the glucan synthases Bgs4 and Ags1 along the cleavage furrow. Ync13 subsequently interacts with the SM protein Sec1 for vesicle fusion. Mutations in this pathway disrupt septum integrity and lead to cell lysis. Our work provides key insights into how membrane trafficking is tightly controlled to maintain cell integrity during cytokinesis. Funding: The work was supported by the National Institute of General Medical Sciences of the National Institutes of Health grants R01 GM118746 and GM118746-06S1 to J-QW and by Swiss National Science Foundation grant 310030_191990 to SGM. J-QW was supported by fellowships from the Swiss National Science Foundation (grant ISZEZ0_200027) and Herbette Foundation at UNIL to SGM during his faculty professional leave. SZ was supported by the Pelotonia Postdoctoral Fellowship Program. Y-HZ was supported by the Pelotonia Graduate Fellowship Program. KJZ was supported by the Pelotonia Undergraduate Fellowship Program. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. In this study, we aimed to map out some key spatiotemporal pathways for plasma membrane deposition and septum formation during cytokinesis using fission yeast as a model system by mistargeting proteins to mitochondria, co-immunoprecipitation, in vitro binding assays, genetic and cellular methods, live-cell confocal microscopy, and electron microscopy. We find that Ync13 regulates exocytosis by interacting with Rga7–Rng10, the SM protein Sec1, and the TRAPP-II complex. The Ync13–Rga7–Rng10 module selectively controls the accumulation and distribution of glucan synthases involved in secondary septum formation, such as Bgs4 and Ags1; rather than Bgs1, which is essential for the primary septum assembly. Consistently, the secondary septum is defective in ync13 mutants, which leads to cell lysis during daughter-cell separation. Collectively, we find that the Ync13–Rga7–Rng10 module and the TRAPP-II complex are central players for plasma membrane dynamics and secondary septum formation during cytokinesis. Rga7 is a Rho2 GAP in fission yeast that plays crucial roles in cytokinesis [ 73 – 76 ]. It contains an F-BAR domain necessary for its membrane association and a Rho GAP domain at its C-terminus. Rga7 localizes to the plasma membrane at the cell tips during interphase and relocates to the division site during cytokinesis, a process dependent on its interaction with the coiled-coil protein Rng10 and membrane lipids [ 73 – 76 ]. Without Rng10, Rga7 essentially disappears from the division site, leading to defective septum formation and cell lysis [ 74 , 75 ]. Rga7 collaborates with F-BAR proteins Cdc15 and Imp2, C2 domain-containg protein Fic1, and the paxillin Pxl1 to maintain actomyosin ring stability and ensure successful septum formation and separation [ 32 , 73 , 77 – 79 ]. Similar to Ync13, Rga7 and Rng10 collaboratively regulate the accumulation and dynamics of glucan synthases [ 74 , 75 ]. Rga7 is reported to facilitate the trafficking of β-glucan synthase Bgs4 from the Golgi to the plasma membrane [ 76 ]. Cells lacking Rga7 show defects similar to those observed in bgs4 and ync13 mutant cells [ 74 , 75 ]. However, the relationships between Ync13, Rga7–Rng10, the TRAPP-II complex, and Sec1 in cytokinesis were unknown. Endocytosis also plays essential and dynamic roles in cytokinesis, particularly in membrane remodeling, signal transduction, and recycling membrane proteins. Cells internalize membrane lipids and proteins from the plasma membrane mainly through the clathrin-mediated endocytosis from the growth and division sites [ 59 – 65 ]. During cytokinesis, endocytosis helps retrieve old/inactive or excess membrane proteins and recycle materials to be reused for septum formation or membrane expansion [ 5 , 61 , 63 , 66 ]. Thus, endocytosis works hand-in-hand with exocytosis to maintain the proper levels of lipids, glucan synthases, and other proteins needed for plasma membrane deposition and septum formation. Yeast endocytosis is actin-dependent and many endocytic mutants in genes such as the endocytic adaptor ede1, fimbrin fim1, and clathrin light chain clc1 result in furrow ingression defects, cell wall abnormalities, or cytokinesis failure [ 59 , 67 – 73 ]. The coordination of exocytosis and endocytosis is essential for successful cytokinesis. Exocytosis delivers the proteins and membranes needed for furrow ingression and extracellular matrix (including cell wall) formation or remodeling to the division site [ 16 – 18 , 43 ]. Exocytosis, a highly regulated process, involves several sequential stages: vesicle delivery/trafficking, tethering and docking onto the plasma membrane, priming of the fusion machinery, and membrane fusion via SNARE complex assembly [ 5 , 44 – 48 ]. In fission yeast cytokinesis, the TRAPP-II complex recognizes and tethers secretory vesicles along the whole cleavage furrow, while the octameric tethering complex exocyst mainly tethers vesicles that contain the glucanases Eng1 and Agn1 and other cargos at the rim of the division plane although it also functions along the furrow [ 6 , 42 , 49 ]. During mammalian synaptic exocytosis, after vesicle tethering, three SNARE proteins—synaptobrevin-2 (Syb2) on synaptic vesicles, and syntaxin-1 (Syx1) and SNAP-25 (SN25) on the plasma membrane form a ternary trans-SNARE complex [ 50 – 52 ]. This complex brings the vesicle and plasma membrane into close proximity to facilitate membrane fusion. Vesicle tethering and fusion are regulated by Rab GTPases and several other proteins including: the Sec1/Munc18 (SM) family protein Munc18-1, which initially locks Syx1 in a closed conformation to inhibit SNARE complex assembly; and the Munc13/UNC-13 family protein Munc13-1, which catalyzes the transition from the Munc18-1/Syx1 complex to the SNARE complex in the presence of SN25 and Syb2 [ 53 – 57 ]. In S. pombe, Ync13 is the homolog of Munc13 and UNC-13. Ync13 localizes to cell tips during interphase and the plasma membrane at the cleavage furrow during cytokinesis [ 58 ]. Deletion of Ync13 (ync13Δ) results in defective exocytosis, impaired endocytosis, uneven distribution of cell wall enzymes at the division site, and extensive cell lysis during cell separation [ 58 ]. However, the regulatory mechanisms of Ync13 in membrane dynamics, its binding partners, and the exact cause of cell lysis upon its deletion remain unclear. The septum is primarily constructed by three essential transmembrane glucan synthases. The β-Glucan synthase Bgs1/Cps1 synthesizes the linear (1,3)β-glucan for the primary septum [ 7 – 11 ], while the β-glucan synthase Bgs4/Cwg1 and the α-glucan synthase Ags1/Mok1 are mainly involved in the formation of the secondary septum [ 9 , 12 – 15 ]. Once the septum is complete and mature, daughter cells separate via digesting the primary septum mainly by the glucanases Eng1 and Agn1 [ 16 – 18 ]. Proper plasma membrane deposition, septum formation, and daughter-cell separation are essential for maintaining cell integrity and viability during cytokinesis, especially due to the high cellular turgor pressure in fungal cells [ 19 – 22 ]. The F-BAR protein Cdc15, transmembrane protein Sbg1, Rho1 GTPase, and other proteins help recruit and/or activate Bgs1 behind the contractile ring to build the primary septum [ 10 , 23 – 31 ]. Cdc15 binds to the plasma membrane through its F-BAR domain and helps deliver Bgs1 to the plasma membrane while the paxillin Pxl1 mediates the interaction of Bgs1 to the contractile ring [ 25 , 32 , 33 ]. Septins, the anillin Mid2, the exocyst complex, the Rho3 and Rho4 GTPases, and Rho4 GEF Gef3 concentrate the glucanases Eng1 and Agn1 to the rim of the division plane for daughter-cell separation [ 34 – 42 ]. In addition, the Transport Particle Protein-II (TRAPP-II) complex is also important for Eng1’s localization at the centroid of the division plane [ 6 , 10 , 23 – 29 ]. However, the mechanisms that control the precise spatiotemporal localizations of Bgs4 and Ags1 on the plasma membrane at the division plane for secondary septum formation remain poorly understood. Cytokinesis is a highly conserved cellular process occurring at the late stage of the cell division cycle, resulting in the generation of two daughter cells. From fungi to humans, this process involves coordinated actions of division site selection, actomyosin contractile ring assembly and constriction, plasma membrane deposition and invagination at the cleavage furrow, and extracellular matrix formation or remodeling [ 1 – 6 ]. In fungal cells, including the fission yeast Schizosaccharomyces pombe, two layers of new plasma membranes and a division septum form behind the constricting ring during cytokinesis. The septum is a three-layered cell wall structure that consists of a middle primary septum flanked by secondary septa on each side. Results Ync13 is a key functional effector of the Rga7-Rng10 complex for vesicle targeting during cell division We found that Ync13, Rga7, and Rng10 form a complex that helps the TRAPP-II complex tether vesicles associated with secondary, but not primary, septum formation. To test whether Ync13 serves as a functional effector of the Rga7–Rng10 subcomplex, we mildly overexpressed Ync13 using the strong 3nmt1 promoter under repressing condition (YE5S rich medium with thiamine) and/or artificially retained Ync13 at the division site using the GBP-GFP system in rga7Δ cells. Specifically, 3nmt1-mECitrine-ync13 was retained at the division site by GBP-tagged Pmo25, which is an MO25 family protein that mostly colocalizes with Ync13 on the division plane, although Pmo25 also localizes to one of the spindle pole bodies [97–100]. Ync13 is not an abundant protein [58] and its division-site level is reduced by ~85% in rga7Δ cells (Fig 3B), thus mild overexpression is necessary for these assays. rga7Δ cells had severe lysis and negligible growth on plates with Phloxine B (a red dye accumulated in lysed or unhealthy cells because they cannot pump it out) at both 25 and 36°C (Fig 6A–6D). Mild overexpression of Ync13, but not expressing Pmo25-GBP alone, reduced cell lysis of rga7Δ cells from 78% to 51% at 36°C and promoted more growth at both 25 and 36°C (Fig 6A–6D, compare strains 1, 5, and 6). As expected, microscopy revealed robust localization of Ync13 at both the division site and the spindle pole body in rga7Δ cells expressing both 3nmt1-mECitrine-ync13 pmo25-GBP, and this tethered Ync13 persisted along the cleavage furrow throughout cytokinesis and septum formation (Fig 6A and 6B, compare strains 3, 4, 5, and 7). Importantly, enforced division-site localization of Ync13 dramatically rescued the cytokinesis and cell lysis defects and growth of rga7Δ cells at both 25 and 36°C, as evidenced by further decreased cell lysis to 15% in rga7Δ 3nmt1-mECitrine-ync13 pmo25-GBP cells at 36°C (Fig 6A–6D; compare strains 1, 5–7). Together, these findings indicate that Ync13 is a key functional effector acting downstream of the Rga7–Rng10 complex at the division site. PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 6. Mild overexpression and forced retention of Ync13 at the division site rescue rga7Δ. (A–D) The genotypes of the seven strains used are listed between panels (C) and (D). (A and B) Micrographs showing cell morphology and/or Ync13 localization in the indicated strains in rga7+ or rga7Δ cells at 25°C (A) or 36°C (B). Cells were grown for 36–40 h at 25°C in YE5S + thiamine medium and then imaged directly (A) or shifted to 36°C for 3.5 h before imaging. Arrowheads mark the examples of mECitrine-Ync13 mistargeting to the SPB by Pmo25-GBP in strains 4 and 7. Note, mECitrine-Ync13 tends to form irregular aggregates at 36°C but not 25°C. Scale bars, 5 µm. (C) Quantification of cell lysis for cells grown at 36°C for 3.5 h as in (B). (D) Growth assay illustrating the rescue of rga7Δ by tethering mECitrine-Ync13 to the division site using Pmo25-GBP or by mildly overexpressing Ync13 using the 3nmt1 promoter. Cells were grown on YE5S + PB plates for 24 h at 25°C or 36°C. The underlying data (for panel C) can be found in S1 Raw Data file. https://doi.org/10.1371/journal.pbio.3003466.g006 To test if there is a correlation between Ync13 levels and vesicle fusion at the division site, we mildly overexpressed Ync13 from the 3nmt1 promoter in YE5S rich medium without additionally added thiamine to obtain cells with different Ync13 levels (the rich medium has some residual amount of thiamine, which partially represses the nmt1 promoter) and then tracked the Rab11 GTPase Ypt3 labeled vesicles. This resulted in increased levels of Ync13 as well as Ypt3 at the division site (S8B Fig). We measured the Ync13 intensity at division site and counted the number of Ypt3 vesicles reaching the division site in 2-min continuous movie at the middle focal plane. We observed that increasing Ync13 level promoted the tethering and accumulation of more Ypt3 vesicles at the division site until it reached a plateau (S8B Fig). Collectively, Ync13, working with Rga7 and Rng10, plays an important role in vesicle targeting and fusion on the plasma membrane at the division site during cytokinesis. [END] --- [1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3003466 Published and (C) by PLOS One Content appears here under this condition or license: Creative Commons - Attribution BY 4.0. via Magical.Fish Gopher News Feeds: gopher://magical.fish/1/feeds/news/plosone/