Septins Are Involved at the Early Stages of Macroautophagy in S

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RESEARCH ARTICLE Septins are involved at the early stages of macroautophagy in S. cerevisiae Gaurav Barve1, Shreyas Sridhar1, Amol Aher1, Mayurbhai H. Sahani1, Sarika Chinchwadkar1, Sunaina Singh1, Lakshmeesha K. N.1, Michael A. McMurray2 and Ravi Manjithaya1,*

ABSTRACT blocks, such as amino acids, back to the cytoplasm. The biogenesis Autophagy is a conserved cellular degradation pathway wherein of autophagosomes remains incompletely understood. double-membrane vesicles called autophagosomes capture long-lived In budding yeast cells, the site of autophagosome formation proteins, and damaged or superfluous organelles, and deliver them to is known as the pre-autophagosomal structure (PAS) and is the lysosome for degradation. Septins are conserved GTP-binding perivacuolarly located. Recent work has shown that the PAS proteins involved in many cellular processes, including phagocytosis is tethered to endoplasmic reticulum (ER) exit sites where multiple and the autophagy of intracellular bacteria, but no role in general autophagy proteins colocalize in a hierarchical sequence (Graef autophagy was known. In budding yeast, septins polymerize into ring- et al., 2013; Suzuki et al., 2007). The membrane source for the shaped arrays of filaments required for cytokinesis. In an unbiased developing autophagosome is contributed by the trafficking of Atg9 – – – – genetic screen and in subsequent targeted analysis, we found along with its transport complex (Atg1 Atg11 Atg13 Atg23 – – – autophagy defects in septin mutants. Upon autophagy induction, Atg27 Atg2 Atg18 TRAPIII) to help build the initial cup-shaped pre-assembled septin complexes relocalized to the pre- structure, the phagophore (Legakis et al., 2007; Reggiori et al., 2004; – – autophagosomal structure (PAS) where they formed non-canonical Tucker et al., 2003). Additional recruitment of the Atg5 Atg12 septin rings at PAS. Septins also colocalized with autophagosomes, Atg16 complex as well as Atg8 allows the completion of the where they physically interacted with the autophagy proteins Atg8 and autophagosome (Feng et al., 2014). Atg9. When autophagosome degradation was blocked in septin- Septin proteins bind guanine nucleotides and co-assemble in mutant cells, fewer autophagic structures accumulated, and an hetero-oligomers capable of polymerizing into cytoskeletal filaments autophagy mutant defective in early stages of autophagosome (Mostowy and Cossart, 2012). Septin filaments associate directly biogenesis (atg1Δ), displayed decreased septin localization to the with membranes in a curvature-dependent manner (Bridges et al., PAS. Our findings support a role for septins in the early stages of 2016) and regulate membrane dynamics, including vesicle fusion budding yeast autophagy, during autophagosome formation. events (Mostowy and Cossart, 2012). In immune cells, septins also localize transiently to the phagocytic cup and are functionally This article has an associated First Person interview with the first involved in phagocytosis (Huang et al., 2008). Septins have been author of the paper. implicated in autophagy in mammalian cells infected by intracellular bacteria, where they form cage-like structures around the bacterial KEY WORDS: Autophagy, Noncanonical ring, Septin, cells that colocalize with the autophagosome marker autophagosome Autophagosome biogenesis, Pre-autophagosomal structure, PAS, marker MAP1LC3A, the homolog of yeast Atg8. It is believed that Atg9 trafficking these structures entrap bacteria, restricting their motility and targeting them for autophagy-mediated degradation (Mostowy et al., 2009, INTRODUCTION 2010). During Shigella infection, assembly of septin cages and the Macroautophagy (herein autophagy) is an evolutionarily conserved autophagosome in the host mammalian cells are interdependent intracellular waste disposal and recycling process that is critical for (Mostowy et al., 2010, 2011; Sirianni et al., 2016). Despite these normal cellular and organismal homeostasis. Autophagy involves findings, it remains unclear to what extent septins contribute to the formation of double-membrane vesicles called autophagosomes autophagy outside the context of bacterial infection (Torraca and that engulf intracellular material destined for degradation. Mostowy, 2016). Autophagosomes eventually fuse with vacuoles or lysosomes, In S. cerevisiae cells undergoing mitotic proliferation, five septin resulting in cargo degradation and recycling of cellular building proteins – Cdc3, Cdc10, Cdc11, Cdc12 and Shs1 – comprise an array of filaments that is directly associated with the plasma membrane at the mother–bud neck, and controls cell polarity, bud 1Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India. 2University of Colorado, morphogenesis and cytokinesis (Glomb and Gronemeyer, 2016; Oh Anschutz Medical Campus, Department of Cell and Developmental Biology, and Bi, 2011). Upon nitrogen starvation, diploid yeast cells undergo Aurora, CO 80045, USA. meiosis and sporulation, during which a cup-shaped double- *Author for correspondence ([email protected]) membrane structure, the prospore membrane (PSM), engulfs haploid nuclei and other organelles to form stress-resistant spores M.H.S., 0000-0001-8534-2197; M.A.M., 0000-0002-4615-4334; R.M., 0000- (Neiman, 2005, 2011). Yeast septins are required for proper PSM 0002-0923-5485 biogenesis (Heasley and McMurray, 2016), but there was no known This is an Open Access article distributed under the terms of the Creative Commons Attribution role for septins in yeast autophagy. Here, we describe autophagy License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. defects in septin-mutant strains and physical interactions between septins and established autophagy factors that support a functional

Received 29 July 2017; Accepted 10 January 2018 role for septins in yeast autophagy. Journal of Cell Science

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Fig. 1. See next page for legend. Journal of Cell Science

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Fig. 1. Septins migrate from the pre-existing bud-neck ring to cytoplasm (Weems et al., 2014). These findings indicate that the septin during starvation. (A) Pexophagy was affected in cdc10P3SG44D (cdc10-5) complexes involved in pexophagy are composed of the same septins cells as compared to WT cells at the non-permissive temperature (37°C). that were previously synthesized when nutrients were available and (B) Microscopy images of Cdc10–GFP, Cdc11–GFP and Shs1–GFP cells under nutrient rich, nutrient deficient and rapamycin (0.4 µg/ml) treatment supported cytokinesis in budding cells. conditions. Cells from log phase (0.6 to 0.8 OD) were transferred to starvation To ask whether septins are more generally involved in autophagy, medium (1 OD/ml) and imaged at different time points. (C) Quantification of the we examined the processing of GFP–Atg8, which is processed in number of cells showing rings and puncta grown in rich, starvation or the vacuole during autophagy (Cheong and Klionsky, 2008). rapamycin treatment medium for 24 h. For quantification, cells showing only Similar to the pexophagy results obtained with Pot1–GFP, we ring or only dots were considered. Images acquired were converted into noticed considerable slowdown of autophagic flux in septin mutant maximum intensity projections, deconvolved and a total of 100 cells were – quantified. (D) Cdc10, Cdc11 and Shs1 all colocalize as puncta during cells, as evidenced by slower processing of GFP Atg8 (Fig. S2). In starvation. Strains JTY5396 and JTY5397 were grown as in B and imaged. addition to mutant alleles harboring substitutions in specific (E) Septin localization in presence of cycloheximide (C) and rapamycin (R). residues, conditionally viable septin-mutant cells can be obtained Cells were grown as described in Fig. 1B in presence of cycloheximide by deleting the CDC10 gene (Flescher et al., 1993; Frazier et al., (50 µg/ml) and rapamycin (YPD+C+R) and in presence of rapamycin 1998; McMurray et al., 2011). In cells lacking Cdc10, septin (0.4 µg/ml) alone (YPD+R). Scale bars: 5 µm. filament assembly and functions essential for mitotic proliferation require septin hetero-hexamers formed via non-native Cdc3 RESULTS homodimerization (McMurray et al., 2011). Cells lacking Cdc10 Autophagy defects in septin mutants are temperature-sensitive for mitotic proliferation due to inefficient To identify autophagy defects in viable mutant yeast strains, we Cdc3 homodimerization at high temperatures (McMurray et al., introduced into a collection of temperature-sensitive (Ts−) mutants 2011). If the same septin complexes that function in cytokinesis are in a POT1-GFP strain, which expresses a marker of pexophagy also involved in autophagy, then we would expect to find autophagy (Kondo-Okamoto et al., 2012), a specialized form of autophagy in defects in cdc10Δ cells at 37°C. Indeed, this was the case, as assayed which peroxisomes are degraded (Oku and Sakai, 2016). Targeting by both Pot1–GFP and GFP–Atg8 processing (Fig. S3A,B). of Pot1–GFP to the vacuole during starvation-induced pexophagy Similarly, cells lacking Cdc11 require non-native Cdc12 results in destruction of the Pot1 part of the fusion protein and homodimerization for survival, but septin function is more accumulation of free GFP, which is readily detected by severely compromised in cdc11Δ mutants because Shs1 occupies immunoblotting (Fig. 1A; Fig. S1A,B). Unlike in wild-type (WT) the same position as Cdc11 in a subset of hetero-octamers and, in cells, where free GFP accumulated at both 22°C and 37°C, in cells doing so, prevents efficient Cdc12 homodimerization (McMurray expressing any of several Ts− mutant alleles of the septin CDC10 et al., 2011). We found that Pot1–GFP processing was more (G100E or P3S G44D) or CDC11 (G29E, G34D or S31F S100P) severely compromised in cdc11Δ than in cdc10Δ mutants more free GFP was detected at 22°C, compared to what was seen at (Fig. S3A,B), consistent with the relative magnitude of the effects 37°C, and the Pot1–GFP fusion remained intact at 37°C (Fig. 1A; on mitotic proliferation. Also consistent was the relatively minor Fig. S1A). These results were also corroborated by using effect of deleting SHS1 (Fig. S3A,B), which has only mild effects fluorescence microscopy to visualize the delivery of GFP-labeled on mitotic proliferation at 37°C (Finnigan et al., 2015; Mino et al., peroxisomes to the vacuole as diffuse GFP inside the vacuolar 1998). Finally, deleting SHS1 in a cdc11Δ background improves lumen (Fig. S1B). At 37°C the number of starved septin-mutant mitotic proliferation, presumably because Shs1 no longer interferes cells showing free GFP inside the vacuole was reduced significantly with Cdc12 homodimerization (McMurray et al., 2011). Pexophagy when compared to the numbers of starved WT cells, and also when and autophagy defects in cdc11Δ shs1Δ cells were equivalent to the compared to numbers of mutant cells incubated at 22°C (Fig. S1C). defects in cdc10Δ cells (Fig. S3A,B), providing additional support These data point to a requirement for septin function in pexophagy. for our conclusion that cytokinesis and autophagy share similar In nutrient-replete conditions, the Ts− mutants of CDC10 and requirements for septin complex assembly. CDC11 in which we found pexophagy defects arrest cell division In WT cells, the induction of autophagy triggers a coalescence of with failed cytokinesis (Hartwell, 1971). Interestingly, we did multiple small vacuoles into a single organelle (Baba et al., 1994), not observe Pot1–GFP-processing defects in cells expressing Ts− which is readily observed by visualizing FM4-64 labeling of the mutant versions of CDC3 (G365R) or CDC12 (G247E) (Fig. S1D), vacuolar membrane (Fig. S3C). We noticed that in septin mutant which were originally isolated in the same cell division screen cells, particularly the viable deletion mutants, vacuolar coalescence (Hartwell, 1971) as the CDC10 and CDC11 mutants that caused was largely defective (Fig. S3C). The defect in pexophagy in pexophagy defects. To explain this discrepancy, we considered that cdc11Δ cells was rescued by introduction of a plasmid encoding in cdc3(G365R) or cdc12(G247E) cells, high temperature prevents WT Cdc11, confirming that the pexophagy defect resulted from the de novo assembly of septin complexes but does not destabilize absence of Cdc11 (Fig. S3E). existing structures (Dobbelaere et al., 2003; Kim et al., 1991; Weems If septin mutant cells have defects in autophagy, then they should et al., 2014). Since pexophagy, like autophagy in general, occurs in be sensitive to starvation, survival during which requires a starved non-dividing cells, we hypothesized that a functional functional autophagy pathway (Suzuki et al., 2011). Indeed, contribution of septins to pexophagy may not require assembly of heterozygous diploid strains lacking one copy of CDC10 are new septin complexes, and instead utilizes pre-existing complexes known to be sensitive to nutrient deprivation (Davey et al., 2012), a assembled prior to the nutrient withdrawal and temperature upshift. previously unexplained phenotype. Haploids lacking Cdc10 are Indeed, yeast septins are exceedingly long-lived proteins, even also sensitive to rapamycin (Chan et al., 2000), which could reflect a during starvation (McMurray and Thorner, 2008). In support of this requirement for autophagy to survive the starvation-like metabolic model, Pot1–GFP processing was compromised in cdc12-td cells conditions that result from inhibition of Tor1/2, as autophagy is (Fig. S1D), which express a temperature-degron-tagged Cdc12 that induced by rapamycin even in nutrient-replete conditions (Noda and is known to cause rapid disassembly of pre-existing filamentous Ohsumi, 1998). Finally, we note that a high-throughput genetic septin structures associated with the bud neck in dividing cells interaction study previously reported negative interactions between Journal of Cell Science

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Fig. 2. See next page for legend. Journal of Cell Science

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Fig. 2. Septins colocalize with autophagosomes and form ring like (Fig. 2C,D). Similar results were obtained using GFP-tagged septins structures. (A) Septins colocalize with the mCherry–Atg8 (mCh-Atg8)-labeled and an RFP-tagged version of Ape1/Lap4, an aminopeptidase that is PAS. Cells were grown as described in Fig. 1B and imaged. All images are of a tethered to the PAS in its precursor form prior to proteolytic single z-section and are deconvolved. (B) Quantification of the number of PASs that colocalize with septins. More than 300 cells were counted. From activation in the vacuolar lumen (Delorme-Axford et al., these cells, only 20–30% cells showed a PAS and cells that showed 2015) (Fig. 2E,F). Careful imaging revealed that septin–GFP colocalization between the PAS and septin dot were quantified. Quantification puncta could often be resolved as rings surrounding the PAS was performed manually by using the cell counter plugin of Fiji at every (Fig. 2G; Fig. S4A–E). The dimensions of these rings (400–600 nm z-section. (C) Representative images showing colocalization of septins and in diameter) are about half the size of septin rings at the bud neck autophagosomes (autophagosomes are highlighted by white arrowheads). (Okada et al., 2013) and are instead similar to the size of Cells were grown as in Fig. 1B and were imaged. (D) Quantification of the autophagosomes (400–900 nm in diameter) (Suzuki and Ohsumi, number of cells showing multiple colocalizations between septins and ∼ mCherry–Atg8 puncta. For quantification, images were deconvolved and 2010). The autophagy protein Atg9 is also known to form 500- background subtracted, and then colocalization was checked by using the nm-wide rings around mCherry–Atg-marked autophagosomes colocalization highlighter plugin. Colocalized points were then quantified by (Yamamoto et al., 2012). Septin–GFP localization at the PAS was using the cell counter plugin of Fiji. More than 150 cells were quantified. transient (Movie 2), providing an explanation for our observations (E) Colocalization of septins with Ape1–RFP. Cdc10-GFP, Cdc11–GFP and that not every PAS was associated with septins. These results – Δ – Shs1 GFP ypt7 cells expressing Ape1 RFP were grown as in Fig. 1B and support a model in which septins associate with the developing PAS imaged. (F) Quantification of the number of septin puncta colocalized with Ape1–RFP puncta. (G) Formation of a non-canonical ring around mCherry– and remain associated with mature autophagosomes. Atg8 by Shs1–GFP. Cells were grown as in Fig. 1B and were imaged. Eight z-sections of the same image at 0.2 µm each are shown. Scale bars: 2 µm. Septins interact with autophagy proteins To ask whether septin localization at the PAS involves physical cdc10 mutants and mutations in the autophagy genes ATG3, ATG8 interactions between septins and known autophagy proteins, we and ATG9 (Costanzo et al., 2010). These findings provide employed two parallel approaches. Immunoprecipitation of GFP- independent support for a functional requirement for septins tagged septins using the GFP tag as an epitope resulted in the co- in autophagy. precipitation of untagged Atg8, and the amount of co-precipitated Atg8 increased in the absence of Ypt7 (Fig. 3A), consistent with a Septins move from the bud neck to the PAS during starvation prolonged interaction due to stabilization of autophagosomes. and associate with mature autophagosomes Negligible Atg8 was precipitated by the GFP antibody when GFP Our genetic findings indicate a functional role for septin complexes was not fused to a septin (Fig. 3A). Furthermore, Cdc10 interacted in autophagy and further suggested that the same septin complexes in vivo with Atg9 in a bimolecular fluorescence complementation assembled prior to induction of autophagy are utilized during (BiFC) assay (Fig. 3B); other septins were not tested. In addition to autophagy, without a requirement for new septin synthesis or single cytoplasmic puncta in starved cells, which we confirmed to assembly. Based on these findings, we predicted that, during be the PAS because they colocalized with Ape1–RFP, a Cdc10– starvation, septins should re-localize from the bud neck to sites of Atg9 BiFC signal was observed at the necks of budding cells autophagosome assembly (i.e. the PAS). Consistent with this (Fig. 3B,C; Movie 3). Since Atg8 and Atg9 are also involved in the prediction, upon starvation GFP-tagged Cdc10, Cdc11 and Shs1 ‘cytoplasm to vacuole targeting’ (Cvt) pathway, a biosynthetic form present at the bud neck quickly (within 5 h) transitioned to cytosolic of selective autophagy active even in rich medium conditions puncta (Fig. 1B,C; Movie 1). Addition of rapamycin to cells in rich (Reggiori and Klionsky, 2013), these observations likely represent medium had the same effect (Fig. 1B,C). mCherry-tagged Cdc10 otherwise transient associations between septins and the autophagy colocalized in these cytosolic puncta with Cdc11–GFP and Shs1– machinery during Cvt that are prolonged by the essentially GFP (Fig. 1D). We could not obtain conclusive results with irreversible BiFC event. The autophagy proteins are thereby fluorescently tagged Cdc3 or Cdc12 due to the propensity in artificially tethered to the septin ring at the bud neck, which starvation conditions of these fusion proteins to form, and incorporate facilitates detection of the BiFC event, but does not faithfully report other septins into, aberrant rod-shaped structures (Fig. S1E; data not on where the interaction first took place. Taken together, these shown). Crucially, relocalization of septins from the bud neck to findings provide strong evidence that septins physically interact cytosolic puncta did not require new protein synthesis, since with the core autophagy machinery both during Cvt and starvation- equivalent results were observed in the presence of the translation associated autophagy. inhibitor cycloheximide (Fig. 1D). These findings demonstrate that, upon starvation, pre-existing septin proteins re-localize from the site Septins are involved in autophagosome biogenesis of cytokinesis to cytosolic puncta, consistent with a role in a cytosolic To determine at which stage of autophagosome assembly septins process in these conditions. function, we first examined septin-mutant cells to identify the To ask whether the cytosolic puncta to which septins relocalize step in autophagosome formation that fails when septins are upon starvation included the PAS, we examined septin–GFP dysfunctional. We combined the cdc10(P3S G44D) mutation localization in cells also expressing an mCherry-tagged version of with ypt7Δ, to block autophagosome degradation, and shifted Atg8, which marks the PAS prior to its processing within the starved cells to 37°C. We found a decrease in the number of vacuole (Delorme-Axford et al., 2015). Approximately 30% of mCherry–Atg8 foci per cell compared to that in ypt7Δ cells with WT cytosolic septin–GFP puncta in starved cells were also labeled by septins (Fig. 3D,E), indicating that septin dysfunction perturbs mCherry–Atg8 (Fig. 2A,B). Whereas in WT cells autophagosomes autophagosome biogenesis, rather than delivery of autophagosomes disappear as they fuse with the vacuole, in cells lacking Ypt7, to the vacuole and degradation. Next, we examined septin–GFP and a Rab GTPase required for autophagosome-vacuole fusion, mCherry–Atg8 localization in cells lacking Atg1, in which PAS autophagosomes persist (Ishihara et al., 2001; Kim et al., 1999). assembly begins but no mature autophagosomes are produced We observed a corresponding increase in the number of septin foci (Suzuki et al., 2007). In atg1Δ cells, colocalization between GFP- that were also marked by mCherry–Atg8 in cells lacking Ypt7 tagged septins and mCherry–Atg8 decreased significantly Journal of Cell Science

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Fig. 3. See next page for legend. Journal of Cell Science

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Fig. 3. Septins are involved in autophagosomes biogenesis. (A) Western 2016), but these studies did not determine whether septins blot showing the septin–Atg8 interaction in WT and in ypt7Δ strains expressing participate in the assembly of autophagocytic membrane – – – Cdc10 GFP, Cdc11 GFP and Shs1 GFP. Cells were grown as described in structures per se, and defects in general autophagy (i.e. not the Materials and Methods. IB, immunoblot; IP, immunoprecipitation. (B,C) BiFC experiments. A strain expressing Cdc10-Vc (Cdc10 C-terminus associated with bacterial infection) have not been reported. tagged with C-terminus of Venus) and Atg9-Vn (Atg9 C-terminus tagged with The Atg9 retrograde transport defects we found in septin mutants N-terminus of Venus) with or without Ape1–RFP was grown as described in suggest that septins may help Atg9 molecules to deliver membrane Fig. 1B and imaged after 5 h of incubation in starvation medium. source for developing autophagosomes. Additionally, our findings (D) Representative images and (E) quantification showing autophagosome of dynamic septin localization to a subset of PAS structures after number per cell at 37°C. All the images are maximum intensity projections, and initial PAS formation, septin rings of diameters consistent with those more than 50 cells were quantified manually with Fiji. *P<0.05 (comparison between non-Ts and Ts at 37°C); **P<0.01 (comparison between 22°C and of the autophagosomal membranes, interactions between septins and 37°C in Ts) (two-way ANOVA). (F) Representative images and the autophagosomal membrane protein Atg9, and defects in (G) quantification of colocalization events between mCherry–Atg8 and the autophagosome maturation in septin mutants are all consistent three GFP-tagged septins in the atg1Δ strain. A total of 50 cells were quantified with roles for septins in guiding isolation membrane extension. In manually at every z-plane. *P<0.05 for Cdc10–GFP, **P<0.01 for Cdc11–GFP this regard, the autophagy defects we observed in septin mutant cells and Shs1–GFP (two-way ANOVA). Scale bars: 2 µm (C,F); 5 µm (B,D). are reminiscent of defects in extension of the yeast PSM (Heasley and McMurray, 2016), another double-bilayer membrane that engulfs cytoplasmic components prior to fusion of its leading edges. compared to that in WT cells (Fig. 3F,G), suggesting that septins Notably, whereas proper septin function in PSM extension appears arrive at the nascent autophagosome after the PAS has already to require the de novo assembly of hetero-octamers containing two begun to assemble. sporulation-specific septin proteins (Garcia et al., 2016), our The autophagy protein Atg9 accumulates at the PAS in atg1Δ cells findings suggest that the same septin hetero-octamers assembled due to a defect in retrograde Atg9 transport to the sources during mitotic proliferation are sufficient to support autophagosome of membrane trafficking (Sekito et al., 2009). In WT cells, maturation. Emerging studies (Bridges et al., 2016) suggest that the Atg9–mCherry colocalized primarily with GFP-tagged septins ability of rod-shaped septin hetero-oligomers to interact with (Fig. 4A,B). Interestingly, a single bright punctum of GFP–Atg9 membranes of specific micron-scale curvatures and polymerize was also observed in cdc10(P3S G44D) cells incubated at 37°C into filaments may be key to septin function in various contexts. The (Fig. 4C). This punctum colocalized with the PAS marker Ape1 details of interactions between septins and the established autophagy (Fig. 4C–E), indicating that septin dysfunction prevents proper machinery, particularly membranes, and whether post-translational Atg9 retrograde transport away from the PAS, a phenocopy of the modifications to the septins drive their departure from the site of absence of Atg1. We further noticed that Ape1 was mislocalized in cytokinesis are compelling subjects for future research. septin mutant cells, as only a few Ape1–RFP puncta colocalized with GFP–Atg8 in cdc10(P3S G44D) cells incubated at 37°C (Fig. 4F,G). MATERIALS AND METHODS Taken together, these experiments point to a role for septin complexes Yeast strains and media in biogenesis of autophagosomes following PAS assembly. Wild-type (WT) and autophagy knockout mutant yeast strains used in this study are derived from BY4741, BY4742, and S288C. These strains were obtained from EUROSCARF. Strain and primer details are listed in Tables DISCUSSION S1 and S2, respectively. The WT Pot1–GFP strains are laboratory strains The original budding yeast mutants defective in autophagy were with GFP tagged genomically to the C-terminus of Pot1, and were obtained identified by unbiased genetic screens based on phenotypes of from Prof. Richard Rachubinski, University of Alberta, Canada. Septin Ts− ‘ ’ failure to accumulate autophagic bodies , to survive during mutants were kindly provided by Prof. Charlie Boone, Toronto, into which a nitrogen starvation (Tsukada and Ohsumi, 1993) or to degrade POT1-GFP cassette was transformed to obtain strains used for pexophagy specific cytoplasmic enzymes (Thumm et al., 1994), with an assays. Septin knockout mutants were prepared using the standard underlying assumption that autophagy is non-essential for colony transformation protocols (Baudin et al., 1993). GFP-ATG8 pRS316 and growth in rich medium. Another study systematically searched for 2xmCherry-ATG8 pRS316 plasmids were a kind gift from Prof. Yoshinori autophagy defects in a collection of mutants harboring Ohsumi, Tokyo Institute of Technology, Tokyo. GFP-Atg9 pRN295 was a hypomorphic alleles of essential genes (Shirahama-Noda et al., kind gift from Prof. Michael Thumm, University of Stuttgart, Germany. 2013), but by definition these alleles provide sufficient function to Vector pSUN5 was created by amplifying Atg9 promoter and the open- reading frame (ORF) from a WT strain and cloned into the pRS316vector at support proliferation. We report here the identification of septin the SacII and NotI sites. The tandem repeat of mCherry separated by a 45 bp mutants in what is, to our knowledge, the first unbiased screen for linker region was cloned in two steps between the Not1, XmaI and HindIII autophagy (pexophagy) defects among conditionally lethal sites. A linker region of 13 bp was also included between the Atg9 ORF and mutants. We observed severe defects under conditions non- start codon of mCherry. permissive for proliferation, which explains why septin mutants WT cells and mutants were grown in YPD medium (1% yeast extract, were not isolated in previous autophagy screens. 2% peptone and 2% dextrose) at 30°C and 22°C, respectively. For Septins are functionally important for numerous processes pexophagy assays, oleate medium (0.25% yeast extract, 0.5% peptone, involving changes in membrane shape, ranging from cytokinesis 1% oleate, 5% Tween-40 and 5 mM phosphate buffer) was used to induce (Hartwell, 1971; Kim et al., 2011) to mitochondrial fission peroxisome formation, and synthetic defined medium (0.17% yeast nitrogen (Pagliuso et al., 2016) to the retraction of membrane blebs base lacking amino acids and ammonium sulphate plus 2% dextrose) was used to induce autophagy. For Ts− mutants and knockout mutants, 22°C and (Gilden et al., 2012). Macroautophagy requires the synthesis and ‘ ’ 37°C were used as the permissive and non-permissive temperatures, directed extension of a double-bilayer isolation membrane which, respectively. when its leading edges fuse together, becomes an autophagosome, and is destined for fusion with the vacuole or lysosome. In metazoan Microscopy and cytology cells, septins form cage-like assemblies around intracellular bacteria Cells were grown in respective media, centrifuged (20817 g for 2 min). and recruit autophagocytic machinery (Torraca and Mostowy, and were mounted on agarose (2% w/v) pads for microscopy. Images Journal of Cell Science

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Fig. 4. See next page for legend. were taken in z-sections of 0.2 µm step size using a Delta Vision colocalized entities were either quantified manually by using the cell microscope (GE Healthcare) fitted with 100×1.4 NA objective and Cool- counter plugin or automatically by using the colocalization highlighter SNAP HQ2 camera. Images were acquired using FITC and TRITC plugin in Fiji for all z-sections. Representative colocalizations filters. Image processing and quantification were performed with events quantified manually were also confirmed by line profile and SoftWorx (GE Healthcare) and Fiji (NIH) software. For colocalization colocalization measurement options in SoftWorx (GE Healthcare). All analysis, images were de-convolved and background subtracted, and the supplementary movies are of a single z-plane. Journal of Cell Science

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Fig. 4. Septins colocalize with Atg9 and play a role in Atg9 retrograde Statistics transport. (A) Representative images showing colocalization between septins All statistical analysis was performed using GraphPad Prism. To calculate and Atg9. Cdc10–GFP, Cdc11–GFP and Shs1–GFP cells expressing significance levels two-way ANOVA and Student’s t-tests were used. The Atg9–mCherry (Atg9-mCh) were grown as in Fig. 1B and were imaged. mean±s.e.m. is shown in all graphs. (B) Quantification of the number of cells showing colocalization between – septins and Atg9 mCherry puncta. Quantification was performed as in Acknowledgements Fig. 3G. More than 150 cells were quantified. (C) Atg9 retrograde transport is We would like to thank Prof. Charles Boone, Prof. Yoshinori Ohsumi, Prof. Kausik affected in the cdc10-5 strain at 37°C. Cells were grown in starvation medium Chakraborthy, Prof. Michael Snyder, Prof. Michael Thumm, Prof. Richard for 6 h and were imaged. (D) Quantification of the number of cells showing a Rachubinski, Prof. Jeremy Thorner, for generously sharing strains, plasmids and bright Atg9 punctum at 22°C (permissive temperature) and 37°C (non- reagents. Critical reading of the manuscript and inputs from Prof. M. R. S. Rao, permissive temperature). Quantification was performed manually by using Fiji Aparna Hebbar, and members of the autophagy lab. We thank Veena A. and software, and a total of 50 cells were quantified in each of the three Priyadarshini Sanyal for technical help. experiments. ***P<0.001, 22°C versus 37°C in WT and cdc10-5 cells (two-way ANOVA). (E) Quantification of the number of cells showing colocalization Competing interests between the bright GFP–Atg9 punctum and Ape1–RFP. Quantification was The authors declare no competing or financial interests. performed manually by using Fiji software at each z-section, and a total of 30 cells were quantified in each of the three experiments. ***P<0.001, atg1Δ Author contributions versus cdc10-5 cells (unpaired t-test.). (F) Colocalization between GFP–Atg9 Conceptualization: R.M., G.B., S. Sridhar, A.A., M.A.M.; Methodology: G.B., and mCherry–Atg8, and GFP–Atg8 and Ape1–RFP. The cdc10-5 cells S. Sridhar, A.A., M.H.S., S.C., S. Singh, L.K.N.; Software: G.B.; Validation: G.B.; expressing either GFP–Atg9 with mCherry–Atg8 or GFP–Atg8 with Ape1– Formal analysis: G.B., S. Sridhar, A.A., M.H.S., S.C., S. Singh, L.K.N., M.A.M.; RFP were grown in SD Ura or SD −His −Ura medium at 22°C. Logarithmically Investigation: R.M.; Resources: R.M., M.A.M.; Data curation: R.M., M.A.M.; Writing - growing cells were then incubated in starvation medium (1 OD/ml) for 3 h at original draft: R.M., G.B., S. Sridhar; Writing - review & editing: R.M., G.B., M.A.M.; 22°C and 37°C. (G) Quantitation of the colocalization of GFP–Atg9 and Visualization: R.M., G.B., A.A., M.A.M.; Supervision: R.M.; Project administration: mCherry–Atg8 puncta with GFP–Atg8 and Ape1–RFP puncta. 30 cells were R.M.; Funding acquisition: R.M. quantified in each of the three experiments. **P<0.01 (paired t-test). Scale bars: 2 µm (A,F); 5 µm (C). Funding This work was supported by a Wellcome Trust DBT India Alliance Intermediate Fellowship (5009159-Z-09-Z) and Jawaharlal Nehru Centre for Advanced Scientific Research intramural funds to R.M. S. Sridhar was supported by a fellowship from the Western blot analysis and quantification Council of Scientific and Industrial Research (CSIR). Deposited in PMC for Whole cell extracts were prepared via the trichloroacetic acid (12.5% TCA immediate release. w/v) precipitation method followed by ice-cold acetone washes (twice). 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