Strains, growth conditions, primers and plasmids
All yeast strains (Table S1) are congenic to W303 (ade2-1, trp1-1, leu2-3,112, his3-11, 15 ura3). W303 bears a single nucleotide deletion in the BUD4 gene (bud4-G2459fs) that results in a premature stop codon. The bud4-G2459fs gene produces a truncated protein of 838 aminoacids that lacks 609 aminoacids and carries 18 non-natural aminoacids at C-terminus (https://www.yeastgenome.org). This mutation leads to septin ring disassembly, instead of splitting, at mitotic exit 90. In order to properly visualize septin ring splitting, the genotype of most strains has been corrected to carry full-length BUD428,91.
Yeast cultures were grown at 30°C in either synthetic medium (SD) supplemented with the appropriate nutrients or YEP (1% yeast extract, 2% bactopeptone, 50 mg/l adenine) medium. Raffinose was supplemented to 2%, glucose to 2% and galactose to 1%. NAA (1-Naphthaleneacetic acid) and IAA (3-indoleacetic acid) were dissolved in ethanol as 1000X and 500X stock solutions, respectively, and used at a final concentration 0.1mM and 1 mM, respectively, unless otherwise indicated. Cells were synchronized in G1 by alpha factor (4 µg/ml) in YEP medium containing the appropriate sugar at 25°C. G1 arrest was monitored under a transmitted light microscope and cells were released in fresh medium at 30°C (typically after 120–135 min of alpha factor treatment) after being collected by centrifugation at 2000 g and washed with YEP containing the appropriate sugar. Nocodazole was used at 15 µg/ml.
One-step tagging techniques were used to generate strains bearing HOF1 deletion, HOF1-3XminiAID, eGFP- and 3HA-tagged HOF1, 6Gly-3Flag-tagged CDC10, K.l.TRP1-marked RCH1, as well as to introduce the kanMX gene marker upstream or downstream of HOF1 for CRISPR/Cas9 gene editing. All HOF1-3XminiAID strains also express the O.s.TIR1 gene from the ADH1 promoter to induce protein degradation 92.
To generate the HOF1 phosphomutants and HOF1 F-BAR(2-279) deleted mutants by CRISPR/Cas9 gene editing, pSP1712 (Cas9 and gRNA against kanMX co-expression vector) and a double-stranded DNA repair template were co-transformed into yeast cells bearing the kanMX marker upstream or downstream of HOF1. DNA repair templates were amplified from synthetic DNAs cloned in pTwist plasmids (Twist Bioscience). After transformation, edited cells were cured from the co-expression vector and gene editing was confirmed by PCR followed by restriction enzyme digestion and sequencing.
The plasmid used for expression of MBP-Hof1-6His (1–669; pBG2163) was a gift from B. Goode (Brandeis University, Waltham, MA) and has been previously described 54. Gibson assembly (New England Biolabs) was used to construct a plasmid for expression of the phosphomimetic MBP-Hof1-P4E-6His (pSP1785): a HOF1 DNA fragment (coding for aa 257–587 of Hof1) containing the 10 substitutions of Hof1-P4E phospho-mimetic mutant (S313E, S314E, S337E, T341E, T350E, S366E, S421E, S423E, S424E, S563E) was amplified from pSP1722 (pTwist bearing a HOF1-P4E fragment from 1254 bp of the ORF to 121 bp after the stop codon) using primers MP1257 and MP1218, and cloned into the backbone vector amplified from pBG2163 using primers MP1444 and MP1454. The complete list of primers and plasmids used in this study can be found in Tables S2 and S3.
Live cell imaging
For time-lapse video microscopy, cells were mounted on 1% agarose pads in SD medium on fluorodishes and filmed at controlled temperature. For scoring septin ring splitting (Fig. 1B,D,F,G, 2E,F,H, 4D, and S3A,B), cells were filmed with a 100X 1.49 NA oil immersion objective mounted on a Nikon Eclipse Ti microscope equipped with an EMCCD Evolve-512 Camera (Photometrics) and iLAS2 module (Roper Scientific) and controlled by Metamorph. Z-stacks of 8 planes were acquired every 4 min with a step size of 0.4 µm and a binning of 1. Z-stacks were max-projected with ImageJ or Metamorph. For quantifying fluorescence intensities associated to mCherry-Cdc3 and Hof1-eGFP and for resolving single from double Hof1 rings, cells were filmed with a 60X UPLXAPO 1.42 NA oil immersion objective mounted on an Olympus IX83 inverted microscope coupled to Yokogawa W1 spinning disk unit, equipped with a sCMOS Fusion BT camera (Hamamatsu) and controlled by the CellSens software. Z-stacks of 8 or 15 planes were acquired every 1–2 min with a step size of 0.24 µm, and max-projected using ImageJ. Alternatively (Fig. 3C, S5B), cells were filmed with a 100X Plan Apo lambda 1.45 NA oil immersion objective mounted on a Nikon inverted microscope coupled to the Andor Dragonfly spinning disk, equipped with an EMCCD iXon888 Life (Andor) camera and controlled by Andor fusion software. Focus was maintained using the Perfect Focus System (Nikon). Z-stacks of 8 planes were acquired every 1–2 min with a step size of 0.4 µm, and deconvolved (6 iterations, denoising filter size = 1 and pre-sharpening = 2). Z-stacks were max-projected using ImageJ.
For still images of septin collars in hof1Δ mutant (Fig. 1C), live cells were washed with PBS, mounted on slides with coverslips and immediately imaged at room temperature with a 100X 1.4 NA Plan Apochromat oil immersion objective mounted on an epifluorescent widefield Zeiss Axioimager Z2 microscope equipped with an sCMOS ZYLA (Andor) camera and controlled by the Zen software. Z-stacks of 15 planes were acquired with a step size of 0.3 µm and max-projected with ImageJ.
Quantification of septin- and Hof1-associated fluorescence intensities.
Fluorescence intensities associated to mCherry-Cdc3 and Hof1-eGFP were quantified with ImageJ on max-projected z-stacks. Intensities were measured within a rectangular ROI defining the bud neck. A rolling ball radius of 50 pixels was set to subtract the background and fluorescent intensities were determined using ImageJ Analyze Particles tool after applying a threshold. The highest fluorescence integrated intensity reached at any time point within the time frame of interest was set at 100%, while all the other fluorescence intensities were relative to this reference value. Relative intensities were then averaged after setting as time 0 the time immediately preceding septin ring splitting. The number of samples analysed (n) is indicated in the figures. Error bars correspond to standard deviations.
Protein extracts, co-immunoprecipitation assays and western blotting
For TCA protein extracts, 10–15 ml of cell culture in logarithmic phase (OD600 = 0.5–1) were collected by centrifugation at 2000 g, washed with 1 ml of 20% TCA and resuspended in 100 µl of 20% TCA before breakage of cells with glass beads (diameter 0.5–0.75 mm) on a Vibrax VXR (IKA). After addition of 400 µl of 5% TCA, lysates were centrifuged for 10 min at 845 g. Protein precipitates were resuspended in 100 µl of 3X SDS sample buffer (240 mM Tris–Cl pH 6.8, 6% SDS, 30% glycerol, 2.28 M β-mercaptoethanol, 0.06% bromophenol blue), denatured at 99°C for 3 min, and loaded on SDS–PAGE after elimination of cellular debris by centrifugation (5 min at 20,000 g).
For coimmunoprecipitations, cells were lysed in TBSN buffer (100 mM NaCl, 25 mM Tris-Cl pH 7.4, 2mM EDTA, 0.1% NP-40, 1mM DTT, supplemented with 1 mM sodium orthovanadate, 60 mM β-glycerophosphate and a cocktail of protease inhibitors (Complete EDTA-free; Roche) at 4°C with 14 cycles of 30" breakage followed by 30" in ice. 10 ODs of cleared extracts were diluted in 300 µl of TBSN supplemented with inhibitors, out of which 5 µl were withdrawn and denatured in 3 x SDS sample buffer for inputs. The remaining IP mix was incubated for 1h at 4°C on a nutator with 25 µl of protein G dynabeads (Invitrogen) preadsorbed for 90 min with 1 µl of anti-Flag M2 antibody and washed with TBSN to eliminate the excess of antibody. Magnetic beads were washed six times with TBSN buffer and bound proteins eluted with an excess of 3XFlag peptide (0.5 mg/ml in 50 mM Tris-Cl pH 8.3, 1mM EDTA, 0.1% SDS) upon incubation at R.T. for 25 min with vigorous shaking. 12.5 µl of 3X SDS sample buffer (240 mM Tris–Cl pH 6.8, 6% SDS, 30% glycerol, 2.28 M β-mercaptoethanol, 0.06% bromophenol blue) were added to the eluted proteins, followed by denaturation at 99°C for 3 min and loading on SDS–PAGE. Proteins were wet-transferred to Protran membranes (Schleicher and Schuell) overnight at 0.2 A and treated for western blotting as above.
For western-blotting, proteins were wet-transferred on Protran membranes (Schleicher and Schuell) overnight at 0.2 A. Specific proteins were detected with monoclonal anti-AID (M214-3 Medical and Biological Laboratories; 1:1000), anti‐Pgk1 (22C5D8 Invitrogen Molecular Probes, 1:40000) anti-GFP (3H9 Chromotek; 1:1000), anti-Pkc1 (sc-6801 Santa Cruz; 1:5000), anti‐HA 12CA5 (AgroBio custom made; 1:5000), anti-Flag M2 (F3165 Sigma, 1:5000) or polyclonal anti-Cdc11 (sc-7170 Santa Cruz; 1:2000). Antibodies were diluted in 5% low‐fat milk (Regilait) dissolved in TBST (25 mM Tris-Cl pH 7.5, 137 mM NaCl, 2.68 mM KCl, 0.1% Tween-20). Secondary antibodies were purchased from GE Healthcare, and proteins were detected by a home‐made luminol/p-coumaric acid enhanced chemiluminescence system.
FACS analysis of DNA contents
For DNA quantification by fluorescence-activated cell sorting (FACS), 1–2 X 107 cells were collected, spun at 10000 g and fixed with 1 ml of 70% ethanol for at least 30 min at RT. After one wash with 50 mM Tris-Cl pH 7.5, cells were resuspended in 0.5 ml of the same buffer containing 0.05 ml of a preboiled 10 mg/ml RNAse solution and incubated overnight at 37°C. The next day cells were spun at 10,000 g and resuspended in 0.5 ml of 5 mg/ml pepsin freshly diluted in 55 mM HCl. After 30‐min incubation at 37°C, cells were washed with FACS buffer (200 mM Tris-Cl pH 7.5, 200 mM NaCl, 78 mM MgCl2) and resuspended in the same buffer containing 50 µg/ml propidium iodide. After a short sonication, samples were diluted (1:8) in 200 µl of 50 mM Tris-Cl pH 7.5. 20000 events were scored and analysed for each sample on an ACEA NovoCyte cytometer instrument controlled by NovoExpress software (Agilent).
Protein purification
Yeast Cdc11-capped septin octamers were purified as previously described 72. Briefly, E.coli strain BL21 (DE3) Rosetta cells were transformed with two bicistronic plasmid pFM453 (colE1, Ampr, CDC10, CDC11) and pFM455 (p15A, Kanr, MBP-CDC12, His6-CDC3) or with pFM453 and pFM873 (p15A, Kanr, MBP-CDC12, His6-CDC3-yeGFP)73,93. Cells were grown in LB containing 50 µg/ml of ampicillin, 25 µg/ml of kanamycin, 34 µg/ml of chloramphenicol, 0.2% glucose and induced at OD600 = 1 by addition of 0.2 mM IPTG. After 20h at 16°C, cells were harvested by centrifugation and resuspended in Buffer A (25 mM NaHPO4 pH 7.8, 300 mM NaCl, 0.5 mM MgCl2, 5% glycerol) supplemented with 5 mM β-mercaptoethanol, 1 mg/ml of lysozyme and a cocktail of protease inhibitors (Complete EDTA-free Roche). Cells were sonicated 3x with 1’30’’ cycles of 8” pulses / 8’’ ice and extracts cleared at 30000 g for 30’ at 4°C. Lysates were incubated 2 hours at 4°C with 1 ml of amylose resin (New England Biolabs), pre-washed 3x with buffer A, on a rotating wheel. After incubation with the protein extracts, the slurry was washed 3 times with buffer A and loaded on a Polyprep column (Bio-Rad). Fractions of 0.5 ml were eluted with buffer A supplemented with 10 mM maltose and quantified by Nanodrop. The most concentrated fractions were pulled together and MBP was cleaved from the complex using thrombin at 5 U/mg of protein overnight at 6°C. Proteins were then diluted in 10 ml of buffer B (25 mM NaHPO4 pH 7.8, 300 mM NaCl, 0.5 mM MgCl2, 5% glycerol, 0.1% Triton X100, 6 mM imidazole) supplemented with 5 mM β-mercaptoethanol and incubated 2 hours at 4°C with 1 ml of Ni-NTA agarose beads (Qiagen), pre-washed 3x with buffer B, on a rotating wheel. The slurry was then washed 3 times with buffer C (25 mM NaHPO4 pH 7.8, 500 mM NaCl, 0.5 mM MgCl2, 5% glycerol, 0.15% Triton X100, 5 mM β-mercaptoethanol, 8 mM imidazole) and loaded on a Polyprep column (Bio-Rad). Fractions of 0.5 ml were eluted with buffer A supplemented with 200 mM imidazole and quantified by Nanodrop. The most concentrated fractions were dialysed in a buffer containing 20 mM Tris-Cl pH 8.2, 300 mM NaCl, 0.2 mM MgCl2 and 2 mM DTT. Proteins were finally concentrated through Amicon Ultra filter units (10 kDa cut-off).
For Hof1 and Hof1-P4E purification, E.coli strain BL21 (DE3) Rosetta cells were transformed with pBG2163 (pMAL-C2 MBP-Hof1-6His 54) or pSP1785 (pMAL-C2 MBP-Hof1-P4E-6His). Cells were grown in LB containing 50 µg/ml of ampicillin, 34 µg/ml of chloramphenicol, and 0.2% glucose, to late log phase (OD600 = 0.7–0.9). Expression was induced with 0.8 mM IPTG overnight at 18°C, and then cells were pelleted and stored at -80°C. Pellets were thawed, and resuspended in lysis buffer (20 mM Tris-Cl, pH 8, 300 mM NaCl) supplemented with 1 mg/ml of lysozyme and a cocktail of protease inhibitors (Complete EDTA-free, Roche). Cells were sonicated 3x with 1’30’’ cycles of 8” pulses / 8’’ ice and extracts cleared at 30000 g for 30 min at 4°C. Lysates were incubated 1 hr at 4°C with 1.5 ml of Ni-NTA agarose beads (Qiagen), pre-washed 3x with 20 mM Tris-Cl pH 8, 300 mM NaCl, 6 mM imidazole at 4°C on a rotating wheel. After incubation with the protein extracts, the slurry was washed 3 times with 20 mM Tris-Cl pH 8, 300 mM NaCl, 8 mM imidazole and loaded on a Polyprep column (Bio-Rad). Fractions of 0.5 ml were eluted with 20 mM Tris-Cl pH 8, 300 mM NaCl, 300 mM imidazole and quantified by Nanodrop. The most concentrated eluted fractions were pooled and diluted fourfold with lysis buffer lacking imidazole, mixed with 1.5 ml amylose beads and incubated at 4°C for 1h. The beads were washed five times with 10 ml lysis buffer and loaded on a Polyprep column (Bio-Rad). Fractions of 0.5 ml were eluted with lysis buffer supplemented with 20 mM maltose and 1 mM DTT and quantified by Nanodrop. The most concentrated fractions were dialysed in a buffer containing 50 mM Tris-Cl pH 7.5, 50 mM KCl, 150 mM NaCl, 1mM EDTA, 1mM DTT. Proteins were finally concentrated through Amicon Ultra filter units (50 kDa cut-off), snap frozen in liquid N2 and stored at − 80°C.
Dynamic light scattering
For dynamic light scattering, we used a a Litesizer 500 setup (Anton Paar; scattering angle of 90° in-vacuo laser wavelength 658 nm, maximum power 40 mW). Recombinant septin octamers at 180 nM were polymerised in solution by lowering the salt concentration to 30 mM NaCl, and immediately measured. After 3 min, 180 nM of either recombinant MBP-Hof1 or MBP-Hof1-P4E were added. The hydrodynamic radius was obtained from a standard second-order cumulant fit.
Transmission Electron Microscopy
Cdc11-capped septin octamers were diluted to 360 nM in low salt buffer (20 mM Tris-Cl pH 8, 30 mM NaCl, 2 mM MgCl2, 40 µM GTP) and let polymerise for at least 30 min at 4°C. Then, septins were mixed with recombinant Hof1 or Hof1-P4E (molar ratio 1:2, 1:1 or 1:0.5), diluted in same buffer to 180 nM final concentration. 3 µl of samples were absorbed on glow-discharged formvar carbon-coated grids (Delta Microscopies FCF100-Cu) and stained with 1% uranyl acetate. Grids were observed using a JEOL 1400 Flash transmission electron microscope at 120kV. Micrographs were recorded using a One View camera (Gatan Inc.) at different magnifications.
Fluorescence microscopy and supported lipid bilayers
In all experiments, glass coverslips and slides were cleaned by three successive rounds of sonication for 30 min in 1 M NaOH, 100% ethanol and milliQ water, with extensive rinsing in double-distilled water between each step. Coverslips were rinsed in 100% ethanol, dried and finally plasma cleaned just before using.
To visualize formation of septin assemblies in solution, a glass coverslip was attached to a glass slide and several ~ 20 µl flow chambers were made by placing strips of parafilm parallel to the shorter length of the slide followed by a short incubation of the slides at 100°C to seal the edges. A mix of recombinant eGFP-tagged and untagged septin octamers (ratio 1:1) was diluted to 200 nM in polymerisation buffer (20 mM Tris-HCl pH 8, 30 mM NaCl, 2 mM MgCl2, 40 mM GTP). Then, septins were mixed with 50% mol of recombinant Hof1 or Hof1-P4E in the same buffer to 50 nM final concentration. 20 µl of sample was added to a flow chamber that was immediately sealed with modelling clay. Images were acquired at room temperature with a 100X 1.4 NA Plan Apochromat oil immersion objective mounted on an epifluorescence widefield Zeiss Axioimager Z2 microscope equipped with an sCMOS ZYLA (Andor) camera and controlled by the Zen software. Z-stacks of 10 planes were acquired with a step size of 0.3 µm and max-projected with ImageJ. Background was subtracted and denoised using ImageJ tools.
Supported lipid bilayers were composed of either 95,8% (w/w) DOPC, 4% (w/w) DGS-NTA(Ni), 0,2% (w/w) Rhodamine-PE or 77% (w/w) Egg-PC, 2.8% (w/w) brain PI(4, 5)P2, 20% (w/w) liver PI and 0.2% (w/w) TopFluor-TMR-PI(4, 5)P2. Lipids dissolved in methanol/chloroform (1:2) were mixed and dried for 1h in a vacuum oven at 60°C. Small unilamellar vesicles (SUVs, diameter ∼ 100nm) were obtained by extrusion of multilamellar vesicles in citrate buffer (citric acid - trisodium citrate 20 mM pH 4.6, KCl 50 mM, EGTA 0.5 mM) heated to 44°C. 40 µl of SUVs were deposited on a two-well silicone insert (IBIDI), and incubated 1h at 40°C. Bilayers were carefully rinsed with washing buffer (20 mM Tris-Cl pH 8, 300 mM NaCl) and then equilibrated with equilibration buffer (20 mM Tris-Cl pH8, 30 mM NaCl, 1 mg/ml lipid-free BSA). Cdc11-capped yeGFP-tagged and untagged septin octamers were separately diluted in polymerisation buffer (20 mM Tris-Cl pH 8, 30 mM NaCl, 2 mM MgCl2, 40 mM GTP), mixed in a 1:1 ratio (eGFP-tagged:untagged), and incubated for at least 30 min on ice. Purified Hof1 or Hof1-P4E were diluted in the same buffer. 50 nM of septins octamers in 20 µL were applied on the lipid bilayer and immediately visualised with a Zeiss LSM880 Airyscan confocal microscope. Then, 50, 100 or 250 nM of either Hof1 or Hof1-P4E in a volume of 20 µl were added. PI(4, 5)P2-containing SLBs were visualised with a Zeiss LSM880 Airyscan confocal microscope using an Argon laser for 488 nm and 514 nm as excitation sources. Acquisitions were performed with a 63x/1.4 NA objective. Multidimensional acquisitions were acquired via an Airyscan detector (32-channel GaAsP photomultiplier tube (PMT) array detector), controlled by Zeiss Zen software. DOPC-containing SLB were visulalised with a 60X UPLXAPO 1.42 NA oil immersion objective mounted on an Olympus IX83 inverted microscope coupled to Yokogawa W1 spinning disk unit, equipped with a sCMOS Fusion BT camera (Hamamatsu) and controlled by the CellSens software. Images were analysed by ImageJ and 3D projections were created with the Imaris software using the normal shading projection tool.