EMBO/SourceData huggingface dataset:
Figure 7Sub-populations of WT and TM6 cells, grown separately and mixed in different fractions as indicated in percentages, can easily be distinguished by flow cytometry, FL1 YFP channel, FL3 mCherry channel. Histogram of single-cell ratios of FL1/FL3 fluorescence intensities of mixed WT and TM6 populations analyzed by flow cytometry. Here, a sub-population of minimally 5% can be distinguished.Tukey boxplots showing the YFP/mCherry ratio of individual cells measured by microscopy as a function of glycolytic flux. At least 35 cells were analyzed in each condition. The glycolytic flux is here reported as the flux between the metabolites fructose 6-phosphate (F6P) and FBP. Glycolytic fluxes were estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al, 2019).YFP/mCherry ratio measured by microscopy in co-existing dividing (high-flux) versus non-dividing (low-flux) isogenic TM6 cells on 10 gL-1 glucose.Brightfield (BF), YFP and mCherry microscopy images for a co-existing dividing (high-flux) and a non-dividing (low-flux) TM6 cell expressing the flux sensor in 10g L-1 glucose minimal medium.The production rates of YFP and mCherry are uncoupled during the cell cycle in the biosensor-expressing strain (F), which reflects the cell-cycle dynamics of intracellular FBP concentration and glycolytic flux. In a control strain, lacking CggR, the production rates of YFP and mCherry are coupled (G). The uncoupling was calculated for individual cell-cycle trajectories as the difference between the YFP and mCherry production rates normalised to have the same scale (see more details in Materials and methods; Appendix Figure S10). Each curve represents the mean across the indicated number of cell cycles in a replicate experiment. The corresponding shaded areas denote the 95% confidence intervals of the means (bootstrapping with 5 000 iterations). We smoothed the single-cell-cycle trajectories of YFP and mCherry signals as well as cell volume via the Gaussian process regression, and used these trajectories to derive the YFP and mCherry production rates, accounting for fluorescent-protein maturation in a first-order kinetics model. To align the cell-cycle trajectories and to calculate the phase, we used the array of three cell cycle events E {cytokinesis (cyt), budding, next cyt} as reference points. The cell cycles used for the analysis had the duration in the interval between 150 and 300 minutes, with the mean duration presented in parentheses for each replicate experiment. The cells belonged to the TM6 strain and were cultivated on 20 gL-1 glucose in the microfluidic device.
Figure 2(A) HOIL-1 activity towards purified bovine liver glycogen was measured using Cy5-labelled ubiquitin. Control assays lacking ATP, UBE2L3, HOIL-1 and glycogen are indicated, as are subsequent treatments with 50 μg/mL human salivary α‑amylase and 1.5 M hydroxylamine (NH2OH). The gel has been stained using the periodic acid-Schiff (PAS) method to visualise glycogen (purple-magenta) and Coomassie Blue stain to visualise protein.(B) Prior to PAS and Coomassie staining, the gel shown in (A) was stained with Flamingo fluorescent protein stain (shown in red) and glycogen ubiquitylation observed by visualising the fluorescent Cy5-ubiquitin signal (shown in green).(D) In vitro ubiquitylation of maltoheptaose by HOIL-1. Assays were treated with α‑amylase or hydroxylamine as indicated. Reaction products were detected by Coomassie staining.
Figure 3(F) HOIL-1 activity against the indicated maltosaccharides (2 mM) was assayed and visualised by staining with Coomassie protein stain. Where indicated, assays were treated with 1.5 M hydroxylamine. Met1-linked ubiquitin tetramer was also included in these assays.(H) In vitro ubiquitylation of the indicated cyclodextrins (2 mM) by HOIL-1, visualised by Coomassie staining. Met1-linked ubiquitin tetramer was also included in these assays.
Figure 4(A) 1 μg of each of the indicated recombinant proteins was incubated with amylose resin for 60 min, the beads were pelleted by centrifugation, and proteins in the pellet (P) and supernatant (S) were compared to the protein input (I) by means of western blotting with the appropriate antibodies.(B) Bovine liver glycogen and potato starch-derived amylopectin were incubated for 60 min with the indicated recombinant proteins, centrifuged for 90 min at 100,000 × g, and protein in the resulting supernatants (S) and pellets (P) analysed by western blotting with the appropriate antibodies. Protein without polysaccharide was used as a negative control (Buffer).(C) Amylose binding assays of full-length (1-1072) and truncated HOIP protein, visualised by immunoblotting. Shown on the right are schematic representations of the truncated HOIP constructs used. Images not drawn to scale. PUB, peptide N-glycanase and UBA- and UBX-containing protein domain; ZF, zinc finger domain; NZF, Npl4 zinc finger domain; UBA, ubiquitin-associated domain; RBR, RING-in-between-RING domain; LDD, linear ubiquitin chain determining domain.(D) Amylose binding assays of full-length (1-387) and truncated Sharpin mutants along with schematic representations of the Sharpin constructs used. Images not shown to scale. PH, pleckstrin homology domain; LTM, LUBAC-tethering motif; UBL, ubiquitin-like domain.
Figure 5(A) Maltoheptaose ubiquitylation by HOIL-1 was assayed at 30°C in the presence of Met1- or Lys63-linked ubiquitin dimers for the indicated times and visualised by Coomassie staining.(B) Maltoheptaose ubiquitylation by HOIL-1 was assayed at 30°C in the presence of Met1- or Lys63-linked ubiquitin tetramers for the indicated times and visualised by Coomassie staining.(C) HOIL-1[T203A/F204A/R210A] was assayed at 30°C in the absence or presence of the indicated ubiquitin dimers and tetramers. These mutations have been reported to impair the binding of ubiquitin to the NZF domain and their approximate location in HOIL-1 is indicated (schematic not to scale).(D) Assay of HOIL-1[Δ194-222] lacking the NZF core domain at 30°C in the absence or presence of the indicated ubiquitin oligomers.(E) Allosteric activation of HOIL-1[233-510] (lacking the LTM, UBL and NZF domains at the N‑terminus) by ubiquitin oligomers.
Figure 6(B) Time-course assay performed at 30°C featuring wild-type HOIL-1 in the absence or presence of the indicated ubiquitin oligomers, visualised by Coomassie staining.(C) HOIL-1[I358R], assayed as in (B). (D) HOIL-1[F381A/E383A], assayed as in (B).(E) The truncated HOIL-1[233-510; I358R] mutant was assayed as in (B). (F) The truncated HOIL-1[233-510; F381A/E383A] mutant was assayed as in (B).
Figure 7(A) Requirements for en bloc transfer of linear ubiquitin dimer to maltoheptaose were investigated by leaving out the reaction components indicated. Reaction products were detected by staining with Coomassie.(B) Requirements for en bloc transfer of Met1-linked ubiquitin tetramer to maltoheptaose were investigated by leaving out the reaction components indicated.(C) Time-course assay performed at 30°C in the presence of Met1-linked ubiquitin oligomers in which the two most C-terminal residues were either glycine (normal ubiquitin) or alanine to prevent conjugation to substrates (GG/AA).(D) Maltoheptaose ubiquitylation was assayed for 60 s at 30°C in the presence of the indicated ubiquitin tetramers.
Figure 1(A) Using denaturing immunoprecipitation (IP), Munc18-1 was isolated from cell lysate of HEK293T cells expressing M18WT, M18Y145F or M18Y473F together with or without n-Src. Munc18-1 was immunoblotted for tyrosine phosphorylation using the 4G10 antibody (αPtyr), after which the blot was stripped and re-blotted for Munc18-1. Total lysate (TL) is shown as loading control. N-Src was tagged with a flag-tag for visualization.(B) Same as (A) but using lysate from HEK293T cells expressing M18WT or M18Y473F together with or without n-Src, c-Src or Fyn. The first two lanes are IPs performed using empty beads to control for non-specific binding of Munc18 or kinases to the beads.(F) Lipid mixing was monitored by the increase of Atto 488 fluorescence for 12 minutes. Data were normalized to the maximum fluorescence after liposome lysis by detergent. Error bars = s.e.m., N = 3.(G) Same as (E-F) but the reactions were preincubated on ice for 5 minutes in the presence of Complexin II (CpxII 600 pmol). Error bars = s.e.m., N =5.(I) Lipid mixing was monitored by the increase of Atto 488 fluorescence for 60 minutes. Data were normalized to the maximum fluorescence after liposome lysis by detergent. Error bars = s.e.m., N =3.
Figure 2Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using confocal microscopy (A) Typical confocal images from neurons stained for MAP2, Synaptobrevin/VAMP2 and Munc18-1.Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using confocal microscopy (B) Average synaptic Munc18-1 intensity (M18WT: 389 ± 85 a.u., n=21; M18Y473D: 261 ± 49 a.u., n=20, t-test with Welch correction, p = 0.2000), somatic Munc18-1 intensity (M18WT: 328 ± 49 a.u., n=21; M18Y473D: 188 ± 21 a.u., n=20, t-test with Welch correction, p = 0.0138), synapse number (M18WT: 129 ± 14, n=21; M18Y473D: 159 ± 14, n=20, t-test, p = 0.1203) and dendrite length (M18WT: 0.433 ± 0.028, n=21; M18Y473D: 0.454 ± 0.037, n=20, t-test, p = 0.6613).Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology(C) Left: typical examples of evoked release upon action potential stimulation.Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology (D) Left: typical responses to hyperosmotic sucrose application (500 mM, 3.5 sec.) used to assess the RRP. Average RRP charge (M18WT: 1.68 ± 0.64 nC, n=13; M18Y473D: 0.08 ± 0.02 nC, n=10, t-test with Welch correction, p = 0.0274).Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology (E) Average Pves (EPSC charge / RRP charge) (M18WT: 6.75 ± 0.77%, n=13; M18Y473D: 0.24 ± 0.20%, n=10; Mann-Whitney U test, p < 0.0001).Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology (F) Left: example traces of spontaneous release of single SVs (mEPSCs). Average mEPSC frequency (M18WT: 20.18 ± 5.18 Hz, n=15; M18Y473D: 0.28 ± 0.13 Hz, n=17, Mann-Whitney U test, p < 0.0001), mEPSC amplitude (M18WT: 26.0 ± 1.7 pA, n=15; M18Y473D: 27.8 ± 2.2 pA, n=17, t-test, p = 0.5372) and mEPSC decay time (M18WT: 1.98 ± 0.10 ms, n=15; M18Y473D: 2.84 ± 0.41 ms, n=17, t-test with Welch correction, p = 0.0549).
Figure 4 Synaptic transmission was assessed during and immediately after sustained stimulation in autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473D. (A) Rundown of EPSC amplitude during 100 pulses at 10 Hz. Top: example traces of the current evoked by 40Hz stimulation. Synaptic transmission was assessed during and immediately after sustained stimulation in autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473D.(B) EPSC charge during 100 pulses at 40 Hz. Top: example traces of the current evoked by 40Hz stimulation. Insert shows total charge transferred during entire train (M18WT: 2.63 ± 0.38 nC; M18Y473D: 1.48 ± 0.24 nC, t test with Welch correction, p = 0.0177).Synaptic transmission was assessed during and immediately after sustained stimulation in autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473D.(C) Synaptic transmission was assessed before (naive(n)) and two seconds after (a) 40Hz stimulation. Average EPSC charge M18WT (naive: 70.9 ± 19.5 pC; after HFS: 75.4 ± 17.4 pC, n=12, paired t-test, p = 0.4049). Average EPSC charge M18Y473D (naive: 0.888 ± 0.342 pC, after HFS: 25.6 ± 9.3 pC, n=9, paired t-test, p = 0.0271).Synaptic transmission was assessed during and immediately after sustained stimulation in autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473D.(D) A hypertonic sucrose solution (500 mM, 3.5 sec.) was applied to release the RRP before (naive (n)) or two seconds after (a) HFS (100 pulses at 40 Hz).Synaptic transmission was assessed during and immediately after sustained stimulation in autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473D.(E) Quantification of RRP and Pves (EPSC charge / RRP charge * 100) from C-D. Average RRP size M18WT (naive: 1.73 ± 0.69 nC, after HFS: 1.34 ± 0.54 nC, n=12, paired t-test, p = 0.0557). Average RRP size M18Y473D (Naive: 0.061 ± 0.019 nC, after HFS: 0.207 ± 0.050 nC, n=9, paired t-test, p = 0.0246). Average Pves M18WT (naive: 7.07 ± 0.76 %, after HFS: 10.6 ± 1.6 %, n=12, paired t-test, p = 0.0201). Average Pves M18Y473D (naive: 0.27 ± 0.22 %, after HFS: 10.8 ± 2.2%, n=9, paired t-test, p = 0.0012).Synaptic transmission was assessed during and immediately after sustained stimulation in autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473D.(F) PDBu application (1 µM) enhanced EPSC size in neurons expressing M18Y473D.
Figure 5(B) GST-Munc18-1 constructs were immobilized on glutathione beads and incubated with the indicated molar excess of Synaptobrevin/VAMP2 (over Munc18-1) for 1.5 h at 4°C. Bound Synaptobrevin/VAMP2 was analyzed by Western blot and immune decorating with an anti-Synaptobrevin/VAMP2 antibody and quantified by the LICORE system and ImageJ software. Synaptobrevin/VAMP2 bound to the GST-Munc18-1 mutants is represented as percentage of Synaptobrevin/VAMP2 bound to GST-Munc18-1 WT. Error bars indicate s.e.m., n=3.(D) Lipid mixing was monitored by the increase of Atto 655 fluorescence for 30 minutes. Data were normalized to the maximum fluorescence after liposome lysis by detergent. The minimum fluorescence was set to 0%. Error bars = s.e.m., N = 3.(E) Trans-SNARE Formation Assay: t- and v-SNARE SUVs were incubated in the presence or absence of different Munc18-1 constructs for 30min at 4°C. After solubilization and precipitation of the t-SNAREs using nickel beads, presence of full-length VAMP2 in the precipitates was probed by immunoblotting, which was used as an indicator for trans-SNARE assembly between SUVs.
Figure 6(A) Typical examples of single EPSCs. EPSC amplitude (M18WT: 2.98 ± 0.76 nA, n=11; M18Y473D: 0.042 ± 0.018 nA, n=11, M18Y473D/P335A: 2.02 ± 0.67 nA, n=7, Kruskal-Wallis test, p < 0.0004).Evoked and spontaneous release in autaptic munc18-1 null hippocampal neurons expressing M18Y473D, M18Y473D/P335A or wild-type Munc18-1 as control.(B) Typical traces of spontaneous release.Evoked and spontaneous release in autaptic munc18-1 null hippocampal neurons expressing M18Y473D, M18Y473D/P335A or wild-type Munc18-1 as control.(C) mEPSC frequency (M18WT: 8.23 ± 2.29 Hz, n=11; M18Y473D: 0.096 ± 0.056 Hz, n=8, M18Y473D/P335A: 2.18 ± 1.02 Hz, n=11, Kruskal-Wallis test, p = 0.2390), mEPSC amplitude (M18WT: 16.7 ± 1.0 pA, n=11; M18Y473D: 21.6 ± 5.2 pA, n=10, M18Y473D/P335A: 14.8 ± 1.2 pA, n=6, ANOVA, p = 0.1465) and decay time (M18WT: 2.53 ± 0.29 ms, n=11; M18Y473D: 3.79 ± 0.71 ms, n=10, M18Y473D/P335A: 2.85 ± 0.36 pA, n=6, Kruskal-Wallis test, p < 0.0003).Evoked and spontaneous release in autaptic munc18-1 null hippocampal neurons expressing M18Y473D, M18Y473D/P335A or wild-type Munc18-1 as control.(D) Depression of EPSC amplitude during a 10 Hz train. Inset shows first 4 pulses normalized to the first EPSC.
Figure 7 Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology. (A) Evoked release upon action potential stimulation. Average EPSC amplitude (M18WT: 3.78 ± 0.58 nA, n=22; M18Y473F: 4.10 ± 0.61 nA, n=20, Mann-Whitney U test, p = 0.7915). Typical responses are depicted on the top. Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(B) Average mEPSC frequency (M18WT: 5.42 ± 1.86 Hz, n=17; M18Y473F: 4.42 ± 1.01 Hz, n=14, t-test with Welch correction, p = 0.6424), mEPSC amplitude (M18WT: 20.72 ± 0.7 pA, n=17; M18Y473F: 20,5 ± 0.9 pA, n=14, t-test, p = 0.8373) and mEPSC decay time (M18WT: 2.54 ± 0.20 ms, n=17; M18Y473F: 2.46 ± 0.18 ms, n=14, t-test, p = 0.7550). Example traces of spontaneous release are depicted on top.Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(C) EPSC depression during 10 Hz stimulation is normal.Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(D) Depression of EPSC charge during 40 Hz stimulation is normal. Inset shows the first 5 pulses.Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(E) EPSC amplitude recovery two seconds after an RRP depleting 40 Hz train. Average EPSC recovery (M18WT: 82.2 ± 6.7 %, n=17; M18Y473F: 65.5 ± 11.0 %, n=12, t-test, p = 0.1805).Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(F) The amount of DSE-triggered EPSC depression was quantified by the ratio of the average EPSC amplitude (taken over 4 pulses given at 0.2 Hz) before or immediately following 10 seconds depolarization to 0mV. Typical EPSCs before and following DSE induction are depicted on the left. Average DSE (M18WT: 0.57 ± 0.04 fold, n=20; M18Y473F: 0.53 ± 0.04 fold, n=16, t-test, p = 0.5227).Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(G) PDBu was bath applied during 0.05 Hz stimulation. Maximum potentiation of EPSC amplitude (M18WT: 2.56 ± 0.48 fold, n=7; M18Y473F: 2.82 ± 0.98 fold, n=8, t-test with Welch correction, p = 0.8168).
Figure 8(A) Using denatured IP, Munc18-1 was pulled-down from cell lysate of HEK293T cells expressing M18WT or M18Y473A together with SFK kinases or an empty vector as control. Munc18-1 was then immunoblotted for tyrosine phosphorylation using the 4G10 antibody. The total amount of Munc18-1 was detected after stripping and reblotting for Munc18-1. (B-G) Munc18-1 null hippocampal neurons were rescued with wildtype Munc18-1a or a Munc18-1 mutant, Y473A, in which Y473 was replaced with alanine. (B) Typical confocal images of neurons stained for MAP2, Synaptobrevin /VAMP2 and Munc18-1. Scale bar = 50πm.(B-G) Munc18-1 null hippocampal neurons were rescued with wildtype Munc18-1a or a Munc18-1 mutant, Y473A, in which Y473 was replaced with alanine.(C) Quantification of confocal images. Average synaptic Munc18-1 intensity (M18WT: 389 ± 85 a.u., n=21; M18Y473A: 321 ± 48 a.u., n=20, t-test with Welch correction, p = 0.4868), somatic Munc18-1 intensity (M18WT: 328 ± 49 a.u., n=21; M18Y473A: 245 ± 42 a.u., n=20, t-test, p = 0.2810), synapse number (M18WT: 129 ± 14, n=21; M18Y473A: 159 ± 16, n=20, t-test, p = 0.1548) and dendrite length (M18WT: 0.433 ± 0.028, n=21; M18Y473A: 0.461 ± 0.037, n=20, t-test, p = 0.5473).(B-G) Munc18-1 null hippocampal neurons were rescued with wildtype Munc18-1a or a Munc18-1 mutant, Y473A, in which Y473 was replaced with alanine.(D) Evoked release upon action potential stimulation. Average EPSC amplitude (M18WT: 7.96 ± 0.94 nA, n=30; M18Y473A: 1.44 ± 0.34 nA, n=33, t-test with Welch correction, p < 0.0001). Typical responses are depicted on the right.(B-G) Munc18-1 null hippocampal neurons were rescued with wildtype Munc18-1a or a Munc18-1 mutant, Y473A, in which Y473 was replaced with alanine.(E) Release by hyperosmotic sucrose application (500mM, 3.5sec.) was used to assess the RRP. Left: RRP charge (M18WT: 1.68 ± 0.64 nC, n=13; M18Y473A: 0.27 ± 0.08 nC, n=13, t-test with Welch correction, p = 0.0487). Typical responses are depicted in the middle. Right: vesicular release probability per neuron (EPSC charge / RRP charge). Mean Pves (M18WT: 6.75 ± 0.77%, n=13; M18Y473A: 1.98 ± 0.72%, n=13, t-test, p = 0.0001).(B-G) Munc18-1 null hippocampal neurons were rescued with wildtype Munc18-1a or a Munc18-1 mutant, Y473A, in which Y473 was replaced with alanine.(F) Top: example traces of spontaneous release of single vesicles (mEPSCs). Average mEPSC frequency (M18WT: 20.18 ± 5.18 Hz, n=15; M18Y473A: 0.48 ± 0.15 Hz, n=20, t-test with Welch correction, p = 0.0019), amplitude (M18WT: 26.0 ± 1.7 pA, n=15; M18Y473A: 24.5 ± 1.6 pA, n=20, t-test, p = 0.5334) and decay time (M18WT: 1.98 ± 0.10 ms, n=15; M18Y473A: 2.13 ± 0.17 ms, n=20, t-test with Welch correction, p = 0.4499).(B-G) Munc18-1 null hippocampal neurons were rescued with wildtype Munc18-1a or a Munc18-1 mutant, Y473A, in which Y473 was replaced with alanine.(G) Typical electron microscopy images from rescued autaptic hippocampal Munc18-1 null neurons (scale bar = 100 nm). Neurons expressing M18Y473A have less synaptic vesicles (SV) docked at the number of docked SV. Average