EMBO/soda-nel huggingface dataset:
Mice deficient in Epg5 exhibit selective neuronal vulnerability to degeneration. (2013)
Figure 2.

Defects in brain and spinal cord in Epg5−/− mice. (A) Nissl staining of the fifth layer of the cortex in 10-mo-old Epg5+/− and Epg5−/− mice. (B) The number of pyramidal cells in the fifth layer per millimeters squared. (C and D) H&E staining of the hippocampus shows reduced pyramidal cell numbers in Epg5−/− mice. Means ± SEM of three mice are shown in B and D. (E) The normally developed cerebellum in Epg5−/− mice. (F) The thickness of the molecular layer in the cerebellum in mutant and control mice. (G and H) Purkinje cells, stained by calbindin, are not reduced in number in Epg5−/− mice. Means ± SEM of five mice are shown in F and H. (I) Arrows indicate Pyknotic neurons in the anterior horn of Epg5−/− mice. (J) The number of motor neurons in the spinal cord (SC) in Epg5+/− and Epg5−/− mice. (K) The numbers of Nissl-stained interneurons in the spinal cord are similar in control and Epg5−/− mice. Means ± SEM of three mice are shown in J and K. (L) Eosinophilic spheroids (arrows) accumulate in the thoracic spinal cord of Epg5−/− mice. (M) Eosinophilic spheroids are swollen degenerating axons (arrow). (N) EM pictures showing degenerated axons (arrows) in the spinal cord of Epg5−/− mice. (O and P) GFAP staining in Epg5+/− and Epg5−/− mice. DCST, dorsal corticospinal tract. Bars: (A, C, L, and O) 50 µm; (E) 500 µm; (G) 20 µm; (I) 100 µm; (M and P) 10 µm; (N) 2 µm.
Mice deficient in Epg5 exhibit selective neuronal vulnerability to degeneration. (2013)
Figure 3.

Accumulation of LC3-II, p62 aggregates, and ubiquitin-positive inclusions in Epg5 knockout mice. (A) Levels of LC3 and p62 in brain and spinal cord extracts from Epg5+/− and Epg5−/− mice. SC, spinal cord. (B) Polyubiquitinated proteins in detergent-soluble (S) and -insoluble (I) fractions from brain and spinal cord homogenates of Epg5+/− and Epg5−/− mice. (C) p62 aggregates and ubiquitin-positive aggregates are absent from Epg5+/− mice but dramatically accumulate and are colocalized (arrows) in Epg5−/− mice. (D–F) p62 aggregates are detected in neurons (arrows; D) and oligodendrocytes (arrows; E) but absent from astrocytes (F) in Epg5−/− mice. (G) Cytoplasmic TDP-43 aggregates accumulate in motor neurons in the anterior horn of spinal cord in Epg5−/− mice. Arrows show cytoplasmic TDP-43 aggregates. DSCT, dorsal corticospinal tract. (H) Percentages of cytoplasmic TDP-43 aggregate-positive motor neurons in the fifth layer of cortex and anterior horn of the spinal cord in Epg5+/− and Epg5−/− mice. Means ± SEM of three mice are shown. LC, lateral column. Bars, 10 µm.
Mice deficient in Epg5 exhibit selective neuronal vulnerability to degeneration. (2013)
Figure 4.

Epg5 knockout mice show muscle atrophy. (A and B) Electromyography of the gastrocnemius muscle from a 3-mo-old Epg5−/− mouse showing fibrillations (A) and positive sharp waves (B). These defects were absent in control mice. (C) At 10 mo, Epg5−/− mice showed high-amplitude and long-duration action potentials when conducting MUAP tests. The blue lines show the start and end of an action potential. Dur, duration; Amp, amplitude. (D–F) H&E staining of gastrocnemius muscles showed features of muscle degeneration in Epg5−/− mice. The arrow in E indicates centrally nucleated fibers. (G) The transcription levels of the atrophy-related genes were up-regulated in gastrocnemius muscles of Epg5−/− mice. (H and I) Electron micrographs of Epg5+/− and Epg5−/− muscles. The arrows indicate abnormal enlarged mitochondria. (J) Levels of LC3-II and p62 in muscle extracts from Epg5+/− and Epg5−/− mice. (K) Percentage of p62 aggregate-positive myofibers in Epg5+/− and Epg5−/− mice. Means ± SEM of three mice are shown. (L) Accumulation and colocalization of p62 and ubiquitin aggregates in the gastrocnemius muscles of Epg5−/− mice but not controls. Bars: (D–F and L) 10 µm; (H and I) 1 µm.
Mice deficient in Epg5 exhibit selective neuronal vulnerability to degeneration. (2013)
Figure 5.

Epg5 deficiency causes a defect in autophagosome maturation and impairs endocytic trafficking. (A) Number of LC3 puncta in Epg5+/− (control) and Epg5−/− MEFs. (B) Western blot showing levels of LC3-I and LC3-II in control and Epg5−/− MEFs upon indicated treatment (Starv., starvation; Rapa., rapamycin; Bafilo., bafilomycin A1). (C) Proteinase K (ProK) protection assay in different MEFs. PNS, postnuclear supernatant; P, pellet fractions. (D) Colocalization ratio of LC3 and LAMP-1 in control and Epg5−/− MEFs. 50 cells were examined per time point in A and D. (E) Colocalization of LC3 puncta and lysosomes in control and Epg5−/− MEFs 4 h after starvation. (F) Under nutrient repletion conditions, almost no autophagic elements are detected in Epg5+/− MEFs. RER, rough ER; M, mitochondrion; N, nucleus. The arrowhead indicates a vacuole with a late residual body-like appearance (aAV-III). (G) Under nutrient repletion conditions, Epg5−/− MEFs accumulate a large number of autophagic vacuoles. Red arrow, a likely autophagosome; red arrowheads, complex vacuoles of the aAV-I type; white arrowheads, aAV-II vacuoles. (H and I) High magnification of autophagic elements in unstarved Epg5−/− MEFs. (H) The red arrow indicates a complex autophagic vacuole containing multilayered membrane structures (white arrowheads) and a lipid-like region (L). (I) The red arrow indicates an autophagosome containing cytoplasm and ribosomes. The red arrowhead points to an autophagosome containing a mitochondrion and cytoplasmic material with membranous structures. The white arrow shows an aAV-III vacuole. (J) Compared with control cells, Rab5-labeled early endosomes are significantly enlarged in Epg5 siRNA-treated cells. (K and L) EGFR degradation in control and Epg5 knockdown cells. (M and N) Distribution of Alexa Fluor 488–conjugated transferrin at different time points in control siRNA- and Epg5 siRNA-treated cells. Higher magnification views are shown in the insets. Error bars indicate SEMs. Bars: (E, J, M and N, main images) 10 µm; (F and G) 2 µm; (H and I) 100 nm; (J, M, and N, insets) 1 µm.
NAADP-regulated two-pore channels drive phagocytosis through endolysosomal Ca2+ nanodomains, calcineurin and dynamin (2020)
Figure 1

Fig. 1. FcγR-mediated phagocytosis requires local not global Ca2+ signals

(A, B) [Ca2+]i changes in individual BMDMs were measured (A) using fura-2 during frustrated phagocytosis (n = 46-113) and (B) by dropping beads onto adherent cells loaded with Calbryte 520 (n = 64-159), in 1.8 mM extracellular Ca2+ (+Ca2+o) or Ca2+-free medium containing 100 μM EGTA (-Ca2+o) with or without prior loading with 25 μM EGTA/AM or 25 μM BAPTA/AM. The maximum fura-2 ratio changes (∆ 350/380) or fluorescence changes normalized to initial fluorescence (∆ F/F0) are plotted.

(C) Schematic depicting the buffering ranges of BAPTA (local Ca2+) and EGTA (global Ca2+).

(D) FcγR-mediated phagocytosis was monitored using beads conjugated to Alexa Fluor 488 (in green). External beads were differentially labelled with anti-IgG (in red) and distinguished by their dual labelling (in yellow). Cytochalasin D (10 μM, Cyt D) prevented bead internalization.

(E) Bead uptake in 15 min, under conditions as in (A, B), n = 80-91 cells with representative images of individual cells delineated by a dotted line.

(F-K) Experiments conducted in -Ca2+o: (F) 1 μM thapsigarin (Tg) or 10 μM CPA pre-treatment, all but eliminated the 1 μM ionomycin (iono) response; single-cell Ca2+ traces and mean ∆ F/F0, n = 450-603 cells. (G) FcγR activation (as in B) evoked Ca2+ oscillations that were blocked with Tg or CPA treatment, whilst bead phagocytosis was not affected (H), n = 312-428 cells. (I) 1 μM bafilomycin A1 (Baf) for 3h, or Ca2+- binding dextran (Cal520-dx) and non-Ca2+-binding dextrans (FITC-dx and Texas red-dx) trafficked to lysosomes, minimally affected global Ca2+ signals evoked by FcγR activation (J); in parallel, bead uptake was only reduced by bafilomycin A1 or by chelating Ca2+ within the lysosome with Cal520-dx (K), n = 100-162 cells. All cells are WT BMDM and all beads are 3-μm IgG-opsonized.

Data information: Scale bars = 5 μm (D, G, I) or 10 μm (E). Bar charts presented as the mean ± S.E.M. and probability determined using one-way ANOVA (*P<0.05, ***P<0.001).
NAADP-regulated two-pore channels drive phagocytosis through endolysosomal Ca2+ nanodomains, calcineurin and dynamin (2020)
Figure 2

Fig. 2. NAADP and TPCs are essential for efficient FcγR-dependent Ca2+ signals and phagocytosis

(A) The Ca2+ signal elicited by frustrated phagocytosis was significantly reduced in Ned-19 (10 μM)-treated WT and in all TPC-KO BMDMs; maximum Ca2+ amplitude, expressed as a percentage of WT control (n = 314-641 cells).

(B-D) Phagocytosis of opsonized 3-µm (B and C) and 6-μm (D) beads was inhibited by Ned-19 (B) and in BMDMs from Tpcn-/- mice compared to WT (n = 134-280 cells).

(E, F) (E) Phagocytosis of 3-µm beads was unchanged in MCOLN1-/- BMDMs, but was impaired using 6-µm beads (F), n = 89-157.

(G, H) Heterologous expression of mouse TPCs tagged with TagRFP-T (in red) rescues the phagocytic defect of Tpcn1-/- and Tpcn2-/- BMDM, but a pore-dead TPC2-mutant (N257A) does not, n = 39-94. Images taken 40 min post- 3-μm bead addition.

(I) Raising cytosolic Ca2+ with ionomycin (1 μM) rescues the Tpcn1-/- and Tpcn2-/- phagocytic defect, whilst UTP (100 μM) and ML-SA1 (50 μM) only weakly rescue (n = 84-192 cells).

Data information: Scale bars = 5 μm (G). Graphs presented as the mean ± S.E.M. and probability determined using one-way ANOVA (*** P<0.001, **P<0.01, *P<0.05 vs WT). See also Appendix Figures S2 and S3.
NAADP-regulated two-pore channels drive phagocytosis through endolysosomal Ca2+ nanodomains, calcineurin and dynamin (2020)
Figure 5

Fig. 5. Peri-lysosomal Ca2+ nanodomains around TPCs drive phagocytosis

(A-F) RAW 247.6 macrophages loaded with a cytosolic chemical dye (Cyto dye) to detect global cytosolic Ca2+, and heterologously expressing (B) TPC1-G-GECO1.2 (TPC1-GG) or (C-E) TPC2-G-GECO1.2 (TPC2-GG) to detect peri-endo-lysosomal Ca2+ domains, which were not detected by (F) a TPC2 mutant (D276K)-G-GECO1.2 that blocks ion permeation or (A) unfused cytosolic G-GECO1.2 (Cyto GG). (A-F) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm beads in -Ca2+o expressed as a percentage of the maximum fluorescence of the indicator, determined by adding 2 μM ionomycin and 10 mM CaCl2 at the end of each experiment (% Fmax) (D) TPC2-G-GECO1.2 activity was inhibited by 10 µM Ned-19, but was unaffected by (E) chelating global Ca2+ with EGTA/AM (100 μM for 2 min prior to bead addition).

(G) Summary data of the first spike from cells A-F (subsequent spikes show the same pattern; Appendix Fig. S4B), n = 87-274 cells; ***P<0.001 vs Cyto GG, ### P<0.001 vs TPC2-GG ctrl, ††† P<0.001 vs Cyto GG.

(H) Fusion of G-GECO1.2 to TPC2 did not alter the affinity of G-GECO1.2 for Ca2+ determined in permeabilized cells (n = 34-71 cells).

(I, J) (I) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm beads in -Ca2+o expressed as a percentage of Fmax. (I, J) Pre-emptying the ER stores with CPA or thapsigargin (Tg) abolished the global cytosolic Ca2+ response evoked by beads, but a TPC2-G‑GECO1.2 component remained, n = 17 - 121 cells.

(K-M) No local Ca2+ signal was detected with TPC2-G-GECO1.2 when Ca2+ spiking was evoked by ER-Ca2+ release with 100 μM UTP (K, L) or 10 μM CPA (M); only a bystander response equivalent to unfused cytosolic G-GECO1.2 was detected (n = 121-182 cells; *P<0.05).

Data information: Graphs presented as the mean ± S.E.M. and probability determined using one-way ANOVA. See also Appendix Figures S4 and S5.
NAADP-regulated two-pore channels drive phagocytosis through endolysosomal Ca2+ nanodomains, calcineurin and dynamin (2020)
Figure 6

Fig. 6 Bead uptake requires lysosomal Ca2+ nanodomains, but different stimuli recruit different lysosomal Ca2+ channels

(A) Cartoon depicting the rapalog CID system (FKBP12/FRB*) to acutely bring tandem Ca2+-binding proteins, Calbindin-2 (CB), to lysosomes.

(B) Translocation of CB, a non-Ca2+ binding CB mutant (CB mut) or a triple mTagBFP2 (BFP) to the lysosome (LAMP1), as indicated by the increase in the Pearson's correlation coefficient was induced by 250 nM rapalog (30 mins) compared with ethanol (EtOH) control-treated RAW 247.6 cells. n = 11-25 cells; ***P<0.001.

(C) Images of a single RAW 247.6 cell displaying yellow puncta indicative of successful re-positioning of CB (in green) to the lysosome (in red) in the presence of rapalog but not EtOH.

(D) When CB was brought to the lysosome (+rapalog) uptake of beads was inhibited, whilst the non-Ca2+ binding CB (CB mut; n = 46-187 cells) or triple mTagBFP2 (BFP; n = 38-52 cells) did not block phagocytosis;***P<0.001.

(E) Bead uptake in BMDMs with and without loading of 25 µM EGTA/AM and 10 μM Ned-19. EGTA-insensitive phagocytosis of beads was inhibited by Ned-19 (n= 75-102 cells) in WT BMDM; ***P<0.001.

(F, G) RAW 247.6 macrophages expressing cytosolic G-GECO1.2 (Cyto GG), TPC2-G-GECO1.2 (TPC2-GG) or TRPML1-G-GECO1.2 (TRPML1-GG). (F) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm or 6-μm beads in -Ca2+o expressed as a percentage of the maximum indicator fluorescence (% Fmax = 2 μM ionomycin and 10 mM CaCl2). (G) Summary of first Ca2+ spike; n = 69-121 (3-μm bead), 22-46 (6-μm beads) cells; ***P<0.001, **P<0.01 vs Cyto GG, ### P<0.001 TPC2-GG vs TRPML1-GG.

Data information: Scale bars = 10 μm (C). Bar charts presented as the mean ± S.E.M. and probability determined using one-way ANOVA.
NAADP-regulated two-pore channels drive phagocytosis through endolysosomal Ca2+ nanodomains, calcineurin and dynamin (2020)
Figure 7

Fig. 7. TPC-mediated Ca2+ release recruits calcineurin which in turn activates dynamin

(A) Dynamin inhibitors (80 μM Dynasore or 20 μM Dyngo-4a) inhibit phagocytosis of opsonized 3-μm beads in WT BMDM; n = 101-149 cells, ***P<0.001.

(B) Rescue of the TPC-KO phagocytic defect by the dynamin activator, Ryngo-1-23 (80 μM): mean number of internalised beads per cell as a percentage of the WT control; n = 128-241 cells, **P<0.01, ***P<0.001.

(C) In WT BMDM, expression of a dominant-negative dynamin-2 mutant (Dyn2 K44A) reduced phagocytosis (n = 68-184). Expression of wildtype dynamin-2 (Dyn2) or constitutively active dynamin-2 (S764A mutant, mimicking the dephosphorylated state) in Tpcn1-/- macrophages restored phagocytosis of 3-μm beads after 10 min, n = 10-76 cells; ***P<0.001 vs WT control, ### P<0.001 vs Tpcn1-/- control.

(D) Single WT BMDM expressing HA-tagged dynamin-2 (immunolabelled with anti-HA, in red) undergoing phagocytosis of IgG-3-μm beads (blue). Images were taken 10 or 30 min post-addition of beads; actin (green) labelled with Phalloidin Alexa 488.

(E) Cartoon indicating sites of action of pharmacological agents.

(F) Calcineurin inhibitors, FK506 (10 μM) or cyclosporin A (CsA, 10 μM) inhibited phagocytosis in WT cells, n = 65-159 cells.

(G) Ryngo (80 μM) rescued phagocytosis defects in Tpcn1-/- BMDM even in the presence of FK506 or CsA, n = 103-208 cells.

(H, I) Calcineurin activity and cytosolic Ca2+ responses simultaneously monitored in single RAW 247.6 using CaNAR2 and jRGECO1a, respectively, upon addition of 3-μm beads. CaNAR2 signals (FRET Y/C) were inhibited by FK506 (10 μM) and Ned-19 (10 μM); (H) single-cell traces and (I) mean maximum responses. n = 65-169; ***P<0.001 vs control.

(J) Phagocytosis induced dephosphorylation of dynamin-2 (ratio of phospho-dynamin to total dynamin-2 expressed as a percentage of WT at time zero) in WT BMDM, but not in Tpcn1-/- BMDM; **P<0.01, ***P<0.001 (time-point matched unpaired t-test) n = 10-15. Measured using in-cell western assay, representative wells of plate image: total dyn2 (red) and phospho-dyn2 (green). See also Appendix Figures S6 and S7.

Data information: Scale bars = 2 μm (D). Graphs presented as the mean ± S.E.M. and probability determined using one-way ANOVA.
NAADP-regulated two-pore channels drive phagocytosis through endolysosomal Ca2+ nanodomains, calcineurin and dynamin (2020)
Figure 8

Fig. 8. Lysosomal clustering inhibits dynamin-dependent phagocytosis

(A-C) RAW 246.7 macrophages expressing the dynein-binding protein (mTagBFP2-BicD2-FKBP12) for rapalog-induced lysosome repositioning to the MTOC, also co-expressed HsLAMP1-mCherry with or without a C-terminal FRB* (LAMP-FRB* or LAMP, in red). Cells were treated with 0.1% ethanol or 250 nM rapalog for 2 h. Only the tripartite complex of FKBP12-rapalog-FRB induced clustering of lysosomes at the MTOC (A, B). This clustering was selective for lysosomes because ER and mitochondria (KDEL-GFP and MitoTracker Green) were not affected (C). (A, B) Phagocytosis of IgG-3-μm beads was reduced by immobilizing lysosomes at the MTOC, n = 80-564 cells; ***P<0.001.

(D, E) (D) Activation of dynamin-2 with Ryngo 1-23 or expression of constitutively active dynamin-2 (S764A mutant, expression confirmed by the grayscale image) restored phagocytosis of IgG-3-μm beads (n = 34-51 cells), but had no effect on the phagocytosis of IgG-6-μm beads, n = 36-72 cells (E); ***P<0.001 EtOH Ctrl vs rapalog Ctrl, ###P<0.001 rapalog Ctrl vs rapalog Ryngo/Dyn2S764A.

Data information: All scale bars = 5 μm. Bar charts presented as the mean ± S.E.M. and probability determined using one-way ANOVA.
A plant-specific SWR1 chromatin remodeling complex couples histone H2A.Z deposition with nucleosome sliding. (2020)
Figure 1

Figure 1. Identification of all components of the SWR1 complex in Arabidopsis.

(A) The list of co-precipitated proteins as determined by immunoprecipitation followed by mass spectrometry (IP-MS). The SWR1 subunits not shared by the ISWI complexes are shown in blue; The DDT domain-containing ISWI specific subunits are shown in orange; CHR11 and CHR17 are shared subunits of the SWR1 and ISWI complexes and are shown in green.

(B) Schematic representation of the protein-protein interaction as determined by IP-MS. Red lines represent the interaction among the SWR1 components; grey lines indicate the interaction among the ISWI components. Arrows point to the interaction proteins that were identified by IP-MS.

(C) Determination of the interaction of MBD9 with HTA9, HTA11, PIE1, CHR11, and CHR17 by co-immunoprecipitation.
A plant-specific SWR1 chromatin remodeling complex couples histone H2A.Z deposition with nucleosome sliding. (2020)
Figure 2

Figure 2. CHR11 and CHR17 are recruited to the SWR1 complex by MBD9.

(A) Schematic representation of the interaction of SWR1 components as determined by Y2H assays. Proteins shown in blue are the SWR1 subunits that are not shared by the ISWI complex; the ISWI catalytic subunits CHR11 and CHR17 are shown in green.

(B, C) Determination of the interaction of PIE1-Flag, MBD9-Myc, and CHR11-HA by in vitro pull down assays. PIE1-Flag, MBD9-Myc, and CHR11-HA were expressed from yeast and subjected to the pull down assays. Anti-Flag (B) and anti-HA (C) agaroses were independently used in the pull down assays.

(D) The effect of the pie1 and mbd9-1 mutations on the formation of the SWR1 complex as determined by IP-MS. Transgenic plants expressing Flag-tagged MBD9, PIE1, and CHR11 in the WT and mutant backgrounds were subjected to the IP-MS experiment with anti-Flag agarose.

(E) Diagrams showing the interaction regions of CHR11, MBD9, and PIE1 as determined by Y2H assays. The interaction regions are labelled by red frames. Conserved domains are shown.
A plant-specific SWR1 chromatin remodeling complex couples histone H2A.Z deposition with nucleosome sliding. (2020)
Figure 3

Figure 3. The effect of the SWR1 mutations on development and flowering time. (A) Morphological phenotypes of the WT, chr11/17, pie1, swc2, and rin2 mutant plants. Shown are 24-day-old plants. The top panel shows the phenotypes of plants in the soil; the bottom panel shows the phenotypes of plants on the black cloth.

(B) Shown are rosette leaves of the WT, pie1, swc2, and rin2 mutant plants. Rosette leaves were cut from bolting plants.

(C) The early-flowering phenotype of the mbd9-1, mbd9-3, mbd9-4 and hta9-1/11-1 mutants compared to the WT. 24-day-old plants in soil are shown.

(D) Numbers of rosette leaves of the WT and mutant plants shortly after bolting. Rosette leaves from at least 20 plants were counted for each genotype. Values are mean ± SD. ** P<0.01, student's t test.

(E) Determination of the effect of the arp4-1, arp6 and swc6-1 mutations on flowering time. The top panel shows the number of rosette leaves in the WT, arp4-1, arp6, and swc6-1 mutant plants; the bottom panel shows a photograph of the plants. Rosette leaves from at least 20 plants were counted for each genotype. Values are mean ± SD. ** P<0.01, student's t test.

(F) Determination of the effect of the tra1a and tra1b mutations on flowering time. The top panel shows numbers of rosette leaves of the WT, tra1a-1, tra1a-2, and tra1b mutant plants; the bottom panel shows a photograph of the plants. Rosette leaves from at least 20 plants were counted for each genotype. Values are mean ± SD. ** P<0.01, student's t test.
A plant-specific SWR1 chromatin remodeling complex couples histone H2A.Z deposition with nucleosome sliding. (2020)
Figure 4

Figure 4. The effect of chr11/17, hta9-1/11-1, mbd9-1, and pie1 on gene expression as determined by RNA-seq.

(A) Venn diagrams showing overlaps of differentially expressed genes in the mutants relative to the WT. Up- and down-regulated genes were independently analyzed. The data were generated from two biological replicates. Differentially regulated genes were called when log2(fold change)>1 or <-1; P<0.01.

(B) Heat maps showing the effect of indicated mutations on the expression of differentially expressed genes identified in the mbd9-1 mutant. The color bar shows log2(fold change). Up- and down-regulation are shown by red and blue, respectively.

(C) Scatter plots showing that the expression pattern in the hta9-1/11-1 mutant is correlated with that in the chr11/17, mbd9-1, and pie1 mutants. The differentially expressed genes identified in the hta9-1/11-1 mutant were subjected to the analysis.