EMBO/SourceData huggingface dataset:
Figure 1Time lapse images of T6SS activity in tagged parental strain (VipA-mCherry2) and ∆tssA mutant background. The parental strain forms multiple dynamic structures per cell. ∆tssA mutant predominantly forms dynamic sheath spots (white arrow) and few WT-like sheath structures (blue arrow). Scale bars: 2 µm.H2-T6SS dynamics in tagged parental strain (∆retS TssB2-mCherry2, referred to as WT H2) and ΔtssA2PA strain. Blue arrow indicates extending and contracting T6SS sheath structures, white arrow points to dynamic sheath spots. The WT H2 strain harbors multiple dynamic sheath structures per cell, while the ΔtssA2PA strain displays very few dynamic spots or extended and contracting structures. Scale bars: 2 µm.
Figure 2Time lapse images of T6SS activity in tagged strain (VipA-mCherry2 TssAVC-mNeonGreen). White arrow indicates path of TssAVC localization during one cycle of T6SS dynamics. Scale bar: 1 µm.Kymographs of dynamics observed for TssAVC. Frequency of specific dynamics is indicated in the upper right corner of the merge image. (B) Sheath and TssAVC dynamics from A. TssAVC localizes to T6SS initiation site and to distal end of growing sheath (white arrow). (C) Once sheath reaches cell periphery, TssAVC dissociates from distal end (white arrow) and sheath stays extended a prolonged period of time. (D) TssAVC dissociates from growing sheath structure and re-associates at a later time point (white arrows) to continue catalysis of polymerization. (E) TssAVC stays attached to sheath distal end after contraction (white arrow).Fluorescence microscopy of T6SS dynamics in a double tagged strain (∆retS TssB2-mCherry2 TssA2PA-mNeonGreen). White arrow indicates full cycle of extending and contracting T6SS sheath. Scale bar: 1 µm.Kymographs of TssB2 and TssA2PA dynamics. TssA2PA localizes at sheath initiation site and on distal end during polymerization (G), dissociates before contraction (H), dissociates and re-associated during sheath extension (I) or stays attached to distal end after contraction (J).
Figure 3Top view (XY-plane) and side view (XZ-plane) of the TssAVC Nt2 domain cryo-EM reconstruction (pink, non transparent) shown with fitted Nt2-dimer model (left). Partial Nt2-CTD and Nt1-Nt2 linker densities are highlighted with black arrows. Top and side views of ribbon diagram of Nt2-dimer (middle). Representative 2D class averages of TssAVC particles with visible connections between Nt2-dimer and CTD (top right), representative 2D class averages of Nt2-dimer particles, Nt2-CTD linker and Nt1-Nt2 linker densities are highlighted with blue and yellow arrows (bottom right).
Figure 4The ∆tagA mutant displays long and bent but still dynamic structures. Scale bar: 2 µm. Frames from time lapse movies showing sheath dynamics in the ∆tagA mutant. Sheath polymerization does not stop when the distal end reaches opposing membrane (white arrow). Continued polymerization causes the sheath to tilt and distal end to move towards the bacterial cell pole, ultimately effecting bending of the whole structure. C. Sheath detaches from its membrane anchor (orange arrow), keeps extending to maximum cell length and then contracts. Scale bars: 1 µm.Time lapse series of T6SS activity in tagged strain (VipA-mCherry2 TagAVC-mNeonGreen). Orange arrow indicates sheath distal end, white arrow indicates initiation site of another T6SS sheath. Scale bar: 1 µm.Kymographs of dynamics observed for TagAVC. E. TagAVC localizes at the distal end when sheath reaches the cell periphery. Signal of TagAVC-mNeonGreen disappeared and reappeared on this site repeatedly. F. TagAVC localizes at the distal end when sheath reaches the cell periphery and stays associated to this site until contraction. G. TagAVC briefly localizes at T6SS initiation site. H. TagAVC briefly localizes at T6SS initiation site and at distal end, where it stays attached after contraction.
Figure 5H1-T6SS dynamics in tagged parental strain (∆retS TssB1-mCherry2, referred to as WT H1) and ΔtssA1PA strain. Blue arrow indicates extending and contracting T6SS sheath structures, white arrow points to dynamic sheath spots. Scale bars: 2 µm.Fluorescence microscopy of T6SS dynamics in ∆retS TssB1-mCherry2 strain harboring pPSV35 plasmid with TssA1PA-mNeonGreen fusion. Scale bar: 2 µm.Kymographs of TssB1 and TssA1PA dynamics. TssA1PA localizes exclusively at sheath initiation site (B-E), disappears after contraction (C,D) or remains in place for longer timeframe (C,E). TssA1PA signal appears in neighboring cell shortly after T6SS attack (B,C).
Figure 1(D-E) Western blot analysis of RIPA soluble-fraction and RIPA-insoluble, urea-soluble fraction of proteins prepared from HEK293T cells expressing empty plasmid (control), FLAG-KIF5A WT, FLAG-ΔExon27. Insoluble/soluble KIF5A fractions were detected with anti-FLAG and actin was used as a loading control, three biological replicates. Statistical analysis was performed using student t-test (*p < 0.05, **p<0.01, ***p<0.001). Data represent the mean ± s.d.
Figure 2(B) Pull down analysis showing the association of ΔExon27 with WT KIF5A, two biological replicates.
Figure 5(A) Sanger sequencing chromatograms of cDNA from control and patient-derived iMNs (P1-P3) showing the effect of mutations on KIF5A transcripts. While the c.3020+2T>C mutation causes exon 27 skipping, the c.2993-1G>A mutation results in an aberrant transcript with a splice acceptor shifted only by a single nucleotide, leading to a frameshift but containing exon 27. The respective protein product predicted for both mutations comprise the same C-terminal aberrant 39 amino acid end.(B) Western blot analysis for validation of the custom mutant-specific antibody using transfected HEK293T cells. While an antibody against the total KIF5A (lower panel) recognized both wild type and mutant protein, the custom antibody targeting the aberrant "neopeptide" detected ΔExon27 specifically (upper panel). NT: No transfection.(C) Mutant KIF5A proteins are endogenously expressed in iMNs, detected by using the custom mutant specific antibody directed against the C-terminal aberrant 39 amino acid sequence. CTL: Control, P1: Patient 1, P2: Patient 2, P3: Patient 3, two independent experiments.(D) Representative images of KIF5A distribution (arrowheads) in control and patient iMNs. Scale bar: 20 µm. (E) Abundance of KIF5A inclusions after normalizing to total neuron numbers. Graph represents data from n=146 control 1 (CTL1), n = 134 control 2 (CTL2), n = 154 patient 1 (P1), n = 162 patient 2 (P2) and n = 133 patient 3 (P3) iMNs from 3 individual experiments. Bars indicate mean ± s.d. Statistical analysis was performed using Mann Whitney Wilcoxon Test (*p<0.05).
Figure 1B, ITC experiment shows the CBS-pair domain of CNNM3 binds PRL2 with a nanomolar Kd and one-to-one stoichiometry.D, Structure of CBS-pair domain of CNNM2 (PDB 4IY0) with the residues DPFYE indicated.F, ITC data of CBS-pair domain mutants binding to PRL2 identifies aspartic acid 426 as essential for binding.G, Kinetics of hydrolysis of a synthetic substrate by PRL2 shows burst kinetics. Addition of the wild-type CBS-pair domain but not the D426A mutant inhibits phosphatase activity.
Figure 2A, Treatment of PRL2 with a synthetic substrate leads to complete phosphorylation of the catalytic residue, cysteine 101. Recombinant PRL2 was treated with 3-O-methylfluorescein phosphate (OMFP) and analyzed by SDS-PAGE with the Phos-tag reagent. Phosphocysteine is hydrolyzed by boiling.B, Detection of phosphorylated by electrospray mass spectrometry. OMFP-treatment increased the mass of the intact protein by 79.99 amu but had no effect on the C101A mutant.C, 31P NMR spectrum of the thioPO3 signal from phosphocysteine in PRL2. The broad peak is TCEP in the buffer.D, Cysteine phosphorylated PRL2 does not bind to CNNM3. Recombinant PRL2 was phosphorylated by treatment with OMFP and subjected to pull-down by the CBS-pair domain of CNNM3 as a GST fusion. Only unphosphorylated PRL2 was observed in the pull-down lanes while phosphorylated PRL2 was in the flow-through lanes.E, ITC measurement of the affinity of PRL2 for the CNNM3 GST-CBS-pair domain. Phosphorylation by OMFP completely blocked binding while oxidization of PRL2 by diamide decreased the affinity 200-fold.F, The cysteine residues in the PRL2 active site are not required for binding the CNNM3 CBS-pair domain.
Figure 3A, Mutations in the CBS-pair loop of CNNM4 prevent binding to PRL3. Lysates of COS7 cells transfected with the indicated constructs were subjected to immunoprecipitation (IP) and immunoblotting (IB) with the indicated antibodies.B C, Wild-type CNNM4-dependent magnesium efflux is inhibited by co-transfection by PRL3 but the efflux by the CNNM4 D485A mutant, which is unable to bind PRL3, is resistant to inhibition. HEK293 cells transfected with the indicated constructs were loaded with Magnesium Green and Mg2+ removed from the medium at the indicated time point (arrowhead). The means of relative fluorescence intensities of 10 cells are graphed as a function of time in panel B and the mean and s.e.m. at 5 min plotted in panel C. P-values were calculated using one way ANOVA followed by Bonferroni's multiple comparison test. n.s., not significant.B C, Wild-type CNNM4-dependent magnesium efflux is inhibited by co-transfection by PRL3 but the efflux by the CNNM4 D485A mutant, which is unable to bind PRL3, is resistant to inhibition. HEK293 cells transfected with the indicated constructs were loaded with Magnesium Green and Mg2+ removed from the medium at the indicated time point (arrowhead). The means of relative fluorescence intensities of 10 cells are graphed as a function of time in panel B and the mean and s.e.m. at 5 min plotted in panel C. P-values were calculated using one way ANOVA followed by Bonferroni's multiple comparison test. n.s., not significant.
Figure 4A, The CBS-pair domain (yellow) forms a dimer and binds two molecules of PRL2 via an extended loop. The positions of the PRL2 C-terminal CAAX prenylation motif [42] and CNNM3 N-terminus suggest that the membrane would sit above the complex.B, Contacts between the CNNM3 CBS2 loop and the PRL2 active site.C, Stereoview of the 2FO-FC omit map of the PRL2 binding loop of CNNM3 contoured at 1 .D, Detail of complex with reduced PRL2, showing how CNNM3 Asp426 inserts into the pocket adjacent to the catalytic cysteine Cys101.E, Model of phosphocysteine preventing CNNM binding. Crystal structure of PRL1 with a bound sulfate shows phosphocysteine would prevent Asp426 binding. Residues are numbered according to the PRL1 structure (PDB 1XM2).
Figure 5A, PRLs expressed in HEK293 cells are intrinsically phosphorylated on cysteine. Phos-tag analysis of cell lysates shows close to 50% of transiently expressed FLAG-PRL1, -2, and -3 are phosphorylated. The phosphorylated PRL bands are marked by an asterisk.B, Immunoblots of PRL1 expressed in HEK293 cells show phosphorylation requires the catalytic cysteine (C104) but not the adjacent cysteine (C49) in the active site.C, Co-immunoprecipitation of Myc-PRL by FLAG-CNNMs shows only the unphosphorylated forms of PRL1 binds CNNM4.D, Endogenous PRLs in HeLa cells are cysteine phosphorylated.E, Levels of unphosphorylated PRL increase in cultured cells in response to removal of magnesium from the cultured medium.
Figure 1A) In our chemoattraction-assay setup, nematode chemoattraction behavior is quantified using a measurable chemoattraction index (C.I.); the higher the C.I. measured in the assay, the stronger the attraction of the males to the sex pheromone. Data information: We assayed 400 males from each strain by using the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value.B) After vaporization (VAC) at 4°C for 24 h, the C. remanei female extract failed to attract wild-type C. elegans males. Conversely, a drop of the sex pheromone extract placed on the lid of a 60-mm Petri dish evoked chemoattractive behavior in wild-type C. elegans males (volatility test). Thus, the functional components in C. remanei female sex pheromone are volatile substances. Data information: We assayed 400 males from each strain by using the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.C) Mutant males with established defects in amphid neurons responded to C. remanei sex pheromone to a lesser degree relative to wild-type, demonstrating that some or all of the amphid neurons are required for sex pheromone perception. Data information: We assayed 400 males from each strain by using the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.
Figure 2A) Inspection of specific amphid-neuron-defective mutants revealed that AWA neurons are most likely responsible for pheromone perception. Here, an edge connects neurons to a genetic mutant if the neurons are documented to be affected in the mutant. The color code of edges represents the average C.I. of multiple genetic mutants with impaired neuronal functions or specified abnormal neuron identities. The region colored green shows the range of wild-type sex pheromone response in the male. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.B) AWA-laser-ablated males were not attracted by the sex pheromone in single-worm chemoattraction assays. Both AWA-defective mutant males (lin-11 and odr-7) and AWA-laser-ablated males failed to reach the sex pheromone within 30 min. Wild-type C. elegans males responded normally to the sex pheromone and wild-type C. elegans hermaphrodites were not attracted by same-sex pheromone. The single-worm chemoattraction assay was performed as described in Materials and Methods. Sample size of males in AWA-laser-ablation experiment: 12; all other experiments: 100 worms.
Figure 3B) Mutant males of 4 GPCR signaling cascade components failed to respond to C. remanei sex pheromone, suggesting that the sex pheromone receptor is a GPCR and that both aforementioned signaling pathways are involved in pheromone perception. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.C) In males from both AWA-specific-rescue odr-3 mutant strain and odr-3 rescued mutant strain, the sex pheromone response was significantly restored in single male worm arrival assays. However, males from the CEM-specific-rescue odr-3 mutant strain failed to reach the sex pheromone within 30 min. In odr-3 and him-5 strain experiments, the sample sizes were 12 and 20 worms, respectively; in all other experiments, 40 worms were used.
Figure 4A) srd-1 mutant males were not attracted to the sex pheromone but were attracted to the positive-control chemoattractants, 1:1000 diacetyl and 10 mg/mL pyrazine. This demonstrated that SRD-1 is required and specific for sex pheromone perception. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.B) Summary of srd-1 expression pattern. The transcriptional reporter Psrd-1::gfp was detected in both male and hermaphrodite ASI neurons in the head region, and only in male AWA and ADF neurons.C) SRD-1::GFP fusion construct revealed predominant SRD-1 localization at the cilia of AWAs in C. elegans males. Scale bar = 5 μm.D) Examination of srd-1 transcriptional reporter revealed that srd-1 is expressed in AWA neurons in males but not hermaphrodites. A transcriptional reporter for odr-7 indicated the locations of AWAs (arrowheads). Scale bar = 50 μm.F) AWA-specific rescue of srd-1 restored sex pheromone perception ability in both males and hermaphrodites. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.G) Ectopic expression of srd-1 in AWB neurons elicited a distinct repulsive behavior toward the sex pheromone in both males and hermaphrodites. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.
Figure 5A) The GCaMP5 reporter was used to visualize activation of the SRD-1 receptor by the sex pheromone in AWA neurons. To facilitate real-time imaging of the process, the heads of the transgenic worms were immobilized on soft-agar pads permeable to sex pheromone.B) Excitation of the GCaMP5 reporter in AWA neurons could be observed through both lateral and top views but the top view was clearer and more suitable for quantification. Arrowheads: AWA neurons. Scale bar = 50 μm.C) Relative fluorescence-intensity change was quantified through calcium imaging. Wild-type males showed receptor excitation by sex pheromones from C. elegans hermaphrodites or C. remanei females. This response was lost in srd-1-mutant-strain males but was restored following AWA-specific rescue of srd-1. Red triangles: time points of sex pheromone addition. Three males were assayed from each strain.
Figure 6B) Wild-type C. elegans males are less attracted to sex pheromone than wild-type C. remanei males. Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.  C) Expression level of srd-1 cDNA in srd-1 transgenic lines. srd-1 mutant males were rescued using, separately, wild-type C. elegans and wild-type C. remanei srd-1 cDNAs. Transgenic lines expressing the molecule at a similar level were selected for use in chemoattraction assays. Four independent transgenic lines were selected, and the expression-level data were combined into a single bar. The expression levels were measured using qPCR. Data information: For transgenic-line studies, each bar represents a total of 1200 males from 4 independent transgenic lines; 20 assays were performed for 2 independent transgenic lines and 10 assays were performed for the other 2 independent transgenic lines, and the tests were conducted over 2-4 separate days. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.  D) srd-1 mutant males rescued with Ce-srd-1 cDNA showed lower responsiveness than those rescued with Cre-srd-1 cDNA, which indicates that sex pheromone perception ability depends primarily on the protein coding region and not the regulatory sequences in the promoter. The sex pheromone perception defect of srd-1 mutants was not rescued in males expressing CT-truncated SRD-1 proteins. The findings show that the CT region is essential for sex pheromone perception. Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.  E) srd-1 mutant males rescued by using Ce-srd-1 cDNA together with Cre-srd-1 CT cDNA were more responsive to sex pheromone than those rescued using Cre-srd-1 cDNA with Ce-srd-1 CT cDNA. The results of the CT-swapping experiment demonstrated that sex pheromone perception ability is determined by the CT region and not the rest of the SRD-1 protein. Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.
Figure 2PS decarboxylase activity in wild-type (WT, W303) and the indicated mutant strains expressed as (B) the molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE), after 20 min incubation with 13C315N-serine.(C) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.
Figure 3PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as (B) the molecular species composition of *PS and *PE after 20 min incubation with 13C315N-serinePS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as ; (C) the corresponding molecular signatures of PSD activity.
Figure 4(B) Acyl chain composition of WT and psd1∆ cells overexpressing SCT1 (pSCT1) vs. empty vector control (pEmpty). Data is presented as mean ± SD (n>10). **** p < 0.0001, multiple t-test.(C) Ratio of the proportions of saturated over unsaturated acyl chains (SFA/UFA) of WT, psd1∆, crd1∆ and psd2∆ overexpressing SCT1 vs. empty vector control. Data is presented as mean ± SD (n≥3). ** p<0.01, **** p < 0.0001, unpaired two-tailed t-test.(D) OLE1 transcript levels in WT and psd1∆ transformed with pSCT1 vs. pEmpty as determined by RT-qPCR. Data were normalized to endogenous ACT1 mRNA, and to the average of WT pEmpty (n=3).
Figure 5(A) SFA/UFA ratios of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. control (pEmpty). Data is presented as mean ± SD (n≥3). * p < 0.05, ** p < 0.01, **** p < 0.0001, n.s. not significant, unpaired two-tailed t-test of the indicated bar compared to WT pSCT1.(B) Growth of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. pEmpty. Serial dilutions (10-1 - 10-4) were spotted on SGal + 0.05% glucose and incubated for 3 days at 30°C.PS decarboxylase activity in wild-type (WT, BY4741) and the indicated mutant strains transformed with pEmpty, expressed , and (D) molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE) after 20 min incubation with 13C315N-serine. The differences in molecular species profiles of *PS and *PE between the BY4741 WT shown here and those obtained by 2H3-serine labeling (Figure 4A) are attributed to the different culture media, lipid extraction procedures and mass spectrometry techniques used.(E) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.
f2(b) The CD spectra of the Beclin 1 CC domain at different temperatures.(c). Dynamic exchange assay of wild-type Beclin 1. His-tagged and untagged Beclin 1 CC domain were co-incubated at the indicated temperatures and coprecipitated at preset temperatures by Ni2+- nitrilotriacetic acid (NTA) agarose beads. The presence of heterodimeric Beclin 1 CC domain, consisting of one monomer with his-tag and the other monomer without tag, was checked by SDS gel and indicated by the arrow.(d) Self-association pattern of Beclin 1 in vivo. Both GFP- and AsRed-tagged full-length Beclin 1 are co-transfected into HEK 293T cells and their self-association is probed by co-IP using GFP antibody for immunoprecipitation, followed by western blot with anti-AsRed antibody.