Figures and data
Deletion of Dip2 leads to increased cell wall stress resistance.
(A) A model depicting the possible role of Dip2 in regulating DAG species required for PKC activation which governs the CWI pathway in yeast by activating the downstream MAPK cascade. Activation of the MAPK cascade results in increased cell wall synthesis thereby strengthening the cell wall. Dip2 has been depicted in its three-domain architecture, harbouring DMAP-binding domain 1 (DBD1), tandem fatty acyl-AMP ligase-like domains (FLD1 and FLD2).
(B) Serial dilution assay for WT, two biological replicates of Δdip2 (Δdip2_Colony1 and Δdip2_Colony2; Δdip2_Colony1 has been used for further experiments). pDip2::Δdip2 represents Δdip2_Colony1 complemented with Dip2 expressed under its native promoter. Control plate contains synthetic complete (SC) media and cell wall (CW) stress plate has either congo red (CR) (100µg/mL) or calcofluor white (CFW) (50µg/mL). N=3.
(C) Colony forming units of WT and Δdip2 grown in SC media control and cell wall stress induced by CR (100µg/mL). Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test; n = 3; ***p < 0.001; ns = not significant). N=3.
(D) Representative western blot showing pSlt2 (56 kDa) levels in WT and Δdip2 compared to the WT treated with CFW for 30 min, used as a positive control. Bar graph showing quantification of fold change in the pSlt2 levels, normalised with total Slt2. Triose phosphate isomerase (Tpi1) (27 kDa) has been used as a loading control. Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test; n > 3; ***p < 0.001; ns = not significant.
(E) Serial dilution assay image in synthetic complete (SC) media control plate and in the presence of congo red (100µg/mL) for WT and Δdip2 compared with Δdip2 complemented with wildtype and catalytically inactive Dip2 mutants (Dip2D523A and Dip2L687A), expressed in a plasmid pYSM7, under its native promoter. N=3.
(F) Serial dilution assay for WT and Δdip2 grown in SD media control and in the presence of CR (100µg/mL) and cercosporamide (2µg/mL). N=3.
(G) Representative western blot for WT, Δdip2 and cercosporamide treated Δdip2 showing the levels of pslt2 (56 kDa). Quantification of pSlt2 levels for Δdip2 treated with cercosporamide (5µg/mL) compared to WT and Δdip2 control, normalised with total Slt2. Phosphoglycerate kinase (Pgk1) (45 kDa) has been used as a loading control. Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test; N=3; **p < 0.01; ns = not significant).
Dip2-regulated selective DAG subspecies are associated with the activation of PKC signalling.
(A) Lipidomic analysis of WT treated with CFW (50µg/mL) for 30 minutes. DAG intensities normalized to the total protein content in WT+CFW have been compared to the intensities from the same species in WT control sample. Data are represented as mean ± standard error of mean (SEM) (unpaired, two-tailed Student’s t-test; N=6). ****p < 0.0001; ***p < 0.001 ns = not significant.
(B) A flowchart for the liposome sedimentation assay explaining the incubation of liposomes with protein, followed by ultracentrifugation and western blotting for supernatant (unbound protein) and pellet (bound protein) fraction.
(C) Representative western blot for liposome sedimentation assay with different DAGs showing supernatant fraction (unbound protein) and pellet fraction (liposome bound protein), probed for C1 domain using anti-GFP antibody (43k Da). N > 3.
(D) Western blot probing for C1 domain bound to different concentrations of C36:0 DAG containing liposomes (pellet fraction) and the unbound protein (supernatant fraction) using anti GFP antibody. Structure of respective DAGs is shown on the right side. N > 3.
(E) Western blot probing for C1 domain at different concentrations of C34:1 DAG containing liposomes in both pellet and supernatant fractions. Structure of respective DAGs have been shown on the right side. N > 3.
(F) Bar graph showing quantification of percentage binding of C1-GFP with increasing concentrations of specific (C36:0) and non-specific (C34:1) DAGs. Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test; n=3; ***p < 0.001; ns = not significant), N=3.
PKC activation is independent of bulk DAG metabolism pathway.
(A-C) Lipidomic analysis of DAGs in Δdga1, Δlro1 and Δdgk1, compared to WT. Fold change values are mentioned below and are represented as green colour gradient. Data are represented as mean ± SEM (unpaired, two-tailed Student’s t-test; N = 5) ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns = not significant.
(D) Serial dilution assay for single knockouts of bulk DAG acting enzymes (Dga1, Lro1 and Dgk1) in the presence of CFW (50µg/mL), compared to SC media control plate. N=3.
(E) Representative western blot and quantification of pSlt2 (56 kDa) levels in deletion strains of DAG metabolizing enzymes. Fold change has been quantified with respect to the total Slt2. Triose phosphate isomerase (Tpi1) (27 kDa) has been used as a loading control. Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test); N=4; ****p < 0.0001; *p < 0.05; ns = not significant.
(F) DAG subspecies quantification using lipidomics for the double knockout of LRO1 and DGA1, compared to WT and Δdip2. Data are represented as mean ± SEM (unpaired, two-tailed Student’s t-test); n = 6; ****p < 0.0001; ***p < 0.00; *p < 0.05; ns = not significant.
(G) Serial dilution assay image for the cell wall stress sensitivity of Δdga1Δlro1, compared to WT and Δdip2, in the presence of CFW (50µg/mL), compared to SC media control plate. N=3. SC media control image is reused in Figure 3-figure supplement 2.
(H) Representative western blot for pSlt2 (56 kDa) estimation in Δdga1Δlro1, compared to total Slt2 and a loading control Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) (36 kDa). Fold change has been quantified with respect to the total Slt2. Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test); N=3; **p < 0.01; ns = not significant.
Psi1-Plc1-Dip2 triad axis regulates selective DAG subspecies to modulate PKC signalling.
(A) Schematic showing various pathways (green arrows) that feed into the production of DAGs in the presence of various enzymes (shown in blue) and different chemical inhibitors (red) blocking the respective pathways.
(B) Quantification of selective DAGs in WT and Δdip2 samples treated with two different concentrations, i.e., 0.5 and 1 µM of U73122. Data are represented as mean ± SEM (unpaired, two-tailed Student’s t-test; N = 5). ****p < 0.0001; ***p < 0.001; ns = not significant.
(C) Specific DAG quantification using lipidomics for deletion of Δplc1 and Δplc1Δdip2. Data are represented as mean ± SEM (unpaired, two-tailed Student’s t-test; n = 6). ****p < 0.0001; ***p < 0.001 ns = not significant. N=6.
(D) Representative western blot image and quantification of pSlt2 (56 kDa) levels for indicated samples. Fold change has been quantified with respect to the total Slt2. Gapdh (36 kDa) has been used as a loading control. Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test; N>4; ****p < 0.0001; **p < 0.01; ns = not significant.
(E) Lipidomic analysis of specific DAGs in double knockout of PSI1 and DIP2, compared with WT and Δpsi1. Data are represented as mean ± SEM (unpaired, two-tailed Student’s t-test; **p < 0.01; *p < 0.05; ns = not significant. N = 5).
(F) Representative western blot image and quantification of pSlt2 (56 kDa) levels for indicated samples. Two data points for controls (WT and Δdip2) are same as that of Figure 4D. Fold change has been quantified with respect to the total Slt2. Phosphoglycerate kinase (Pgk1) (45 kDa) has been used as a loading control. Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test; N>3; ****p < 0.0001; ***p < 0.001; ns = not significant.
(G) Schematic showing Psi1-Plc1-Dip2 axis to regulate Pkc1 signalling where Psi1 remodels phosphoinositides (PI) to enrich it with C36:0 and C36:1 containing acyl chain, which is channelled to the selective DAGs via Plc1 and in turn activate Pkc1.
Dip2 and PKC share a parallel history of co-emergence coevolution across Opisthokonta.
(A) Emergence and distribution of DAG effector proteins across tree of life. Green circles represent presence, while red circles represent absence of effector proteins. Yellow circle represents the primitive form of PKC. List of DAG effectors is taken from a previous study (Colon-Gonzalez & Kazanietz, 2006).
(B) Phylogenetic profiling of Dip2 and PKC shows their co-emergence in Opisthokonta. Inset shows presence of Dip2 and PKC in different fungal branches (Ocana-Pallares et al., 2022).
Green circle represents presence, red represents absence, while yellow circle represents a primitive form of PKC harbouring only C1 and kinase domain.
(C) Interspecies coevolution plot (using patristic distance) between fungal Dip2 and PKC using three different phylogenetic algorithms, with the correlation coefficient value represented by r. Number of organisms and significance levels are indicated.
(D) Interspecies correlation coefficient values (r) for Dip2 compared with PKC and control proteins Gapdh and Pgk using various algorithms for calculating phylogenetic distances. r value is calculated for Gapdh and Pgk the same way. The table contains the exact correlation coefficient values for all the above-mentioned protein pairs using different algorithms.
(E) Coevolution analysis (using genetic distance) between Dip2 and PKC along with their individual comparison with control protein Gapdh. Number of organisms and significance levels are indicated.
(F) Correlation coefficient values represented as bar graph for Dip2 compared with a set of housekeeping genes and lipid metabolizing proteins.
A graphical representation depicting the emergence of Dip2-PKC axis and the evolution of selective DAG-based PKC signalling in Opisthokonta.
Dip2 is recruited parallel to PKC in the tree of life at the root of Opisthokonta evolution ∼1.2 billion years ago. This Dip2-PKC axis remains conserved across Fungi and Metazoan. Two distinct DAG pools are sourced from de novo or salvage pathways and acyl chain remodelling of PIs, leading to metabolic and signalling DAG pool, respectively. Remodelled phosphoinositides (PI, PIP, PIP2) by Psi1 are enriched with 18:0 acyl chains and channelled to corresponding PIP2, which forms respective DAG species (C36:0, C36:1) upon hydrolysis by Plc1. These selective DAG subspecies act as secondary messenger for PKC signalling and therefore regulate the CWI pathway in yeast. Dip2 maintains the levels of these selective DAGs by facilitating their conversion to TAGs, thereby creating a diversification of DAG function into metabolic and signalling pathways.
Serial dilution assay in YPD media for WT, Δdip2, pDip2::Δdip2 represents Δdip2 complemented with Dip2 expressed under its native promoter. Control plate contains YPD media and cell wall (CW) stress plate has either congo red (CR) (100μg/mL) or calcofluor white (CFW) (50μg/mL). N=3.
(A) Western blot image showing expression of wildtype Dip2-GFP-His-HA (219 kDa) and mutant Dip2 expressed in Δdip2. WT serves as a negative control and Pgk1(45 kDa) blot serves as the loading control. (B) Representative western blot image showing pSlt2 (56 kDa) levels on complementing Δdip2 with wildtype and catalytic mutant Dip2 (Dip2D523A and Dip2L687A). Quantification of pSlt2 levels for WT and mutant Dip2 complemented Δdip2 strain, normalised with total Slt2. Phosphoglycerate kinase (Pgk1) (45 kDa) has been used as a loading control. Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test; N=3; *p < 0.05; ns = not significant).
(A) Microscopy image showing WT cells under normal condition (upper panel) and treated with calcofluor white (50µg/mL) (lower panel). Endogenous Dip2 has been tagged with GFP, while mitochondria have been visualized by using a mitochondria-targeted mCherry plasmid transformed in Dip2-GFP cells. The enlarged images of representative bud cells for control and treated, marked with 1 and 2 respectively are shown in inset. Scale bar = 5µm.
(B-C) Line scan of signal intensity along the white line shown in inset images of (A), marked 1 and 2, respectively.
(A) Representative western blot showing increasing activation of Pkc1 pathway (the level of pSlt2) in a time-dependent manner upon CFW treatment (50μg/mL). Tpi1 (27 kDa) has been used as a loading control.
(B) Lipidomic quantification of TAGs for WT cells under cell wall stress in the presence of CFW (50µg/mL) compared to WT under normal growth condition.
(A) Coomassie stained SDS-PAGE gel image of purified Pkc1-C1 domain, expressed and purified from yeast.
(B) Western blot showing no binding (in the pellet fraction) in case of incubation of liposomes with only GFP as a negative control.
(A) SDS-PAGE gel stained with Coomassie showing purified C1 domain of Drosophila melanogaster (C198E-GFP) and Rattus novergicus (C1δ-GFP).
(B) Liposome binding assay for Pkc98E C1 domain from Drosophila melanogaster for multiple DAGs, probed using anti-GFP antibody. Bar graph showing the percentage of DAG binding of Pkc98E C1 domain for different DAG subspecies.
(C) Liposome binding assay for Pkcδ C1 domain from Rattus novergicus for multiple DAGs, probed using anti-GFP antibody. Bar graph showing the percentage of DAG binding of Pkcδ C1 domain for different DAG subspecies.
(A) Serial dilution assay for single deletions of DAG acting enzyme genes (DGA1, LRO1 and DGK1) in the presence of CR (100μg/ml). N=3.
(B) Serial dilution assay for the cell wall stress sensitivity of Δdga1Δlro1, compared to WT and Δdip2, in the presence of CR (100µg/mL), compared to SC media control plate. N=3. SC media control image is same as that of Fig 3G.
Live cell microscopy images showing the DIC images (left), Dip2 tagged with GFP (green), Mitotracker Red (red) and merged images for red and green channels (right) for (A) WT, (C) Δlro1 and (E) Δdga1. Scale bar represents 5 µm.
Line scan of signal intensity along the white lines shown in the respective merged images of (B) WT, (D) Δlro1 and (F) Δdga1. Green and red peaks represent Dip2-GFP and mitotracker red, respectively.
(A) Lipidomic analysis showing the levels of bulk DAGs in WT and Δdip2 on treating with 0.5 and 1 μM of U73122. Data are represented as mean ± SEM (unpaired, two-tailed Student’s t-test). N = 6.
(B) Lipidomic analysis for the DAG subspecies in WT and Δdip2 in the presence and absence of Propranolol and (C) Aureobasidin. Data are represented as mean ± SEM (unpaired, two-tailed Student’s t-test; ns = not significant). N = 6.
(A) Bar graph showing quantification of DAG subspecies level in Δpah1 and Δpah1Δdip2 DKO. Significance in red shows increase in DAG species while the black arrows show decrease in DAGs. Data are represented as mean ± SEM (unpaired, two-tailed Student’s t-test; n = 6). ****p < 0.0001; ***p < 0.001 ns = not significant. N=6.
(B) Cell wall stress assay for Δpah1 and Δpah1Δdip2, compared with Δdip2, in the presence of congo red (100µg/mL).
(C) Western blot and quantification of pSlt2 (56 kDa) in Δpah1 and Δpah1Δdip2, normalized to total Slt2 (56 kDa). Pgk1 (45 kDa) is used as a loading control. Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test; n=3; **p < 0.01; ns = not significant).
(D) Live cell microscopy image for Dip2-GFP in Δpah1 co-stained with Mitotracker Red (red). Scale bar represents 5 µm.
(E) Line scan of signal intensity along the white line shown in the merged image. Green and red peaks represent Dip2-GFP and mitotracker red, respectively.
(A) Microscopy image panel showing DIC image (left), fluorescence for Plc1 tagged with GFP, mitotracker-red for staining mitochondria and the merged image for both the fluorophores (right). Scale bar represents 5 µm.
(B) Line scan plotted for the fluorescence intensity of Plc1-GFP along with mitotracker-red across the line shown in the merged panel.
(C) Microscopy image panel for Plc1-GFP stained with FM4-64 for visualizing vacuoles. Th merged image for red and green channel is sown on the right. Scale bar represents 5 µm.
(D) Fluorescence intensity plotted for GFP and FM4-64 along the line showing colocalization of Plc1 with vacuole.
DAG subspecies quantification for Δpsi1 measured via lipidomics. Data are represented as mean ± SEM (unpaired, two-tailed Student’s t-test; N=5). ****p < 0.0001; ***p < 0.001, **p < 0.01, ns = not significant.
(A) A schematic showing domain architecture and distribution of PKC isoforms (green circles) across Unikonta. Further classification of PKC isoforms (conventional, novel, atypical and Protein Kinase C-Related Kinase (PKN)) and domain annotations are indicated.
(B) Phylogenetic profiling of PKC showing its distribution among protists (grey), microsporidia (blue), fungi (yellow) and metazoan (green). Types of PKC isoforms found in metazoan are mentioned in bracket.
(C) Distribution of Psi1 and Plc1 and their domains across tree of life. Domain organizations of both Psi1 and Plc1 are shown on the top. Green and red colours represent the presence and absence of the proteins in respective supergroups mentioned on the left side.
(A) Interspecies correlation plot for Dip2 & Pkc1 and Gapdh with Pkc1 and Dip2 each from all the fungal sequences present in KEGG Genome Database.
(B) Correlation coefficient values represented as a bar graph for Gapdh compared to other protein controls. Pink colour window behind the bar graphs shows the empirical cut-off of 0.8 correlation coefficient.
(C) Interspecies correlation plot between Dip2 and various control proteins like Ribosomal 60s subunit protein L5 (Rpl5), Cytochrome C (CytC) and Actin.
