Journal: Nature Communications

Article Title: Acute diacylglycerol production activates critical membrane-shaping proteins leading to mitochondrial tubulation and fission

doi: 10.1038/s41467-025-57439-9

Figure Lengend Snippet: a Representative images of HEK293A cells (5 μm scale bar) showing the localization of the OMM-targeted FRB recruiter (OMM-FRB-mRFP, magenta), endophilin B1 (EndoB1-mEGFP, green), and the GTPase-deficient mutant of Drp1 (mCherry-Drp1 K38A ) after 10 min of rapamycin-induced (100 nM) recruitment of the catalytically active Bc PI-PLC 3A (emiRFP670-FKBP- Bc PI-PLC 3A ) to the cytosolic membrane leaflet of the mitochondria. The composite images present an overlay of the OMM-FRB together with EndoB1. In addition, a more detailed time series showing the individual fluorescent channels associated with the images presented here is provided as Supplementary Fig. and Supplementary Movie . b Representative images of Drp1 KO HeLa cells (5 μm scale bar) showing the localization of the OMM-targeted FRB recruiter (OMM-FRB-mRFP, magenta) and EndoB1 (EndoB1-mEGFP, green) after 10 min of rapamycin-induced (100 nM) recruitment of the catalytically active Bc PI-PLC 3A (emiRFP670-FKBP- Bc PI-PLC 3A ) to the cytosolic membrane leaflet of the mitochondria. The composite images present an overlay of the OMM-FRB together with EndoB1. The complete presentation of the individual fluorescent channels from this representative image is provided as Supplementary Fig. , while a complete time series for a replicate of this experiment is provided as Supplementary Movie . c , d Representative images of HEK293A cells (5 μm scale bar) showing the localization of the OMM-targeted FRB recruiter (OMM-FRB-ECFP, magenta) and either EndoB1 ( c ; EndoB1-mEGFP, green) or the isolated N-BAR domain ( d ; residues 1–270, EndoB1 N-BAR -mEGFP, green) in response to rapamycin-induced (100 nM) recruitment of the catalytically active Bc PI-PLC 3A (mRFP-FKBP- Bc PI-PLC 3A , gray) to the cytosolic membrane leaflet of the mitochondria. The composite images shown present an overlay of the OMM-FRB together with EndoB1. In addition, the complete time series (Supplementary Movie ) as well as enlarged still images (bottom row panels; 2.5 μm scale bar) are presented for ( c ), which shows Bc PI-PLC 3A -induced translocation of the full-length EndoB1 to the OMM.

Article Snippet: Fig. 8 Deletion of the amphipathic H 0 and H 1i helices or disruption of self-assembly significantly reduces Bc PI-PLC 3A -induced translocation of EndoB1 to the OMM. a Domain architecture of the isolated N-BAR domain from EndoB1 (middle panel; AlphaFold2 prediction AF-Q9Y371-F1; DeepMind) , presented along with a comparison of the physiochemical properties (HeliQuest) of the N-terminal amphipathic H 0 (right side panel) and H 1i (left side panel) helices of EndoB1 (UniProt: Q9Y371).

Techniques: Mutagenesis, Membrane, Isolation, Translocation Assay

Journal: Nature Communications

Article Title: Acute diacylglycerol production activates critical membrane-shaping proteins leading to mitochondrial tubulation and fission

doi: 10.1038/s41467-025-57439-9

Figure Lengend Snippet: a , b Representative images of a planar bilayer (magenta) containing 5% PI ( a ) or 5% DAG ( b ) with 15% CL, before and after incubation with EndoB1-mEGFP (green). c Representative images of a planar bilayer (magenta) of the indicated lipid composition after incubation with EndoB1 N-BAR (Residues 1–270)-mEGFP (green). White arrows in magnified panels mark protein clusters that coincide with regions of high membrane fluorescence indicating tubulation. d Representative images of membrane tubes (magenta) of the indicated lipid composition incubated with EndoB1 N-BAR -mEGFP (green). e Membrane densities of EndoB1 N-BAR -mEGFP are reported as the ratio of EGFP and membrane fluorescence across a range of tube sizes as well as on the planar bilayer island of the indicated lipid composition. Data are normalized to the membrane density of EndoB1 N-BAR -mEGFP on tubes of < 15 nm radius containing 5% DAG and 15% CL and represents the mean ± SD of 63 tubes and 31 different regions on planar bilayers for DAG-containing and 106 tubes and 35 different regions on planar bilayers for PI-containing membranes. Statistical significance was estimated using unpaired Mann-Whitney’s test. *** denotes p < 0.0001, ** denotes p = 0.074, * denotes p = 0.0238. f Representative time-lapse images of a planar bilayer responding to flowing EndoB1 N-BAR -mEGFP, which are also presented as Supplementary Movie . The inset shows a magnification of a single representative tubulation event. Images are acquired in the membrane fluorescence channel and are inverted in contrast for clarity. White arrows mark a single tubule, yellow arrows mark events of coiling of the tubule, and green arrows mark the coalescence of other single tubules in the vicinity. g Representative time-lapse images showing fluorescence recovery after photobleaching the intrinsic fluorescent lipid probe in a large area of the planar bilayer displaying EndoB1 N-BAR -mEGFP-coated tubules, which are also presented as Supplementary Movie . The region above the dotted line is where the lipid probe was bleached.

Article Snippet: Fig. 8 Deletion of the amphipathic H 0 and H 1i helices or disruption of self-assembly significantly reduces Bc PI-PLC 3A -induced translocation of EndoB1 to the OMM. a Domain architecture of the isolated N-BAR domain from EndoB1 (middle panel; AlphaFold2 prediction AF-Q9Y371-F1; DeepMind) , presented along with a comparison of the physiochemical properties (HeliQuest) of the N-terminal amphipathic H 0 (right side panel) and H 1i (left side panel) helices of EndoB1 (UniProt: Q9Y371).

Techniques: Incubation, Membrane, Fluorescence

Journal: Nature Communications

Article Title: Acute diacylglycerol production activates critical membrane-shaping proteins leading to mitochondrial tubulation and fission

doi: 10.1038/s41467-025-57439-9

Figure Lengend Snippet: a Domain architecture of the isolated N-BAR domain from EndoB1 (middle panel; AlphaFold2 prediction AF-Q9Y371-F1; DeepMind) , presented along with a comparison of the physiochemical properties (HeliQuest) of the N-terminal amphipathic H 0 (right side panel) and H 1i (left side panel) helices of EndoB1 (UniProt: Q9Y371). Protein structures were prepared using the PyMOL Molecular Graphics System (Version 3.0; Schrödinger, LLC). b – d Representative images of HEK293A cells (10 μm scale bar) showing the localization of the OMM-targeted FRB recruiter (OMM-FRB-ECFP, magenta) and EndoB1 mutants with deletions of either the H 0 helix ( b , EndoB1 △H0 (△Residues 5–30)-mEGFP, green), H 1i helix ( c , EndoB1 △H1i (△Residues 69–88)-mEGFP, green), or both amphipathic segments ( d , EndoB1 △H0,△H1i (△Residues 5–30 + △Residues 69–88)-mEGFP, green) before (top row panels) or 10 min after (bottom row panels) rapamycin-induced (100 nM) recruitment of the catalytically active Bc PI-PLC 3A (mRFP-FKBP- Bc PI-PLC 3A , gray) to the cytosolic membrane leaflet of the mitochondria. e Kinetics and ( f ) quantified area under the curve measured for the OMM localization of the wild-type EndoB1 (green), isolated N-BAR domain (EndoB1 N-BAR , Residues 1–270, magenta), L227D point-mutant (EndoB1 L227D , blue), as well as the EndoB1 △H0 (△Residues 5–30, orange), EndoB1 △H1i (△Residues 69–88, yellow), and EndoB1 △H0,△H1i (△Residues 5–30 + △Residues 69–88, gray) deletion mutants in HEK293A cells following recruitment of FKBP- Bc PI-PLC 3A to the cytosolic membrane leaflet of the mitochondria, as measured using EndoB1 variant-specific OMM-EndoB1 BRET biosensors (AKAP TM -mVenus-tPT2A-EndoB1(or variants)-sLuc). To allow for direct comparisons between OMM-EndoB BRET variants, the BRET values obtained for recruitment of the active iRFP-FKBP- Bc PI-PLC 3A enzyme were normalized to measurements made using the corresponding catalytically-inactive iRFP-FKBP- Bc PI-PLC DEAD control. BRET measurements are presented as mean values ± SEM from three independent experiments carried out using triplicate wells.

Article Snippet: Fig. 8 Deletion of the amphipathic H 0 and H 1i helices or disruption of self-assembly significantly reduces Bc PI-PLC 3A -induced translocation of EndoB1 to the OMM. a Domain architecture of the isolated N-BAR domain from EndoB1 (middle panel; AlphaFold2 prediction AF-Q9Y371-F1; DeepMind) , presented along with a comparison of the physiochemical properties (HeliQuest) of the N-terminal amphipathic H 0 (right side panel) and H 1i (left side panel) helices of EndoB1 (UniProt: Q9Y371).

Techniques: Isolation, Comparison, Membrane, Mutagenesis, Variant Assay, Control