Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: ( A ) Human endosomal SNARE proteins were aligned with human IFITM proteins, and a 53-residue stretch of sequence is shown. Residues in red text are the central polar arginine or glutamine residues characteristic of canonical SNARE proteins and are labeled as the central “0” layer. Surrounding heptad repeats of hydrophobic residues are labeled from “−7” to “+8” in relation to the “0” layer, and sites highlighted in yellow correspond to hydrophobic residues. Black underlined residues in IFITM3 were mutated for functional experiments. Green underlined residues in IFITM3 correspond to the previously described amphipathic alpha helix. ( B ) HEK293T cells were transfected with IFITM3-FLAG (WT, F75/78A, R85Q, or G95L) and, 48 h later, challenged with IAV (A/PR/8/34 (PR8), H1N1) at a multiplicity of infection of 0.10. 18 h post-infection, cells were fixed, permeabilized, immunostained with anti-FLAG and anti-NP, and analyzed by flow cytometry. The percentage of NP+ cells was measured as a fraction of FLAG+ cells and shown as mean and standard error (normalized relative to Empty Vector, which was set to 100%). Infections were performed independently three times (biological replicates). Differences that were statistically significant from WT as determined by one-way ANOVA are indicated by (*). Exact p values are as follows (from left to right): p < 0.0001, p < 0.0001, p < 0.0001. ( C ) Structural prediction of the R-SNARE-like motif of IFITM3 was performed with Alphafold and FATCAT. Residues 55–113 of IFITM3 were modeled against a template of the R-SNARE motif of VAMP8 which was previously crystallized as part of the STX7-STX8-Vti1b-VAMP8 trans-SNARE complex (PDB: 1GL2). ( D ) Left: HEK293T cells were co-transfected with IFITM3-FLAG or Empty Vector and a construct encoding a single SNARE protein: STX7-HA, STX8-HA, Vti1b-HA, VAMP8-HA, or VAMP7-myc. SDS-PAGE and immunoblotting were performed with anti-HA, anti-myc, and anti-FLAG in whole cell lysates. Anti-actin was used as loading control. Right: From co-transfected cells, IFITM3-FLAG was immunoprecipitated with anti-FLAG followed by SDS-PAGE and immunoblotting with anti-HA, anti-myc, and anti-FLAG. Heavy chain immunoglobulin chain was used as loading control. Co-transfection of Empty Vector and STX7-HA was used as a negative control for immunoprecipitation. ( E ) Left: HEK293T cells were co-transfected with STX7-HA and either IFITM3-FLAG (WT, F75/78A, or R85Q) or Empty Vector. SDS-PAGE and immunoblotting were performed with anti-HA and anti-FLAG in whole cell lysates. Anti-actin was used as loading control. Right: From co-transfected cells, IFITM3-FLAG was immunoprecipitated with anti-FLAG followed by SDS-PAGE and immunoblotting with anti-FLAG and anti-HA. Light chain immunoglobulin was used as loading control. Co-transfection of Empty Vector and STX7-HA was used as a negative control for immunoprecipitation. The HA/FLAG ratio was calculated for the indicated lanes and shown as mean and standard error (normalized relative to WT, which was set to 100%). Differences that were statistically significant from WT as determined by one-way ANOVA are indicated by (*). Exact p values are as follows (from left to right): p < 0.0001, p < 0.0001. Numbers and tick marks left of blots indicate position and size (in kilodaltons) of protein standard in ladder. Immunoblots were performed independently three times, and a representative example is shown. IAV Influenza A virus, Ig immunoglobulin, IP immunoprecipitation, EV Empty Vector, WT wild-type. .

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Residue, Sequencing, Labeling, Functional Assay, Transfection, Infection, Flow Cytometry, Plasmid Preparation, Construct, SDS Page, Western Blot, Control, Immunoprecipitation, Cotransfection, Negative Control, Virus

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: ( A ) The R-SNARE-like motif of IFITM3 and the R-SNARE motif of VAMP8 (53 residues each) were compared by predictive protein structure alignment and comparison algorithm TM-align. A TM score of 0.75847 was recorded, indicating that the two regions are likely to adopt the same alpha helical protein fold. ( B ) Structural prediction of the R-SNARE-like motif of IFITM3 was performed with Alphafold and FATCAT. Residues 55–113 of IFITM3 were modeled against a template of the R-SNARE motif of VAMP8 which was previously crystallized as part of the STX7-STX8-Vti1b-VAMP8 trans-SNARE complex (PDB: 1GL2). VAMP8 was then swapped with the predicted structure of IFITM3 and shown in relation to the coiled-coiled structure formed with STX7, STX8, and Vti1b. Top: the side chains of residues F75, F78, and R85 of IFITM3 are depicted in yellow and labeled. Bottom: alternative view of the side chains of F75, F78, and R85 of IFITM3. ( C ) Top: examination of the central polar “0” layer of the Q-SNAREs STX7, STX8, Vti1b in relation to the R85 residue in the predicted configuration of IFITM3. Bottom: examination of the central polar “0” layer of the STX7-STX8-Vti1b-VAMP8 complex (PDB: IGL2). .

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Comparison, Labeling, Residue

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: ( A ) Top: HEK293T cells were co-transfected with STX8-HA, STX7-HA, or Vti1b-HA and either Empty Vector or IFITM3-FLAG (WT, F75/78A, or G95L). SDS-PAGE and immunoblotting were performed with anti-HA and anti-FLAG in whole cell lysates. Anti-tubulin was used as loading control. Bottom: from co-transfected cells, STX8-HA, STX7-HA, or Vti1b-HA were immunoprecipitated with anti-HA followed by SDS-PAGE and immunoblotting with anti-HA and anti-FLAG. Heavy chain immunoglobulin was used as loading control. Co-transfection of Empty Vector and STX8-HA was used as a negative control for immunoprecipitation. The FLAG/HA ratio was calculated for the indicated lanes and shown as mean and standard error (normalized relative to WT with STX7-HA, which was set to 100%). Differences that were statistically significant from WT with STX7-HA as determined by one-way ANOVA are indicated by (*). Exact p values are as follows (from left to right): p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001. Numbers and tick marks left of blots indicate position and size (in kilodaltons) of protein standard in ladder. Immunoblots were performed independently three times, and a representative example is shown. ( B ) HEK293T cells stably expressing Empty Vector, IFITM3 WT-FLAG, or IFITM3 G95L-FLAG were transfected with STX7-GFP, fixed, immunostained with anti-FLAG, and analyzed by confocal immunofluorescence microscopy. Colocalization was measured between FLAG and STX7-GFP by calculating the Pearson’s correlation coefficient using Fiji software. Coefficients were calculated from medial Z-slices from three fields of view containing 5–15 cells per condition and presented as means and standard error. Scale bar = 15 microns. Ig immunoglobulin, IP immunoprecipitation, EV Empty Vector, WT wild-type, PCC Pearson’s correlation coefficient. .

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Transfection, Plasmid Preparation, SDS Page, Western Blot, Control, Immunoprecipitation, Cotransfection, Negative Control, Stable Transfection, Expressing, Immunofluorescence, Microscopy, Software

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: ( A ) Left: HeLa cells (WT or IFITM3 KO) were transfected with STX7-HA. SDS-PAGE and immunoblotting were performed with anti-HA and anti-IFITM3 in whole cell lysates. Anti-actin was used as loading control. Right: STX7-HA was immunoprecipitated with anti-HA antibody followed by SDS-PAGE and immunoblotting with anti-HA and anti-IFITM3. Heavy chain immunoglobulin was used as loading control. An isotype matched antibody was used as a control for immunoprecipitation. ( B ) Endogenous STX8 was immunoprecipitated from HeLa (WT or IFITM3 KO) with anti-STX8 followed by SDS-PAGE and immunoblotting with anti-STX8 and anti-IFITM3. Heavy chain immunoglobulin was used as loading control. An isotype matched antibody was used as a control for immunoprecipitation. The (#) symbol denotes the presence of light chain immunoglobulin. ( C ) Endogenous Vti1b was immunoprecipitated from HeLa (WT or IFITM3 KO) with anti-Vti1b followed by SDS-PAGE and immunoblotting with anti-Vti1b and anti-IFITM3. Light chain immunoglobulin was used as loading control. ( D ) Endogenous VAMP8 was immunoprecipitated from HeLa (WT or IFITM3 KO) with anti-VAMP8 followed by SDS-PAGE and immunoblotting with anti-VAMP8 and anti-IFITM3. Light chain immunoglobulin was used as loading control. ( E ) Endogenous VAMP7 was immunoprecipitated from HeLa (WT or IFITM3 KO) with anti-VAMP7 followed by SDS-PAGE and immunoblotting with anti-VAMP7 and anti-IFITM3. Two forms of VAMP7 were detected following immunoprecipitation: one form of ~25 kD and another of ~15 kD (Wojnacki et al, ). Light chain immunoglobulin was used as loading control. ( F ) Left: Recombinant STX7 and recombinant IFITM3 (WT, F75/78A, or G95L) were mixed together in vitro and reaction inputs were visualized by SDS-PAGE and immunoblotting with anti-STX7 and anti-IFITM3. Right: recombinant STX7 was immunoprecipitated with anti-STX7 followed by SDS-PAGE and immunoblotting with anti-STX7 and anti-IFITM3. Heavy chain immunoglobulin was used as loading control. Numbers and tick marks left of blots indicate position and size (in kilodaltons) of protein standard in ladder. Immunoblots were performed independently two times and a representative example is shown. Ig immunoglobulin, IP immunoprecipitation, KO knockout, WT wild-type, r recombinant. .

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Transfection, SDS Page, Western Blot, Control, Immunoprecipitation, Recombinant, In Vitro, Knock-Out

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: ( A ) HeLa cells (WT or IFITM3 KO) were subjected to whole cell lysis, SDS-PAGE, and immunoblotting with anti-IFITM2/3, anti-STX8, anti-Vti1b, anti-VAMP8, anti-VAMP7, and anti-actin. Red and green arrows indicate IFITM3 and IFITM2, respectively, recognized by the anti-IFITM2/3 antibody. ( B ) STX7-HA was transfected into HeLa (WT or IFITM3 KO) and immunoprecipitated with anti-HA followed by SDS-PAGE and immunoblotting with anti-HA, anti-IFITM3, anti-STX8, and anti-Vti1b. Heavy chain immunoglobulin was used as loading control. An isotype matched antibody was used as a control for immunoprecipitation. The (#) symbol denotes the presence of light chain immunoglobulin. ( C ) Purified recombinant IFITM3 protein (WT, F75/78A, or G95L) of varying inputs (6, 12, or 18 µg) were subjected to SDS-PAGE and visualized by Coomassie stain. ( D ) 20 ng of purified recombinant IFITM3 protein (WT, F75/78A, or G95L) were subjected to SDS-PAGE and immunoblotting with anti-IFITM3. ( E ) Recombinant STX7 of varying inputs (0.5, 1, or 2 µg) were subjected to SDS-PAGE and visualized by Coomassie stain. ( F ) Recombinant STX7 was subjected to SDS-PAGE and immunoblotting with anti-STX7. Numbers and tick marks left of blots indicate position and size (in kilodaltons) of protein standard in ladder. Immunoblots were performed independently two times and a representative example is shown (for ( A ) and ( B )) or once (for ( C ), ( D ), ( E ), and ( F )). Ig immunoglobulin, IP immunoprecipitation, EV Empty Vector, WT wild-type, r recombinant. .

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Lysis, SDS Page, Western Blot, Transfection, Immunoprecipitation, Control, Purification, Recombinant, Staining, Plasmid Preparation

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: ( A ) Size exclusion chromatography was performed on recombinant IFITM3 proteins (WT, F75/78A, and G95L) and eluted fractions were analyzed by SDS-PAGE and Coomassie staining. 150 µg recombinant protein was loaded as input and fraction numbers correspond to mL volumes eluted from column. ( B ) Left: HEK293T cells stably expressing Empty Vector, IFITM3 WT-FLAG, or IFITM3 G95L-FLAG were co-transfected with STX7-HA and VAMP7-Myc. Whole cell lysates were subjected to SDS-PAGE and immunoblotting with anti-HA, anti-FLAG, and anti-Myc. Actin was used as a loading control. Right: STX7-HA was immunoprecipitated with anti-HA followed by SDS-PAGE and immunoblotting with anti-HA, anti-FLAG, and anti-Myc. The Myc/HA ratio was calculated for the indicated lanes and shown as mean and standard error (normalized relative to Empty Vector, which was set to 100%). Differences that were statistically significant from Empty Vector as determined by one-way ANOVA are indicated by (*). Exact p values are as follows (from left to right): p = 0.0099, p = 0.8782. Numbers and tick marks left of blots indicate position and size (in kilodaltons) of protein standard in ladder. Immunoblots were performed independently three times, and a representative example is shown (except ( A ), which was performed once). ( C ) HeLa (WT or IFITM3 KO) were transfected with STX7-GFP and VAMP8-mCherry and confocal immunofluorescence microscopy was performed. Colocalization between STX7-GFP and VAMP8-mCherry was measured by calculating the Pearson’s correlation coefficient using Fiji software. Coefficients were calculated from medial Z-slices from three fields of view containing 5–15 cells per condition and presented as means and standard error. Scale bar = 15 microns. IP immunoprecipitation, WT wild-type, r recombinant. .

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Size-exclusion Chromatography, Recombinant, SDS Page, Staining, Stable Transfection, Expressing, Plasmid Preparation, Transfection, Western Blot, Control, Immunoprecipitation, Immunofluorescence, Microscopy, Software

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: ( A ) HEK293T cells stably expressing Empty Vector, IFITM3 WT-FLAG, or IFITM3 G95L-FLAG were co-transfected with STX7-HA and VAMP8-Myc. Whole cell lysates were subjected to SDS-PAGE and immunoblotting with anti-HA, anti-FLAG, and anti-Myc. Actin was used as a loading control. Right: STX7-HA was immunoprecipitated with anti-HA followed by SDS-PAGE and immunoblotting with anti-HA, anti-FLAG, and anti-Myc. The Myc/HA ratio was calculated for the indicated lanes and shown as mean and standard error (normalized relative to Empty Vector, which was set to 100%). Differences that were statistically significant from Empty Vector as determined by one-way ANOVA are indicated by (*). Exact p values are as follows (from left to right): p = 0.0016, p = 0.4318. ( B ) HEK293T cells stably expressing Empty Vector, IFITM3 WT-FLAG, or IFITM3 G95L-FLAG were transfected with STX7-GFP and VAMP8-mCherry, fixed, and analyzed by confocal immunofluorescence microscopy. Colocalization was measured between STX7-GFP and VAMP8-mCherry by calculating the Pearson’s correlation coefficient using Fiji software. Coefficients were calculated from medial Z-slices from three fields of view containing 5–15 cells per condition and presented as means and standard error. Scale bar = 15 microns. ( C ) HEK293T cells stably expressing Empty Vector, IFITM3 WT-FLAG, or IFITM3 G95L-FLAG were inoculated with IAV (+) or medium (−) for 18 h and subjected to whole cell lysis. Endogenous STX8 was immunoprecipitated with anti-STX8 followed by SDS-PAGE and immunoblotting with anti-STX8, anti-FLAG, anti-VAMP8, and anti-VAMP7. The VAMP8/STX8 and VAMP7/STX8 ratios were calculated for the indicated lanes and shown as mean and standard error (normalized relative to No Virus Empty Vector, which was set to 100%). Differences that were statistically significant from No Virus Empty Vector as determined by one-way ANOVA are indicated by (*). Exact p values are as follows (from left to right, top to bottom): p = 0.0007, p = 0.6986, p = 0.0093, p = 0.4523, p < 0.0001, p = 0.7960. ( D ) Endogenous STX8 was immunoprecipitated from HeLa cells (WT or IFITM3 KO) with anti-STX8 followed by SDS-PAGE and immunoblotting with anti-STX8, anti-IFITM3, and anti-VAMP8. Light chain immunoglobulin was used as loading control. An isotype matched antibody was used as a control for immunoprecipitation. The VAMP8/STX8 ratio was calculated for the indicated lanes and shown as mean and standard error (normalized relative to WT, which was set to 100%). Differences that were statistically significant from WT as determined by student’s T test are indicated by (*). Exact p value: p = 0.0009. Numbers and tick marks left of blots indicate position and size (in kilodaltons) of protein standard in ladder. The (#) symbol denotes the presence of light chain immunoglobulin. Immunoblots were performed independently three times, and a representative example is shown. Ig immunoglobulin, IP immunoprecipitation, EV Empty Vector, WT wild-type, PCC Pearson’s correlation coefficient. .

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Transfection, SDS Page, Western Blot, Control, Immunoprecipitation, Immunofluorescence, Microscopy, Software, Lysis, Virus

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: ( A ) HEK293T stably expressing Empty Vector, IFITM3 WT-FLAG, or IFITM3 G95L-FLAG were co-transfected with STX7-HA, STX8-HA, Vti1b-HA, and VAMP8-Myc. Following whole cell lysis, samples were either boiled at 100 °C (+) or not (−). SDS-PAGE and immunoblotting was performed with anti-Myc and anti-FLAG. Actin was used as loading control. Numbers and tick marks left of blots indicate position and size (in kilodaltons) of protein standard in ladder. Immunoblots were performed independently twice, and a representative example is shown. ( B ) HEK293T cells stably expressing Empty Vector, IFITM3 WT-FLAG, or IFITM3 G95L-FLAG were pulsed with Dextran Alexa Fluor 488 for 2 h followed by addition of Magic Red for 5 min. Living cells were then analyzed immediately by confocal immunofluorescence microscopy. Colocalization between Dextran and Magic Red was measured by calculating the Pearson’s correlation coefficient using Fiji software. Coefficients were calculated from medial Z-slices from three fields of view containing 5–15 cells per condition and presented as means and standard error. Scale bar = 15 microns. PCC Pearson’s correlation coefficient. .

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Transfection, Lysis, SDS Page, Western Blot, Control, Immunofluorescence, Microscopy, Software

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: IFITM3 selectively regulates the trans-SNARE assembly driving late endosome-late endosome fusion (STX7-STX8-Vti1b-VAMP8), while enabling late endosome-lysosome fusion. As a result, endocytic cargos (including viruses) are more efficiently trafficked to lysosomes in IFITM3-expressing cells. LE late endosome, Lys lysosome.

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Expressing

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Recombinant, Plasmid Preparation, Sequencing, Protease Inhibitor, Immunoprecipitation, Virus, Software