O-GlcNAcylation of AMFR stabilizes TSPAN4 to regulate migrasome formation for viral release

Abstract

Migrasomes are migration-dependent organelles, serving as delivery packets to mediate release of cytoplasmic contents. Tetraspanin 4 (TSPAN4) acts as a marker for migrasomes and is essential for their formation. However, the regulatory mechanism(s) underlying TSPAN4-mediated migrasome biogenesis and its physiological functions remain to be elucidated. Here, we identified AMFR, an ER-resident E3 ligase, regulates migrasome formation through catalyzing the K48-linked polyubiquitination of TSPAN4 for degradation. Further, O-GlcNAcylation of AMFR by OGT at threonine 643 disrupts AMFR-TSPAN4 interactions, thereby stabilizing TSPAN4 and promoting migrasome formation. Additionally, viruses dynamically regulate migrasome formation by modulating AMFR O-GlcNAcylation and TSPAN4 ubiquitination. During the early stages of VSV or HSV-1 infection, viruses enhance migrasome formation and exploit these structures to spread among neighboring cells, whereas abolish migrasome formation during the late stages of infection. Our findings reveal a negatively regulatory mechanism governing migrasome biogenesis, and highlight how VSV and HSV-1 manipulate this process to facilitate their release.

Fig. 1. Migrasomes facilitate viral release.

a NRK cells stably expressing Vector or TSPAN4-Flag were infected with VSV-GFP (Multiplicity of infection (MOI) = 0.01) or HSV-1 (MOI = 0.01) and incubated for an additional 12 or 36 h. Culture supernatants were collected, and viral titers were quantified using plaque assays. Data represent the mean ± SD from three biological replicates. Statistical significance was determined using a two-tailed Student’s t-test. Source data are provided as a Source Data file. b Wild-type (WT) or Tspan4 Knockout (KO) NRK cells were infected with VSV-GFP (MOI = 0.01) or HSV-1 (MOI = 0.01) and incubated for an additional 12 or 36 h. Culture supernatants were collected, and viral titers were quantified using plaque assays. Data represent the mean ± SD from three biological replicates. Statistical significance was determined using a two-tailed Student’s t-test. Source data are provided as a Source Data file. c L929 cells stably expressing TSPAN4-Flag were infected with lentivirus carrying constructs with non-specific shRNA or shRNA targeting Cert, Cers5, or Sgsm2, cells were infected with VSV-GFP (MOI = 0.01) or HSV-1 (MOI = 0.01) and incubated for an additional 12 or 36 h. Culture supernatants were collected, and viral titers were quantified using plaque assays. Data represent the mean ± SD from three biological replicates. Statistical significance was determined using a two-tailed Student’s t-test. Source data are provided as a Source Data file. d WT, PIP5K1A or Rab35 KO NRK-TSPAN4-Flag cells were infected with VSV-GFP (MOI = 0.01) or HSV-1 (MOI = 0.01) and incubated for an additional 12 or 36 h. Culture supernatants were collected, and viral titers were quantified using plaque assays. Data represent the mean ± SD from three biological replicates. Statistical significance was determined using a two-tailed Student’s t-test. Source data are provided as a Source Data file. e L929 cells stably expressing TSPAN4-mCherry were infected with VSV-GFP at an MOI of 0.2. Samples from cell bodies and purified migrasomes were analyzed via WB using antibodies against VSV-M, HSP90, Calnexin, Tubulin, and identified migrasome-specific marker ITGA5, PIGK, and EOGT (n = 3 independent experiments). Source data are provided as a Source Data file. f NRK cells stably expressing TSPAN4-mCherry were incubated with purified migrasomes from. e (n = 3 independent experiments). Cells were imaged by confocal microscopy. Green, VSV-GFP; red, TSPAN4-mCherry. Scale bar represents 10 μm. g Representative transmission electron micrograph (TEM) images of VSV-GFP (at an MOI of 0.2) infected NRK cells (n = 3 independent experiments). Red arrows indicated virions in migrasomes. Mito, mitochondria. Scar bar represents 500 nm. h Time-lapse imaging of VSV-GFP (at an MOI of 0.2) infected NRK cells stably expressing TSPAN4-mCherry. Images were captured every 6 min by confocal microscopy. Green, VSV-GFP; red, TSPAN4-mCherry. Scale bar, 10 μm. i Representative images from plaque assay of three biological replicates from purified migrasomes (from VSV-GFP infected TSPAN4-mCherry stably expressing L929 cells) treated with either Proteinase K, Proteinase K and Triton ×-100, or PBS. Source data are provided as a Source Data file. j, k NRK cells stably expressing TSPAN4-mCherry were infected with VSV-GFP at an MOI of 0.2 for the indicated time. Cells were imaged by confocal microscopy. Green, VSV-GFP; red, TSPAN4-mCherry. Scar bar represents 10 μm. Number of migrasomes from the cells in (i); n = 150 cells for 0 h, n = 150 cells for 3 h, n = 149 cells for 6 h, n = 150 cells for 9 h, and n = 150 cells for 12 h per group from three independent experiments. Error bars, mean ± SD. Source data are provided as a Source Data file. l, m HeLa cells stably expressing TSPAN4-Flag were infected with VSV-GFP or HSV-1 at an MOI of 1 for indicated time. Lysates were analyzed via WB. Relative intensity of TSPAN4 normalized to Tubulin of three independent experiments was quantified. Error bars, mean ± SD. Source data are provided as a Source Data file.

Fig. 2. TSPAN4 is ubiquitinated for degradation.

a HeLa cells stably expressing TSPAN4-Flag were treated with 100 μM CHX for indicated time. Lysates were analyzed via WB. Relative intensity of TSPAN4 normalized to GAPDH of three independent experiments was quantified. Error bars, mean ± SD. Source data are provided as a Source Data file. b HeLa cells stably expressing TSPAN4-Flag were treated with 100 μM CHX for 1 h, and further treated with 100 μM CQ or 10 μM MG132 for 6 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 normalized to GAPDH of three independent experiments was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. c, d HeLa cells stably expressing TSPAN4-Flag were infected with or without VSV-GFP or HSV-1 at an MOI of 1 for indicated time, and were further treated with or without 10 μM MG132 or 100 μM CQ for 6 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 normalized to Tubulin of three independent experiments was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. e HEK293T cells were transfected with HA-Ub^K48 with or without TSPAN4-Flag for 36 h. Interactions were analyzed by co-immunoprecipitation (n = 3 independent experiments). Source data are provided as a Source Data file. f HEK293T cells were transfected with HA-Ub^K48 and TSPAN4-Flag mutants for 36 h. Interactions were analyzed by co-immunoprecipitation (n = 3 independent experiments). Source data are provided as a Source Data file. g Amino acid sequence from 106 to 238 of TSPAN4 and point mutations used in this study. h 6 K (K110, K120, K121, K187, K195, K232) are the main ubiquitin site of TSPAN4. HEK293T cells were transfected with HA-Ub^K48 and TSPAN4-Flag or its mutants for 36 h. Interactions were analyzed by co-immunoprecipitation (n = 3 independent experiments). Source data are provided as a Source Data file. i HeLa cells were transfected with TSPAN4-Flag or TSPAN4^6KR-Flag for 24 h, and cells were further treated with 100 μM CHX for indicated time. Lysates were analyzed via WB. Relative intensity of TSPAN4 normalized to Tubulin of three independent experiments was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file.

Fig. 3. AMFR mediates the K48-linked TSPAN4 ubiquitination to regulate migrasome formation.

a Co-immunoprecipitation analysis of TSPAN4-Flag with AMFR-Myc in HEK293T cells (n = 3 independent experiments). Source data are provided as a Source Data file. b Purified His-AMFR was incubated with purified GST or GST-TSPAN4, and analysis of the interactions between AMFR and TSPAN4 by in vitro GST pull-down was performed (n = 3 independent experiments). Source data are provided as a Source Data file. c AMFR overexpression enhances the ubiquitin level of TSPAN4. HEK293T cells were transfected with TSPAN4-Flag, HA-Ub^K48, and AMFR-Myc or mutant AMFR^3CS-Myc for 36 h. Lysates were subjected to Flag immunoprecipitation and analyzed via WB (n = 3 independent experiments). Source data are provided as a Source Data file. d HeLa cells were transfected with indicated plasmids for 24 h, and lysates were analyzed via WB. Relative intensity of TSPAN4 and TSPAN4^6KR of three independent experiments normalized to Tubulin, was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. e HeLa cells stably expressing TSPAN4-Flag were transfected with gradient AMFR-Myc or point mutant AMFR^3CS-Myc for 24 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 of three independent experiments normalized to Tubulin, was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. f WT or Amfr KO HeLa cells stably expressing TSPAN4-Flag were treated with 100 μM CHX for indicated time. Lysates were analyzed via WB. Relative intensity of TSPAN4 of three independent experiments normalized to Tubulin, was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. g Amfr KO HeLa cells stably expressing TSPAN4-Flag were transfected with AMFR-Myc or AMFR^3CS-Myc for 24 h, and harvested before 1 h with or without 100 μM CHX treatment. Lysates were analyzed via WB. Relative intensity of TSPAN4 of three independent experiments normalized to Tubulin, was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. h Subcellular fractions were isolated from HeLa cells stably expressing TSPAN4-Flag transfected with AMFR-Myc or AMFR^3CS-Myc. Calnexin represent the ER, Rab11 represent endosomes, and Tubulin represents cytoplasm, respectively. Source data are provided as a Source Data file. i Quantification of the migration speed of GFP or AMFR-GFP expressing NRK cells. 20 cells of two independent experiments were counted. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. j, k NRK-ITGA5-mCherry cells were transfected with GFP, AMFR-GFP, or AMFR^3CS-GFP. Cells were imaged by confocal microscopy. Green, GFP, AMFR-GFP, and AMFR^3CS-GFP; red, ITGA5-mCherry; yellow, Merge. Number of migrasomes from the cells in (j); n = 150 cells per group from three independent experiments. Scar bar represents 10 μm. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. l, m Tspan4 KO NRK cells were transfected with TSPAN4-mCherry or TSPAN4^6KR-mCherry, with or without GFP, AMFR-GFP, or AMFR^3CS-GFP. Wheat germ agglutinin (WGA) was used to stain migrasomes. Green, Vec, AMFR-GFP and AMFR^3CS-GFP; red, TSPAN4-mCherry and TSPAN46^KR-mCherry; white, Migrasomes. Scar bar represents 10 μm. Cells were imaged by confocal microscopy. Number of migrasomes from the cells in (l); n = 150 cells per group from three independent experiments. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. n Tspan4 KO Hela cells were transfected with the indicated plasmids for 24 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 and TSPAN4^6KR of three independent experiments normalized to Tubulin, was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file.

Fig. 4. O-GlcNAcylation regulates TSPAN4 degradation and migrasome formation.

a, b NRK cells expressing ITGA5-mCherry were treated with 10 μM TMG or 50 μM OSMI-1 for 6 h. Cells were imaged by confocal microscopy. Red, ITGA5-mCherry. Number of migrasomes from the cells in (a); n = 150 cells per group from three independent experiments. Scar bar represents 10 μm. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. c, d NRK-TSPAN4-mCherry cells were transfected with negative siRNA or OGT siRNA for 24 h, and cells were further transfected with GFP, RNAi-resistant OGT(R)-GFP or OGT(R)^K852M-GFP for another 24 h. Cells were imaged by confocal microscopy. Green, GFP, OGT(R)-GFP and OGT(R)^K852M-GFP; red, TSPAN4-mCherry; yellow, Merge. Number of migrasomes from the cells in (c); n = 150 cells per group from three independent experiments. Scar bar represents 10 μm. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. e HeLa cells stably expressing TSPAN4-Flag were transfected with indicated plasmids for 24 h, and cells were treated with or without 100 μM CHX for 1 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 of three independent experiments normalized to Tubulin, was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. f HeLa cells stably expressing TSPAN4-Flag were transfected with or without OGT siRNA for 48 h, and before harvested, cells were supplemented with or without 10 μM MG132 for 6 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 of three independent experiments normalized to Tubulin, was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. g HeLa cells stably expressing TSPAN4-Flag were transfected with or without OGT siRNA for 24 h, and cells were further transfected with OGT(R)-HA or OGT(R)^K852M-HA for 24 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 of three independent experiments normalized to Tubulin, was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. h, i Analysis of the interactions between TSPAN4-Flag and Ub^K48-HA treated with 10 μM TMG or 50 μM OSMI-1 for 6 h via co-immunoprecipitation assay (n = 3 independent experiments). Source data are provided as a Source Data file. j HEK293T cells were transfected with or without OGT siRNA for 12 h, and cells were further transfected with Ub^K48-HA and TSPAN4-Flag for 36 h. The interactions between TSPAN4-Flag and Ub^K48-HA were analyzed via co-immunoprecipitation assay (n = 3 independent experiments). Source data are provided as a Source Data file. k HEK293T cells were transfected with Ub^K48-HA and TSPAN4-Flag, with OGT-Myc or OGT^K852M-Myc for 36 h. The interactions between TSPAN4-Flag and Ub^K48-HA were analyzed via co-immunoprecipitation assay (n = 3 independent experiments). Source data are provided as a Source Data file.

Fig. 5. O-GlcNAcylation of AMFR impairs AMFR-TSPAN4 interactions.

a HEK293T cells were transfected with AMFR-Flag or TSPAN4-Flag and OGT-Myc. Flag antibody immunoprecipitated or cell lysates were subjected to WB (n = 3 independent experiments). O-GlcNAc antibody RL2 was used. Source data are provided as a Source Data file. b Co-immunoprecipitation assay for the interactions between AMFR-HA and OGT-Flag in HEK293T cells (n = 3 independent experiments). Source data are provided as a Source Data file. c GST pull-down analysis of GST-OGT with His-AMFR (n = 3 independent experiments). Source data are provided as a Source Data file. d OGT directly O-GlcNAcylates AMFR in vitro. In vitro glycosylation assay was performed with His-AMFR as substrate, which was incubated with purified GST-OGT, and the GlcNAcylated AMFR was blotted with RL2 antibody and the total His-AMFR and purified GST-OGT proteins were detected by WB (n = 3 independent experiments). Source data are provided as a Source Data file. e AMFR O-GlcNAcylation is reversed by OGA via in vitro glycosylation assay (n = 3 independent experiments). Source data are provided as a Source Data file. f HEK293T cells were transfected with or without OGT siRNA for 12 h, and cells were further transfected with AMFR-Flag for 36 h. Cell lysates were subjected to immunoprecipitation with Flag antibody and analyzed via WB (n = 3 independent experiments). Source data are provided as a Source Data file. g HEK293T cells were transfected with plasmids as indicated. Cell lysates were subjected to immunoprecipitation with Flag antibody and analyzed via WB (n = 3 independent experiments). Source data are provided as a Source Data file. h Diagram showing the amino acid sequence of AMFR and six potential O-GlcNAcylated sites. i HEK293T cells were transfected with indicated plasmids, and AMFR O-GlcNAcylation was analyzed by immunoprecipitation with Flag antibody and WB with the indicated antibodies. Quantification signal of AMFR and its mutants O-GlcNAcylation of immunoprecipitation from three independent experiments. Error bars represent the mean ± SD. Source data are provided as a Source Data file. j Purified WT or His-AMFR^T643A was used as substrates for in vitro glycosylation assay (n = 3 independent experiments). Source data are provided as a Source Data file. k LC-MS/MS spectrum of the O-GlcNAcylated peptide of AMFR. The Y1 ions confirm that O-GlcNAcylation occurs at T643. l, m NRK-ITGA5-mCherry cells were transfected with indicated plasmid. Cells were imaged by confocal microscopy. Green, GFP and OGT-GFP; red, ITGA5-mCherry; white, Halo, AMFR-Halo and AMFR^T643A-Halo. Number of migrasomes from the cells in (l); n = 150 cells per group from three independent experiments. Scar bar represents 10 μm. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. n HeLa cells stably expressing TSPAN4-Flag were transfected with indicated plasmids for 24 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 of three independent experiments normalized to Tubulin, was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. o HEK293T cells were transfected with or without OGT siRNA for 12 h, and cells were further transfected with AMFR-Myc and TSPAN4-Flag for 36 h. Cell lysates were subjected to immunoprecipitation with Flag antibody and analyzed via WB. Quantification signal of AMFR-Myc of co-immunoprecipitation from three independent experiments. Error bars represent the mean ± SD. Source data are provided as a Source Data file. p HEK293T cells were transfected with the indicated plasmids for 36 h. Cell lysates were subjected to immunoprecipitation with Flag antibody and analyzed via WB. Quantification signal of AMFR-HA of co-immunoprecipitation from three independent experiments. Error bars represent the mean ± SD. Source data are provided as a Source Data file. q HEK293T cells were transfected with the indicated plasmids for 36 h. Cell lysates were subjected to immunoprecipitation with GFP antibody and analyzed via WB. Quantification signal of AMFR-Flag and its mutant of co-immunoprecipitation from three independent experiments. Error bars represent the mean ± SD. Source data are provided as a Source Data file. r O-GlcNAcylation of AMFR decrease AMFR-TSPAN4 interactions in vitro. His-AMFR was first incubated with OGT and UDP-GlcNAc for 4 h, then incubated with GST-tagged TSPAN4. Immunoprecipitation was performed with anti-His antibody. Quantification signal of GST-TSPAN4 of Pull-down from three independent experiments. Error bars represent the mean ± SD. Source data are provided as a Source Data file.

Fig. 6. Viruses dynamically regulate AMFR O-GlcNAcylation and TSPAN4 ubiquitination.

a NRK cells stably expressing TSPAN4-Flag were transfected with or without AMFR-Flag and infected with VSV-GFP (MOI = 0.01) or HSV-1 (MOI = 0.01) for 12 or 36 h and treated with or without 10 μM TMG for 6 h. Culture supernatants were collected, and viral titers were quantified using plaque assays. Data represent the mean ± SD from three biological replicates. Statistical significance was determined using a two-tailed Student’s t-test. Source data are provided as a Source Data file. b NRK cells stably expressing TSPAN4-Flag were transfected with or without AMFR-Flag, OGT-HA, or OGT^K852M-HA and infected with VSV-GFP (MOI = 0.01) or HSV-1 (MOI = 0.01) for 12 or 36 h. Culture supernatants were collected, and viral titers were quantified using plaque assays. Data represent the mean ± SD from three biological replicates. Statistical significance was determined using a two-tailed Student’s t-test. Source data are provided as a Source Data file. c WT and OGT KD NRK cells stably expressing TSPAN4-Flag were transfected with or without OGT(R)-HA, or OGT(R)^K852M-HA and infected with VSV-GFP (MOI = 0.01) or HSV-1 (MOI = 0.01) for 12 or 36 h. Culture supernatants were collected, and viral titers were quantified using plaque assays. Data represent the mean ± SD from three biological replicates. Statistical significance was determined using a two-tailed Student’s t-test. Source data are provided as a Source Data file. d, e Analysis of interactions between TSPAN-Flag and HA-Ub^K48 under VSV-GFP or HSV-1 infection at an MOI of 1 for indicated time by co-immunoprecipitation assay (n = 3 independent experiments). Source data are provided as a Source Data file. f, g Analysis of interaction between AMFR-Myc and OGT-Flag with VSV-GFP or HSV-1 infection at an MOI of 1 for indicated time by co-immunoprecipitation assay. Quantification signal of AMFR-Myc of co-immunoprecipitation from three independent experiments. Error bars represent the mean ± SD. Source data are provided as a Source Data file. h, i Analysis the AMFR O-GlcNAcylation with VSV-GFP or HSV-1 infection at an MOI of 1 for indicated time by immunoprecipitation assay and analyzed by WB using anti-O-GlcNAc antibody. Quantification signal of AMFR O-GlcNAcylation of immunoprecipitation from three independent experiments. Error bars represent the mean ± SD. Source data are provided as a Source Data file. j, k Analysis of interaction between AMFR-Myc and TSPAN4-Flag with VSV-GFP or HSV-1 infection at an MOI of 1 for indicated time by co-immunoprecipitation assay. Quantification signal of AMFR-Myc of co-immunoprecipitation from three independent experiments. Error bars represent the mean ± SD. Source data are provided as a Source Data file.

Fig. 7. Viruses regulate migrasome formation via AMFR-TSPAN4 axis.

a Amfr WT and KO HeLa cells stably expressing TSPAN4-Flag were infected with or without VSV-GFP at an MOI of 1 for 12 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 of three independent experiments normalized to Tubulin, was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. b, c Amfr KO NRK cells stably expressing TSPAN4-mCherry were infected with or without VSV-GFP at an MOI of 0.2 for 12 h. Cells were imaged by confocal microscopy. Green, VSV-GFP; red, TSPAN4-mCherry; yellow, Merge. Number of migrasomes from the cells in (b); n = 150 cells per group from three independent experiments. Scar bar represents 10 μm. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. d Amfr KO HeLa cells stably expressing TSPAN4-Flag were transfected with or without AMFR-Myc or AMFR^3CS-Myc for 24 h and then infected with VSV-GFP at an MOI of 1 for another 12 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 of three independent experiments normalized to Tubulin, was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. e, f Amfr KO NRK cells stably expressing ITGA5-mCherry with or without AMFR-Halo or AMFR^3CS-Halo were infected with VSV-GFP at an MOI of 0.2 for 12 h. Cells were imaged by confocal microscopy. Green, VSV-GFP; red, ITGA5-mCherry; white, Halo, AMFR-Halo, and AMFR^3CS-Halo. Number of migrasomes from the cells in (e); n = 150 cells per group from three independent experiments. Scar bar represents 10 μm. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. g HeLa cells stably expressing TSPAN4-Flag or TSPAN4^6KR-Flag were infected with or without VSV-GFP at an MOI of 1 for 12 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 of three independent experiments normalized to Tubulin was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. h, i NRK cells stably expressing TSPAN4^6KR- mCherry were infected with or without VSV-GFP at an MOI of 0.2 for 12 h. Green, VSV-GFP; red, TSPAN46^KR-mCherry; yellow, Merge. Cells were imaged by confocal microscopy. Number of migrasomes from (h); n = 149 no infection, n = 149 VSV-GFP from three independent experiments. Scar bar represents 10 μm. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. j HeLa cells stably expressing TSPAN4-Flag were transfected with or without OGT-Myc or OGT^K852M-Myc for 24 h and then infected with or without VSV-GFP at an MOI of 1 for another 12 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 of three independent experiments normalized to Tubulin, was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. k, l NRK cells stably expressing ITGA5-mCherry transfected with or without OGT-Halo or OGT^K852M-Halo were infected with or without VSV-GFP at an MOI of 0.2 for 12 h. Cells were imaged by confocal microscopy. Green, VSV-GFP; red, ITGA5-mCherry; blue, OGT-Halo and OGT^K852M-Halo. Number of migrasomes from the cells in (k); n = 150 cells per group from three independent experiments. Scar bar represents 10 μm. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. m Proposed model for virus infection dynamically regulates migrasome formation via AMFR O-GlcNAcylation and TSPAN4 ubiquitination.