Reagents and antibodies

Fibronectin (FN, #PHE0023), WGA (#W7024), Puromycin (#A1113803), Prolong Live Antifade Reagent (#P36975) and Lipofectamine 3000 (#L3000001) were purchased from Thermo Fisher Scientific. BAPTA-AM (#A1076), Diaminobenzidine (#D12384) and Proteinase K (#P8811) were purchased from Sigma-Aldrich. GLPG0187 (#HY-100506) was purchased from MCE. Vigofect (#T001) was purchased from Vigorous. G418 (#E859) was purchased from Amresco. Hygromycin B (#10843555001) was purchased from Roche.

Anti-Rab5 (#ab218624), anti-Rab8a (#ab188574), anti-Cellubrevin (#ab5789), anti-SNAP23 (#ab4114), anti-M-CSF (#ab233387) and anti-MCP1 (#ab7202) antibodies were from Abcam. Anti-Myosin-5a (#3402), anti-Rab8a (#6975), anti-Rab10 (#8127), anti-Rab11 (#5589), anti-VAMP2 (#13508), anti-Itg α5 (#4705) and anti-Itg β1 (#4706) antibodies were from Cell Signaling Technology. Anti-Cellubrevin (#ET7108-31) and anti-M-CSF (#ET1609-1) antibodies were from HuaAn Biotechnology. Anti-GAPDH (#60004-1-Ig) and anti-VAMP7 (#22268-1-AP) antibodies were from Proteintech Group. Anti-CCL2 (#AF-479-NA) antibody was from R&D SYSTEMS. Anti-syntaxin4 (#MA5-38156) antibody was from Thermo Fisher Scientific. Anti-CPQ (#HPA023235-100UL) antibody was from Sigma-Aldrich. Goat anti-Rabbit IgG (#111-035-003) and goat anti-mouse IgG (#115-035-003) were from Jackson.

Cells

L929 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, #C11995500BT, Gibco) supplemented with 10% FBS (#04-001-1A, Biological Industries), 2 mM GlutaMAX (#35050-061, Gibco) and 100 U/mL penicillin–streptomycin (#GNM15140, GENOM). Cells were cultured at 37 °C in an incubator with 5% CO2.

DPSCs are a gift from Songling Wang’s Lab (Beijing Stomatological Hospital). DPSCs were grown in MEM-ALPHA (#C3060-0500, VivaCell) supplemented with 10% FBS, 2 mM Glutamax, 100 U/mL penicillin–streptomycin and 1% Antibiotic-Antimycotic (15240-062, Gibco). Cells were cultured at 37 °C in an incubator with 5% CO2.

Cell transfection and virus infection

Cell transfection was conducted using Vigofect according to the manufacturer’s manual. The PiggyBac Transposon Vector System was used to generate stably expressing cell lines as previously described.32 Briefly, various proteins were cloned into pB-CAG (transposon vector) as the expressing plasmid backbone. The pB-CAG constructs combined with pBASE (transposase vector) were co-transfected into L929 cells at a ratio of 1:3 using the above Vigofect transfection protocol. After 24 h, the cells were treated with 600 µg/mL G418 or 200 µg/mL hygromycin B for selection (3–5 days). Single cells were sorted into 96-well plates by flow cytometry. These single-cell clones were cultured and expanded, followed by confocal analysis.

Gene knockdown was achieved with short hairpin RNA (shRNA) in the lentivirus-based vector pLKO.1-puro. Lentiviral production and infection were performed as previously described.33 Briefly, for lentiviral production, lentiviral vectors (pLKO.1, psPAX2 and pMD2.G) were co-transfected into 293T cells at a ratio of 4:3:1. After 48 h, the supernatant was centrifuged at 600× g for 5 min to remove cell debris. Viruses were harvested and used in the following experiments. For virus infection, the indicated cells seeded to 50%–60% confluence were co-cultured with virus containing 8 μg/mL polybrene for 24 h. The cells were placed in fresh medium containing 5 µg/mL puromycin for selection until drug-resistant colonies become visible. Sequences of the shRNAs were as follows: mouse SNAP23: 5′-GAACAACTAAATCGCATAGAA-3′, mouse Stx4: 5′-GAGTCCTGTCCCAGCAATTTG-3′, mouse VAMP2: 5′-CCTCAAGATGATGA-TCATCTT-3′, mouse Itgβ1: 5′-GCACGATGTGATGATTTAGAA-3′.

We used the CRISPR/Cas9-2hit KO system to generate the Myosin-5a knockout L929 cells. Two guide RNA (sgRNA) coding sequences were cloned into PX458M (5′-GTGCCGGTATGCGCCAGGCA-3′ and 5′-AGTTCGCTTCATCGATTCCA-3′). L929 cells were transfected with PX458M containing the Myosin-5a-targeting sequences. After single-cell sorting by flow cytometry, the single-cell clones were further analyzed by PCR and western blotting.

Isolation of migrasomes from cultured cells

Crude migrasomes were collected by differential centrifugation as previously described.34 Briefly, cells and migrasomes in 15-cm dishes were gently harvested into 50-mL tubes after trypsin digestion. All subsequent manipulations were conducted at 4 °C. After double centrifugation at 600× g for 10 min at 4 °C, the supernatant was further centrifuged at 2000× g for 20 min at 4 °C to remove the cell bodies and large debris. Crude migrasomes were then acquired as the pellet by centrifugation at 18,000× g for 30 min at 4 °C.

High-purity migrasome isolation was performed by iodixanol–sucrose (#LYSISO1, Sigma-Aldrich) density gradient centrifugation following a published protocol with minor modifications.35 Briefly, the crude migrasome pellet was resuspended in 800 µL buffer (400 µL extraction buffer mixed with 400 µL 10% Optiprep) and then fractionated at 150,000× g for 4 h at 4 °C in a multistep Optiprep dilution gradient. The step gradient was 50% (500 µL), 40% (500 µL), 35% (500 µL), 30% (500 µL), 25% (500 µL), 20% (500 µL), 15% (500 µL), 10% (500 µL), 5% (500 µL) and crude migrasomes (5%, 800 µL). After centrifugation, samples were collected from top to bottom gently (500 µL per fraction). Fractions 4, 5 and 6 were each mixed with 500 μL PBS and then centrifuged at 18,000× g for 30 min at 4 °C. The pellets were washed with PBS and centrifuged again at 18,000× g for 30 min to pellet the migrasomes. The samples were immediately available for downstream applications such as western blot and TEM analyses.

Imaging and image analysis

10 µg/mL FN was used to precoat confocal dishes at 37 °C for at least 1 h. For confocal snapshot images, cells were cultured in FN-precoated confocal dishes for 10–12 h, and imaged by a NIKON A1RSiHD25 laser scanning confocal microscope at 1024 × 1024 pixels. Z-stack imaging of cells and migrasomes was performed with a NIKON A1 microscope. SIM images were acquired using a Nikon N-SIM Super Resolution Microscope.

For long-term time-lapse images, cells were grown in FN-precoated confocal dishes for 4–6 h before imaging. Cells were then maintained in the living cell system (37 °C, 5% CO2), and monitored by a NIKON A1 microscope. Ultra-fast super-resolution time-lapse images were collected using a GI-SIM. NIS-Elements analysis 5.4 software was used to deconvolute images acquired by the NIKON A1 microscope. Z-projection and 3D reconstruction were performed with NIS-Elements 5.4. Images were processed using ImageJ and Imaris software 8.1.4, and statistical analyses were conducted by GraphPad Prism 8.

TEM

The preparation of TEM samples was conducted following the protocol that we set up previously.32 Briefly, cells were grown in 35-mm dishes precoated with FN (10 µg/mL) for 10–12 h. Cells were pre-fixed with a 1:1 mixture of growth medium and 2.5% glutaraldehyde for 5 min, and were further fixed in 2.5% glutaraldehyde in PB buffer for 2 h at room temperature. After three gentle washes with PBS, cells were dehydrated through a graded ethanol series (50%, 70%, 90%, 95% and 100%) for 8 min per step. The samples were subsequently infiltrated and embedded in SPON12 resin, polymerized at 60 °C for 48 h. Ultrathin 70-nm sections were cut with a diamond knife, collected on Formvar-coated copper grids (100 mesh). These sections were then double-stained with uranyl acetate and lead citrate. After air-drying, samples were examined using an H-7650B TEM at 80 kV.

For APEX2-based intracellular-specific protein imaging by TEM, the procedure was based on the previous protocol with minor modifications.36 Briefly, cells were fixed with 2% glutaraldehyde (in 100 mM sodium cacodylate buffer with 2 mM CaCl2, pH 7.4) at room temperature, then moved to ice for 1 h. All subsequent manipulations were conducted at 4 °C until resin infiltration. After three gentle washes with chilled buffer, cells were treated with 20 mM glycine for 5 min to quench unreacted glutaraldehyde. A freshly diluted DAB solution (0.5 mg/mL DAB in HCl combined with 0.03% H2O2) was added to cells for 5 min. The local deposition of DAB catalyzed by APEX2 could be monitored by light microscopy, and the reaction was stopped by washing three times with chilled buffer. Post-fixation staining with 2% osmium tetroxide (#1250423, SPI) was conducted for 30 min in chilled buffer, followed by washing and soaking in 2% uranyl acetate (#22400, Electron Microscopy Sciences) overnight. Dehydration was performed in cold ethanol (20%, 50%, 70%, 90%, 100%, 100%) for 2 min per step, followed by infiltration with Durcupan ACM resin (Electron Microscopy Sciences) mixed with anhydrous ethanol (1:1) for 30 min, then 100% resin twice for 1 h each. Samples were embedded in fresh resin and polymerized under vacuum at 60 °C for 48 h. DAB-stained areas of embedded cultured cells were identified using transmitted light. After sawed out with a jeweler’s saw, the areas of interest were mounted on dummy acrylic blocks with cyanoacrylic adhesive (Krazy Glue, Elmer’s Products). Ultrathin 70-nm sections were cut as described above, and samples were examined using an H-7650B TEM at 80 kV.

Total membrane protein isolation

Total membrane proteins from plasma membranes and from migrasomes were isolated using the Pierce Cell Surface Protein Isolation Kit (#89881, Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, cells were cultured in FN-precoated confocal dishes for 10 h or 12 h. After being washed with PBS, cells were then treated with Sulfo-NHS-SS-Biotin to biotinylated cell membrane proteins. Biotin-labeled membrane proteins were subsequently isolated from cell bodies or migrasomes using NeutrAvidin Agarose, respectively. Membrane proteins from cell bodies and migrasomes were normalized to equal total protein loading for western blot analysis.

Secretion analysis

Cargo secretion analysis was conducted following a published protocol with minor modifications.37 Briefly, equal numbers of the indicated cells were seeded into FN-precoated dishes for 10 or 16 h, and the medium was gently collected into 50 mL tubes. After double centrifugation at 600× g for 10 min at 4 °C, the supernatant was further centrifuged at 2000× g for 20 min at 4 °C to remove the cell bodies and large debris. Soluble proteins in the medium were concentrated by a 10 KD Amicon filter (Millipore), and the cell lysates were collected, respectively. These concentrated medium was normalized with the numbers of cells, and then subjected to western blot analysis.

Western blot analysis

The details of western blot analysis were described before.32 Briefly, the indicated cells or migrasomes were lysed by 2.5% SDS lysis buffer and boiled for 10–20 min at 95 °C. The protein concentration of each sample was determined using the BCA kit. Proteins were separated on SDS–PAGE gels of an appropriate percentage according to the molecular weight of the target proteins, followed by electrophoretic transfer onto PVDF membranes. After blocking with 5% non-fat milk in TBST buffer, membranes were incubated with primary antibody overnight at 4 °C. Membranes were then incubated with secondary antibody (HRP) for 1 h at room temperature, and signals were detected with a WESTAR ηC 2.0 kit (CYANAGEN).

The following primary antibodies were used for western blot analysis at the indicated dilution: anti-Myosin-5a (1:1000), anti-SNAP23 (1:2000), anti-M-CSF (1:1000), anti-CCL2 (1:1000), anti-PIGK (1:1000), anti-CPQ (1:1000), anti-Itg α5 (1:1000), anti-Itg β1 (1:1000), anti-Syntaxin4 (1:1000), anti-VAMP2 (1:1000) and anti-GAPDH (1:5000).

Statistical analysis

Statistical analyses were conducted using the unpaired two-tailed t-test in Graphpad Prism 5 (or 8) software (Graphpad Software). Data are the means ± SEM. Significance is indicated by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant. Statistical parameters and significance are reported in the figures and the figure legends.