Release of cholesterol-rich particles from the macrophage plasma membrane during movement of filopodia and lamellipodia
- David Geffen School of Medicine, University of California, Los Angeles, United States
- University of California, Los Angeles, United States
- University of Western Australia, Australia
Figures
Figure 1 with 3 supplements
Macrophages release plasma membrane–derived particles onto the substrate during extension and retraction of filopodia and lamellipodia, as judged by correlative live-cell imaging and SEM.
Cells were plated onto poly-D-lysine–coated gridded glass-bottom Petri dishes, and videos were recorded for 24 hr at 5 min intervals (see Figure 1—videos 1–2). The ‘Live cell’ images show the final …
Figure 1—figure supplement 1
Macrophages release particles from the plasma membrane of filopodia and lamellipodia by a process that resembles budding.
(A) Upper left, scanning electron micrograph (SEM) of a mouse peritoneal macrophage (yellow arrow), revealing a lawn of ~30-nm particles on the surrounding substrate. A higher magnification image of …
Figure 1—video 1
Mouse peritoneal macrophages release vesicular particles onto the surrounding substrate during the extension and retraction of filopodia and lamellipodia.
Video shows a macrophage that was imaged by SEM in the top row of Figure 1. Macrophages were plated onto poly-D-lysine–coated gridded glass-bottom Petri dishes, and videos were recorded for 24 h at …
Figure 1—video 2
Shows a macrophage imaged by SEM in the bottom row of Figure 1.
Video shows the final 15 h of a 24-h period of live-cell imaging.
Figure 2 with 6 supplements
Inhibiting macrophage movement with latrunculin A or blebbistatin abolishes particle release onto the surrounding substrate.
Scanning electron micrographs (SEMs) of mouse peritoneal macrophages that had been treated with latrunculin A (A) or blebbistatin (B). Macrophages were treated with latrunculin A, blebbistatin, or …
Figure 2—video 1
Treatment of mouse peritoneal macrophages with latrunculin A or blebbistatin prevent projection and retraction of filopodia/lamellipodia.
Video shows macrophages treated with vehicle (DMSO) alone. Macrophages were plated on glass-bottom Petri dishes and incubated in medium containing latrunculin A (5 μM), blebbistatin (30 μM), or …
Figure 2—video 2
Shows macrophages treated with vehicle (DMSO) alone.
https://doi.org/10.7554/eLife.50231.008
Figure 2—video 3
Shows macrophages treated with latrunculin A.
https://doi.org/10.7554/eLife.50231.009
Figure 2—video 4
Shows macrophages treated with latrunculin A.
https://doi.org/10.7554/eLife.50231.010
Figure 2—video 5
Shows a macrophage treated with blebbistatin.
https://doi.org/10.7554/eLife.50231.011
Figure 2—video 6
Shows macrophages treated with blebbistatin.
https://doi.org/10.7554/eLife.50231.012
Figure 3 with 2 supplements
Correlative live-cell, scanning electron microscopy (SEM), and NanoSIMS imaging, revealing that particles released onto the substrate during movement of filopodia and lamellipodia are enriched in accessible cholesterol.
RAW 264.7 macrophages were plated onto iridium- and poly-D-lysine–coated gridded glass-bottom Petri dishes. Videos were recorded for 24 hr at 5 min intervals (see Figure 3—videos 1–2). The ‘Live …
Figure 3—video 1
RAW 264.7 macrophages release particles during projection and retraction of lamellipodia.
Video shows a macrophage imaged by SEM and NanoSIMS in the top row of Figure 3. RAW 264.7 macrophages were plated onto iridium- and poly-D-lysine–coated glass-bottom Petri dishes and incubated in …
Figure 3—video 2
Shows a macrophage imaged by SEM and NanoSIMS in the bottom row of Figure 3.
Video shows the final 15 h of a 24-h period of live-cell imaging.
Figure 4 with 2 supplements
Particles released from the plasma membrane of biotinylated mouse peritoneal macrophages can be detected with streptavidin and ALO-D4 (a cytolysin that binds to the accessible pool of cholesterol).
(A) Biotinylated macrophage particles can be detected with fluorescently labeled streptavidin as judged by confocal microscopy. Macrophages in suspension were biotinylated with Sulfo-NHS-SS-biotin. …
Figure 4—figure supplement 1
Particles released from non-biotinylated macrophages do not bind streptavidin-conjugated gold nanoparticles as judged by scanning electron microscopy (SEM).
Non-biotinylated macrophages were plated onto glass-bottom Petri dishes. On the following day, the cells were incubated with streptavidin-conjugated 40-nm gold nanoparticles. Cells were then fixed …
Figure 4—figure supplement 2
Correlative SEM and NanoSIMS imaging of macrophages and plasma membrane–derived particles on the surrounding substrate.
Mouse peritoneal macrophages were plated onto iridium- and poly-D-lysine–coated gridded glass-bottom Petri dishes and incubated in medium containing 10% FBS for 24 hr. Cells were then incubated with …
Figure 5 with 1 supplement
Enrichment in focal adhesion proteins in the particle preparations from RAW 264.7 macrophages.
The most abundant proteins (the top 75th percentile by spectral counts) were analyzed by Enrichr and categorized by GO Cellular Components 2018. (A–B) Analysis of proteins in macrophage particles (n …
Figure 5—figure supplement 1
Isolation of particles released onto the substrate by RAW 264.7 macrophages.
(A) SEM images of RAW 264.7 macrophages plated onto poly-D-lysine–coated silicon wafers, revealing large numbers of particles on the substrate surrounding the macrophages. Higher magnification …
Figure 6 with 8 supplements
Particles released by mouse peritoneal macrophages onto the surrounding substrate are enriched in accessible cholesterol but not sphingomyelin-sequestered cholesterol.
Mouse peritoneal macrophages were plated onto poly-D-lysine–coated glass coverslips and incubated overnight in medium containing 10% FBS and either an FAK inhibitor (CAS 4506-66-5, 2 μM) or vehicle …
Figure 6—figure supplement 1
Inhibiting focal adhesion kinase in mouse peritoneal macrophages is accompanied by large lawns of particles on the surrounding substrate.
Macrophages were plated onto poly-D-lysine–coated glass-bottom Petri dishes and incubated with a focal adhesion kinase (FAK) inhibitor, 2 μM)] or vehicle (DMSO) alone. In some experiments, …
Figure 6—figure supplement 2
Incubating mouse peritoneal macrophages with latrunculin A alters the distribution of ALO-D4 binding.
Macrophages were plated onto poly-D-lysine–coated glass coverslips and incubated with latrunculin A (5 μM) or vehicle alone (DMSO control). The incubation of latrunculin A was initiated either 1 hr …
Figure 6—figure supplement 3
Particles released by mouse peritoneal macrophages are enriched in accessible cholesterol but not in sphingomyelin.
Mouse peritoneal macrophages were plated onto poly-D-lysine–coated glass coverslips and incubated overnight in medium containing 10% FBS and either a focal adhesion kinase (FAK) inhibitor (2 μM) or …
Figure 6—figure supplement 4
Sphingomyelinase treatment reduces OlyA and lysenin binding to the plasma membrane.
(A) Sphingomyelinase markedly reduces OlyA binding to the macrophage plasma membrane. Mouse peritoneal macrophages were plated onto poly-D-lysine–coated glass coverslips and incubated overnight in …
Figure 6—video 1
Movement of mouse peritoneal macrophages under different experimental conditions.
Video shows macrophages treated with vehicle (DMSO) alone. Macrophages were plated onto glass-bottom Petri dishes and incubated in medium containing an FAK inhibitor (2 μM), acetylated LDL (acLDL; …
Figure 6—video 2
Shows macrophages treated with an FAK inhibitor.
https://doi.org/10.7554/eLife.50231.027
Figure 6—video 3
Shows acLDL-loaded macrophages treated with vehicle (DMSO) alone.
https://doi.org/10.7554/eLife.50231.028
Figure 6—video 4
Shows acLDL-loaded macrophages treated with an FAK inhibitor.
https://doi.org/10.7554/eLife.50231.029
Figure 7 with 10 supplements
Correlative live-cell, SEM, and NanoSIMS imaging of mouse peritoneal macrophages, demonstrating that particles released onto the substrate during movement of filopodia and lamellipodia are enriched in accessible cholesterol but not sphingolipid-sequestered cholesterol.
Macrophages were plated onto iridium- and poly-D-lysine–coated gridded glass-bottom Petri dishes, and videos of cell movement were recorded for 24 h at 5-min intervals (see Figure 7—video 1). The red…
Figure 7—figure supplement 1
Correlative live-cell, SEM, and NanoSIMS imaging of mouse peritoneal macrophages, demonstrating that particles released onto the substrate during movement of filopodia and lamellipodia are enriched in accessible cholesterol but not sphingolipid-bound cholesterol.
Macrophages were plated onto iridium- and poly-D-lysine–coated gridded glass-bottom Petri dishes, and videos of cell movement were recorded for 24 hr at 5 min intervals (see Figure 7—video 2). The re…
Figure 7—figure supplement 2
Correlative SEM and NanoSIMS imaging, demonstrating that particles released onto the substrate by macrophages are enriched in accessible cholesterol but not sphingolipid-bound cholesterol.
Mouse peritoneal macrophages were plated onto iridium- and poly-D-lysine–coated gridded glass-bottom Petri dishes. Cells were then incubated at 4°C with [15N]ALO-D4 (which binds accessible …
Figure 7—figure supplement 3
The binding of fluorescently labeled OlyA to the plasma membrane overlaps the staining of the actin cytoskeleton with phalloidin.
Mouse peritoneal macrophages were plated onto poly-D-lysine–coated glass coverslips and incubated in macrophage growth medium containing 10% FBS. On the next day, cells were incubated at 4°C with …
Figure 7—figure supplement 4
Correlative live-cell, SEM, and NanoSIMS imaging of mouse peritoneal macrophages treated with an FAK inhibitor, demonstrating that particles released onto the substrate during movement of filopodia and lamellipodia are enriched in accessible cholesterol but not sphingolipid-bound cholesterol.
Mouse peritoneal macrophages were plated onto iridium- and poly-D-lysine–coated gridded glass-bottom Petri dishes and incubated in medium containing an FAK inhibitor (2 μM). Videos of cell movement …
Figure 7—figure supplement 5
Correlative live-cell, SEM, and NanoSIMS imaging of mouse peritoneal macrophages treated with an FAK inhibitor, demonstrating that particles released onto the substrate during movement of filopodia and lamellipodia are enriched in accessible cholesterol but not sphingolipid-bound cholesterol.
Mouse peritoneal macrophages were plated onto iridium- and poly-D-lysine–coated gridded glass-bottom Petri dishes and incubated in medium containing an FAK inhibitor (2 μM). Videos of cell movement …
Figure 7—figure supplement 6
Correlative SEM and NanoSIMS imaging, demonstrating that particles released onto the substrate by macrophages treated with an FAK inhibitor are enriched in accessible cholesterol but not sphingolipid-bound cholesterol.
(A) Mouse peritoneal macrophages were plated onto iridium- and poly-D-lysine–coated gridded glass-bottom Petri dishes and incubated in medium containing an FAK inhibitor (2 μM). Cells were then …
Figure 7—video 1
Macrophage release particles during movement of filopodia and lamellipodia movement.
Video shows a macrophage treated with vehicle (DMSO) imaged by SEM and NanoSIMS in Figure 7. Mouse peritoneal macrophages were plated onto iridium- and poly-D-lysine–coated glass-bottom Petri dishes …
Figure 7—video 2
Shows a macrophage treated with vehicle (DMSO) imaged by SEM and NanoSIMS in Figure 7—figure supplement 2.
Video shows a 24-h period of live-cell imaging.
Figure 7—video 3
Shows a macrophage treated with an FAK inhibitor imaged by SEM and NanoSIMS in Figure 7—figure supplement 4.
Video shows the final 10 h of a 24-h period of live-cell imaging.
Figure 7—video 4
Shows a macrophage treated with an FAK inhibitor imaged by SEM and NanoSIMS in Figure 7—figure supplement 5.
Video shows the final 10 h of a 24-h period of live-cell imaging.
Figure 8 with 2 supplements
Distribution of distinct pools of cholesterol in the macrophage plasma membrane.
Mouse peritoneal macrophages were plated onto poly-D-lysine–coated MatTek dishes and incubated in medium containing 10% FBS. On the next day, cells were incubated with [15N]ALO-D4 and [13C]OlyA (20 …
Figure 8—figure supplement 1
Reduced efflux of [3H]cholesterol from [3H]cholesterol-loaded macrophages when the release of particles was blocked with blebbistatin.
Mouse peritoneal macrophages were loaded with [3H]cholesterol (1 μCi/ml) and acLDL (20 μg/ml) in macrophage growth media containing 1% lipoprotein-deficient serum (LPDS) for 24 hr. On the next day, …
Figure 8—figure supplement 2
Increased efflux of [3H]cholesterol from [3H]cholesterol-loaded macrophages in the presence of HDL.
Mouse peritoneal macrophages were loaded with [3H]cholesterol (1 μCi/ml) and acLDL (20 μg/ml) in macrophage growth media containing 1% LPDS for 24 hr. On the next day, cells were washed, released …
Figure 9 with 1 supplement
Mouse peritoneal macrophages release accessible cholesterol–enriched particles onto a polymerized collagen IV matrix.
After biotinylating the cell-surface proteins of macrophages with Sulfo-NHS-SS-biotin, the cells were plated onto glass-bottom Petri dishes that had been coated with polymerized Alexa Fluor …
Figure 9—figure supplement 1
Mouse peritoneal macrophages release particles onto a polymerized collagen IV matrix.
After biotinylating the cell-surface proteins of macrophages with Sulfo-NHS-SS-biotin, the cells were plated onto glass-bottom Petri dishes coated with polymerized collagen IV. SEM images show …
Figure 10 with 2 supplements
Biotinylated mouse peritoneal macrophages release plasma membrane–derived material onto the surface of dead endothelial cells.
Mouse brain endothelial cells (bEnd.3) were plated onto glass- bottom Petri dishes and allowed to grow to confluency. After fixing the endothelial cells with 0.1% glutaraldehyde in PBS, they were …
Figure 10—figure supplement 1
Correlative live-cell and SEM images, revealing the release of particles by a live macrophage onto the surface of a dead macrophage.
RAW 264.7 macrophages were plated onto poly-D-lysine–coated gridded glass-bottom Petri dishes, and videos of cell movement were recorded for 20 hr at 5 min intervals (see Figure 10—video 1). …
Figure 10—video 1
Release of vesicular particles by a live RAW 264.7 macrophage onto the surrounding substrate and onto a dead RAW 264.7 macrophage.
RAW macrophages were plated onto poly-D-lysine–coated glass-bottom Petri dishes and incubated in media containing 10% FBS. Cells were imaged by live-cell microscopy for 20 hr at 5 min intervals. Whit…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (M. musculus) | RAW 264.7 | ATCC | Catalog No. TIB-71 RRID: CVCL_0493 | |
| Cell line (M. musculus) | bEnd.3 | ATCC | Catalog No. CRL-2299 RRID: CVCL_0170 | |
| Recombinant DNA reagent | ALO-D4 plasmid | PMID: 25809258 | Dr. Arun Radhakrishnan (UT Southwestern) | |
| Recombinant DNA reagent | OlyA plasmid | PMID: 30712872 | Dr. Arun Radhakrishnan (UT Southwestern) | |
| Chemical compound, drug | N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (carbodiimide) | Millipore-Sigma | Catalog No. 03449 | |
| Chemical compound, drug | Glutaraldehyde25% solution | Electron Microscopy Sciences | Catalog No. 16220 | |
| Chemical compound, drug | Osmium tetroxide4% solution | Electron Microscopy Sciences | Catalog No. 18459 | |
| Chemical compound, drug | Paraformaldehyde16% solution | Electron Microscopy Sciences | Catalog No. 15170 | |
| Chemical compound, drug | EMbed 812 | Electron Microscopy Sciences | Catalog No. 14120 | |
| Chemical compound, drug | Sodium cacodylate trihydrate | Electron Microscopy Sciences | Catalog No. 12300 | |
| Chemical compound, drug | Uranyl acetate | SPI-Chem | Catalog No. 02624AB | |
| Chemical compound, drug | Latrunculin A | Sigma | Catalog No. L5163 | |
| Chemical compound, drug | Blebbistatin | Abcam | Catalog No. ab120425 | |
| Chemical compound, drug | FAK inhibitor | Calbiochem | Catalog No. CAS 4506-66-5 | |
| Chemical compound, drug | LXR agonist | Sigma | Catalog No. G6295 | |
| Chemical compound, drug | HDL | Alfa Aesar | Catalog No. J64903 | |
| Chemical compound, drug | [3H]cholesterol | PerkinElmer | Catalog No. NET139250UC | |
| Chemical compound, drug | Gold–streptavidin conjugation reagent | Abcam | Catalog No. ab186864 | |
| Chemical compound, drug | EZ-Link Sulfo-NHS-SS-biotin | ThermoFisher | Catalog No. 21331 | |
| Chemical compound, drug | Sphingomyelinase from Staphylococcus aureus | Sigma | Catalog No. S8633 | |
| Other | 35 mm glass-bottom gridded MatTek dish | MatTek | Catalog No. P35G-1.5–14-CGRD |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.50231.049
