Lipid packing contributes to the confinement of caveolae to the plasma membrane
- Department of Medical and Translational Biology and Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, SciLifeLab, Umeå University, Sweden
- Department of Pharmacy, Uppsala University, Uppsala Biomedical Center, Sweden
- Department of Clinical Microbiology, Wallenberg Centre for Molecular Medicine and Umeå Centre for Microbial Research, Umeå University, Sweden
- Institute for Molecular Bioscience, The University of Queensland, Australia
- Centre for Microscopy and Microanalysis, The University of Queensland, Australia
- Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Sweden
Figures
Figure 1 with 1 supplement
The drug Dyngo-4a constrains both axial and lateral mobility of caveolae independently of dynamin.
(A) Immunoblots of dynamin triple knockout cells showing dynamin2 expression after 5 days of tamoxifen treatment (TKO) or no treatment (ctrl) as indicated. Sizes depicted in kDa. (B) Representative immunofluorescent staining of Caveolin1 in dyn triple knockout (TKO) cells treated as in (A) as indicated, Scale bar, 5 μm. (C) Quantification of the number of caveolae per square micrometer counted in the basal plasma membrane (PM) in immunofluorescent-labeled dyn TKO cells treated as in (A) as indicated. Mean ± SD from at least 20 cells per condition. Significance was assessed using t-test, p=0.7421. (D) Color-coded trajectories showing caveolae movements in the PM. Cav1-GFP was transiently expressed in dyn TKO cells after 5 days of tamoxifen treatment. Cells were serum-starved 1 hr prior to 30 min treatment with DMSO (ctrl) or 30 μM Dyngo-4a before imaged on total internal reflection fluorescence (TIRF) over 5 min. Red trajectories represent caveolae with long duration times, and purple represents caveolae with short duration times as indicated. Scale bar, 10 μm. (E–F) Quantification of Cav1-GFP track duration time (E) and track displacement length (F). Numbers were related to ctrl-treated cells, and track mean from at least 23 cells per condition are shown ± SD (E) and track length mean from at least 15 cells per condition are shown ± SD (F). Significance was assessed using t-test, ****p≤0.0001. (G) Visualization of the track displacement of color-coded trajectories in (D) by alignment of the starting position of all tracks, representing lateral mobility. Scale bar, 1 μm. (H) Kymographic visualization of the track duration of color-coded trajectories in (D) that were present at time zero, representing axial stability.
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Figure 1—source data 1
Original uncropped immunoblots for Figure 1.
- https://cdn.elifesciences.org/articles/108369/elife-108369-fig1-data1-v1.zip
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Figure 1—source data 2
Annotated uncropped immunoblots for Figure 1, indicating lane identities and bands used in analysis.
- https://cdn.elifesciences.org/articles/108369/elife-108369-fig1-data2-v1.zip
Figure 1—figure supplement 1
Cavin1 and EH domain containing 2 (EHD2) expression in dynamin triple knockout fibroblasts.
Ball-and-stick representations of Dyngo-4a (top left panel), Dynasore (bottom left panel), and cholesterol (top right panel). Carbon atoms are shown in gray, hydrogen in white, oxygen in red, and nitrogen in blue. (B–C) Representative immunofluorescent staining of dynamin triple knockout cells showing Cavin1 (B) and EHD2 (C) expression after 5 days of tamoxifen treatment (KO) or no treatment (ctrl) as indicated. Scale bar, 5 μm. (D–E) Representative immunofluorescent staining of Cav1-GFP expressing KO cells showing Cavin1 (D) and EHD2 (E) expression after 30 min treatment with (0.1%) DMSO (top panels) or 30 μM Dyngo-4a (bottom panels). Scale bar, 10 μm. (F) Illustration of caveola mobility and detection in total internal reflection fluorescence (TIRF) imaging.
Figure 2 with 1 supplement
Caveolae internalization is inhibited by Dyngo-4a.
(A) Representative electron micrographs of caveolae in Dyngo-4a or ctrl-treated cells. Black arrowheads show caveolae and stars show clathrin-coated vesicles (CCVs) in cells treated with Dyngo-4a. Yellow arrows mark the positions where neck (N), width (W), and height (H) measurements were acquired. Scale bar, 100 nm. (B) Scatter plots show the ratios of height or neck to width measurements of 60 caveolae per treatment, mean ± SD. (C) Representative immunofluorescent maximum-intensity projections of immunofluorescent cholera toxin B subunit (CTxB) uptake in ctrl-treated cells. Bottom panels show the masks generated from each fluorescent marker which were used to quantify the number of Cav1-GFP and CTxB-647 colocalizing spots devoid of EEA1, indicated by the white arrows in the magnified view. Scale bar, 10 μm.(D) Quantification of the number of Cav1-GFP/CTxB colocalizing spots negative for EEA1 per cell in ctrl- or Dyngo-4a-treated cells. Mean ± SD from 15 cells per condition (n=3). Significance was assessed using a t-test, *p≤0.05.
Figure 2—figure supplement 1
Caveolae internalization is inhibited by Dyngo-4a.
(A) Representative immunofluorescent maximum-intensity projections of fluorescent cholera toxin B subunit (CTxB) uptake in Dyngo-4a-treated cells. Bottom panels show the masks generated from each fluorescent marker which were used to quantify the number of Cav1-GFP and CTxB-647 colocalizing spots devoid of EEA1. Scale bar, 10 μm. (B) Representative confocal FRAP time-lapse series showing repletion of cytosolic Cav1-GFP in a photobleached area in ctrl- or Dyngo-4a-treated KO cells as indicated. Intensity recovery was monitored for 10 min after photobleaching. Cav1-GFP fluorescence intensities are intensity-coded using LUT. Scale bar, 5 μm. (C) Recovery curves of Cav1-GFP corresponding to (B). Cav1-GFP fluorescence intensity was normalized to background and reference. n=12, mean ± SEM.
Figure 3 with 1 supplement
Dyngo-4a binds and inserts into membranes.
(A) Top, illustration of Langmuir-Blodgett experiment. Bottom, representative Dyngo-4a adsorption to 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) monolayer. Dyngo-4a (30 μM) (black line) or DMSO (gray line) was injected underneath the lipid film with the starting surface pressure of 20 mN m–1 and the surface pressure shift (Δπ) was recorded over time. (B) Top, illustration of the QCM-D setup. Bottom, QCM-D measurements of supported lipid bilayer (SLB) formation and Dyngo-4a (30 μM) (black line) or DMSO (gray line) adsorption to a POPC SLB, followed by rinsing. Arrows depict time of injection of DMSO (equivalent to the concentration that Dyngo-4a is dissolved in), sample injection (Dyngo-4a or DMSO), DMSO injection, and saline buffer wash (150 mM NaCl, pH 7.4) injection, respectively.
Figure 3—figure supplement 1
Dyngo-4a adsorbs to and affects the softness of the supported lipid bilayer (SLB).
QCM-D measurements of the softness (ΔD) during SLB formation and Dyngo-4a (30 mM) (red line) or DMSO (0.2%) (black line) adsorption or desorption over time. Arrows depict time of injection of DMSO (0.2%, equivalent to the concentration that Dyngo-4a is dissolved in), sample injection (Dyngo-4a or DMSO), 0.2% DMSO injection, and saline buffer wash (150 mM NaCl, pH 7.4) injection, respectively.
Figure 4 with 1 supplement
Dyngo-4a inserts underneath the phospholipid headgroups at a similar position as cholesterol.
(A) Molecular dynamics simulation of Dyngo-4a in a membrane. Snapshot of a single Dyngo-4A molecule (cyan stick and red volume) in 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayer (phospholipid headgroups in dark gray and lipid chains as light gray sticks, dark blue represents the aqueous phase). (B–C) Accelerated weight histogram (AWH) simulations of Dyngo-4a (B) or chol (C) in POPC membrane. Potential of mean force (PMF) profile shows the energy required to move a Dyngo-4a molecule or a chol molecule from the membrane center to the aqueous phase, respectively. (D) Molecular dynamics simulation of Dyngo-4a in a membrane. Snapshot of a single Dyngo-4A molecule (cyan stick and red volume) in POPC:Chol (70:30%) bilayer (phospholipid headgroups in dark gray and lipid chains as light gray sticks, chol as orange sticks, dark blue represents water). (E) AWH simulations of Dyngo-4a in POPC:Chol (70:30%) membrane. The PMF profile shows the energy required to move a Dyngo-4a molecule from the membrane center to the aqueous phase. In the PMF profiles, the lines represent the mean, and the shaded regions represent the SD from triplicate simulations. (F) Molecular dynamics simulation snapshot of multiple Dyngo-4a molecules in POPC:Chol (70:30%) bilayer (color coding as in (D)). Dyngo-4a molecules did not cross the membrane throughout the simulation, so they were placed randomly at the beginning of the simulations and could translocate from one leaflet to another via periodic boundary condition. (G) Deuterium order parameter profiles for POPC tails in POPC:Chol (70:30%) membrane. Left shows sn1 tail (saturated, 16 carbons) and right shows sn2 tail (unsaturated, 18 carbons). No Dyngo-4a (black), single Dyngo-4a (gray), multiple Dyngo-4a (red); dashed lines depict the outer leaflet, and solid line the inner leaflet. (H) Contour plot based on heatmaps in Figure 4—figure supplement 1E, highlighting the predominant configuration with Dyngo-4a (red) localized adjacent to a chol (orange) cluster in the outer leaflet.
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Figure 4—source code 1
Python scripts used to produce the contour plots.
- https://cdn.elifesciences.org/articles/108369/elife-108369-fig4-code1-v1.zip
Figure 4—figure supplement 1
Dyngo-4a triggers lipid packing frustration in POPC:Chol membranes.
(A) Normalized density profiles for POPC (black), POPC:Chol (30:70, orange) membranes without and with inserted Dyngo-4a (red) molecules. The center of the membrane is located at 8 nm on the x-axis of the graphs. (B) Snapshot of multiple Dyngo-4a molecules (cyan stick and red volume) in POPC bilayer. Dyngo-4a molecules did not cross the membrane throughout the simulation, so they were placed randomly at the beginning of the simulations and could translocate from one leaflet to another via periodic boundary condition. Phospholipid headgroups shown in dark gray and lipid chains as light gray sticks, dark blue represents the aqueous phase. (C) Deuterium order parameter profiles for POPC tails in a POPC membrane. Left shows sn1 tail (saturated, 16 carbons) and right shows sn2 tail (unsaturated, 18 carbons). No Dyngo-4a (black), single Dyngo-4a (gray), multiple Dyngo-4a (red); dashed lines depict the outer leaflet, and solid line the inner leaflet. (D) Plot showing how the area per lipid changes upon addition of a single or multiple Dyngo-4a molecules in a POPC (black) or POPC:Chol (70:30, orange) membrane. (E) Heatmaps depict the spatial distribution of Dyngo-4a in POPC or POPC:Chol (70:30) membrane (left) or the spatial distribution of Chol in POPC:Chol (70:30) membrane with or without Dyngo-4a (right) as indicated. Dyngo-4a displays higher mobility in a pure POPC membrane compared to when it neighbors a chol cluster in a POPC:Chol membrane. Chol displays less uniform distribution in the membrane in the presence of Dyngo-4a.
Figure 5 with 1 supplement
Dyngo-4a decreases the lipid packing in the outer leaflet of the plasma membrane (PM), which is counteracted by increased levels of cholesterol.
(A) Visualization of the PM mobility of a ctrl-treated cell (top) and a Dyngo-4a-treated cell (bottom) from 5 min total internal reflection fluorescence (TIRF) movies. The outline of the PM at the start (0 s, red), middle (60, 120, 180, and 240 s, gray), and end (300 s, black) are depicted. Insets to the right show magnifications of the indicated areas. Scale bar, 10 μm. (B) Quantification of the PM mobility of cells following treatment with DMSO (ctrl) or Dyngo-4a at different concentrations as indicated. The means are shown from five cells per condition ± SD. (C) Young’s modulus calculated from force-indentation curves from the representative curves in (Figure 5—figure supplement 1D) over 31 and 33 cells treated with DMSO and Dyngo-4a, respectively. Each data point is an average value calculated from at least 40 individual force curves on each cell. Statistical analysis: parametric Student’s t-test with Welch’s correction. ns = nonsignificant. (D) Representative deconvolved fluorescent micrographs of HeLa cells stained with C-Laurdan captured at 442 nm (left panel) and 483 nm (middle panel). Yellow arrows depict analyzed stretch of PM with no neighboring fluorescent internal membrane as seen in the right panel. Red area corresponds to membrane with high membrane order, and purple corresponds to membrane with low membrane order. Scale bar, 5 μm. (E) Quantification of the mean GP-values for DMSO (ctrl) or Dyngo-4a-treated cells as indicated. All data points from at least 26 cells per condition are shown, n=3, mean ± SD. Significance was assessed using t-test, **p≤0.0065. (F) Quantification of Cav1-mCh track duration time following chol:MβCD treatment as indicated. Numbers were related to ctrl-treated cells. All data points from at least 26 cells per condition are shown, n=3, mean ± SD. Significance was assessed using t-test, ****p≤0.0001. The source code for data analysis of the C-Laurdan experiments is previously published and available via DOI: https://doi.org/10.1016/j.softx.2023.101570 or GitHub https://github.com/Parmryd/Find-and-measure-plasma-membrane/tree/src (Parmryd, 2023).
Figure 5—figure supplement 1
Analysis of the effect of Dyngo-4a on plasma membrane (PM) mobility, caveolae displacement, and transferrin (Tfn) uptake, atomic force microscopy (AFM) force-indentation curves, and incorporation of cholesterol (chol) using fusogenic liposomes and MBCD.
(A) Representative total internal reflection fluorescence (TIRF) micrographs and illustration of how the PM motion was retained from a 5 min TIRF movie. Basal PM outline at start (red line) and at the end (yellow line). Black and white images illustrate the area change from the start image to the end image. (B-C) Quantification of the caveolae displacement length (red) and Tfn-647 uptake (blue) in cells treated with DMSO (ctrl) or Dyngo-4a at different concentrations (B) and different time points (C) as indicated. Analysis of caveolae mobility was performed using Imaris software, and track means from at least 25 cells per condition are shown ± SD. Quantification of Tfn-647 intensity was performed using ImageJ, and relative intensity means from at least 500 cells are shown ± SD. (D) Representative AFM force curves detected on glass, on DMSO-treated cells (ctrl), and Dyngo-4a-treated cells. Inset shows example fitting of the force curve with Hertz model. (E) Quantification of Cav1-GFP track duration time in cells treated with DMSO (ctrl) or Dyngo-4a in the presence and absence of C-Laurdan. Numbers were related to ctrl-treated cells. Analysis was performed using Imaris software, and track mean from at least 30 cells per condition are shown ± SD. Significance was assessed using t-test, ****p≤0.0001. (F) Representative TIRF micrographs of ctrl- or Dyngo-4a-treated Cav1-mCh cells 5 min after addition of fusogenic liposomes containing Bodipy-chol or no addition as indicated. (G) Representative TIRF micrographs of ctrl- or Dyngo-4a-treated Cav1-mCh cells 5 min after addition of Bodipy-chol:MBCD or no addition as indicated. All scale bars, 10 μm.
Author response image 1
Videos
Video 1
Cav1-GFP dynamics in a DMSO-treated TKO cell.
Video 2
Cav1-GFP dynamics in a Dyngo-4a-treated TKO cell.
Video 3
Cav1-GFP and mCherry-cavin1 colocalization in a Dyngo-4a-treated TKO cell.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | A431 | ATCC | Cat# CRL-1555; RRID:CVCL_0037 | |
| Cell line (Mus musculus) | Dynamin triple knockout cell line; TKO cells | Park et al., 2013 | ||
| Cell line (Homo sapiens) | HeLa Flp-In T-REx Caveolin1-mCherry | Hubert et al., 2020b | ||
| Antibody | Anti-PTRF (Rabbit polyclonal) | abcam | Cat# ab48824; RRID:AB_88224 | IF (1:100) |
| Antibody | Anti-EHD2 (rabbit polyclonal) | Morén et al., 2012 | RRID:AB_2833022 | IF (1:200) |
| Antibody | Anti-EEA1 (mouse monoclonal) | BD Biosciences | Cat# 610456; RRID:AB_397830 | IF (1:100) |
| Antibody | Anti-Caveolin1 (Rabbit polyclonal) | abcam | Cat # ab2910; RRID:AB_303405 | IF (1:500) |
| Antibody | Anti-beta actin (mouse monoclonal) | Cell Signaling Technology | Cat# 3700; UniProt ID P60709 | WB (1:1000) |
| Antibody | Anti-Dyn2 (rabbit polyclonal) | Thermo Fisher Scientific | Cat# PA1-661; RRID:AB_2293040 | WB (1:1000) |
| Antibody | Anti-Rabbit IgG (H&L), HRP-conjugated (goat polyclonal) | Agrisera | Cat# AS09 602; RRID:AB_1966902 | WB (1:50,000) |
| Antibody | Anti-mouse IgG (Fab specific)-Peroxidase (goat polyclonal) | Merck | Cat# A9917; RRID:AB_258476 | WB (1:10,000) |
| Antibody | Anti-mouse IgG secondary antibody coupled to Alexa Fluor 568 (Goat polyclonal) | Thermo Fisher Scientific | Cat# A11031; RRID:AB_144696 | IF (1:300) |
| Antibody | Anti-rabbit IgG secondary antibody coupled to Alexa Fluor 488 (Goat polyclonal) | Thermo Fisher Scientific | Cat# A11034; RRID:AB_2576217 | IF (1:300) |
| Recombinant DNA reagent | mCherry-cavin1 | This study | Murine cavin1 cloned under the control of PCMV IE promoter in pmCherry-C1 vector | |
| Recombinant DNA reagent | Cav1-GFP | This study | Dog Cav1 cloned under the control of PCMV IE promoter in pEGFP-N2 vector | |
| Chemical compound, drug | Dyngo-4a | abcam | Cat# 120689 | |
| Chemical compound, drug | 4-Hydroxytamoxifen | Merck | Cat# H6278 | |
| Chemical compound, drug | C-Laurdan | Kim et al., 2007 | ||
| Chemical compound, drug | Methyl-β-cyclodextrin; MβCD | Merck | Cat# C4555 | |
| Chemical compound, drug | Bodipy-Cholesterol | Avanti Polar Lipids | Cat# 810255 | |
| Chemical compound, drug | 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) | Avanti Polar Lipids | Cat# 850725 | |
| Chemical compound, drug | 1,2-Dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) | Avanti Polar Lipids | Cat#890890 | |
| Chemical compound, drug | 1-Palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) | Avanti Polar Lipids | Cat# A80557 | |
| Chemical compound, drug | Alexa 647-conjugated CTxB | Thermo Fisher Scientific | Cat# C34778; | |
| Chemical compound, drug | Transferrin Alexa Fluor 647-conjugate | Invitrogen | Cat# T23366; RRID:AB_2337114 | |
| Chemical compound, drug | Lipofectamine 2000 | Thermo Fisher Scientific | Cat# 11668019 | |
| Chemical compound, drug | Doxycycline hyclate | Merck | Cat# D9891 | |
| Chemical compound, drug | Hygromycin B | Thermo Fisher Scientific | Cat# 10687010 | |
| Chemical compound, drug | Blasticidin S HCl | Thermo Fisher Scientific | Cat# R21001 | |
| Software, algorithm | Imaris software 10.0.1 | Bitplane | RRID:SCR_007370 | https://imaris.oxinst.com |
| Software, algorithm | ImageJ/Fiji | Schindelin et al., 2012 | RRID:SCR_002285 | http://fiji.sc/ |
| Software, algorithm | Prism 10.0.2 | GraphPad | RRID:SCR_002798 | https://www.graphpad.com |
| Software, algorithm | Huygens Professional | Scientific Volume Imaging | RRID:SCR_014237 | https://svi.nl/Huygens-Professional |
| Software, algorithm | AWS suite | AWSensors Technology | https://awsensors.com | |
| Software, algorithm | JPK data processing 7.0.165 | Bruker | https://www.bruker.com/en.html | |
| Software, algorithm | Find_plasma_membrane | Adler et al., 2023 | ||
| Other | Mini Extruder | Avanti Research | Cat# A81023 | |
| Other | X4 unit | AWSensors Technology | Cat# AWS X4 000041A | |
| Other | Microtrough G1 | Kibron | Cat# 6860 | |
| Other | Wilhelmy plate | Kibron | Cat# 1540 | |
| Other | CS-25R17 coverlips (TIRF) | Warner Instruments | Cat# 64-0735 | |
| Other | CS-25R15 coverlips | Warner Instruments | Cat# 64-0715 | |
| Other | Precision cover glasses thickness No. 1.5H | Paul Marienfeld GmbH & Co. KG | Cat# 0117520 |
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Lipid packing contributes to the confinement of caveolae to the plasma membrane
eLife 14:RP108369.
https://doi.org/10.7554/eLife.108369.3
