Biomimetic actin cortices shape cell-sized lipid vesicles
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Abstract
Animal cells are shaped by a thin layer of actin filaments underneath the plasma membrane known as the actin cortex. This cortex stiffens the cell surface and thus opposes cellular deformation, yet also actively generates membrane protrusions by exerting polymerization forces. It is unclear how the interplay between these two opposing mechanical functions plays out to shape the cell surface. To answer this question, we reconstitute biomimetic actin cortices nucleated by the Arp2/3 complex inside cell-sized lipid vesicles. We show that thin Arp2/3-nucleated actin cortices strongly deform and rigidify the shapes of giant unilamellar vesicles and impart a shape memory on time scales that exceeds the time of actin turnover. In addition, actin cortices can produce finger-like membrane protrusions, showing that Arp2/3-mediated actin polymerization forces alone are sufficient to initiate protrusions in the absence of actin bundling or membrane curving proteins. Combining mathematical modeling and our experimental results reveals that the concentration of actin nucleating proteins, rather than actin polymerization speed, is crucial for protrusion formation. This is because locally concentrated actin polymerization forces can drive a positive feedback loop between recruitment of actin and its nucleators to drive membrane deformation. Our work paints a picture where the actin cortex can either drive or inhibit deformations depending on the local distribution of nucleators.
Significance Statement
The cells in our body must actively change shape in order to migrate, grow and divide, but they also need to maintain their shape to withstand external forces during tissue development. Cellular shape control results from an interplay between the plasma membrane and its underlying cortex, a shell composed of crosslinked actin filaments. Using cell-free reconstitution and mathematical modelling, we show that minimal biomimetic actin cortices can mechanically rigidify lipid vesicles while at the same time driving membrane protrusion formation. Our observations suggest that the spatial distribution of actin nucleation determines whether the actin cortex drives or inhibits membrane deformations.
Article activity feed
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most of the cortex-bearing GUVs were frozen in highly irregular shapes, showing sharp corners (pink arrows in Fig. 2A), bleb-like protrusions (yellow arrows), long and thin protrusions (cyan arrows), or highly anisotropic shapes (yellow asterisk).
How often did you see these different morphologies? I'm curious if some are more common than others.
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time-lapse imaging revealed that GUV shapes often remained unchanged over many minutes
Interesting.. I'd expect these structures to be really dynamic. Is the turnover of actin at the membrane decreased in these deflated GUVs?
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No protrusions occurred when actin polymerized in the GUV lumen, showing that Arp2/3-driven actin polymerization at the membrane drives protrusion formation (Fig. S7 B).
Did you do any experiments where you polymerized actin filaments with the Arp2/3 complex, but with non-membrane anchored VCAs?
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Introduction
This is a really interesting paper with a really cool technique, some beautiful images, and a ton of data that tells us a lot about the mechanics of cell protrusions. Additionally, their GUVs with minimal actin cortices could be a super useful tool for better understanding what different proteins and combinations of proteins are doing at the cortex.
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To selectively restrict actin nucleation to the membrane as in cells (26), we used a 10xHis-tagged VCA construct that binds to nickel-chelating lipids in the membrane.
Really clever way to drive formation of an actin cortex at the membrane of the GUVs. But obviously VCAs aren't attached to the membrane like this in biological systems, so are there biological consequences that might affect some of this data? I think it would be interesting to hear a little bit about this in the discussion maybe?
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fragmented the actin cortex by laser ablatio
I was still kind of questioning if the GUV misshaping was really due to the actin vs just having extra membrane around (even though you had the bare GUVs being more spherical), but this experiment and the CytoD experiment are really convincing. Although I wonder if you could include a quantification of roundness or a similar parameter pre and post ablation?
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Importantly, the width of the bleached region did not change significantly over time (Fig. S2)
In figure 1D, it looks like the width of the bleached region didn't change, but it looks like the overall intensity of the signal around the whole GUV deceases - do you think this is from cycling through depolymerization, then monomeric actin, then repolymerizing at the bleached spot?
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This indicates that the fluorescence recovery was dominated by exchange of actin monomers or filaments with the GUV lumen via turnover, rather than by lateral diffusion of actin along the membrane.
I think this FRAP experiment is really cool, and definitely important to show that the actin at the cortex turns over. Also, really cool that the turnover is close to that of living cells even without debranching, severing, disassembling proteins! Can you distinguish between filamentous actin and monomeric actin with your labelling technique? (It might be worth mentioning how you're labelling the actin here.) I'm curious what the ratio of G-actin to F-actin looks like and how this might affect turnover. You have a really great paragraph about this in discussion, but might be useful to mention briefly here.
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Importantly, the width of the bleached region did not change significantly over time (Fig. S2)
In figure 1D, it looks like the width of the bleached region didn't change, but it looks like the overall intensity of the signal around the whole GUV deceases - do you think this is from cycling through depolymerization, then monomeric actin, then repolymerizing at the bleached spot?
-
Introduction
This is a really interesting paper with a really cool technique, some beautiful images, and a ton of data that tells us a lot about the mechanics of cell protrusions. Additionally, their GUVs with minimal actin cortices could be a super useful tool for better understanding what different proteins and combinations of proteins are doing at the cortex.
-
No protrusions occurred when actin polymerized in the GUV lumen, showing that Arp2/3-driven actin polymerization at the membrane drives protrusion formation (Fig. S7 B).
Did you do any experiments where you polymerized actin filaments with the Arp2/3 complex, but with non-membrane anchored VCAs?
-
fragmented the actin cortex by laser ablatio
I was still kind of questioning if the GUV misshaping was really due to the actin vs just having extra membrane around (even though you had the bare GUVs being more spherical), but this experiment and the CytoD experiment are really convincing. Although I wonder if you could include a quantification of roundness or a similar parameter pre and post ablation?
-
time-lapse imaging revealed that GUV shapes often remained unchanged over many minutes
Interesting.. I'd expect these structures to be really dynamic. Is the turnover of actin at the membrane decreased in these deflated GUVs?
-
most of the cortex-bearing GUVs were frozen in highly irregular shapes, showing sharp corners (pink arrows in Fig. 2A), bleb-like protrusions (yellow arrows), long and thin protrusions (cyan arrows), or highly anisotropic shapes (yellow asterisk).
How often did you see these different morphologies? I'm curious if some are more common than others.
-
To selectively restrict actin nucleation to the membrane as in cells (26), we used a 10xHis-tagged VCA construct that binds to nickel-chelating lipids in the membrane.
Really clever way to drive formation of an actin cortex at the membrane of the GUVs. But obviously VCAs aren't attached to the membrane like this in biological systems, so are there biological consequences that might affect some of this data? I think it would be interesting to hear a little bit about this in the discussion maybe?
-
This indicates that the fluorescence recovery was dominated by exchange of actin monomers or filaments with the GUV lumen via turnover, rather than by lateral diffusion of actin along the membrane.
I think this FRAP experiment is really cool, and definitely important to show that the actin at the cortex turns over. Also, really cool that the turnover is close to that of living cells even without debranching, severing, disassembling proteins! Can you distinguish between filamentous actin and monomeric actin with your labelling technique? (It might be worth mentioning how you're labelling the actin here.) I'm curious what the ratio of G-actin to F-actin looks like and how this might affect turnover. You have a really great paragraph about this in discussion, but might be useful to mention briefly here.
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