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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane.

Streszczenie

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

Wprowadzenie

The actin cytoskeleton plays a fundamental role in constructing the intracellular architecture of the cell by coordinating molecular-level contractility and force generation1,2,3. As a result, it mediates numerous essential cellular activities, including cell deformation4,5, division6, migration7,8, and adhesion9. The in vitro reconstitution of actin networks has gained tremendous attention in recent years10,11,12,13,14,15,16,17. The goal of reconstitution is to build a minimal model of the cell devoid of the complex biochemical regulation that exists within live cells. This offers a controllable environment to probe specific intracellular activities and facilitates the identification and analysis of different components of the actin cytoskeleton18,19. Further, the encapsulation of in vitro actin networks inside phospholipid giant unilamellar vesicles (GUVs, liposomes) provides a confined but deformable space with a semi-permeable boundary. It mimics the physiological and mechanical microenvironment of the actin machinery within the cell9,20,21,22.

Among various methods to prepare liposomes, the lipid film hydration method (also known as the swelling method) is one of the earliest techniques23. The dry lipid film hydrates with the addition of buffers, forming membranous bubbles that eventually become vesicles24. To produce larger vesicles with a higher yield, an improved method advancing from the film hydration method, known as the electroformation method25, applies an AC electric field to efficiently promote the hydration process26. The major limitations of these hydration-based methods for actin encapsulation are that it has low encapsulation efficiency of highly concentrated proteins, and it is only compatible with specific lipid compositions24. The inverted emulsion technique, in comparison, has fewer limitations for lipid components and protein concentrations20,27,28,29. In this method, a mixture of proteins for encapsulation is added to the inner aqueous buffer, which is later emulsified in a lipid-containing mineral oil solution, forming lipid-monolayer droplets. The monolayer lipid droplets then cross through another lipid/oil-water interface through centrifugation to form bilayer lipid vesicles (liposomes). This technique has proven to be one of the most successful strategies for actin encapsulation24,30. Separately, there are some microfluidic device methods, including pulsed jetting31,32, transient membrane ejection33, and the cDICE method34. The similarities between the inverted emulsion method and the microfluidic method are the lipid solvent (oil) that is utilized and the introduction of lipid/oil-water interface for the formation of the outer leaflet of liposomes. By contrast, the generation of liposomes by the microfluidic method requires a set-up of microfluidic devices and is accompanied by oil trapped between the two leaflets of the bilayer, which requires an extra step for oil removal35.

In this manuscript, we used the inverted emulsion technique to prepare liposomes encapsulating a polymerized F-actin network as used previously22. The protein mixture for encapsulation was first placed in a buffer with nonpolymerizing conditions to maintain actin in its globular (G) form. The whole process was carried out at 4 °C to prevent early actin polymerization, which was later triggered by allowing the sample to warm to room temperature. Once at room temperature, the actin polymerizes into its filamentous (F) form. A variety of actin-binding proteins can be added to the inner aqueous buffer solution to study protein functionalities and properties, thus, further providing insights into its interaction with the actin network and membrane surface. This method can also be applied to the encapsulation of various proteins of interest36 and large objects (microparticles, self-propelled microswimmers, etc.) close to the size of the final liposomes28,37.

Protokół

1. Preparation of buffers and protein solutions

  1. Prepare the aqueous Inner Non-Polymerization (INP) Buffer in a total volume of 5 mL by mixing 0.1 mM CaCl2, 10 mM HEPES (pH 7.5), 1 mM DTT, 0.5 mM Dabco, 320 mM sucrose, and 0.2 mM ATP.
  2. Prepare the Protein Mix (PM) by adding proteins to the INP buffer at 4 °C with the following concentrations: 11.2 µM non-fluorescent G-actin, 2.8 µM fluorescently labeled actin, and 0.24 µM Arp2/3 (Table of Materials). To form an F-actin layer, add 100 nM gelsolin, 4 µM cofilin, and 2.2 µM VCA-His to the PM. As a control experiment, replace PM by 100 µg/mL fluorescent dye (Table of Materials).
  3. Prepare the aqueous Inner Polymerization (IP) Buffer (aqueous) in a total volume of 5 mL by mixing 100 mM KCl, 4 mM MgCl2, 10 mM HEPES (pH 7.5), 1 mM DTT, 0.5 mM Dabco, 10 mM ATP, and 80 mM sucrose.
  4. Prepare the Final Buffer (FB) by mixing INP buffer (containing PM) and IP buffer at a volume ratio of 1:1 to yield the inner aqueous solution in a total volume of 30 µL that will be encapsulated within the liposome.
  5. Prepare the aqueous Outside Buffer (OB) in a total volume of 150 µL by mixing 10 mM HEPES (pH 7.5), 50 mM KCl, 2mM MgCl2, 0.2 mM CaCl2, 2mM ATP, 1 mM DTT, 0.5 mM Dabco, 212 mM glucose, and 0.1 mg/mL β-casein.
    NOTE: The osmolarity of the OB can be adjusted with glucose to ensure the osmotic pressure of the OB is slightly larger than that of the FB (20-60 mOsm). The density of the FB should be slightly higher than the OB.

2. Preparation of liposomes based on the inverted emulsion techniques

  1. Prepare the lipid-oil mixture
    1. Add 100 µL of 25 mg/mL L-α-phosphatidylcholine (non-fluorescent Egg PC, also called EPC, including 1% DHPE) into a glass vial. Evaporate chloroform with argon gas, leaving a dry solid lipid film (2.5 mg) at the bottom of the vial.
      1. To form an F-actin layer, add 1,2-dioleoyl-sn-glycero-3-{[n(5-amino-1-carboxypentyl) iminodiacetic acid] succinyl} nickel salt (DOGS-NTA-Ni) at a 10:1 ratio of EPC to DOGS-NTA-Ni and mix before the evaporation of chloroform.
    2. Add 2 mL of mineral oil and sonicate the lipid-oil mixture at room temperature in a bath for 1 h to re-suspend the lipids.
      NOTE: The lipid-oil mixture after sonication can be kept for 1 week at 4 °C. Re-sonication is suggested before use.
  2. Prepare a Final Buffer in oil (FB/oil) emulsion for preparing monolayer lipid droplets containing the protein of interest.
    1. To 100 µL of lipid-oil mixture taken in a plastic tube, add 10 µL of FB. Ensure that FB is in one droplet.
    2. Using a glass syringe, draw the lipid-oil-FB mixture and gently aspirate up and down multiple times to emulsify it; draw a small amount of lipid-oil mixture first, and then the FB droplet by placing the tip of the syringe at the periphery of the droplet to break it up into tiny droplets. Repeat the up and down aspiration until a whitish and cloudy emulsion is formed.
  3. Put 30 µL of OB in a separate plastic tube. Place 30 µL of the lipid-oil mixture on top of OB and let it sit for ~10 mins to develop a lipid monolayer at the interface.
    NOTE: If the lipids are charged or the protein is incorporated, the duration of this step should be extended38.
  4. Prepare the liposomes
    1. Carefully add 50 µL of the FB/oil emulsion (step 2.2) to the top oil phase of the tube from step 2.3.
    2. Centrifuge the plastic tube at 100 x g for 15 mins at 4 °C. Vary time and centrifugation speed to optimize for liposome formation.
      NOTE: After centrifugation, the top oil phase should be clear, and the bottom OB (containing liposomes) should be slightly cloudy.
    3. Carefully remove the oil phase away by pipette. Aspirate extra volume if needed. Ensure not to put the pipette tip at the side of the tube to avoid creating a meniscus of oil on top of the liposome phase.
    4. With a new pipette, slowly stick the pipette tip into the remaining bottom phase and aspirate the aqueous volume to collect liposomes.
      NOTE: It is better to lose volume than to incorporate the oil. The inclusion of oil will cause liposomes to rupture, and the internal components will be released into the external buffer. Cut the tip of a pipette to reduce shear.

3. Microscopy observation

  1. Pour 100 µL of OB into the well of an incubation chamber (4-well plate containing 12 mm round coverslips). Gently deposit the collected liposomes (step 2.4.4) into OB, and then place another coverslip on top of the chamber.
    NOTE: The OB contains the β-casein to passivate the glass surface and minimize sticking between the liposomes20.
  2. Observe liposomes with a confocal microscope using a 63x oil-immersion objective. Use 488 nm, 647 nm, and 561 nm laser lines to observe fluorescently labeled lipid, encapsulated fluorescent dye, and encapsulated fluorescently labeled actin, respectively. Capture the frames of interest and save the images in TIFF format.
  3. Process and analyze images in ImageJ/Fiji39. For new users of ImageJ/Fiji, an online tutorial is available (see Table of Materials).
    1. Open the TIFF file using ImageJ/Fiji software.
    2. Go to Image > Adjust > Brightness/Contrast. Adjust the brightness and contrast of the image to the desired scale by adjusting the Minimum and Maximum settings, and the Brightness, and Contrast sliders.
    3. Click on the Rectangle selection tool in the toolbar and select the region of interest (ROI). Go to Image > Crop to crop the ROI.
    4. Go to Analyze > Set Scale. In the pop-up window, enter 1.0 in the Pixel Aspect Ratio field and set μm as the Unit of Length. In the Distance in Pixels field, enter the image width in pixels. In the Known Distance field, enter the real image width in microns.
    5. To measure the size of the liposome, Using the Oval selection tool in the toolbar, draw a circle along the edge of the liposome. Go to Analyze > Measure to measure the area of the circle, from which the diameter of the liposome can be calculated.

Wyniki

The preparation of liposomes based on the inverted emulsion technique is illustrated graphically and schematically in Figure 1.

First, empty (bare) liposomes (~5-50 µm in diameter) that were composed of phospholipid (EPC) and fluorescent lipid (DHPE) were prepared. A bright, far-red fluorescent dye was encapsulated within bare liposomes as a control experiment. Whether a lipid monolayer has successfully formed in the peripheral of the droplet could be determi...

Dyskusje

Several key steps determine the success of a high yield of liposomes during the preparation process. To completely dissolve the lipid film in the oil, the sample must be sonicated until the lipid film at the bottom of the glass vial disappears completely. After the sonication, the lipid-oil mixture must be stored overnight at room temperature under dark conditions for the lipid molecules to disperse further29. The mixture can be stored at 4 °C for up to a week. When preparing an FB/oil emulsi...

Ujawnienia

The authors declare no conflicts of interest.

Podziękowania

We acknowledge funding ARO MURI W911NF-14-1-0403 to M.P.M., the National Institutes of Health (NIH) R01 1R01GM126256 to M.P.M., the National Institutes of Health (NIH) U54 CA209992, NIH RO1 GM126256, NIH U54 CA209992, University of Michigan / Genentech, SUBK00016255 and Human Frontiers Science Program (HFSP) grant number RGY0073/2018 to M.P.M. Any opinion, findings, conclusions, or recommendations expressed in this material are those of the authors(s) and do not necessarily reflect the views of the ARO, NIH, or HFSP. S.C. acknowledges fruitful discussions with V. Yadav, C. Muresan, and S. Amiri.

Materiały

NameCompanyCatalog NumberComments
1,2-dioleoyl-sn-glycero-3-{[n(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DOGS-NTA-Ni) Avanti Polar Lipids Inc.231615773Nickel Lipid
1,4-Diazabicyclo[2.2.2]octaneSigmaD27802-25GDABCO
Actin protein (>99% pure): rabbit skeletal muscleCytoskeleton, IncAKL99-Dnon-fluorescent G-actin
Actin protein (rhodamine): rabbit skeletal muscleCytoskeleton, IncAR05fluorescently labeled actin
Adenosine 5′-triphosphate disodium salt hydrateSigmaA2383-10GATP
Alexa Fluor 647 dyeThermoFisherfluorescent dye
Andor iQ3Andor Technologiescontrol and acquisition software for confocal microscope
Arp2/3 Protein Complex: Porcine BrainCytoskeleton, IncRP01P-AArp 2/3
Calcium chloride dihydrateSigma10035048CaCl2
Chamlide Chambers (4-well for 12 mm round coverslip)Quorum Technologiesincubation chamber
Cofilin protein: human recombinantCytoskeleton, IncCF01-Ccofilin
Confocal Microscope (63× oil-immersion objective)Andor TechnologiesLEICA DMi8
D-(+)-GLUCOSE BIOXTRASigmaG7528glucose
DithiothreitolDOT Scientific DSD11000-10DTT
Gelsolin Protein: Homo Sapiens RecombinantCytoskeleton, IncHPG6gelsolin
Hamilton 1750 Gastight Syringe, 500 µL, cemented needle, 22 G, 2" conical tipCole-ParmerUX-07940-53glass syringe
HEPESAmericanBio7365-45-9
ImageJ/Fijihttps://imagej.net/tutorials/
L-alpha-PhosphatidylcholineAvanti Polar Lipids Inc.97281442EPC
Magnesium chlorideSigma7786303MgCl2
Mineral oil, BioReagent, for molecular biology, light oilSigma8042475mineral oil
N-WASP fragment WWA (aa400–501, VCA-His)VCA-His is purified using lab protocol. The protocol can be provided upon reasonable requests
Oregon Green 488 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (Oregon Green 488 DHPE)Thermo FisherO12650DHPE
Potassium chlorideSigma7447407KCl
SucroseSigma57-50-1sucrose
β-Casein from bovine milkSigmaC6905-250MG

Odniesienia

  1. Murrell, M., Oakes, P. W., Lenz, M., Gardel, M. L. Forcing cells into shape: the mechanics of actomyosin contractility. Nature Reviews Molecular Cell Biology. 16 (8), 486-498 (2015).
  2. Stricker, J., Falzone, T., Gardel, M. L. Mechanics of the F-actin cytoskeleton. Journal of Biomechanics. 43 (1), 9-14 (2010).
  3. Linsmeier, I., et al. Disordered actomyosin networks are sufficient to produce cooperative and telescopic contractility. Nature Communications. 7 (1), 1-9 (2016).
  4. Yousafzai, M., Yadav, V., Amiri, S., Errami, Y., Murrell, M. Active regulation of pressure and volume defines an energetic constraint on the size of cell aggregates. Physical Review Letters. 128 (4), 048103 (2022).
  5. Simon, C., et al. Actin dynamics drive cell-like membrane deformation. Nature Physics. 15 (6), 602-609 (2019).
  6. Munro, E., Nance, J., Priess, J. R. Cortical flows powered by asymmetrical contraction transport PAR proteins to establish and maintain anterior-posterior polarity in the early C. elegans embryo. Developmental Cell. 7 (3), 413-424 (2004).
  7. Bray, D., White, J. Cortical flow in animal cells. Science. 239 (4842), 883-888 (1988).
  8. Beaune, G., et al. Spontaneous migration of cellular aggregates from giant keratocytes to running spheroids. Proceedings of the National Academy of Sciences of the United States of America. 115 (51), 12926-12931 (2018).
  9. Murrell, M. P., et al. Liposome adhesion generates traction stress. Nature Physics. 10 (2), 163-169 (2014).
  10. Lee, G., et al. Myosin-driven actin-microtubule networks exhibit self-organized contractile dynamics. Science Advances. 7 (6), (2021).
  11. Anderson, S., Garamella, J., Adalbert, S., McGorty, R., Robertson-Anderson, R. Subtle changes in crosslinking drive diverse anomalous transport characteristics in actin-microtubule networks. Soft Matter. 17 (16), 4375-4385 (2021).
  12. Janson, M. E., et al. Crosslinkers and motors organize dynamic microtubules to form stable bipolar arrays in fission yeast. Cell. 128 (2), 357-368 (2007).
  13. McCall, P. M., MacKintosh, F. C., Kovar, D. R., Gardel, M. L. Cofilin drives rapid turnover and fluidization of entangled F-actin. Proceedings of the National Academy of Sciences of the United States of America. 116 (26), 12629-12637 (2019).
  14. Köhler, S., Schaller, V., Bausch, A. R. Structure formation in active networks. Nature Materials. 10 (6), 462-468 (2011).
  15. Koenderink, G. H., et al. An active biopolymer network controlled by molecular motors. Proceedings of the National Academy of Sciences of the United States of America. 106 (36), 15192-15197 (2009).
  16. Seara, D. S., et al. Entropy production rate is maximized in non-contractile actomyosin. Nature Communications. 9 (1), 1-10 (2018).
  17. Tabatabai, A. P., et al. Detailed balance broken by catch bond kinetics enables mechanical-adaptation in active materials. Advanced Functional Materials. 31 (10), 2006745 (2021).
  18. Murrell, M. P., Gardel, M. L. F-actin buckling coordinates contractility and severing in a biomimetic actomyosin cortex. Proceedings of the National Academy of Sciences of the United States of America. 109 (51), 20820-20825 (2012).
  19. Murrell, M., Gardel, M. L. Actomyosin sliding is attenuated in contractile biomimetic cortices. Molecular Biology of the Cell. 25 (12), 1845-1853 (2014).
  20. Murrell, M., et al. Spreading dynamics of biomimetic actin cortices. Biophysical Journal. 100 (6), 1400-1409 (2011).
  21. Liu, A. P., et al. Membrane-induced bundling of actin filaments. Nature Physics. 4 (10), 789-793 (2008).
  22. Pontani, L. -. L., et al. Reconstitution of an actin cortex inside a liposome. Biophysical Journal. 96 (1), 192-198 (2009).
  23. Reeves, J. P., Dowben, R. M. Formation and properties of thin-walled phospholipid vesicles. Journal of Cellular Physiology. 73 (1), 49-60 (1969).
  24. Litschel, T., Schwille, P. Protein reconstitution inside giant unilamellar vesicles. Annual Review of Biophysics. 50, 525-548 (2021).
  25. Angelova, M. I., Dimitrov, D. S. Liposome electroformation. Faraday Discussions of the Chemical Society. 81, 303-311 (1986).
  26. Limozin, L., Sackmann, E. Polymorphism of cross-linked actin networks in giant vesicles. Physical Review Letters. 89 (16), 168103 (2002).
  27. Pautot, S., Frisken, B. J., Weitz, D. Production of unilamellar vesicles using an inverted emulsion. Langmuir. 19 (7), 2870-2879 (2003).
  28. Natsume, Y., et al. Preparation of giant vesicles encapsulating microspheres by centrifugation of a water-in-oil emulsion. Journal of Visualized Experiments. (119), e55282 (2017).
  29. Chiba, M., Miyazaki, M., Ishiwata, I. s. Quantitative analysis of the lamellarity of giant liposomes prepared by the inverted emulsion method. Biophysical Journal. 107 (2), 346-354 (2014).
  30. Fujii, S., et al. Liposome display for in vitro selection and evolution of membrane proteins. Nature Protocols. 9 (7), 1578-1591 (2014).
  31. Funakoshi, K., Suzuki, H., Takeuchi, S. Formation of giant lipid vesiclelike compartments from a planar lipid membrane by a pulsed jet flow. Journal of the American Chemical Society. 129 (42), 12608-12609 (2007).
  32. Okushima, S., Nisisako, T., Torii, T., Higuchi, T. Controlled production of monodisperse double emulsions by two-step droplet breakup in microfluidic devices. Langmuir. 20 (23), 9905-9908 (2004).
  33. Ota, S., Yoshizawa, S., Takeuchi, S. Microfluidic formation of monodisperse, cell-sized, and unilamellar vesicles. Angewandte Chemie International Edition. 48 (35), 6533-6537 (2009).
  34. Bashirzadeh, Y., Wubshet, N., Litschel, T., Schwille, P., Liu, A. P. Rapid encapsulation of reconstituted cytoskeleton inside giant unilamellar vesicles. Journal of Visualized Experiments. (177), (2021).
  35. Deshpande, S., Caspi, Y., Meijering, A. E., Dekker, C. Octanol-assisted liposome assembly on chip. Nature Communications. 7 (1), 1-9 (2016).
  36. Bashirzadeh, Y., Liu, A. P. Encapsulation of the cytoskeleton: towards mimicking the mechanics of a cell. Soft Matter. 15 (42), 8425-8436 (2019).
  37. Vutukuri, H. R., et al. Active particles induce large shape deformations in giant lipid vesicles. Nature. 586 (7827), 52-56 (2020).
  38. Lemière, J., Carvalho, K., Sykes, C. . Methods in Cell Biology. 128, 271-285 (2015).
  39. Schindelin, J., et al. Fiji: an open-source platform for biological-image analysis. Nature Methods. 9 (7), 676-682 (2012).
  40. Liman, J., et al. The role of the Arp2/3 complex in shaping the dynamics and structures of branched actomyosin networks. Proceedings of the National Academy of Sciencesof the United States of America. 117 (20), 10825-10831 (2020).
  41. Sun, Y., Leong, N. T., Wong, T., Drubin, D. G. A Pan1/End3/Sla1 complex links Arp2/3-mediated actin assembly to sites of clathrin-mediated endocytosis. Molecular Biology of the Cell. 26 (21), 3841-3856 (2015).
  42. Bretschneider, T., et al. Dynamic actin patterns and Arp2/3 assembly at the substrate-attached surface of motile cells. Current Biology. 14 (1), 1-10 (2004).
  43. Goley, E. D., et al. An actin-filament-binding interface on the Arp2/3 complex is critical for nucleation and branch stability. Proceedings of the National Academy of Sciencesof the United States of America. 107 (18), 8159-8164 (2010).
  44. Svitkina, T. M., Borisy, G. G. Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia. The Journal of Cell Biology. 145 (5), 1009-1026 (1999).
  45. Kelleher, J. F., Atkinson, S. J., Pollard, T. D. Sequences, structural models, and cellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba. The Journal of Cell Biology. 131 (2), 385-397 (1995).
  46. Prehoda, K. E., Scott, J. A., Mullins, R. D., Lim, W. A. Integration of multiple signals through cooperative regulation of the N-WASP-Arp2/3 complex. Science. 290 (5492), 801-806 (2000).
  47. Delatour, V., et al. Arp2/3 controls the motile behavior of N-WASP-functionalized GUVs and modulates N-WASP surface distribution by mediating transient links with actin filaments. Biophysical Journal. 94 (12), 4890-4905 (2008).
  48. Izdebska, M., Zielińska, W., Hałas-Wiśniewska, M., Grzanka, A. Involvement of actin and actin-binding proteins in carcinogenesis. Cells. 9 (10), 2245 (2020).
  49. Schäfer, E., Kliesch, T. -. T., Janshoff, A. Mechanical properties of giant liposomes compressed between two parallel plates: impact of artificial actin shells. Langmuir. 29 (33), 10463-10474 (2013).
  50. Whittenton, J., et al. Evaluation of asymmetric liposomal nanoparticles for encapsulation of polynucleotides. Langmuir. 24 (16), 8533-8540 (2008).
  51. Levine, R. M., Pearce, T. R., Adil, M., Kokkoli, E. Preparation and characterization of liposome-encapsulated plasmid DNA for gene delivery. Langmuir. 29 (29), 9208-9215 (2013).
  52. Moga, A., Yandrapalli, N., Dimova, R., Robinson, T. Optimization of the inverted emulsion method for high-yield production of biomimetic giant unilamellar vesicles. ChemBioChem. 20 (20), 2674-2682 (2019).
  53. Vale, R. . Reconstituting the Cytoskeleton. , (2014).
  54. Pardee, J. D., Spudich, J. A. Mechanism of K+-induced actin assembly. The Journal of Cell Biology. 93 (3), 648-654 (1982).
  55. Zimmerle, C. T., Frieden, C. Effect of pH on the mechanism of actin polymerization. Biochemistry. 27 (20), 7766-7772 (1988).
  56. Wegner, A. M., et al. N-wasp and the arp2/3 complex are critical regulators of actin in the development of dendritic spines and synapses. Journal of Biological Chemistry. 283 (23), 15912-15920 (2008).
  57. Blanchoin, L., et al. Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins. Nature. 404 (6781), 1007-1011 (2000).
  58. Römer, W., et al. Actin dynamics drive membrane reorganization and scission in clathrin-independent endocytosis. Cell. 140 (4), 540-553 (2010).
  59. Popova, A. V., Hincha, D. K. Effects of flavonol glycosides on liposome stability during freezing and drying. Biochimica et Biophysica Acta (BBA)-Biomembranes. 1858 (12), 3050-3060 (2016).
  60. Harrigan, P., Madden, T., Cullis, P. Protection of liposomes during dehydration or freezing. Chemistry and Physics of Lipids. 52 (2), 139-149 (1990).
  61. Szoka, F., Papahadjopoulos, D. Comparative properties and methods of preparation of lipid vesicles (liposomes). Annual Review of Biophysics and Bioengineering. 9 (1), 467-508 (1980).
  62. Campillo, C., et al. Unexpected membrane dynamics unveiled by membrane nanotube extrusion. Biophysical Journal. 104 (6), 1248-1256 (2013).
  63. Deng, N. -. N., Yelleswarapu, M., Huck, W. T. Monodisperse uni-and multicompartment liposomes. Journal of the American Chemical Society. 138 (24), 7584-7591 (2016).
  64. Ricketts, S. N., Ross, J. L., Robertson-Anderson, R. M. Co-entangled actin-microtubule composites exhibit tunable stiffness and power-law stress relaxation. Biophysical journal. 115 (6), 1055-1067 (2018).
  65. Sheung, J. Y., et al. Motor-driven restructuring of cytoskeleton composites leads to tunable time-varying elasticity. ACS Macro Letters. 10 (9), 1151-1158 (2021).
  66. Falzone, T. T., Blair, S., Robertson-Anderson, R. M. Entangled F-actin displays a unique crossover to microscale nonlinearity dominated by entanglement segment dynamics. Soft Matter. 11 (22), 4418-4423 (2015).
  67. Francis, M. L., et al. Non-monotonic dependence of stiffness on actin crosslinking in cytoskeleton composites. Soft Matter. 15 (44), 9056-9065 (2019).
  68. Yadav, V., et al. Filament nucleation tunes mechanical memory in active polymer networks. Advanced Functional Materials. 29 (49), 1905243 (2019).
  69. Murrell, M., Thoresen, T., Gardel, M. . Methods in Enzymology. 540, 265-282 (2014).

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