Langmuir 2004, 20, 4629-4639
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Effect of Actin Concentration on the Structure of Actin-Containing Liposomes Shuliang Li and Andre F. Palmer* Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, Indiana 46556 Received December 29, 2003. In Final Form: March 1, 2004 Liposomes encapsulating monomeric actin (G-actin) were produced via extrusion through 400 and 600 nm pore diameter polycarbonate membranes in low ionic strength buffer (G-buffer). After actin-containing liposomes were subjected to high ionic strength polymerization buffer (F-buffer), morphological changes in the structure of actin-containing liposomes were studied using asymmetric flow field-flow fractionation (AFFF) coupled with multiangle static light scattering (MASLS). The general shape of these liposomes was initially determined by fitting three form factors, which describe the angular distribution of scattered light from a spherical thin shell, thin disk, and thin rod, to the experimentally measured light scattering spectra to regress the dimensions of the liposomes corresponding to the proposed geometry. Light scattering spectra that yielded a best fit to the thin disk model were analyzed further and fit to the ellipsoidal of revolution form factor to regress both the major and minor axis dimensions. The results of this analysis showed that actin-containing liposomes extruded through 400 and 600 nm pore diameter membranes in F-buffer, at a low actin concentration (0.1 mg/mL), assumed a spherical shape, which is also the case for plain liposomes (no actin present) in G-buffer and F-buffer. When the actin concentration was increased to 1 mg/mL, the polymerizing actin filaments stretched the initially spherical liposome into a disklike shape. However, when the actin concentration was further increased to 5 mg/mL, the liposomes reverted back to a spherical shape.
Introduction Liposomes are currently being used as promising vehicles for the delivery of therapeutic drugs, proteins, and genes to diseased tissues.1 However, plain liposomes are fragile when exposed to fluid shear stresses, often leading to pronounced deformation and/or complete destruction of the liposome.2 To increase the mechanical strength of plain unilamellar liposomes, several groups have demonstrated that liposomes could be stabilized by encapsulating a cytoskeletal matrix inside the liposome aqueous core.3-6 This strategy was inspired by the layered architecture of eukaryotic cells. In animal cells, the outermost layer of the cell is composed of a lipid bilayer, which mainly consists of phospholipids, and membrane-bound proteins.7 Beneath the lipid membrane, the cytoskeleton, a 3D network of protein filaments, spans the cytoplasm. The cytoskeleton is a 3D network of protein filaments attached to the inner leaflet of the cell membrane. The cytoskeleton largely determines the mechanical stiffness of cells. The cytoskeleton is comprised of three different types of cytoskeletal filaments, namely, actin filaments, microtubules, and various types of intermediate fila* To whom correspondence should be addressed. (1) Lian, T.; Ho, R. J. Y. J. Pharm. Sci. 2001, 90, 667-678. (2) Reviakine, I.; Brisson, A. Langmuir 2000, 16, 1806-1815. (3) Cortese, J. D.; Schwab, B., III; Frieden, C.; Elson, E. L. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 5773-5777. (4) Hotani, H.; Miyamoto. Adv. Biophys. 1990, 26, 135-156. (5) Barmann, M.; Kas, J.; Kurzmeier, H.; Sackmann, E. A new cell model: actin networks encaged by giant vesicles. In The structure and conformation of amphiphilic membranes; Lipowski, R., Richter, D., Kremer, K., Eds.; Springer-Verlag/Germany: Berlin, 1992. (6) Janmey, P. A.; Cunninghan, C. C.; Oster, G. F.; Stossel, T. Cytoskeletal networks and osmotic pressure in relation to cell structure and motility. In Mechanics of swelling: From clays to living cells and tissues; Karalis, T. K., Ed.; Springer-Verlag: Heidelberg, Germany, 1992. (7) Singer, S. J.; Nicolson, G. L. Science 1975, 175, 720-731.
ments.8 Actin is a major component of the cytoskeleton and is actively involved in a variety of cellular processes such as locomotion, cytokinesis, mitosis, and phagocytosis.9,10 Giant liposomes (approximately 5-10 µm in diameter, comparable in size to real cells) encapsulating actin and actin-binding proteins were first used as a simplified biophysical model to study the morphogenesis of the cell membrane. Cortese et al. 3 encapsulated actin filaments inside the aqueous core of liposomes, and demonstrated that the initially spherical liposomes can be forced into asymmetric, irregular shapes. They also observed that the magnitude of liposome deformation was dependent on the length of encapsulated actin filaments. In their study, the length of actin filaments was controlled by coencapsulating gelsolin, an actin-severing protein. In another study, Miyata and Hotani11 monitored the shape changes of liposomes encapsulating actin by dark-field and differential interference-contrast light microscopy. They observed that G-actin-containing liposomes adopted a spherical shape. However, when G-actin was polymerized inside the aqueous core of giant liposomes, two shapes of F-actin-containing liposomes were observed, namely, dumbbell and disklike shapes. Using giant liposomes (5-20 µm in diameter) with an underlying actin cortex as a model system of the cell membrane, Boulbitch et al.12 observed the formation of blisters and/or invaginations by phase-contrast video microscopy. They developed a theory which predicted that local shape instabilities of composite membranes could (8) Joshi, H. C. Curr. Opin. Cell Biol. 1998, 10, 35-44. (9) Higgs, H. N.; Pollard, T. D. Annu. Rev. Biochem. 2001, 70, 649676. (10) Coulombe, P. A.; Bousquet, O.; Ma, L. L.; Yamada, S.; Wirtz, D. Trends Cell Biol. 2000, 10, 420-428. (11) Miyata, H.; Hotani., H. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 11547-11551. (12) Boulbitch, A.; Simson, R.; Simson, D. A.; Merkel, R.; Hackl, W.; Barmann, M.; Sackmann, E. Phys. Rev. E 2000, 62, 3974-3985.
10.1021/la0364690 CCC: $27.50 © 2004 American Chemical Society Published on Web 04/16/2004
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be triggered by a local softening of the membraneassociated cytoskeleton. In a variation of the previous study, Helfer et al.13 designed an in vitro self-assembled network of actin filaments attached to the outer surface of giant liposomes. They tailored the cytoskeletal network to produce a quasi-2D cross-linked network on the outside surface of the giant liposomes. This study showed that the presence of an external actin shell increases the bending modulus of the liposome, and induces the existence of a 2D shear modulus, which demonstrates the viscoelastic behavior of the composite membrane. Limozin et al.14 extended Boulbitch et al.’s12 work and studied the adsorption of actin networks inside giant vesicles composed of dimyristoyl phosphatidylcholine (DMPC). They observed that actin filaments could spontaneously form a thin cortex beneath the inner leaflet of giant liposomes, provided the liposome diameter is smaller than the filament persistence length. Ribbonlike filaments, parallel-aligned filaments, and fuzzy actin cortices were observed using fluorescence microscopy by doping DMPC with different lipids. They also demonstrated that by doping DMPC with high concentrations of cholesterol (x > 0.3) or with a few mole percent of PEGylated lipid, the actin cortex detached from the lipid membrane. Actin-containing liposomes of increasing complexity can be generated via coencapsulation of any of actin’s many regulatory proteins or small molecules. In particular, actincross-linking proteins (fascin, R-actinin, and filamin) are capable of inducing additional types of shape changes in the structure of actin-containing liposomes by reorganizing individual actin filaments into bundles of actin.15 Actincontaining liposomes coencapsulating fascin formed membrane projections, and exhibited enough strength and rigidity to maintain the physical integrity of the projections, whereas most liposomes coencapsulating R-actinin displayed no associated morphological changes. When actin and filamin were coencapsulated inside liposomes, the resultant liposomes transformed into ellipsoidal or nutlike shapes. Miyata and Kinosita16 observed the transformation of actin-encapsulating liposomes induced by the small molecule cytochalasin D. Liposomes that initially assumed either disk or dumbbell shapes were found to become spindle shaped by the addition of cytochalasin D into the medium containing the actin liposomes. It was surmised that cytochalasin D cleaved the actin filaments into smaller fragments, and initiated depolymerization of actin monomers from the filament ends. In addition to actin, microtubules have also been encapsulated inside the aqueous core of giant liposomes to enhance their mechanical stability. In several experimental studies, it was found that initially spherical liposomes encapsulating the monomer tubulin deformed into rugby-ball-shaped structures, as a result of the force generated by tubulin self-assembly into microtubules.4,17,18 By analyzing the bending free energy of the liposomes, several groups19-21 have theoretically predicted the mor(13) Helfer, E.; Harlepp, S.; Bourdieu, L.; Robert, J.; MacKintosh, F. C.; Chatenay, D. Phys. Rev. E 2001, 63, 021904. (14) Limozin, L.; Barmann, M.; Sackmann, E. Eur. Phys. J. E 2003, 10, 319-330. (15) Honda, M.; Takiguchi, K.; Ishikawa, S.; Hotani, H. J. Mol. Biol. 1999, 287, 293-300. (16) Miyata, H.; Kinosita, K. Biophys. J. 1994, 67, 922-928. (17) Fygenson, D. K.; Marko, J. F.; Libchaber, A. Phys. Rev. Lett. 1997, 79, 4497-4500. (18) Emsellem, V.; Cardoso, O.; Tabeling, P. Phys. Rev. E 1998, 58, 4807-4810. (19) Bozic, B.; Sventina, S.; Zeks, B. Phys. Rev. E 1997, 55, 58345842.
Li and Palmer
phological changes in liposome structure induced by the internal polymerization. In this study, we investigated the morphological changes induced by the internal polymerization of actin inside the aqueous core of small (99% purity was obtained from Avanti Polar Lipids (Alabaster, AL), and stored in a chloroform stock solution at -20 °C. Monomeric actin, G-buffer (5 mM Tris-HCl, pH 8.0, 0.2 mM CaCl2, and 0.2 mM ATP), and F-buffer (50 mM KCl, 2 mM MgCl2, and 1 mM ATP) were obtained from Cytoskeleton Inc. (Denver, CO). K+ ionophore was obtained from Fluka (Buchs, Switzerland). All water was taken from a Barnstead E-Pure (Barnstead/Thermolyne, Dubuque, IA) ultrapure deionized water system with a resistivity of 18.1 MΩ-cm, and filtered through 0.02 µm inorganic membrane filters obtained from Whatman (Maidstone, England). G-buffer was used for all AFFF-MASLS studies of plain liposomes and liposomes encapsulating G-actin. F-buffer was used for all studies of plain liposomes and liposomes encapsulating G-actin under polymerizing conditions. Asymmetric Flow Field-Flow Fractionation Coupled with Multiangle Light Scattering. An asymmetric flow fieldflow fractionator (AFFF) (Eclipse) connected in series with an MASLS photometer (DAWN EOS) (Wyatt Technologies, Santa Barbara, CA) was used to measure the shape and geometric dimensions of plain liposomes and liposomes encapsulating actin. During a typical experiment, the liposome dispersion was injected (20) Heinrich, V.; Bozic, B.; Svetina, S.; Zeks, B. Biophys. J. 1999, 76, 2056-2071. (21) Umeda, T.; Nakajima, H.; Hotani, H. J. Phys. Soc. Jpn. 1998, 67, 682-688. (22) Hackl, W.; Barman, M.; Sackmann, E. Phys. Rev. Lett. 1998, 80, 1786-1789. (23) Miyata, H.; Nishiyama, S.; Akashi, K.; Kinosita, K. J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 2048-2053. (24) Palmer, A. F.; Wingert, P.; Nickels, J. Biophys. J. 2003, 85, 12331247. (25) Nickels, J.; Palmer, A. F. Langmuir 2003, 19, 10581-10587.
AFFF-MASLS of Actin-Containing Liposomes into the AFFF channel, where it was separated. The separated liposome dispersion eluent flowed into the MASLS photometer, which continuously measured the particles’ size and shape as a function of time. The multiangle laser photometer is temperature-controlled to within 0.1 °C, and was maintained at 25 °C for all MASLS measurements. The photometer is equipped with a 30 mW gallium arsenide diode laser that emits a vertically polarized beam with a wavelength of 690 nm in a vacuum, which is scattered by the sample. The scattered light is collimated, and simultaneously detected by a fixed array of 18 transimpedance photodiodes, which span an angular region from 22.5° to 147°. The light scattering intensity profile was recorded as a function of time at a rate of one full profile every 1 s. Here, Q ) (4πn/λ0)sin(θ/2) is the amplitude of the scattering wave vector, n ) 1.3316 is the refractive index of the buffer solution, θ is the scattering angle, and λ0 ) 690 nm is the wavelength of the incident light beam in a vacuum. In preparation for all AFFF-MASLS experiments, all glassware was carefully cleaned, while all mobile phases (G- and F-buffer) were thoroughly filtered through 0.1 µm filters. Asymmetric flow-field fractionation is a chromatographic separation technique which is capable of separating a solution of polydisperse particles, with sizes ranging from 1 nm to 10 µm, using two orthogonal flow fields acting within a trapezoidalshaped channel. The top of the channel is bounded by an optically transparent acrylic block, while the bottom is bounded by a 10000 molecular weight cutoff polymeric membrane, Nadir (Millipore). For all experiments the channel thickness was fixed at 350 µm. One flow field, the channel flow (νc), is parallel to the polymer membrane. The other flow field, the cross-flow (νx), is perpendicular to the membrane. In the focus/inject mode of operation, the liposome dispersion is injected into the channel, where it is subjected to a cross-flow of 3 mL/min and a channel flow of 0 mL/min for 3 min. Liposomes exposed to the cross-flow accumulate toward the membrane surface, where they rapidly equilibrate via molecular diffusion and form into a thin ellipsoidal sample front. The ellipsoidal sample front is perpendicular to the channel flow. In the elution mode of operation, the ellipsoidal sample region is exposed to a channel flow of 1 mL/min and a cross-flow of 1.5 mL/min, which linearly decreases to 0 mL/min over a 30 min interval. The crossflow acts to retain particles longer in the channel, thus separating the dispersion on the basis of particle size. In a typical separation, small particles (
