actin polymerization induces a shape change in actin-containing vesicles

Actin Polymerization Induces a Shape Change in Actin-Containing VesiclesAuthor(s): Jorge Daniel Cortese, Bill Schwab, Carl Frieden and Elliot L. ElsonSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 86, No. 15 (Aug. 1, 1989), pp. 5773-5777Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/34202 .

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Proc. Nail. Acad. Sci. USA Vol. 86, pp. 5773-5777, August 1989 Biochemistry

Actin polymerization induces a shape change in actin-containing vesicles

(actin/actin-binding proteins/lipid vesicles)

JORGE DANIEL CORTESE*, BILL SCHWAB III, CARL FRIEDEN, AND ELLIOT L. ELSON

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110

Contributed by Carl Frieden, May 8, 1989

ABSTRACT We have encapsulated actin filaments in the presence and absence of various actin-binding proteins Into lipid vesicles. These vesicles are approximately the same size as animal cells and can be characterized by the same optical microscopic and mechanical techniques used to study cells. We demonstrate that the initially spherical vesicles can be forced into asymmetric, irregular shapes by polymerization of the actin that they contain. Deformation of the vesicles requires that the actin filaments be on average at least 0.5 ,(m long as shown by the effects of gelsolin, an actin filament-nucleating protein. Filamin, a filament-crossiinking protein, caused the surfaces of the vesicles to have a smoother appearance. Het- erogeneous distribution of actin rdaments within the vesicles is caused by lnterfilament interactions and modulated by gelsolin and filamin. The vesicles provide a model system to study control of cell shape and cytoskeletal organization, membrane- cytoskeleton interactions, and cytomechanics.

The shapes and mechanical properties of animal cells are governed mainly by systems of cytoplasmic filaments col- lectively termed the cytoskeleton (1, 2). Of these systems, the actin microfilament system is the principal determinant of cellular viscoelastic properties (B.S. and E.L.E., in preparation) and is most directly involved in driving mechanical processes such as locomotion, cytokinesis, and phagocytosis (2, 3). As cells perform these functions, the organization of the actin cytoskeleton changes, probably under the control of actin-binding proteins that regulate the length and extent of crosslinking of the filaments (3-5). A model system in which cytoskeletal components could be reconstituted inside vesicles comparable in size to cells would be useful for studies of the regulation of cytoskeletal organization and the determi- nation of cellular mechanical properties by the cytoskeleton.

We have developed a model system of this kind for the actin filament system. Actin and actin-binding proteins have been encapsulated in lipid vesicles large enough (up to 20 ,m in diameter) to be characterized by optical microscopy. The vesicles are large enough to be studied with biophysical techniques that measure, for example, actin diffusion by fluorescence photobleaching recovery (FPR) (6) or mechanical properties (1) of individual vesicles. The reconstitution of actin filaments with actin-binding proteins allows study of the effects of the latter on filament organization and distribution and the ability of the actin gel to drive morphological changes. In this work we have investigated the effects of gelsolin, which restricts the length of actin filaments, and of filamin, which crosslinks actin filaments (5). A striking observation is that vesicles are deformed when the actin inside them polymerizes. The extent of the deformation depends on the lengths of the resulting filaments. This observation provides experimental evidence for the speculation that actin

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filament polymerization can drive lamellar extension during cellular locomotion (7).

MATERIALS AND METHODS Materials. Actin was prepared from rabbit skeletal muscle

by the method of Spudich and Watts (8) and then gel-filtered through Sephadex G-150 (9) and labeled with (iodoacetami- do)tetramethylrhodamine (IATR) (10). Plasma gelsolin was purified from rabbit plasma (4, 11). Filamin was purified by a method (12) modified as described (4). Fluorescein-labeled dextran (Mr 36,500) was purchased from Sigma, dissolved at 16 mg/ml in double-glass-distilled water, dialyzed against 1 mM EDTA overnight, and stored at 20?C. All reagents other than actin and actin-binding proteins were obtained from Sigma.

Incorporation of Actin and Actin-Binding Proteins into Lipid Vesicles. In a modification of a previously described method (13), loaded vesicles were prepared by injection of a diethyl ether aqueous solution into a heated solution of the protein to be encapsulated. To minimize the time for encapsulation and the extent of protein denaturation, we used both a lower diethyl ether/water ratio, 1:10 (vol/vol), than traditional methods (13, 14) and a briefer (<5 min) exposure to 55?C temperatures. A mixture of 80% phosphatidylcholine (Ptd- Cho) and 20% phosphatidylethanolamihe (PtdEtn) was se- lected to minimize problems of lipid-phase formation (15) and maximize K+ and Cl- permeability (16). This mixture also avoided aggregation of vesicles (apparent with PtdCho or PtdEtn/phosphatidylserine vesicles) (17). The negatively charged surface prevented actin polymerization directly from the membrane (18). PtdEtn and PtdCho at a molar ratio of 1:4 together with 0.1 mg of valinomycin per mg of total phos- pholipid (0.35 mM final concentration) were dried down from stock solutions immediately prior to use and dissolved into =100 Al of water-washed diethyl ether. One milliliter of the protein solution (actin with or without filamin or gelsolin in 0.2 mM CaCl2/1.5 mM NaN3/0.2 mM ATP/2 mM Tris HCI, pH 8.0; G buffer) was warmed to 52-55?C in a water bath, and the lipid stock solution was injected in small aliquots with gentle shaking (total time, <5 min). The lipid solution was injected at the bottom of the tubes containing the protein mixtures. In all reconstitutions the actin concentration was 48 ttM. The vesicle-containing solution was cooled to room temperature, and the remaining ether was evaporated by flushing the solution with nitrogen.

Polymerization of Actin Inside Lipid Vesicles. The KCI concentration of the vesicle-containing buffer was increased to 100 mM, and K' was rapidly equilibrated across the membrane by valinomycin, thus polymerizing the encapsulated actin. The high concentration of valinomycin ensured that only a few transport cycles (2-3) were needed to equil-

Abbreviation: FPR, fluorescence photobleaching recovery. *Present address: Department of Cell Biology and Anatomy, Uni- versity of North Carolina, Chapel Hill, NC 27599-7090.

5773



5774 Biochemistry: Cortese et al. Proc. Natl. Acad. Sci. USA 86 (1989)

ibrate K+. Valinomycin uniport avoids exchange of K+ for other cations and the possible perturbation of the actin gel structure from resulting pH changes (19).

Vesicles were assayed for actin polymerization and changes of vesicle shape after diluting 100 gl of the vesicle solution into 900 ,ul of either nonpolymerizing G buffer or polymerizing buffer (G buffer containing 100 mM KCI). This 10-fold dilution reduced the background fluorescence to a negligible level compared to vesicle fluorescence. The diluted vesicle-containing solution was incubated at room temperature for 1 hr to allow actin polymerization. To eliminate background fluorescence in FPR experiments, we separated actin-containing vesicles from the unincorporated actin by washing with an excess of the appropriate nonpolymerizing or polymerizing buffer (10 or more volumes), using Centrex concentrators (1-,um pore diameter, Schleicher & Schuell). There were no detectable differences in the shapes of vesicles prepared by dilution or by using Centrex concentrators.

FPR. FPR was performed as described (4, 6, 20) with a x 16 Zeiss Neofluar objective (numerical aperture, 0.35), which gives a 4-,m Gaussian laser spot. Only vesicles with diameter >10 ,um were used for FPR experiments.

Microscopy. Samples were viewed on a Zeiss universal microscope by using a x40 Zeiss Plan objective (numerical aperture, 0.65). Images were collected with a Dage ISIT camera (MTI 66), averaged, and digitized with a Grinnell GMR-274 image processor. Microphotographs were taken from digitized images shown on a Panasonic WV-5300 video monitor. Scanning confocal microscopy was performed with a Zeiss Axioplan microscope equipped with a Bio-Rad MRC- 500 laser scanning instrument. A x 100 Zeiss Neofluar objective (numerical aperture, 1.30) was used, and images were averaged by using the Kalman model (n = 20) available in the MRC-500 software. Optical section thickness was approximately 0.7 ,um.

RESULTS Incorporation and Polymerization of Actin in Vesicles. A

mixture of 10% rhodamine-labeled/90% unlabeled actin, alone or with actin-binding proteins, was encapsulated by injecting a diethyl ether solution of phospholipids and the ionophore valinomycin into the warmed protein solution. A heterogeneous preparation with a high number of oligolamel- lar vesicles with diameters between 2 and 20 ,tm was obtained. Fluorescence microscopy of the vesicles showed actin encapsulation. When the potassium level within the vesicles was raised by the addition of 100 KCI to the incubation buffer, the initially spherical actin-containing vesicles assumed markedly asymmetric shapes (Figs. 1 and 2). Actin polymerization was confirmed by a decrease of actin mobility as measured by FPR (Fig. 1) (6, 20). There was considerable fluorescence recovery of actin monomer in vesicles incubated in G buffer (Fig. la). Actin was immobi- lized by formation of long filaments (20) in vesicles incubated in G buffer with 100 mM KCI (Fig. lb). In control experiments, vesicle-encapsulated fluorescein-labeled dextran molecules diffused freely regardless of whether the vesicles were incubated in G buffer with 100 mM KCI or G buffer without the KCI (Fig. 1 c and d). Although the FPR measurements showed qualitatively large differences in the diffusion rates of actin under polymerizing and nonpolymerizing conditions, they did not readily yield quantitative values for the diffusion coefficient and fraction of mobile actin molecules because the diameters of the vesicles were not large compared to the diameter of the laser spot. Thus, the apparent mobile fraction was reduced because of depletion of the pool of fluorophore, and the apparent diffusion rate was increased because of the small available volume. Although these effects have been quantitatively analyzed for planar systems (21), the analysis is much more complex for three-dimensional systems and has

a b o 0 to - ~~~0 (N

CV)

C ,-~~~~~~~~~~~~~~~~~-

0 10 20 30 40 0) 10 20 30 40 FIG. 1. FPR traces and sample

phase-contrast photomicrographs of ac- Time (sec) Time (sec) tin- and dextran-containing vesicles. The

actin concentration is 48 AM throughout. c d (a) Actin-containing vesicles incubated in

G buffer, showing recovery of fluores- 8 : 0 cence signal in FPR trace after photo- CN :- -. bleaching; the vesicles also show spher-

ical morphology (Inset). (b) Actin- o ~~~~~~~~~~~~~~~~~~~~~containing vesicles incubated in buffer

? j

-. - with 100 mM KCI; FPR traces no longer

0> : ,,, X - : :: : .: :- . show fluorescence recovery, and the ves- 0 o icles are markedly deformed (Inset). (c)

c _ _ Fluorescein-congugated dextran-containing vesicles (fluorescein-labeled dextran at 2 mg/ml) incubated in G buffer,

Ln eshowing recovery of signal and spherical morphology (Inset). (d) Fluorescein- dextran-containing vesicles incubated in

0 0 G buffer with 100 mM KCI, showing 0 10 20 30 40 0 10 20 30 40 recovery of signal in FPR and spherical

Time (sec) Time (sec) morphology (Inset).



Biochemistry: Cortese et al. Proc. Nati. Acad. Sci. USA 86 (1989) 5775

a ~~~~~~~~b

C d

e f

FIG. 2. Phase-contrast (a, c, e, g, and i) and fluorescence digital-intensified (b, d, f, h, and j) images of actin- and actin/gelsolin-containing vesicles al-

9 - _ h lowed to equilibrate in buffer with 100 mM KCI for 1 hr as described in Fig. 1. The actin concentration is 48 tLM throughout. (a and b) Vesicles containing actin incubated in G buffer are spherical. (c and d) Vesicles containing actin incubated in G buffer with 100 mM KCI have a deformed contour and also a heterogeneous distribution of actin. (e and f) Vesicles containing actin and 24 nM gelsolin (gelsolin/actin molar ratio of J 1:2000) have a deformed contour and heterogeneous distribution of actin. (g and h) Vesicles containing actin and 0.24 utM gelsolin (actin/gelsolin molar ratio of 1:200) have a deformed contour and a homogeneous distribution o'f actin. (i and j) Vesicles containing actin and 0.96 gtM gelsolin (gelsolin/actin molar ratio of 1:50) are spherical, and the distribution of actin is homogeneous. (Bars = 2 ~tm.)

not been attempted for this work. A further problem for quantitative analysis is a lensing effect due to the high vesicle curvature. Nevertheless, the FPR measurements that dem- onstrated immobilization of actin at high K+ concentrations showed that actin is extensively polymerized under these conditions.

Actin Polymerization Deforms Vesicles. Polymerization of encapsulated actin extensively deformed vesicles (Fig. lb Inset and Fig. 3 a and b). Vesicles observed at 5 min were undeformed. Deformation developed slowly over the next 30 min (data not shown). Actin-containing vesicles incubated in G buffer (Fig. la Inset) and vesicles containing equivalent concentrations of fluorescein-labeled dextran or bovine se- rum albumin incubated in G buffer with or without 100 mM KCI (Insets of Fig. 1 c and d) did not deform. Replacement of K+ by a comparable quantity of Na+, which neither supports actin polymerization nor is transported by valinomycin, left the vesicles undeformed (not shown). Actin- containing vesicles made without valinomycin had larger sizes and were insensitive to the presence of 100 mM KCI.

Only vesicles containing actin and able to equilibrate K+ across the membrane responded to potassium-induced polymerization conditions by assuming nonspherical shapes. Hence, an osmotically generated force was not responsible for the deformation. Rather, formation of actin filaments deformed the vesicles.

Actin-Binding Proteins Affect Liposome Deformation. The magnitude of vesicle deformation depended on the length of the actin filaments. Filament length was controlled by mixing gelsolin, an actin-binding protein that nucleates filament formation in defined ratios with the actin (22). Under our conditions (especially where the gelsolin/actin ratio is large), self-nucleation by the actin is slow compared to nucleation by gelsolin-actin complexes. Hence, each gelsolitn molecule should ideally nucleate a filament, and the mean length of these filaments should equal the actin/gelsolin ratio divided by 370 monomers per ,um (23). Therefore, the mean length of our gelsolin/actin filaments was varied between 0.07 ,m (1:25) and 5.4 ,tm (1:2000). It is important to note that these are mean lengths, and the actual distribution of filament



5776 Biochemistry: Cortese et al. Proc. Natl. Acad. Sci. USA 86 (1989)

ab

C~~~~~~~~~~~~~~~

FIG. 3. Confocal images of vesicles containing actin (48 aM) and actin-binding proteins, equilibrated with G buffer with 100 mM KCI. (a and b) Vesicles containing actin, deformed and with an inhomogeneous distribution of fluorescence. (Bar = 2 ,tm.) (c) Vesicle containing actin and 0.96 ,uM gelsolin (gelsolin/actin molar ratio is 1:50), spherical and with a homogeneous fluorescence distribution. (Bar = 1 Am.) (d) Vesicle containing actin and 9.6 ,uM filamin (approximated filamin/actin molar ratio is at least 1:10, considering encapsulation efficiency problems) (9), ellipsoidal and with a peripheral distribution of fluorescence. (Bar = 1 ,um.) Images obtained by phase-contrast and conventional fluorescence microscopy on similar samples (as described in the legend for Fig. 2) experienced the same changes in shape (not shown).

lengths is quite broad and right-skewed (22). Vesicles containing short actin filaments, obtained by mixing gelsolin and actin at molar ratios between 1:25 and 1:50, were spherical after polymerization (Fig. 2 i and j and 3c). Increasing the lengths of the filaments by using gelsolin/actin ratios between 1:200 and 1:1000 produced nonspherical vesicles (Fig. 2 e-h). At a gelsolin/actin ratio of 1:2000, there was substantial deformation (Fig. 2 c and h). The transition between spherical and irregular shapes occurred sharply between gelsolin/actin ratios of 1:100 and 1:200, which corresponds to mean filament lengths of 0.27 and 0.54 ,um, respectively. In addition to its effect on vesicle shape, the length of the filaments also influenced their distribution within the vesicles. As the lengths of the filaments increased, their distribution became more nonuniform. In contrast to the sharp transition of vesicle shape, however, the density heterogeneity dimin- ished gradually as filaments shortened until the distribution of actin appeared to be homogeneous for gelsolin/actin - 1:50 (mean length c 0.14 gm; Fig. 2f, h, andj). Observations by scanning confocal microscopy, which provided a narrower depth of focus, confirmed these findings (Fig. 3). The areas of increased filament density were found throughout the vesicles suggesting that gradients of K+ were probably not responsible. Moreover, K+ gradients within the vesicles should be rapidly dissipated (within about 25 ms) because of diffusion.

A filament-crosslinking protein, filamin, had a different effect on vesicle shape. Vesicles containing actin and filamin showed a smoother surface than those lacking filamin (Fig.

3d). In these experiments the initial molar ratio of filamin to actin was 1:5, but the ratio in the vesicles may have been somewhat lower because of the molecular weight depen- dence of encapsulation efficiency (14).

DISCUSSION Evidence from a variety of sources indicates that cellular motility and resistance to deformation are dominated by the underlying cytoskeleton (1, 3, 24). Thus, local changes in cell shape and viscoelasticity result from changes in the organization and extent of polymerization of cytoskeletal filaments, especially actin microfilaments. In the reconstituted model system that we have developed, the compositions of both the enveloping membrane and the encapsulated cytoskeleton are far simpler than their cellular counterparts. Nevertheless, as we have shown, the model system does provide useful information about the effects of changes of filament length on filament organization and on vesicle shape. It also provides a simple approach for demonstrating the role of specific actin-binding proteins in the control of cell shape and filament organization. Of course the approach is amenable to further elaboration, and we expect that it also will be useful for studies of other aspects of cytoskeletal function.

When sodium is substituted for potassium at equal osmo- larity and when ionophore is omitted, the vesicles do not deform. Hence the osmotic pressure difference between the buffers with and without 100 mM KCl cannot explain the observed vesicle deformation. The role of filament polymer-



Biochemistry: Cortese et al. Proc. Natl. Acad. Sci. USA 86 (1989) 5777

ization and organization in driving vesicle deformation is further confirmed by the effects of gelsolin, which alters the length distribution of the actin filaments, and filamin, which crosslinks the filaments.

Long actin filaments not only spontaneously deformed vesicles but also assumed a dramatically heterogeneous distribution within the vesicles. Heterogeneity in actin gels has already been observed on much larger scales (4, 25), but our results demonstrate spontaneous domain formation at lower concentrations of actin and at sizes comparable to animal cells. A possible explanation of the observed anisot- ropy would be discrete nucleation sites for polymerization. Heterogeneous nucleation of actin polymerization at membrane defects or microphase separations could nucleate small groups of parallel filaments which could act as primers for the formation of a few larger filament bundles (4) or domains (25), perhaps preferentially oriented along the membrane (26), which could then deform a vesicle (27). Actin filaments formed in the presence of gelsolin would probably form numerous shorter and more randomly oriented bundles that would be insufficiently anisotropic to deform a vesicle. That the nonuniformity of actin density depends weakly on filament length whereas vesicle shape depends strongly indicates, however, that the relation between these effects is not simple. The observed heterogeneity of actin density could result from a thermodynamic collapse of the actin gel (28, 29). This occurs when the balance of forces driving gel expansion (i.e., gel osmotic pressure) and gel contraction (i.e., inter- polymeric attraction) shifts towards contraction. Then, the volume of the gel can be greatly reduced (and its density can be correspondingly increased in some areas, producing over- all a nonuniform distribution of actin filaments) by a small change in an external variable such as pH or ion concentration. Gel-collapse theory is consistent with the filamin results. Filamin, which crosslinks actin, might have been expected to increase vesicle deformation by increasing the mechanical strength (30) of the actin gel that deforms the vesicle. However, gel-collapse theory predicts that increases in the elastic modulus of the gel would resist collapse. Our results showed that inclusion of filamin gave a more isotropic actin distribution and less vesicle deformation than actin alone (Fig. 3d). Gel collapse would enable a small change in the conditions (ion concentration, polymer concentration and number, and polymer elasticity) to drive important filament rearrangements (31).

The abrupt effect of small changes in filament length on vesicle shape and heterogeneity of actin density suggests that corresponding changes in a cell could affect its shape and cytoskeletal organization. For example, if actin filament lengths in a cell were in an appropriate range, then the activation or inactivation of a small number of capping proteins (e.g., gelsolin) could change the filament lengths suffi- ciently to trigger a localized change in cell shape such as the extension of a protopod. Spontaneous reorganization of filaments could be stabilized by actin-crosslinking proteins such as filamin (30) or a actinin (32). It is likely, however, that additional processes such as changes in gel osmotic pressure and interactions with the membrane are important (19, 31, 33, 34). These could also depend critically on filament length (35).

Our results demonstrate that actin polymerization per se can drive biological membranes to change shape. We have shown that interactions among actin filaments that vary with filament length are sufficient both to cause the vesicles to change shape and to cause an inhomogeneous distribution of actin in the vesicles. Similar thermodynamic forces could influence dy- namic cytoskeletal processes in living cells. These model systems should also be quite useful for probing the effects of ion fluxes and actin-binding proteins on the assembly and organization of filament systems and the mechanical conse- quences of filament interactions on a cellular scale.

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actin polymerization induces a shape change in actin-containing vesicles

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