Mechanical Properties of Neuronal Growth Cone Membranes Studied by Tether Formation with Laser Optical Tweezers

Transcription

1 988 Biophysial Journal Volume 68 Marh Mehanial Properties of Neuronal Growth Cone Membranes Studied by Tether Formation with Laser Optial Tweezers Jianwu Dai and Mihael P. Sheetz Department of Cell Biology, Duke University Medial Center, Durham, North Carolina 2771 USA ABSTRACT Many ell phenomena involve major morphologial hanges, partiularly in mitosis and the proess of ell migration. For ells or neuronal growth ones to migrate, they must extend the leading edge of the plasma membrane as a lamellipodium or filopodium. During extension of filopodia, membrane must move aross the surfae reating shear and flow. Intraellular biohemial proesses driving extension must work against the membrane mehanial properties, but the fores required to extend growth ones have not been measured. In this paper, laseroptial tweezers and a nanometer-level analysis system were used to measure the neuronal growth one membrane mehanial properties through the extension of filopodia-like tethers with IgG-oated beads. Although the probability of a bead attahing to the membrane was onstant irrespetive of treatment; the probability of forming a tether with a onstant fore inreased dramatially with ytohalasin B or D and dimethylsulfoxide (DMSO). These are treatments that alter the organization of the atin ytoskeleton. The fore required to hold a tetherat zero veloity (Fa) was greaterthan fores generated by single moleular motors, kinesin and myosin; and Fa dereased with ytohalasin B or D and DMSO in orrelation with the hanges in the probability of tether formation. The fore of the tether on the bead inreased linearly with the veloity of tether elongation. From the dependeny of tetherfore on veloity of tether formation, we alulated a parameter related to membrane visosity, whih dereased with ytohalasin B or D, ATP depletion, noodazole, and DMSO. These results indiate that the atin ytoskeleton affets the membrane mehanial properties, inluding the fore required for membrane extension and the visoelasti behavior. INTRODUCTION Cell migration is an important omponent of metastasis, invasion, the immune response, and development. During migration, the plasma membrane is distorted by the mehanial fores of the motility proess (Vasiliev, 1985; Trinkaus, 1985). The role of the membrane is largely passive in responding to ytoskeletal deformations (Sheetz, 1993), although previously an ative role through a membrane flow had been postulated (Bretsher, 1989). In the ase of growth one migration, the axon must elongate, inreasing the plasma membrane area dramatially (Popov et ai., 1993). Even if the membrane is passive, the growth one must work against the load that membrane distortion produes. Consequently, the mehanial properties of the ell membranes ontribute to the ell deformability and movement. Understanding the mehanial properties of growth one membranes will help us better understand ell migration at a fundamental level. Several experimental methods have been used to investigate mehanial properties of ell membranes (Hohmuth et ai., 1973; Evans and Skalak, 1979; Pasternak and Elson, 1982; Bray et ai., 1986; Eo and Waugh, 1987). Using these methods, the mehanial properties ofliposomes, sea urhin eggs, erythroytes, and lymphoytes have been studied. Reeived for publiation 22 August /994 and in final form 28 November /994. Address reprint requests to Dr. Mihael P. Sheetz, Department of Cell Biology, P.O. Box 379, Duke University Medial Center, Durham, NC Tel.: ; Fax: ; mike_sheetz@ ellbio.duke.edu by the Biophysial Soiety /95/3/988/9 $2. However, they are appliable mainly to suspension ells and are inappliable to ells with omplex ell struture suh as neuronal ells. The interpretation of the membrane ontribution in many suh measurements is ompliated by the fat that the ytoskeleton is also deformed in a major way. To irumvent the diret ytoskeletal ontribution, membranous tethers laking a ontinuous ytoskeleton have been studied in erythroytes and pure lipid bilayers. From these studies, the stati and dynami omponents of the membrane mehanial properties were determined. The stati tension on tethers ontains ontributions from the in-plane tension (Hohmuth and Evans, 1982a; Waugh, 1982a) and the bending stiffness of the bilayer, whih is highly urved in the tethers (Waugh and Hohmuth, 1987). When tethers are elongated, a visous fore is introdued that ontains ontributions from the membrane visosity and the interbilayer shear as the lipids flow onto the tether. The fluid nature of suh tethers indiates that they are largely membranous, and the absene of spetrin or atin in erythroyte tethers has shown that even the membrane skeleton is depleted (Berk and Hohmuth, 1992). Nevertheless, ytoskeleton an influene the formation of tethers as evidened by the differenes between erythroytes and pure lipid vesiles. Perhaps even the tether mehanial properties would be modified by alterations of the ytoskeleton. In motile ells and partiularly in the forward portion of migrating ells, atin filament assembly and disassembly is intimately tied to motility (reviewed in Sheetz, 1994; Zigmond, 1993; Cooper, 1991). Further, atin and atinbinding proteins suh as spetrin are losely assoiated with plasma membranes in virtually all ells, inluding neurons (Bennett, 199). The support of plasma membranes by the

2 Dai and Sheetz Tether Formation of Growth Cones by Laser Tweezers 989 membrane skeleton is extensive in the erythroyte but in other ells glyoproteins an diffuse over miron distanes before enountering barriers to lateral diffusion (de Brabander et ai., 1991; Edidin et ai., 1991). The barriers to lateral diffusion have not been identified, but when glyoproteins beome anhored to the ytoskeleton, they are generally anhored to the atin filaments. Atin is onsequently losely apposed to the plasma membrane and may have an important role in determining the membrane properties. Beause tether formation involves the separation of membrane from mostofthe membrane skeleton, there should be dramati effets of the atin ytoskeleton on the probability oftether formation. We will therefore probe the effet of atin depolymerization on tether formation and tether mehanial properties. Previous work (Ashkin and Dziedzi, 1989: Wayne et ai., 1991) found that the laser trap ould produe suffiient fore to pull membranous tethers from ell surfaes. Optial tweezers allow exquisitely fine ontrol of position (-1 nm for trap beam stability) and of fores (-.1 pn resolution) on a wide range of partile sizes (25 nm to 25 f-lm) in a noninvasive manner (Svoboda and Blok, 1994: Kuo and Sheetz, 1992). The laser tweezers were developed for the mirosopi manipulation of ells and organelles with a minimum ofdamage (Ashkin, 197; Ashkin and Dziedzi, 1987, 1989; Ashkin et ai., 1987). They have been used for a variety of appliations, inluding the measurement of membrane barriers (Edidin et ai., 1991, 1994), the fore of single motor moleules (Kuo and Sheetz, 1993; Svoboda et ai., 1994; Finer et ai., 1994), and regional speializations in ell membranes (Kuik et ai., 1991; Shmidt et ai., 1994). We now extend those studies to determine the membrane mehanial properties from the fores applied to the beads for different veloities oftether elongation. With nanometerlevel motion analysis, the instantaneous fore on the tether an be measured. In addition, we an evaluate the relative strength ofthe membrane-skeleton interation from the probability of tether formation at a given fore on the bead. We measured the mehanial properties after treatment by the ytohalasins, DMSO, ethanol, noodazole, and ATP depletion. These treatments altered the membrane-ytoskeleton interation and affeted the membrane mehanial properties. The method we desribed here for determining membrane mehanial properties with the laser tweezers an be generally applied under a variety of different onditions. MATERIALS AND METHODS Cell ulture Chik dorsal root ganglion (DRG) explants were disseted from 12-day hik embryos and plated in the growth wells on treated overslips. To prepare the ell growth wells, 2 x 2 mm (No.1) glass overslips (BDL, Linoln park, NJ) and 1o-mm-diameterloning ylinders (Bello, Vineland, NJ) were leaned by soaking in 2% nitri aid for 2-3 h, followed by rinsing in distilled water for 1 h. Then they were put into 95% ethanol overnight and rinsed in distilled water for at least 2 h. After drying and sterilization, a loning ylinder was seured to the overslip with sterilized silione grease to fonn a growth well. The growth well was oated with.1 % poly-l-lysine for 15 min at room temperature, rinsed 3 times with sterilized water, then dried in a sterile hood. Just before the dissetion, poly-l-lysine-oatedgrowth wells were exposed to a 1:5 Matrigel (Collaborate Researh, Bedford, MA):MEM solution. Explants were maintained at 37 C, 5% CO 2 in lear MEM (Gibo BRL, Grand Island, NY) supplemented with the following: 2 mm HEPES, 6 mg/ml gluose, 5 /LVml pen./strep., 2 mm L-glutamine, 1 /LVml N2 Serum-free supplement (Gibo), and 1 nm/ml nerve growth fator (NGF 2.5s; Gibo). The explants were used after they were inubated for h. Bead preparation In previous studies, we observed that ovaspheres oated with a ontrol IgG preparation would bind to growth one membranes without any apparent perturbation ofgrowth one behavior. To prepare IgG-oated beads, rat IgG (Sigma Chemial Co., St. Louis, MO) was solubilized at a onentration of 1 mg/ml in PBS. Then, 5 /LI ofovaspheres (.5 /Lm, Duke Sientifi, Palo Alto, CA) was added to 5 /LI of the above IgG solution and was inubated at 4 C overnight. The beads were pelleted by entrifugation at 2 x g and 4 C for 1 min. Then the beads were resuspended in 1 mg/ml BSA-PBS solution, rinsed by pelleting and resuspension with MEM medium 3 times and resuspended in 1 /Ll of MEM medium. For the experiments, the bead solution was diluted 3:1 in DRG medium. Calibration of the laser trap The laser optial trap was alibrated by visous drag through the aqueous medium in the mirosope foal plane (Kuo and Sheetz, 1993). The visous fore was generated by osillatory motion of the speimen by a piezoerami-driven stage (Wye Creek Instruments, Federik, MD) at a onstant veloity. The position of the bead in the trap was traked using the nanometer-level traking program (Gelles et ai., 1988) to analyze video reords of the experiments. Positional variation of the partile in the trap with 6 mw was 11 (:!::1.7) nm. The alibration shows a very linear foredistane relationshipfor the optial tweezers (Fig. 1). Tostudy the variation in trap alibration with height above the overslip surfae, latex beads (.5 /Lm in diameter) were trapped with the same laser power at different perpendiular positions. The inrease in partile displaement at 2 /Lm or less from the glass surfae (Fig. 1 B) implies a visous oupling to the overslip surfae. From 2-5 /Lm above the surfae, the fore on the beads in the trap was onstant (Fig. 1 B). This alibration was used to alulate the fores that fonn tethers. All of these experiments were perfonned 3-4 /Lm above the overslip surfae to minimize visous oupling to the glass surfae, and the laser power was simultaneously monitored. Laser optial trap manipulations Just before observation, the loning ylinder was removed and the overslip ontaining the ells was mounted on an aluminum overslip holder using silione grease; then a seond leaned overslip was mounted on top to fonn a flow ell. The IgG-overed latex beads and the treatment solutions suh as ytohalasins and DMSO were exhanged for the nonnal medium. The growth ones were viewed by a video-enhaned differential interferene ontrast (DIC) mirosope (IM-35 mirosope; Zeiss, Oberkohen, Germany) with a fiber opti illuminator. The stage was maintained at 38 C using an air urrent inubator. The laser trap onsisted of a polarized beam from an llw TEMOO-mode near-infrared (164 om) laser (model C-95; CVI Corporation, Albuquerque, NM) that was expanded by a 3 X beam expander (CVI Corporation) then foused through an 8-mm-foal-length ahromati lens (Melles Griot, Irvine, CA) into the epifluoresene port of the Zeiss IM-35 mirosope (Kuo and Sheetz, 1992). In the binding experiments, the IgG-overed beads were added by exhange ofthe ell ulture medium. The bead was trapped with -6 mw of laser power, put on to the ell surfae and held for 4 s before pulling at a onstant veloity. To detennine the probability of bound beads fonning tethers, the beads were held with the laser trap on the ell surfae for 4 s and were pulled out at a veloity of-3 /Lrn/s. We detennined that the perentage oftether fonnation was not altered

3 99 A so.oo -= o.oojt4!lt ljt.jl o FRAME B 12. e- a: 11. l! l- S 1..5 ao.oo ) ao.oo = os 7.. Biophysial Journal Ba8d position above lbe ailde aurlae ()lm) Volume 68 Marh rb-rhrh.,-f--' :I ? r (nm) FIGURE 1 Sequene ofmeasurements to alibrate the radial fore ofthe trap. (A) The radial position ofthe bead (r) in the trap during a series ofalibration displaements. The bead was held with the laser trap -4 /lorn above the overslip surfae, and the piezoerami-driven stage was moved repeatedly at a onstant rate. (B) Calibration of the laser trap at different heights above the glass surfae. The displaement proedure in A was repeated for latex beads (.5 /lorn in diameter) held at different heights above the overslip, at onstant power. Six beads were tested at eah height. The results show that experiments need to be done at least 2 /lorn above the overslip surfae to minimize visous oupling to the glass surfae. (C) Calibration of the laser trap for partiles held -3-4/lom above the overslip surfae by varying the laser power for a onstant veloity of movement. The result is shown as the relationship between the position of the bead in the trap (r) and the normalized light fore F N in pn mw-\ as alulated from Stokes Law and the power of the trap, whih was monitored simultaneously. The slope of the linear fit line is (pn /low-i /lorn-i). by exposing growth ones to even 1 s of laser irradiation. Beads either remained at the ell surfae or a tether was formed. All ofthe samples treated or untreated were manipulated under the same onditions. Analysis of the mehanial properties of the membranes To measure the fore of the tether at zero veloity, F o, the position of the bead in the trap during tether formation was traked using the nanometersale traking program that was developed previously (Gelles, 1988), and the fore (F) of the tether on the bead was alulated from the alibration of the laser trap. After measuring the tether growth rate (V) using a "ruler" program, a plot F vs. V was obtained. Two methods were used to estimate the radius of the tether. First, latex beads.5 /lorn in diameter were used as a standard in the "ruler" program. From the video tape, we measured the relative diameter of the tether and then ompared with the bead. The other way was to determine the relative intensity through orthogonal sans aross the DIC image ofboth the tether and the axon (Shnapp et ai., 1988). After measuring the diameter of the axon with the "ruler" program, we an estimate the radius of the tether (the intensity should be related to the radius squared). The radius of the tether was alulated to be about.2 /lorn in both ases. With inreasing veloity, there was no detetable hange in tether ontrast. To alulate the membrane surfae visosity, we developed a program using the fourth-order Runge Kulla routine to solve the equation (Eq. 21) in Dr. Waugh's paper (Waugh, 1982a) and ompared that value with the formulation desribed by Hohmuth and olleagues (Hohmuth et ai., 1982a). RESULTS Probability of bead binding and tether formation When the IgG-oated beads were held on the DRG growth one surfae with the optial tweezers for 4 s, only a fration bound to the membrane. Those that did bind to the surfae did so irreversibly and would not release under the onditions tested. Pulling on beads with the optial tweezers either produed tubular membrane tethers or the beads were pulled out of the tweezers. To understand the ellular fators that influene binding and tether formation, we measured the effet of the ytohalasins, noodazole, ATP depletion (Sheetz et ai., 199), and organi solvents (dimethylsulfoxide (DMSO) and ethanol) on the probability ofbinding and tether formation. As shown in Fig. 2, there was no hange in the probability ofbead binding with any ofthe hanges in the ellular onditions. We expeted that the probability oftether formation would be related to the ytoskeleton-membrane interation and that 1+-r-+-., h-f ,...+-r-+-.,...,.. al 8 S II 4 ] 2 ' i' i' :z: :z: 1Il ::I. ::I. i ' f :Ii 1Il :Ii 'E a. " iii... u () III III Q.... >. >. I- z () () ( FIGURE 2 Bead binding to the surfae is measured as the probability of IgG overed latex beads binding to the hiken DRG growth one surfae after 4 s using a laser trap of-6 mw (laser power measured in the speimen plane) for a variety of experimental onditions. For the DMSO and ethanol (EtOH) treatment, DMSO or EtOH was added to the MEM medium, and the final onentration for DMSO or EtOH is.5 and 1%, respetively. Cytohalasin B and D were diluted into the MEM medium from DMSO stok solutions to give final onentrations for both Cytohalasin Band D of 1 /lom and for DMSO of -.3%. The noodazole solution was prepared by diluting a 1 mm noodazole stok solution in DMSO to give a 1 /log/ml solution. For ATP depletion, a solution of 2-deoxygluose (3 mm) and sodium azide (1 mm) in the ell medium was used, and the growth ones assayed showed no motility. All of these treatment solutions were added to the ells by exhange flow, and the ells were inubated for 1 min before starting the measurements. For eah experiment, 7 beads were studied.

4 Dai and Sheetz Tether Formation of Growth Cones by Laser Tweezers 991 the probability of tether formation would inrease with the fore of the tweezers (as was observed). On a related issue, those partiles that did not form tethers normally did not diffuse after esaping from the trap, indiating that they were attahed to the ytoskeleton. Beause most fators were likely to derease the affinity of the two strutures, we hose a relatively low fore (-1 pn) where only 36% ofthe beads would form tethers (Fig. 3). Cytohalasin Band D are known to inhibit atin dynamis within growth ones and ells, resulting in a ondensation ofthe atin network and separation of the atin ytoskeleton from muh of the membrane. Both ytohalasins inreased the probability oftether formation to greater than 9%. Controls of the solvent (DMSO) used for adding the ytohalasins (.25% solvent) also showed some inrease, but.5% DMSO only inreased the probability of tether formation to 55% (Fig. 3). Both the disruption of mirotubules by noodazole and the blok of motility by ATP depletion aused no hange in tether formation. Thus, hanges in the organization of the atin ytoskeleton, but not mirotubules or ATP levels, produed an inrease in the ability to form tethers. Stati tether fore (F o ) After tethers are formed on DRG growth ones, they rapidly retrat when the tweezers are turned off, indiating that a signifiant fore is pulling the tether membrane bak onto the growth one. Photographs of the tether-pulling sequene are shown in Fig. 4, inluding bead binding to the ell surfae in the trap, pulling the tether at a onstant rate with the laser trap, and the omplete retration of the tether when the tweezers were turned off. We an utilize the measured displaement of the bead in the trap to estimate the fore of the tether on the bead (Fig. 5 A). The tether fore inreases only slightly with the tether length in other systems where it has been! 1 8 go! 6.t j 4 1.It:: Q 2! '.,. :t:.!! i 1:1 C W Q Q N.,..,..,. Q... II) i" i" :t: en ::1 ::1 i s en W S D. CI) iii' C U.,. II) liu u u >. >.... z FIGURE 3 The probability of tether formation for the beads attahed to the ell surfae. The beads were pulled using the laser trap (6 mw) and at a onstant veloity (3 p.m/s). The treatments are the same as desribed in Fig. 2. For eah experiment, 5 beads were studied. FIGURE 4 Photomirographs of the tether-pulling sequene. (I) Bead was held on the growth one surfae by the laser trap. (2 and 3) The tether was pulled at a onstant veloity by the laser trap. (4) The omplete retration of the tether when the laser trap was turned off. tested (D. B. Wayne, S. C. Kuo, and M. P. Sheetz, unpublished data). The average tether length in the experiments was around 15 /-Lm. Rather we have measured the hange in the tether fore with the veloity of tether elongation and extrapolated to zero veloity to obtain a ritial fore, F o, whih must be exeeded for tether growth. Where we tested it, the F o estimated from extrapolation to zero veloity was within experimental error ofthe value obtained in stati measurements of the tether fore. Fig. 5 A shows the distane between the enter ofthe bead and the enter of the laser trap after the initial extension and then during a onstant veloity elongation. In about 1 perent of the ases, the fore peaked at the start of the elongation and then returned to a plateau level. For these studies we estimated the average plateau level for the fore measurement. After plotting the fore (F) forming the tether vs. the tether growth rate (Y), we performed a linear leastsquares fit and extrapolated to V = to obtain F o (Fig. 5 B). The measurements for Fig. 5 were all made with different tethers that were formed on many different growth ones. For the ontrol sample, we also measured the displaement of the beads at zero veloity and obtained an average value of this fore of5. (1.5)pN, whih is in agreement with the estimation of F o from the F vs. V plot of (N = 3) pn.

5 992 Biophysial Journal Volume 68 Marh 1995 A h r f---,-f I _ 6. Z S; II. 4. B o....j!.j:...l:j-':4..l...jl:l+.:.ilil!-,-.j...j...-t-'-...l..l-+-l-1-",+ o FRAME z e Ii. 1. B ,1.4.. Q i. II> ' e!!!. 'U C N iii' i5" tn tn :z: A- ().;.; tn 2 u >. >. Q Q W Z () () Q '".! 5.. -t-'-'-j-l.-j--l...l..l-'-!-,-..l...i...l...j-'-i...j...-'-t...l...i-'-",+..l...i...l...i-+ o V Cum/se) FIGURE 5 (A) The position of the bead in the trap (r) with time during the tether formation. After the bead was trapped, it was fust held on the ell surfae for 5 s and then pulled out at a onstant veloity, forming a tether. This tether length is -14 M-m. (B) Determination of the fore (F o ) at zero veloity of tether growth from plotting F vs. tether growth rate (V). F o = 6.72 pn was obtained at V = from a linear least-squares fit of the data points (slope of the plot = 1.51). Beause F oshould reflet omponents of the in-plane tension of the plasma membrane and membrane bending rigidity, we expeted that hanges in the ellular organization should alter F o. When the growth ones were treated by ytohalasin Band D, ethanol, and DMSO, F o went from 6.7 pn down to -3 pn in the presene of DMSO and to -4 pn in the presene of ytohalasins and ethanol (Fig. 6 A). However, F o was not affeted by noodazole or ATP depletion. These results are similar to the probability oftether formation and suggest that the ytoskeleton plays a major role in determining the fore needed to extend filopodia and tethers. Visous fores in tethers As the veloity of tether elongation was inreased, there was a linear inrease in the fore of the tether on the bead from the visous omponents of membrane movement into the tether. There are several different analyses of the visous omponents that ontribute to the overall visous fore. Contributions ould ome from urving the membrane and the resulting interbilayer shear, from the bilayer visosity, and from the drag of moving membrane past the assoiated yto i 1. > ' ;:.8 II.I.6 J 'Ii.4 I.2!. i' i' :I. :I. II> i. ' f...!!!. e 'U.. Q - C N ld Q :z: A- tn tn ().;.; 11) 2 u >. >. Q Q W Z () () Q '" FIGURE 6 (A) The ritial fore (F o )ofthe growth ones before and after various treatments. The treatment protools are desribed in Fig. 2. (B) The slope derived from a linear least-squares fit of the tether growth rate vs. the fore of tether formation. The oeffiient of the orrelation in eah experiment in Fig. 6 is given below: (from ontrol to noozadole).84,.81,.86,.82,.71,.89,.76,.66, and.6, respetively. skeleton. Waugh's equation (Waugh, 1982a) allows us to estimate an apparent membrane visosity negleting ontributions of the visous shear between the ell membrane and ytoskeleton and the slip between halves of the bilayer (Fig. 7). Alternate methods ofalulating the membrane visosity inlude other visous terms and, therefore, this value represents an upper limit of the membrane visosity. There were surprisingly large dereases in the slope with ytohalasin Band D, DMSO, and ATP depletion. The pattern of the visosity hanges with these treatments was not the same as hanges in F o. The greatest differene was with ATP depletion. Compared with the ontrol sample, F o is a little bit bigger but the slope is about 3 times smaller after the ATP depletion (Fig. 6 B). Thus, the visous properties of

6 Tether Formation of Growth Cones by Laser Tweezers Dai and Sheetz i!.: ,.-h t--.--t-rl-,--t r-if---, :.2 o :;:...E :I C ;. :!i:!i :t. e., So f/) ::I f/) ::I.. '".. aa So () ()!!!. :I: I '" '" '" Ii....'" Ii. FIGURE 7 The apparent membrane surfae visosity of the growth ones before and after the treatments. The visosity was estimated following Eq. 21 in Waugh (1982). the growth one membranes are dramatially altered by metaboli and ytoskeletal hanges. DISCUSSION The optial tweezers have been proven useful for a number of studies of motor funtion and a wide variety of biologial experiments (Kuo and Sheetz, 1992, 1993; Svoboda and Blok, 1994; Finer et ai., 1994). Here we show that they an be used to provide a relative measure of membraneytoskeleton attahment and the mehanial properties of membranes. The limitation of the tweezers is photodamage, and we have been onerned that photodamage may influene the values obtained. From our experiene with DRG growth ones, there is no observable hange in growth one behavior at these trap levels, whereas at higher irradiation levels (>2 mw) we do observe loss of filopodia and a general inhibition of motility. An additional problem is the potential for forming tethers without beads, but suh tethers are notably different in that they ontain a bulb of ytoplasm at the end (Ashkin and Dziedzi, 1989). Further, without beads we were unable to form tethers at the laser powers used. The trap ould exert some fore diretly on the tether, but it is onsiderably smaller in diameter (-2 nm vs. 5 nm for the bead) and further separated from the trap enter, whih makes it unlikely that the diret fore on the tether is more than 2% of that on the bead. For omparing the relative fores under different ellular onditions, the optial tweezers provides self-onsistent measurements, and the measured fores represent a lower bound for the atual values. Membrane-ytoskeleton attahment The exat nature of the linkages between membranes and the ytoskeleton are still ambiguous, although two general types of linkage have been onsidered. In one ase, attahments would arise from transmembrane glyoproteins that are ultimately linked to atin-binding proteins. Alternatively, y- 993 toskeletally linked proteins suh as spetrin ould weakly assoiate with the bilayer surfae and, beause suh proteins are in an extended onfiguration and part of larger omplexes, the sum of the many weak interations would form a strong linkage. In marosopi terms, the differene here is similar to that between a soap membrane overing but only tied at a few disreet points to a rigid strutural net and a soap film spread aross that same net where it is adsorbed to elements of the net. In the first instane, the disruption of the strutural net (most likely atin-based) would make it easier to separate the membrane from the net; but the fore, F o' needed to hold the membrane off the net should not hange. In the seond ase, alteration of the net should affet both the ease of separation of the membrane from the net; and beause the membrane-net assoiation is reversible, it should also affet the fore, F o, needed to hold the two strutures apart. We find that both the perent tether formation and F o are altered in parallel, whih is most onsistent with the latter hypothesis, namely, that the attahments that prevent tether formation are reversible and ontribute to the tension in the tether or F o. There are several other reasons to believe that the membrane is not extensively tied to the ytoskeleton by strong ontats. Reent estimates of the mehanial fore required to break apart a protein-protein interation with a 1-5 assoiation onstant are large, in the range of 1 pn (Kuo and Lauffenburger, 1993). Breaking of suh interations would result in a dramati derease in the fore on the bead. Although jumps are seen oasionally at the start of tether formation, they are small in magnitude, 6-1 pn, and are only seen in -1% of tethers formed. From many previous studies of the diffusion of partiles on the surfae of growth ones and motile ells, barriers to lateral diffusion are spaed over miron distanes (de Brabander et ai., 1991; Edidin et ai., 1991). We might expet a similar separation between the strutural links that would hold the membrane and ytoskeleton together. Thus, with.5 miron beads, bound beads should be able to fit within the gaps in the network and tethers ould be formed by pulling on those beads without breaking strong membrane-ytoskeletal linkages. Previous studies have indiated that the probability of tether formation is inversely related to the membraneytoskeleton interation (Shmidt et ai., 1993). We know that both ytohalasins B or D an affet the network of atin filaments in the ell, although there is some small differene between them (Edds, 1993). DMSO an also affet the arrangement of ytoskeleton (Yumura and Fukui, 1983; Weiner et ai., 1993). It has been reported that ethanol an influene ell migration and invasion in vitro as well as F-atin organization, and it an affet the membrane onformation and struture (Weiss et ai., 1991; Mooradian and Smith, 1993; Staler et ai., 1993; Ho et ai., 1994). Therefore, a possible explanation for the above result is that the ytohalasins and DMSO will derease the membraneytoskeleton interation through rearrangements of the atin ytoskeleton and thereby inrease the probability of tether formation for a given fore. On the other hand, DMSO, like

7 994 Biophysial Journal Volume 68 Marh 1995 ethanol, might indiretly affet membrane mehanis through an alteration in the interfaial interation energy between the membrane and an otherwise normal atin ytoskeleton. The mirotubule (M])organization in ells appears to have no affet on the membrane-ytoskeleton interation (Wang et ai., 1993) as does ATP depletion. In other growth ones, ATP depletion inhibits the partile movement on the membrane (Sheetz et ai., 199). General alteration in the organization ofthe atin ytoskeleton is linked to parallel hanges in the probability of tether formation and F o. We suggest that the membrane interation with the ytoskeleton is through reversible, weak bonds and not through strong protein-protein bonds. F o ofgrowth one membranes (-6.7 pn) is muh less than that of erythroyte membranes, whih is about 5 pn (Waugh and Bauserman, 1995, in press). The larger membranetensionofthepreswollenred ellsmightontributethe larger F o. There is an extensive interation between the red ell spetrin-atin network and the membrane bilayer that possibly aounts for the major portion of the fore required for tether extension. By impliation, a similar network may be assoiated with the growth one plasma membrane. In omparison with the fores that myosin motors or atin polymerization an develop, the fore for extension is omparable with two myosin moleules or one atin filament, indiating that either mehanism ould generate suffiient fore to produe extension (Sheetz, 1994). In addition to binding fore between membrane and ytoskeleton, other fators that ontribute to the fore required for tether formation inlude the membrane bending stiffness and the inplane membrane tension (Hohmuth, personal ommuniation). The bending stiffness of the membrane plays an important role in proesses beause of the major hanges in membrane urvature. To estimate the membrane bending stiffness, an equation developed by Waugh and Hohmuth (1987) an be used. In this ase, if we assume that the fore (F) forming the tether is 8 pn, the radius (rj of the tether is.2 p.m, then the bending stiffness (B) = F X r127f' = 2.55 X 1-12 dyne m. The growth one membrane bending stiffness is similar to that of the lipid bilayer membranes X 1-12 dyne m. (BO and Waugh, 1989) and erythroyte membranes (Evans, 1983). Beause the bending stiffness of the bilayer should not hange with ytohalasin B or D, we suggest that the reversible membrane ytoskeleton assoiations or possibly in-plane tension is dereased by these treatments. Membrane surfae visosity The visoelasti properties ofthe membrane ould ontribute to the ontrol of growth and extension rates, but these data indiate that visous fores are too small at typial extension veloities (maximally p.rn/s) to ause a signifiant effet. Although there are potentially several fators ontributing to the visoelasti fore, we have used Eq. 21 in Waugh's paper (Waugh, 1982a) to estimate the membrane visosity. Beause the growth one is only a very small part of the whole neuron ell, the ratio of the radius of the tether (rj(-.2 p.m) to the radius of the ell (re> or (rire) is very small «1/5). Following Waugh, the value of FIFo an be used diretly to alulate membrane surfae visosity (Waugh, 1982a), whih is about 2.1 X 1-4 dyn s/m (Fig. 7). In these studies, the apparent visosity ranged from.56 X 1-4 to 4.2 X 1-4 dyn s/m. This value is muh smaller than that Hohmuth obtained for erythroyte membranes (2.8 X 1-3 dyn s/m) and is greater, perhaps very signifiantly, than that reported for egg phosphatidylholine large bilayer vesile membranes (-1.7 X X 1-6 dyn s/m) (Hohmuth et ai., 1982b; Waugh, 1982b). Whih of the fators, inluding the slippage of membrane layers in moving to a highly urved surfae, the planar membrane visosity, and glyoprotein ollisions with the ytoskeleton, makes the major ontribution to the visosity during extension flow is unlear. Evans suggested that the slope ofthe linearfit ofthe tethergrowth rate (V) vs. the fore (F) forming tethers should be related to the frition in the slipping of the bilayers past one another as the bilayer moves from the flat membrane surfae to the highly urved tether (E. A. Evans, Personal ommuniations). Beause we see a major effet ofthe ytohalasins on visosity and they should have little effet on the interfae between the two bilayer halves, we onsider that this fator makes only a small ontribution to the ontrol of membrane visosity. There is a large differene between the visosity of the neuronal membrane and the liposome or red ell membranes using the same formula. Current models of membrane visosity are being hallenged beause they lak terms to onsider the slippage between the two bilayer halves and ytoskeletal ontributions; thus, bettermodelsofthis system are needed to provide testable hypotheses. It is somewhat surprising to find suh large hanges in the visosity with alteration of ell ytoskeleton and partiularly with ATP depletion. Axon elongation and tether formation An interesting question in axon elongation is: where does the axon membrane ome from? In these studies, we are forming tethers at a very rapid rate and lipid is flowing onto the tether at 1-1 times the rate that membrane moves into axons (typial axon elongation rates are 1-4 p.rn/min, and diameters are typially 1 miron, whereas we are pulling tethers of.4 miron at rates of6-6 p.rn/min). Beause the fores needed to produe suh rapid extensions are onsiderably less than those developed by growth ones moving on normal substrata, it should be no problem for growth ones to pull membrane from the ell body and no need to invoke addition at the growth one (Bray and Chapman, 1985; Popov et ai., 1993). In the studies oferythroyte membranes and liposome membranes, the membranes forming tethers were all from the surfae membranes, but in ative growth ones, some of the membrane might ome from the ell body or from internal membrane vesiles. We onsider this last possibility less likely beause normally endoytosis and exoytosis are balaned and we are pulling at suh a high rate that we would

8 Dai and Sheetz Tether Formation of Growth Cones by Laser Tweezers 995 not expet aute adaptation by membrane fusion. Rather, there appears to be a reservoir of membrane that the tethers draw upon either through alteration of the growth one geometry or from strething the membrane. There are a number of biologial proesses for whih tether data are relevant, inluding ell migration, ell volume, and membrane area regulation. The probability oftether formation relates to the ability ofthe ells to migrate beause the release of ell ontats in the rear of the ell often involves formation of retration fibers, i.e., tethers (Shmidt et ai., 1993). How the ell regulates its surfae area and the related parameter of ell volume may well involve an inplane membrane tension that would regulate endoyti versus exoyti rates. Clearly, ytoskeletal fators do affet the physial parameters of tether formation, implying that they are important for ellular funtions. SUMMARY Growth one migration is aompanied by dramati morphologial hanges and an extension of the axonal membrane. From our analyses of the mehanial properties of the membranes, we find that the fores generated by the membrane are on the order ofthose generated by a few myosin motors or atin polymerization or from the breaking of weak protein-protein interations. Beause membraneytoskeleton interations influene the probability of tether formation and the ritial fore required to extend a membrane tether, we suggest that the interations between the membrane and the skeleton are weak and reversible, although pointwise strong interations annot be exluded. The visous properties of the tethers show a strong dependene on solvents and ATP depletion that are not understood. Thus, we find that the laser tweezers an be used to assay the strength of the interation between the ytoskeleton and the membrane bilayer that in tum an influene ell migratory behavior. We thank Drs. 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