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

Transcription

1 988 Biophysicl Journl Volume 68 Mrch Mechnicl Properties of Neuronl Growth Cone Membrnes Studied by Tether Formtion with Lser Opticl Tweezers Jinwu Di nd Michel P. Sheetz Deprtment of Cell Biology, Duke University Medicl Center, Durhm, North Crolin 2771 USA ABSTRACT Mny cell phenomen involve mjor morphologicl chnges, prticulrly in mitosis nd the process of cell migrtion. For cells or neuronl growth cones to migrte, they must extend the leding edge of the plsm membrne s lmellipodium or filopodium. During extension of filopodi, membrne must move cross the surfce creting sher nd flow. ntrcellulr biochemicl processes driving extension must work ginst the membrne mechnicl properties, but the forces required to extend growth cones hve not been mesured. n this pper, lser opticl tweezers nd nnometer-level nlysis system were used to mesure the neuronl growth cone membrne mechnicl properties through the extension of filopodi-like tethers with gg-coted beds. Although the probbility of bed ttching to the membrne ws constnt irrespective of tretment; the probbility of forming tether with constnt force incresed drmticlly with cytochlsin B or D nd dimethylsulfoxide (DMSO). These re tretments tht lter the orgniztion of the ctin cytoskeleton. The force required to hold tether t zero velocity (FO) ws greter thn forces generted by single moleculr motors, kinesin nd myosin; nd Fo decresed with cytochlsin B or D nd DMSO in correltion with the chnges in the probbility of tether formtion. The force of the tether on the bed incresed linerly with the velocity of tether elongtion. From the dependency of tether force on velocity of tether formtion, we clculted prmeter relted to membrne viscosity, which decresed with cytochlsin B or D, ATP depletion, nocodzole, nd DMSO. These results indicte tht the ctin cytoskeleton ffects the membrne mechnicl properties, including the force required for membrne extension nd the viscoelstic behvior. NTRODUCTON Cell migrtion is n importnt component of metstsis, invsion, the immune response, nd development. During migrtion, the plsm membrne is distorted by the mechnicl forces of the motility process (Vsiliev, 1985; Trinkus, 1985). The role of the membrne is lrgely pssive in responding to cytoskeletl deformtions (Sheetz, 1993), lthough previously n ctive role through membrne flow hd been postulted (Bretscher, 1989). n the cse of growth cone migrtion, the xon must elongte, incresing the plsm membrne re drmticlly (Popov et l., 1993). Even if the membrne is pssive, the growth cone must work ginst the lod tht membrne distortion produces. Consequently, the mechnicl properties of the cell membrnes contribute to the cell deformbility nd movement. Understnding the mechnicl properties of growth cone membrnes will help us better understnd cell migrtion t fundmentl level. Severl experimentl methods hve been used to investigte mechnicl properties of cell membrnes (Hochmuth et l., 1973; Evns nd Sklk, 1979; Psternk nd Elson, 1982; Bry et l., 1986; Bo nd Wugh, 1987). Using these methods, the mechnicl properties of liposomes, se urchin eggs, erythrocytes, nd lymphocytes hve been studied. Received for publiction 22 August 1994 nd in finl form 28 November Address reprint requests to Dr. Michel P. Sheetz, Deprtment of Cell Biology, P.O. Box 379, Duke University Medicl Center, Durhm, NC Tel.: ; Fx: ; E-mil: mike_sheetz@ cellbio.duke.edu. C 1995 by the Biophysicl Society /95/3/988/9 $2. However, they re pplicble minly to suspension cells nd re inpplicble to cells with complex cell structure such s neuronl cells. The interprettion of the membrne contribution in mny such mesurements is complicted by the fct tht the cytoskeleton is lso deformed in mjor wy. To circumvent the direct cytoskeletl contribution, membrnous tethers lcking continuous cytoskeleton hve been studied in erythrocytes nd pure lipid bilyers. From these studies, the sttic nd dynmic components of the membrne mechnicl properties were determined. The sttic tension on tethers contins contributions from the in-plne tension (Hochmuth nd Evns, 1982; Wugh, 1982) nd the bending stiffness of the bilyer, which is highly curved in the tethers (Wugh nd Hochmuth, 1987). When tethers re elongted, viscous force is introduced tht contins contributions from the membrne viscosity nd the interbilyer sher s the lipids flow onto the tether. The fluid nture of such tethers indictes tht they re lrgely membrnous, nd the bsence of spectrin or ctin in erythrocyte tethers hs shown tht even the membrne skeleton is depleted (Berk nd Hochmuth, 1992). Nevertheless, cytoskeleton cn influence the formtion of tethers s evidenced by the differences between erythrocytes nd pure lipid vesicles. Perhps even the tether mechnicl properties would be modified by ltertions of the cytoskeleton. n motile cells nd prticulrly in the forwrd portion of migrting cells, ctin filment ssembly nd disssembly is intimtely tied to motility (reviewed in Sheetz, 1994; Zigmond, 1993; Cooper, 1991). Further, ctin nd ctinbinding proteins such s spectrin re closely ssocited with plsm membrnes in virtully ll cells, including neurons (Bennett, 199). The support of plsm membrnes by the

2 Di nd Sheetz Tether Formtion of Growth Cones by Lser Tweezers 989 membrne skeleton is extensive in the erythrocyte but in other cells glycoproteins cn diffuse over micron distnces before encountering brriers to lterl diffusion (de Brbnder et l., 1991; Edidin et l., 1991). The brriers to lterl diffusion hve not been identified, but when glycoproteins become nchored to the cytoskeleton, they re generlly nchored to the ctin filments. Actin is consequently closely pposed to the plsm membrne nd my hve n importnt role in determining the membrne properties. Becuse tether formtion involves the seprtion of membrne from most of the membrne skeleton, there should be drmtic effects of the ctin cytoskeleton on the probbility of tether formtion. We will therefore probe the effect of ctin depolymeriztion on tether formtion nd tether mechnicl properties. Previous work (Ashkin nd Dziedzic, 1989: Wyne et l., 1991) found tht the lser trp could produce sufficient force to pull membrnous tethers from cell surfces. Opticl tweezers llow exquisitely fine control of position (-1 nm for trp bem stbility) nd of forces (-.1 pn resolution) on wide rnge of prticle sizes (25 nm to 25,um) in noninvsive mnner (Svobod nd Block, 1994: Kuo nd Sheetz, 1992). The lser tweezers were developed for the microscopic mnipultion of cells nd orgnelles with minimum of dmge (Ashkin, 197; Ashkin nd Dziedzic, 1987, 1989; Ashkin et l., 1987). They hve been used for vriety of pplictions, including the mesurement of membrne brriers (Edidin et l., 1991, 1994), the force of single motor molecules (Kuo nd Sheetz, 1993; Svobod et l., 1994; Finer et l., 1994), nd regionl speciliztions in cell membrnes (Kucik et l., 1991; Schmidt et l., 1994). We now extend those studies to determine the membrne mechnicl properties from the forces pplied to the beds for different velocities of tether elongtion. With nnometerlevel motion nlysis, the instntneous force on the tether cn be mesured. n ddition, we cn evlute the reltive strength of the membrne-skeleton interction from the probbility of tether formtion t given force on the bed. We mesured the mechnicl properties fter tretment by the cytochlsins, DMSO, ethnol, nocodzole, nd ATP depletion. These tretments ltered the membrne-cytoskeleton interction nd ffected the membrne mechnicl properties. The method we described here for determining membrne mechnicl properties with the lser tweezers cn be generlly pplied under vriety of different conditions. MATERALS AND METHODS Cell culture Chick dorsl root gnglion (DRG) explnts were dissected from 12-dy chick embryos nd plted in the growth wells on treted coverslips. To prepre the cell growth wells, 2 X 2 mm (No. 1) glss coverslips (BDL, Lincoln prk, NJ) nd 1-mm-dimeter cloning cylinders (Bellco, Vinelnd, NJ) were clened by soking in 2% nitric cid for 2-3 h, followed by rinsing in distilled wter for 1 h. Then they were put into 95% ethnol overnight nd rinsed in distilled wter for t lest 2 h. After drying nd steriliztion, cloningcylinder ws secured to the coverslipwith sterilized silicone grese to form growth well. The growth well ws coted with.1% poly-l-lysine for 15 min t room temperture, rinsed 3 times with sterilized wter, then dried in sterile hood. Just before the dissection, poly-l-lysine-coted growth wells were exposed to 1:5 Mtrigel (Collborte Reserch, Bedford, MA):MEM solution. Explnts were mintined t 37 C, 5% CO2 in cler MEM (Gibco BRL, Grnd slnd, NY) supplemented with the following: 2 mm HEPES, 6 mg/ml glucose, 5,d/ml pen./strep., 2 mm L-glutmine, 1 gl/ml N2 Serum-free supplement (Gibco), nd 1 nm/ml nerve growth fctor (NGF 2.5s; Gibco). The explnts were used fter they were incubted for h. Bed preprtion n previous studies, we observed tht covspheres coted with control gg preprtion would bind to growth cone membrnes without ny pprent perturbtion of growth cone behvior. To prepre gg-coted beds, rt gg (Sigm Chemicl Co., St. Louis, MO) ws solubilized t concentrtion of 1 mg/ml in PBS. Then, 5,ulof covspheres (.5,um, Duke Scientific, Plo Alto, CA) ws dded to 5,l of the bove gg solution nd ws incubted t 4 C overnight. The beds were pelleted by centrifugtion t 2 X g nd 4 C for 1 min. Then the beds were resuspended in 1 mg/ml BSA-PBS solution, rinsed by pelleting nd resuspension with MEM medium 3 times nd resuspended in 1,ul of MEM medium. For the experiments, the bed solution ws diluted 3:1 in DRG medium. Clibrtion of the lser trp The lser opticl trp ws clibrted by viscous drg through the queous medium in the microscope focl plne (Kuo nd Sheetz, 1993). The viscous force ws generted by oscilltory motion of the specimen by piezocermic-driven stge (Wye Creek nstruments, Federick, MD) t constnt velocity. The position of the bed in the trp ws trcked using the nnometer-level trcking progrm (Gelles et l., 1988) to nlyze video records of the experiments. Positionl vrition of the prticle in the trp with 6 mw ws 11 (±1.7) nm. The clibrtion shows very liner forcedistnce reltionship for the opticl tweezers (Fig. 1). To study the vrition in trp clibrtion with height bove the coverslip surfce, ltex beds (.5,um in dimeter) were trpped with the sme lser power t different perpendiculr positions. The increse in prticle displcement t 2,um or less from the glss surfce (Fig. 1 B) implies viscous coupling to the coverslip surfce. From 2-5,gm bove the surfce, the force on the beds in the trp ws constnt (Fig. 1 B). This clibrtion ws used to clculte the forces tht form tethers. All of these experiments were performed 3-4,um bove the coverslip surfce to minimize viscous coupling to the glss surfce, nd the lser power ws simultneously monitored. Lser opticl trp mnipultions Just before observtion, the cloning cylinder ws removed nd the coverslip contining the cells ws mounted on n luminum coverslip holder using silicone grese; then second clened coverslip ws mounted on top to form flow cell. The gg-covered ltex beds nd the tretment solutions such s cytochlsins nd DMSO were exchnged for the norml medium. The growth cones were viewed by video-enhnced differentil interference contrst (DC) microscope (M-35 microscope; Zeiss, Oberkochen, Germny) with fiber optic illumintor. The stge ws mintined t 38 C using n ir current incubtor. The lser trp consisted of polrized bem from n 11W TEMOO-mode ner-infrred (164 nm) lser (model C-95; CV Corportion, Albuquerque, NM) tht ws expnded by 3 Xbem expnder (CV Corportion) then focused through n 8-mm-focl-length chromtic lens (Melles Griot, rvine, CA) into the epifluorescence port of the Zeiss M-35 microscope (Kuo nd Sheetz, 1992). n the binding experiments, the gg-covered beds were dded by exchnge of the cell culture medium. The bed ws trpped with -6 mw of lser power, put on to the cell surfce nd held for 4 s before pulling t constnt velocity. To determine the probbility of bound beds forming tethers, the beds were held with the lser trp on the cell surfcefor 4 s nd were pulled out t velocity of -3,um/s. We determined tht the percentge of tether formtion ws not ltered

3 99 Biophysicl Journl Volume 68 Mrch 1995 E 't A 1.-f FGURE 1 A t. f it. 2, $ & 7.. AM %X., _ - _ 2 S B 12. -_ 11. -_ 1- i Sequence of mesurements to clibrte the rdil force of the trp. (A) The rdil position of the bed (r) in the trp during series of clibrtion displcements. The bed ws held with the lser trp -4,um bove the coverslip surfce, nd the piezocermic-driven stge ws moved repetedly t constnt rte. (B) Clibrtion of the lser trp t different heights bove the glss surfce. The displcement procedure in A ws repeted for ltex beds (.5,zm in dimeter) held t different heights bove the coverslip, t constnt power. Six beds were tested t ech height. The results show tht experiments need to be done t lest 2,um bove the coverslip surfce to minimize viscous coupling to the glss surfce. (C) Clibrtion of the lser trp for prticles held -3-4,um bove the coverslip surfce by vrying the lser power for constnt velocity of movement. The result is shown s the reltionship between the position of the bed in the trp (r) nd the normlized light force FN in pn mw-1, s clculted from Stokes Lw nd the power of the trp, which ws monitored simultneously. The slope of the liner fit line is (pn xw-1 Am-'). C u.- uu-r x.5- "...- zz.5- *.. by exposing growth cones to even 1 s of lser irrdition. Beds either remined t the cell surfce or tether ws formed. All of the smples treted or untreted were mnipulted under the sme conditions. Anlysis of the mechnicl properties of the membrnes To mesure the force of the tether t zero velocity, FO, the position of the bed in the trp during tether formtion ws trcked using the nnometerscle trcking progrm tht ws developed previously (Gelles, 1988), nd the force (F) of the tether on the bed ws clculted from the clibrtion of the lser trp. After mesuring the tether growth rte (V) using "ruler" progrm, plot F vs. V ws obtined. Two methods were used to estimte the rdius of the tether. First, ltex beds.5,um in dimeter were used s stndrd in the "ruler" progrm. From the video tpe, we mesured the reltive dimeter of the tether nd then compred with the bed. The other wy ws to determine the reltive intensity through orthogonl scns cross the DC imge of both the tether nd the xon (Schnpp et l., 1988). After mesuring the dimeter of the xon with the "ruler" progrm, we cn estimte the rdius of the tether (the intensity should be relted to the rdius squred). The rdius of the tether ws clculted to be bout.2,am in both cses. With incresing velocity, there ws no detectble chnge in tether contrst. To clculte the membrne surfce viscosity, we developed progrm using the fourth-order Runge Kutt routine to solve the eqution (Eq. 21) in Dr. Wugh's pper (Wugh, 1982) nd compred tht vlue with the formultion described by Hochmuth nd collegues (Hochmuth et l., 1982). RESULTS Probbility of bed binding nd tether formtion When the gg-coted beds were held on the DRG growth cone surfce with the opticl tweezers for 4 s, only frction bound to the membrne. Those tht did bind to the surfce did so irreversibly nd would not relese under the conditions tested. Pulling on beds with the opticl tweezers either produced tubulr membrne tethers or the beds were pulled out of the tweezers. To understnd the cellulr fctors tht influence binding nd tether formtion, we mesured the effect of the cytochlsins, nocodzole, ATP depletion (Sheetz et l., 199), nd orgnic solvents (dimethylsulfoxide (DMSO) nd ethnol) on the probbility of binding nd tether formtion. As shown in Fig. 2, there ws no chnge in the probbility of bed binding with ny of the chnges in the cellulr conditions. We expected tht the probbility of tether formtion would be relted to the cytoskeleton-membrne interction nd tht 8. ~ (A n' 4 n. C. FGURE 2 Bed binding to the surfce is mesured s the probbility of gg covered ltex beds binding to the chicken DRG growth cone surfce fter 4 s using lser trp of -6 mw (lser power mesured in the specimen plne) for vriety of experimentl conditions. For the DMSO nd ethnol (EtOH) tretment, DMSO or EtOH ws dded to the MEM medium, nd the finl concentrtion for DMSO or EtOH is.5 nd 1%, respectively. Cytochlsin B nd D were diluted into the MEM medium from DMSO stock solutions to give finl concentrtions for both Cytochlsin B nd D of 1,uM nd for DMSO of -.3%. The nocodzole solution ws prepred by diluting 1 mm nocodzole stock solution in DMSO to give 1 jig/ml solution. For ATP depletion, solution of 2-deoxyglucose (3 mm) nd sodium zide (1 mm) in the cell medium ws used, nd the growth cones ssyed showed no motility. All of these tretment solutions were dded to the cells by exchnge flow, nd the cells were incubted for 1 min before strting the mesurements. For ech experiment, 7 beds were studied. :' l N z

4 Di nd Sheetz Tether Formtion of Growth Cones by Lser Tweezers 991 the probbility of tether formtion would increse with the force of the tweezers (s ws observed). On relted issue, those prticles tht did not form tethers normlly did not diffuse fter escping from the trp, indicting tht they were ttched to the cytoskeleton. Becuse most fctors were likely to decrese the ffinity of the two structures, we chose reltively low force (- 1 pn) where only 36% of the beds would form tethers (Fig. 3). Cytochlsin B nd D re known to inhibit ctin dynmics within growth cones nd cells, resulting in condenstion of the ctin network nd seprtion of the ctin cytoskeleton from much of the membrne. Both cytochlsins incresed the probbility of tether formtion to greter thn 9%. Controls of the solvent (DMSO) used for dding the cytochlsins (.25% solvent) lso showed some increse, but.5% DMSO only incresed the probbility of tether formtion to 55% (Fig. 3). Both the disruption of microtubules by nocodzole nd the block of motility by ATP depletion cused no chnge in tether formtion. Thus, chnges in the orgniztion of the ctin cytoskeleton, but not microtubules or ATP levels, produced n increse in the bility to form tethers e Sttic tether force (FO) After tethers re formed on DRG growth cones, they rpidly retrct when the tweezers re turned off, indicting tht significnt force is pulling the tether membrne bck onto the growth cone. Photogrphs of the tether-pulling sequence re shown in Fig. 4, including bed binding to the cell surfce in the trp, pulling the tether t constnt rte with the lser trp, nd the complete retrction of the tether when the tweezers were turned off. We cn utilize the mesured displcement of the bed in the trp to estimte the force of the tether on the bed (Fig. 5 A). The tether force increses only slightly with the tether length in other systems where it hs been U'. 1- X o co =. LU. -W ~ ~ ~ c U? ~~~~~~~~ FGURE 3 The probbility of tether formtion for the beds ttched to the cell surfce. The beds were pulled using the lser trp (6 mw) nd t constnt velocity (3,um/s). The tretments re the sme s described in Fig. 2. For ech experiment, 5 beds were studied. 3U FGURE 4 Photomicrogrphs of the tether-pulling sequence. (1) Bed ws held on the growth cone surfce by the lser trp. (2 nd 3) The tether ws pulled t constnt velocity by the lser trp. (4) The complete retrction of the tether when the lser trp ws turned off. tested (D. B. Wyne, S. C. Kuo, nd M. P. Sheetz, unpublished dt). The verge tether length in the experiments ws round 15,um. Rther we hve mesured the chnge in the tether force with the velocity of tether elongtion nd extrpolted to zero velocity to obtin criticl force, Fo, which must be exceeded for tether growth. Where we tested it, the Fo estimted from extrpoltion to zero velocity ws within experimentl error of the vlue obtined in sttic mesurements of the tether force. Fig. 5 A shows the distnce between the center of the bed nd the center of the lser trp fter the initil extension nd then during constnt velocity elongtion. n bout 1 percent of the cses, the force peked t the strt of the elongtion nd then returned to plteu level. For these studies we estimted the verge plteu level for the force mesurement. After plotting the force (F) forming the tether vs. the tether growth rte (V), we performed liner lestsqures fit nd extrpolted to V = to obtin Fo (Fig. 5 B). The mesurements for Fig. 5 were ll mde with different tethers tht were formed on mny different growth cones. For the control smple, we lso mesured the displcement of the beds t zero velocity nd obtined n verge vlue of this force of 5. (± 1.5) pn, which is in greement with the estimtion of Fo from the F vs. V plot of (N = 3) pn.

5 992 Biophysicl Journl Volume 68 Mrch 1995 A l A r E B 5. - ~Ve, 4 t,,9,,^,. U.UU - q' FRAME 25.1,UU' nn y-t z 1. UL. 1.- && 4t ":,,l & & & z. B U nfn _, e L =L $i tng u.uu n m Nu 5. - u.u nn... ls*-- -l V (um/sec) FGURE 5 (A) The position of the bed in the trp (r) with time during the tether formtion. After the bed ws trpped, it ws first held on the cell surfce for -5 s nd then pulled out t constnt velocity, forming tether. This tether length is -14,tm. (B) Determintion of the force (Fo) t zero velocity of tether growth from plotting F vs. tether growth rte (V). Fo = 6.72 pn ws obtined t V = from liner lest-squres fit of the dt points (slope of the plot = 1.51). 6 A L ±L n 7 x il CO) LU z Becuse Fo should reflect components of the in-plne tension of the plsm membrne nd membrne bending rigidity, we expected tht chnges in the cellulr orgniztion should lter Fo. When the growth cones were treted by cytochlsin B nd D, ethnol, nd DMSO, Fo went from 6.7 pn down to -3 pn in the presence of DMSO nd to -4 pn in the presence of cytochlsins nd ethnol (Fig. 6 A). However, Fo ws not ffected by nocodzole or ATP depletion. These results re similr to the probbility of tether formtion nd suggest tht the cytoskeleton plys mjor role in determining the force needed to extend filopodi nd tethers. Viscous forces in tethers As the velocity of tether elongtion ws incresed, there ws liner increse in the force of the tether on the bed from the viscous components of membrne movement into the tether. There re severl different nlyses of the viscous components tht contribute to the overll viscous force. Contributions could come from curving the membrne nd the resulting interbilyer sher, from the bilyer viscosity, nd from the drg of moving membrne pst the ssocited cyto- FGURE 6 (A) The criticl force (FO) of the growth cones before nd fter vrious tretments. The tretment protocols re described in Fig. 2. (B) The slope derived from liner lest-squres fit of the tether growth rte vs. the force of tether formtion. The coefficient of the correltion in ech experiment in Fig. 6 is given below: (from control to nocozdole).84,.81,.86,.82,.71,.89,.76,.66, nd.6, respectively. skeleton. Wugh's eqution (Wugh, 1982) llows us to estimte n pprent membrne viscosity neglecting contributions of the viscous sher between the cell membrne nd cytoskeleton nd the slip between hlves of the bilyer (Fig. 7). Alternte methods of clculting the membrne viscosity include other viscous terms nd, therefore, this vlue represents n upper limit of the membrne viscosity. There were surprisingly lrge decreses in the slope with cytochlsin B nd D, DMSO, nd ATP depletion. The pttern of the viscosity chnges with these tretments ws not the sme s chnges in Fo. The gretest difference ws with ATP depletion. Compred with the control smple, Fo is little bit bigger but the slope is bout 3 times smller fter the ATP depletion (Fig. 6 B). Thus, the viscous properties of

6 Di nd Sheetz Tether Formtion of Growth Cones by Lser Tweezers 993 E ,.2-, E.5-!. ~~l Z FGURE 7 The pprent membrne surfce viscosity of the growth cones before nd fter the tretments. The viscosity ws estimted following Eq. 21 in Wugh (1982). the growth cone membrnes re drmticlly ltered by metbolic nd cytoskeletl chnges. DSCUSSON The opticl tweezers hve been proven useful for number of studies of motor function nd wide vriety of biologicl experiments (Kuo nd Sheetz, 1992, 1993; Svobod nd Block, 1994; Finer et l., 1994). Here we show tht they cn be used to provide reltive mesure of membrnecytoskeleton ttchment nd the mechnicl properties of membrnes. The limittion of the tweezers is photodmge, nd we hve been concerned tht photodmge my influence the vlues obtined. From our experience with DRG growth cones, there is no observble chnge in growth cone behvior t these trp levels, wheres t higher irrdition levels (>2 mw) we do observe loss of filopodi nd generl inhibition of motility. An dditionl problem is the potentil for forming tethers without beds, but such tethers re notbly different in tht they contin bulb of cytoplsm t the end (Ashkin nd Dziedzic, 1989). Further, without beds we were unble to form tethers t the lser powers used. The trp could exert some force directly on the tether, but it is considerbly smller in dimeter (-2 nm vs. 5 nm for the bed) nd further seprted from the trp center, which mkes it unlikely tht the direct force on the tether is more thn 2% of tht on the bed. For compring the reltive forces under different cellulr conditions, the opticl tweezers provides self-consistent mesurements, nd the mesured forces represent lower bound for the ctul vlues. Membrne-cytoskeleton ttchment The exct nture of the linkges between membrnes nd the cytoskeleton re still mbiguous, lthough two generl types of linkge hve been considered. n one cse, ttchments would rise from trnsmembrne glycoproteins tht re ul- timtely linked to ctin-binding proteins. Alterntively, cy- toskeletlly linked proteins such s spectrin could wekly ssocite with the bilyer surfce nd, becuse such proteins re in n extended configurtion nd prt of lrger complexes, the sum of the mny wek interctions would form strong linkge. n mcroscopic terms, the difference here is similr to tht between sop membrne covering but only tied t few discreet points to rigid structurl net nd sop film spred cross tht sme net where it is dsorbed to elements of the net. n the first instnce, the disruption of the structurl net (most likely ctin-bsed) would mke it esier to seprte the membrne from the net; but the force, F, needed to hold the membrne off the net should not chnge. n the second cse, ltertion of the net should ffect both the ese of seprtion of the membrne from the net; nd becuse the membrne-net ssocition is reversible, it should lso ffect the force, Fo, needed to hold the two structures prt. We find tht both the percent tether formtion nd Fo re ltered in prllel, which is most consistent with the ltter hypothesis, nmely, tht the ttchments tht prevent tether formtion re reversible nd contribute to the tension in the tether or Fo. There re severl other resons to believe tht the membrne is not extensively tied to the cytoskeleton by strong contcts. Recent estimtes of the mechnicl force required to brek prt protein-protein interction with 1' ssocition constnt re lrge, in the rnge of 1 pn (Kuo nd Luffenburger, 1993). Breking of such interctions would result in drmtic decrese in the force on the bed. Although jumps re seen occsionlly t the strt of tether formtion, they re smll in mgnitude, 6-1 pn, nd re only seen in - 1% of tethers formed. From mny previous studies of the diffusion of prticles on the surfce of growth cones nd motile cells, brriers to lterl diffusion re spced over micron distnces (de Brbnder et l., 1991; Edidin et l., 1991). We might expect similr seprtion between the structurl links tht would hold the membrne nd cytoskeleton together. Thus, with.5 micron beds, bound beds should be ble to fit within the gps in the network nd tethers could be formed by pulling on those beds without breking strong membrne-cytoskeletl linkges. Previous studies hve indicted tht the probbility of tether formtion is inversely relted to the membrnecytoskeleton interction (Schmidt et l., 1993). We know tht both cytochlsins B or D cn ffect the network of ctin filments in the cell, lthough there is some smll difference between them (Edds, 1993). DMSO cn lso ffect the rrngement of cytoskeleton (Yumur nd Fukui, 1983; Weiner et l., 1993). t hs been reported tht ethnol cn influence cell migrtion nd invsion in vitro s well s F-ctin orgniztion, nd it cn ffect the membrne conformtion nd structure (Weiss et l., 1991; Moordin nd Smith, 1993; Stler et l., 1993; Ho et l., 1994). Therefore, possible explntion for the bove result is tht the cytochlsins nd DMSO will decrese the membrnecytoskeleton interction through rerrngements of the ctin cytoskeleton nd thereby increse the probbility of tether formtion for given force. On the other hnd, DMSO, like

7 994 Biophysicl Journl Volume 68 Mrch 1995 ethnol, might indirectly ffect membrne mechnics through n ltertion in the interfcil interction energy between the membrne nd n otherwise norml ctin cytoskeleton. The microtubule (MT) orgniztion in cells ppers to hve no ffect on the membrne-cytoskeleton interction (Wng et l., 1993) s does ATP depletion. n other growth cones, ATP depletion inhibits the prticle movement on the membrne (Sheetz et l., 199). Generl ltertion in the orgniztion of the ctin cytoskeleton is linked to prllel chnges in the probbility of tether formtion nd Fo. We suggest tht the membrne interction with the cytoskeleton is through reversible, wek bonds nd not through strong protein-protein bonds. Fo of growth cone membrnes (-6.7 pn) is much less thn tht of erythrocyte membrnes, which is bout 5 pn (Wugh nd Busermn, 1995, in press). The lrger membrne tension of the preswollen red cells might contribute the lrger Fo. There is n extensive interction between the red cell spectrin-ctin network nd the membrne bilyer tht possibly ccounts for the mjor portion of the force required for tether extension. By impliction, similr network my be ssocited with the growth cone plsm membrne. n comprison with the forces tht myosin motors or ctin polymeriztion cn develop, the force for extension is comprble with two myosin molecules or one ctin filment, indicting tht either mechnism could generte sufficient force to produce extension (Sheetz, 1994). n ddition to binding force between membrne nd cytoskeleton, other fctors tht contribute to the force required for tether formtion include the membrne bending stiffness nd the inplne membrne tension (Hochmuth, personl communiction). The bending stiffness of the membrne plys n importnt role in processes becuse of the mjor chnges in membrne curvture. To estimte the membrne bending stiffness, n eqution developed by Wugh nd Hochmuth (1987) cn be used. n this cse, if we ssume tht the force (F) forming the tether is 8 pn, the rdius (r,) of the tether is.2,um, then the bending stiffness (B) = F X r/2w = 2.55 X 1-12 dyne cm. The growth cone membrne bending stiffness is similr to tht of the lipid bilyer membrnes X 1-'2 dyne cm. (BO nd Wugh, 1989) nd erythrocyte membrnes (Evns, 1983). Becuse the bending stiffness of the bilyer should not chnge with cytochlsin B or D, we suggest tht the reversible membrne cytoskeleton ssocitions or possibly in-plne tension is decresed by these tretments. Membrne surfce viscosity The viscoelstic properties of the membrne could contribute to the control of growth nd extension rtes, but these dt indicte tht viscous forces re too smll t typicl extension velocities (mximlly.3-.25,um/s) to cuse significnt effect. Although there re potentilly severl fctors contributing to the viscoelstic force, we hve used Eq. 21 in Wugh's pper (Wugh, 1982) to estimte the membrne viscosity. Becuse the growth cone is only very smll prt of the whole neuron cell, the rtio of the rdius of the tether (r) (-.2,um) to the rdius of the cell (rc) or (rl/rc) is very smll (<1/5). Following Wugh, the vlue of F/Fo cn be used directly to clculte membrne surfce viscosity (Wugh, 1982), which is bout 2.1 X 1' dyn s/cm (Fig. 7). n these studies, the pprent viscosity rnged from.56 X 1-4 to 4.2 X 1-4 dyn s/cm. This vlue is much smller thn tht Hochmuth obtined for erythrocyte membrnes (2.8 X 1' dyn s/cm) nd is greter, perhps very significntly, thn tht reported for egg phosphtidylcholine lrge bilyer vesicle membrnes (-1.7 X X 1-6 dyn s/cm) (Hochmuth et l., 1982b; Wugh, 1982b). Which of the fctors, including the slippge of membrne lyers in moving to highly curved surfce, the plnr membrne viscosity, nd glycoprotein collisions with the cytoskeleton, mkes the mjor contribution to the viscosity during extension flow is uncler. Evns suggested tht the slope of the liner fit of the tether growth rte (V) vs. the force (F) forming tethers should be relted to the friction in the slipping of the bilyers pst one nother s the bilyer moves from the flt membrne surfce to the highly curved tether (E. A. Evns, Personl communictions). Becuse we see mjor effect of the cytochlsins on viscosity nd they should hve little effect on the interfce between the two bilyer hlves, we consider tht this fctor mkes only smll contribution to the control of membrne viscosity. There is lrge difference between the viscosity of the neuronl membrne nd the liposome or red cell membrnes using the sme formul. Current models of membrne viscosity re being chllenged becuse they lck terms to consider the slippge between the two bilyer hlves nd cytoskeletl contributions; thus, better models of this system re needed to provide testble hypotheses. t is somewht surprising to find such lrge chnges in the viscosity with ltertion of cell cytoskeleton nd prticulrly with ATP depletion. Axon elongtion nd tether formtion An interesting question in xon elongtion is: where does the xon membrne come from? n these studies, we re forming tethers t very rpid rte nd lipid is flowing onto the tether t 1-1 times the rte tht membrne moves into xons (typicl xon elongtion rtes re 1-4,um/min, nd dimeters re typiclly 1 micron, wheres we re pulling tethers of.4 micron t rtes of 6-6 gm/min). Becuse the forces needed to produce such rpid extensions re considerbly less thn those developed by growth cones moving on norml substrt, it should be no problem for growth cones to pull membrne from the cell body nd no need to invoke ddition t the growth cone (Bry nd Chpmn, 1985; Popov et l., 1993). n the studies of erythrocyte membrnes nd liposome membrnes, the membrnes forming tethers were ll from the surfce membrnes, but in ctive growth cones, some of the membrne might come from the cell body or from internl membrne vesicles. We consider this lst possibility less likely becuse normlly endocytosis nd exocytosis re blnced nd we re pulling t such high rte tht we would

8 Di nd Sheetz Tether Formtion of Growth Cones by Lser Tweezers 995 not expect cute dpttion by membrne fusion. Rther, there ppers to be reservoir of membrne tht the tethers drw upon either through ltertion of the growth cone geometry or from stretching the membrne. There re number of biologicl processes for which tether dt re relevnt, including cell migrtion, cell volume, nd membrne re regultion. The probbility of tether formtion reltes to the bility of the cells to migrte becuse the relese of cell contcts in the rer of the cell often involves formtion of retrction fibers, i.e., tethers (Schmidt et l., 1993). How the cell regultes its surfce re nd the relted prmeter of cell volume my well involve n inplne membrne tension tht would regulte endocytic versus exocytic rtes. Clerly, cytoskeletl fctors do ffect the physicl prmeters of tether formtion, implying tht they re importnt for cellulr functions. SUMMARY Growth cone migrtion is ccompnied by drmtic morphologicl chnges nd n extension of the xonl membrne. From our nlyses of the mechnicl properties of the membrnes, we find tht the forces generted by the membrne re on the order of those generted by few myosin motors or ctin polymeriztion or from the breking of wek protein-protein interctions. Becuse membrnecytoskeleton interctions influence the probbility of tether formtion nd the criticl force required to extend membrne tether, we suggest tht the interctions between the membrne nd the skeleton re wek nd reversible, lthough pointwise strong interctions cnnot be excluded. The viscous properties of the tethers show strong dependence on solvents nd ATP depletion tht re not understood. Thus, we find tht the lser tweezers cn be used to ssy the strength of the interction between the cytoskeleton nd the membrne bilyer tht in turn cn influence cell migrtory behvior. We thnk Drs. Evns nd Hochmuth for their helpful comments on this work. We lso thnk our collegues Denise Wyne, Zhohui Wng, Mingy Jing, Hnry Yu, nd Ron Sterb for their kind help or helpful discussion in prt of this work. This work ws supported by grnts from Ntionl nstitutes of Helth, Humn Frontier Science Progrm, nd Musculr Dystrophy Assocition. REFERENCES Ashkin, A Accelertion nd trpping of prticles by rdition pressure. Phys. Rev. Lett. 24: Ashkin, A Forces of single-bem grdient lser trp on dielectric sphere in the ry optics regime. Biophys. J. 61: Ashkin, A., nd J. M. Dziedzic Opticl trpping nd mnipultion of viruses nd bcteri. Science. 235: Ashkin, A., nd J. M. Dziedzic nternl cell mnipultion using infrred lser trps. Proc. Ntl. Acd. Sci. USA. 86: Ashkin, A., J. M. Dziedzic, J. E. Bjorkholm, nd S. Chu Observtion of single-bem grdient force opticl trp for dielectric prticles. Optics Lett. 19: Ashkin, A., J. M. Dziedzic, nd T. Ymne Opticl trpping nd mnipultion of single cells using infrred lser bems. Nture. 33: Bennett, V Spectrin-bsed membrne skeleton: multipotentil dptor between plsm membrne nd cytoplsm. Physiol. Rev. 7: Berk, D. A., nd R. M. Hochmuth Lterl mobility of integrl proteins in red blood cell tethers. Biophys. J. 61:9-18. Bo, L., nd R. E. Wugh Determintion of bilyer membrne bending stiffness by tether formtion from gint, thin-wlled vesicles. Biophys. J. 55: Bry, D., nd K. Chpmn Anlysis of microspike movements on the neuronl growth cone. J. Neurosci. 5: Bry, D., J. Heth, nd D. Moss The membrne-ssocited "cortex" of niml cells: its structure nd mechnicl properties. J. Cell Sci. Suppl. 4: Bretscher, M. S Endocytosis nd recycling of the fibronectin receptor in CHO cells. EMBO J. 8: Cooper, J. A The role of ctin polymeriztion in cell motility. Annu. Rev. Physiol. 53: Debrbnder, M., R. Nuydens, A. shihr, B. Holifield, K. Jcobson, nd H. Geerts Lterl diffusion nd retrogrde movements of individul cell surfce components on single motile cells observed with nnovid microscopy. J. Cell Bio. 112: Edidin, M., S. Kuo, nd M. P. Sheetz Lterl movements of membrne glycoproteins restricted by dynmic cytoplsmic brriers. Science. 254: Edidin, M., M. C. Zunig, nd M. P. Sheetz Trunction mutnts define nd locte cytoplsmic brriers to lterl mobility of membrne glycoproteins. Proc. Ntl. Acd. Sci. USA. 91: Edds, K. T Effects of cytochlsin nd colcemid on corticl flow in coelomocytes. Cell Motil. Cytoskel. 26: Evns, E. A Bending elstic modulus of red cell membrne derived from buckling instbility in micropipette spirtion tests. Biophys. J. 43: Evns, E. A., nd R. Sklk Mechnics nd thermodynmics of biomembrnes. CRC Crit. Rev. Bioeng. 3: Finer, J. T., R. M. Simmons, nd J. A. Spudich Single myosin molecule mechnics: piconewton forces nd nnometer steps. Nture. 368: Gelles, J., B. J. Schnpp, nd M. P. Sheetz Trcking kinesin-driven movements with nnometer-scle precision. Nture. 331: Gordon-Weeks, P. R Control of microtubule ssembly in growth cones. J. Cell Sci. Suppl. 15: Ho, C., B. W. Willims, M. B. Kelly, nd C. D. Stubbs Chronic ethnol intoxiction induces dptive chnges t the membrne protein/ lipid interfce. Biochim. Biophys. Act. 1189: Hochmuth, R. M, nd E. A. Evns Extensionl flow of erythrocyte membrne from cell body to elstic tether.. Anlysis. Biophys. J. 39: Hochmuth, R. M., N. Mohnds, nd P. L. Blcksher Mesurement of the elstic modulus for red cell membrne using fluid mechnicl technique. Biophys. J. 13: Hochmuth, R. M, H. C. Wiles, E. A. Evns, nd J. T. McCown. 1982b. Extensionl flow of erythrocyte membrne from cell body to elstic tether.. Experiment. Biophys. J. 39: Kucik, D. F., S. C. Kuo, E. L. Elson, nd M. P Sheetz Preferentil ttchment of membrne glycoproteins to the cytoskeleton t the leding edge of lmell. J. Cell Biol. 114: Kuo, S. C., nd D. A. Luffenburger Reltionship between receptor/ lignd binding ffinity nd dhesion strength. Biophys. J. 65: Kuo, S. C., nd M. P. Sheetz Opticl tweezers in cell biology. Trends Cell Biol. 2: Kuo, S. C., nd M. P. Sheetz Force of single kinesin molecule mesured with opticl tweezers. Science. 26: Misw, H., M. Koshiok, K. Sski, N. Kitmur, nd H. Msuhr Lser trpping, spectroscopy nd bltion of single ltex prticle in wter. Chem. Lett. 8: Moordin, A. D., nd T. L. Smith Membrne disordering effect of ethnol cerebrl microvessels of ged rts: brief report. Neurobiol. Aging. 14: Psternk, C., nd E. L. Elson Lymphocyte mechnicl response triggered by cross-linking surfce receptors. J. Cell Biol. 1:

9 996 Biophysicl Journl Volume 68 Mrch 1995 Popov, S., A. Brown, nd M. M. Poo Forwrd plsm membrne flow in growing nerve processes. Science. 259: Schmidt, C. E., T. Chen, nd D. A. Luffenburger Simultion of integrin-cytoskeleton interctions in migrting fibroblsts. Biophys. J. 67: Schmidt, C. E., A. F. Horwitz, D. A. Luffenburger, nd M. P. Sheetz ntegrin-cytoskeleton interctions in migrting fibroblsts re dynmic, symmetric, nd regulted. J. Cell Bio. 123: Schnpp, B. J., J. Gelles, nd M. P. Sheetz Nnometer-scle mesurements using video light microscope. Cell Motil. Cytoskel. 1: Seeger, S., S. Monjembshi, K.-J. Hutter, G. Futtermn, J. Wolfrum, nd K.. Greulich Appliction of lser opticl tweezers in immunology nd moleculr genetics. Cytometry. 12: Sheetz, M. P Glycoprotein motility nd dynmic domins in fluid plsm membrnes. Annu. Rev. Biophys. Biomol. Struct. 22: Sheetz, M. P Cell migrtion by grded ttchment to substrtes nd contrction. Semin. Cell BioL 5: Sheetz, M. P., N. L. Bumrind, D. B. Wyne, nd A. L. Perlmn Concentrtion of membrne ntigens by forwrd trnsport nd trpping in neuronl growth cones. Cell. 61: Schut, T. C., G. Hesselink, B. C. De Grooth, nd J. Greve Experimentl nd theoreticl investigtions on the vlidity of the geometricl optics model for clcultiing the stbility of opticl trps. Cytometry. 12: Stler, S. J., C. Ho, F. J. Tddeo, M. B. Kelly, nd C. D. Stubbs. Contribution of hydrogen bonding to lipid-lipid interctions in membrnes, nd the role of lipid order: effects of cholesterol, incresed phospholipid unsturtion, nd ethnol. Biochemistry. 32: Steubing, R. W., S. Cheng, W. H. Wright, Y. Numjiri, nd M. W. Bems Lser induced cell fusion in combintion with opticl tweezers: the lser cell fusion trp. Cytometry. 12: Svobod, K., nd S. M. Block Biologicl pplictions of opticl forces. Annu. Rev. Biophys. Biomol. Struct. 23: Trinkus, J. B Protrusive ctivity of the cell surfce nd the initition of the cell movement during morphogenesis. Exp. Bio. Med. 1: Vsiliev, J. M Spreding of non-trnsformed nd trnsformed cells. Biochim. Biophys. Act. 78: Wng, N., J. P. Butter, nd D. E. ngber Mechnotrnsduction cross the cell surfce nd through the cytoskeleton. Science. 26: Wugh, R. E Surfce viscosity mesurements from lrge bilyer vesicle tether formtion.. Anlysis. Biophys. J. 38: Wugh, R. E. 1982b. Surfce viscosity mesurements from lrge bilyer vesicle tether formtion.. Experiments. Biophys. J. 38: Wugh, R. E., nd R. M. Hochmuth Mechnicl equilibrium of thick, hollow, liquid membrne cylinders. Biophys. J. 52: Wyne, D. B., S. C. Kuo, nd M. P. Sheetz Actin polymeriztion rtes in novel membrnous extensions (neopodi) formed on neuronl growth cones. J. Cell Biol. 115:12. (Abstr.) Weiner,. H., J. Murphy, G. Griffiths, M. Schleicher, nd A. A. Noegel The ctin-binding protein (p24) is component of the Golgi pprtus. J. Cell Bio. 123: Weiss, L., B. B. Asch, nd G. ELkin Effects of cytoskeletl perturbtion on the sensitivity of Ehrlich scites tumor cell surfce membrnes to mechnicl trum. nvsion & Metstsis. 11: Wright, W. H., G. J. Sonek, Y. Tdir, nd M. W. Berns Lser trpping in cell biology. EEE J. Qunt. Elect. 26: Yumur, S., nd Y. Fukui Filopodelike projections induced with dimethyl sulfoxide nd their relevnce to cellulr polrity in Dictyostelium. J. Cell Biol. 96: Zigmond, S. H Recent quntittive studies of ctin filment turnover during cell locomotion. Cell Motil. Cytoskel. 25: