origin of the unit membrane concept
TRANSCRIPT
P a r t C
Electron Microscopy and X-ray Diffraetion
[nr, ited Papers
Origin o~ the Unit Membrane Concept '
By
J. D. R o b e r t s o n ~
Dep~rtme'n,t of Neurc,patho,l,ogy, H,ar~ard Medica ~. Scho,o~, Belmont, Mass,a&usett.s, U.S.A.
With 22 Figures
The p:ap,er~s i~nelud,ed in :t,h.Ss vo lume are o,f ~two .kin, d,s. F'.irs.t, t,here a re ,short ones p resen t ing the results of recent research as p resen ted at the F ra sea t i Conference . Since, however , it was p l a n n e d tha t the F rasea t i Confe rence wou, ld 5,e the firlst ~of .a serli.es, i t was ,fett t,ha,t ~the eol~eetiion .of !papers ,ought to inc lude a few inv i ted l o n g e r ones of a more genera l su rvey na tu r e to b r ing the sGort, er ~p,a~per:s ,iatto bet,her p.erts?ectiv,e. I wtats ,a,sked t,o wrilt,e such a pap,er ~dea~ing w.~t,h ,the n~i t m,e~nbrane, i ~ceep ted witch istorne r e l u d a n e e beeattse ! ~hav,e revi.ew,ed t'hi,s s:u'b~eet severa l ~li~n,e,s ,already lin r~e,eent ye.ar*s. However , [ .e.or~se~l.t,ed t,o ,do, this in ttle ~n,~,er,e,s~t o~f 'mak!in,g ,the wh.o)e vo lume m,or~e usefu l ,a's a ,ge~er,M ref,er,enee t~o. ;smdent,s. T;o tho,s,e w h o have re~d ,other reviews By me in recent years , there is l i t t le new in the p resen t article. I,t 4s ad~dr~e,s~s.e,d o,nly Lo ~thas,e who a.r.e u~fam,illi,ar ~it,]a 'the ,evohltlioa .of ~t~,e um't bme,~brane .eo,neept.
C u r r e m thou:~hts about m e m b r a n e ~str.ue~ure ~ha~d thei r eiarlies~t ,o r ion 'in the w o r k .of O v , e r t ,o n [1] ab,o,uut the t,urn of 4he .cem,ury. l i e no,ted tha t eerta:i,n ,cell ,naemlbr:a,n.es ~ e r e mo,r,e ea.si.ly '.pene:t'rated b y ,eom*poun~ds wi,th a hligh ,li,pid solub,i}icty ~than ,o.n~es 'th,~t were mare p o l a r ,or wa fe r ,s,o.lu~b~.e. This led J~im t,o p.o~st,ulate t h a t the re migh t be a ~ip:i,d .eo,mp.on.ent 411 ~the anean- b`ra'n, es ,f.hat af fec ted ,eer, ta in f~eature~s ,of me,mb,van.e vem'aea.hi~ity. I,t was a l r eady we}l ,est,ablis,hed f rom ,the !previ.o,us ,eenfu,ry ~that ~ele,ctrieity ~ a s in ,so~e w a y a~s~s,oeiated wi~t.h ceil membranes . T'hu,s ,d n B o. i ,s - t / ,e y :m ,o n d in i849 [2] ,ha,d noted t b a t ,a s~.oltage eouM b.e d.eteefed in nerves a:t ,rest. L a t e r on, B ,e r n s t e i n i:n 1868 [21 r:eati,zed t h a t in ~th'e r,eslt~ing .s,tat,e ,a n.erv,e ,or mu.s,cte fi~b,er .had an exees:s .of po~slit~iv,e ions on tthe ~outsid,e .anLd neg,a,tiv,e ones on ~h,e i~sd~de. I,t i*s r e l evan t t~o O.oin't ~oat t h a t M a t t e u e e i tin 18~2 [2] t~a,d deano,ns,tra~t,ed a r,educt'ion lin t,he st.ea~dy pot,e~i,al ,between t.l~e ,eu~t er~d ,of
1 Supported by Grant B-2665 from the National Institutes of Health and Grant B-5128 from the National Science F.on~dati~n.
2 Present address: Dept. of Anatomy, School of Medicine, Duke University, Durham, North Carolina, U.S.A.
J. D. Robertson: Origin of the Unit Membrane Concept 219
a antrs,cl,e ,art~d 2,t~s< lin,ta'ct s,urfa.ee ,dur ing :~e~a=i,c <a.cti.~'fy. Tlri.s is ~ w n~rd.e,r- ,st,a,rrd.abl,e ~n ,terms ,of rep,e~i,fiwe <dep,ola~i.za~t,i,on o f ,the fkber:s. A ,d r i a n in 1911 [2] ~a,a,d es,f,a~l,is~ed ~ e a.U .or .n~o.t~i~rrg ,rrat'ure ,o,f ?dae .aetqo~n ,p,o~ent~i.al .o,f a .rrerve f iber ~and i:t w a s real, i,z,ed ttkat t~h~s w,a~ .lar:g, e ly ~a m,exrrbran, e ptre- norfmn,on. T?mrs ,ea.rly ~ri ,ttl, e ,p~res.en,t .ee,irfury, a eo.n,eept h,a,d ~grn~wn u,p Ifhat
Fig. 1. Lipid molecules are indicated by a bar and circle. The bar represents the non-poplar carbon chains and the circle, the polar ends o.f the molecule. A mono- mnleeular film of lipid molecule's is depleted .on a water surface indicated by gray
stippling.
FO RC E ~ , ~ 0
AREA
Fig. 2. Fig. 3.
Fig. 2. A diagramat ic force area curve. Force is depicted as increasing on the or.dinant and area on the abcissa. As the force applied t.o the film increases,, the area decreases until there is a break in the curve as shown. This point of dis.c.on- t,inuity is the minimal area of ihe film and from it the area occupied by the head
of each of the lipid molecules can be calculated. Fig. 3. Diagram taken f rom G o r t e r and G r e n d e 1 [~1 showing their conception
of the lipid bi layer in a red blood cell membrane.
~tte.re w.a,s ~a discr,eie ~i.p,i~d c o n f a i~ ing anem br,a.rm a:s ~tre surf~aces ,of ,cel~s wt t i ch i~n ~h,e case .of rrerve,s> v~a~s ,as,s,~ciai,ed w4,t~a ,a~n el~ectrica..1 p,o~e~nili.al ~gra~d,i,e~nf nrs wel~l ,as ,d~iffere~i.al 9er~ne,al~ilify I~o. ~ir~rrs arrd .o.~her oopmp,on~d~s.
I.n 1917 J~ , an g m u i r [31 .l?.ubl,is<hed ~i.s .p.i~o.rre:el~i,rrg e~p.er i~me~s ~o,n ~.oxto- n~ole.cu,l,al: fi.lms a n d pro~i,d,ed la 43rm Ibas,is fo r ~o,u~ ct~rrenrt ,eoEn,ceptlio,n,s o,f rrre.mbr:a~e rstr~rcftrre. I-re ,s,ho,wed .tJhat ,]Jipl~d m~o,~ecufes, ,eo,u~d ibe ~ d e t,o, s ,pread .o~ut ,~n ,a w a t e r : surface a'f .an ,air vza~t,er i infer face w~~lh :~eJir ~pol, a r ,er~d,s 9,o~nt-
220 ]. D. Roberts on
,i.n)g ,~ow.a'rd 'the v~a`ter ~s,trrf~aee, arrd the i r nien-p,olar carb,on ,drains is,tand,ing ,on end nex`t to the ,air int,erf,aee. H e fo,un,d `tha.t 'it was p,o,ss~ible ~o cocrrpres,s these fi.l~s lin ~such ,a w,ay ' that `t,he mo~ec~les ,came v e r y .el,o,se tog,ether w,i`th `the car~b,~n ch~i.r~s et~s,ely ~.ael~.ed ,as 'i~rdli.,cated ,i,n th'e &i, ag ram i,n F~i,g. t. By ',the trse ,o,f .f,orce a rea cu rves l ike t ha t in F,ig. 2, h e ~ a s . ~a!ble 't,o ,show ~th~t the re was a d,efirrit,e ,s~lrf,a,e,e .t3ressur,e ,a~s,s~rci, a~ed wi th 'the film. ~h,e ip,eirrt o f max,inau,m pressure be fore b r e a k a g e is no ted by the d i scon t inu i ty in Fig. 2. At this po in t the molecules are as c losely packed as t hey can be in a single mono laye r .
EXTERIOR
, ~ I ,' I # , @.,.@.,@,.@.,.@., @,
LIPOID
INTERIOR Fig. 4. The origin.a] Danielli-Davs.on pauci-moleeular melnbrane model [71.
Above this po in t the film collapses as the m o n o l a y e r buckles and becomes mul t i l aye red . The area occup ied b y each po la r g roup at the m a x i m u m pressure be fo re b r e a k a g e was ca lcu la ted f rom the chemical ev idence avai l- able about the s t ruc tu re of the molecules involved and f rom knowledge of the exac t n u m b e r tha t had been p l aced on the surface.
T, ke rrext ~s`t,ep d~ .t~lre ~evolut,i,o~n .of ,ou~r ,id,eas ,came Li~n 1925 w~hen G o r t e r arrd G r e n ,d ,e 1 [41 !p.trb~i~s,h~d the i r work ,o,n the ~s`tru,cmre of Jauman red ~blo,od cell ~membranes. T ~ e y ,u,s.ed ~f,h,e ~eeh~iques evo,lved b y L a n,g m ,a i r and by H a r k i n s et al. [5] for the s t udy of monomoleeu l a r l ip id films. T h e y ca lcu la ted ~the ~o.~al a r ea ,of a le~own numlb,er ,o,f red ,ceils a.rrd ex~rae`ted *~e ~,o,t,d .~i,p:id ~pr,es,en{. T..h, ey `tlhe:n ~e.~rs,u~ed ,the to,t,al ,area ,o, ceu,Fi,ed ~ y the ex,$ra'ebed I,~pMs l)y q3~o`t:~ing a f~or'ee ,area curve. T&ey .eo~clud,ed tha`t the re wals j~st ~s!affi, ci,errt ,~ip,~d i,ll ,~a,e red l)lo,o,d cell su r f ace ~o f, arra ,a ~,iln.&ectrlar leaf le t as dep ic t ed in Fig. 3 t aken front the i r pape r . The i r results were sub jec ted t,o c:o~rsi, cLe~albfe .e~i`t,~ci~sm an, d t he re were Chose wh,o h,eLi,ev.ed tha`t r me thod of .cal~eul,ar the :surfa.ee ,area ,o,f the red ceIfs was ne t ~a,ceur, a te aIrd thaX ~he ~met,ho,ds ,u~sed t,e ,ex`tra.ct `the }i'p,i~d were .n,o,t ,a,d,equ~aCe ,f,or ~o,`tal ,ex`tract~on.
Origin of the Unit Membrane Concept 221
Tirer,e w,as an,ot~her so ,~ce ~of e r ro r !i,n ~trat ~hey d id ~ro~ t ake ~m,o accoun t t,h,e defiui.te degrees ,at ,solu~i~i,ty ~f ~some ,of tire l il31id ,eompro,nen,ts dn ~ a t e r . Never - tir, dess , b y a f~ortu~n,a*e eorubirrafli,on ~of ,eircun~s~anees, ~ e i x ,eouclusi,oaas seem, in ~ t r t ,o,f ,pmseut knowlecLge, t.o h a v e l~een ,es;serr%a,lly correc t .
Duri.rrg tN~s o,eri.ud it is ~i,mla,o~ta~t t,o n,o,te ~the ,e:~perimen.t repor~ted b y M u d d and M ~u d d 1931 [6]. q~hey set nla ,an ,oi$I ~ a t e r ~rtter:faee n nde r a cover ,slit 9 o,n ,a m!i,eroseope s~de arrd .s4uffi'ed ~ .e beiravior ,o,f ir~rman bl~o,d eel,~s ~rt ~d~e ~xrterf~aee. T h e y ~r~Sed ~tir,a~t ~ed ,b]~aod ;eel~s ~se~eet, ive ly emt.ered tire o,il pira~se wirile ~hi6e ~blo,od eel~s,e,Ir't.ere,d ;the aqu,eous ,pir~se. T h e y iaa.terpreted /~heir res,ults ,a,s !in,&iea,tilng ~tir~t '~,e red bl~o,od cel l surfa~ce ~i]s irpd~ro.pirobi,e arrd the ~iri.~e ~blo,od :cell ,sur,faee 5Is ir3~dropiril, ie. I .sha,H relier ,to t~is experimma~t ~ater ,o,n ~in eous~der~ng our p resen t c,oamep:ts ,of the s~ructure of fire uni t m,e~brtan,e.
L: lipid Pr: protein
Fig. 5. Diagram from W. J. S ch m i d t [15] sh.owing hi.s conception of the orga- nization of lipid and protein in the myelin sheath based on polarized light studies.
D uri~rg #)re 1930':s '~irere were ,several siguifi,calrt :s,t,e,p,s th,at .led up ~o hire s,o-ealled p.an,ei-,m.o,l,ecular ~theory of cell ,mem~br,an, e stru.cture t irat wa,s advanced ,by D a n i e J l i and D , a v s o n ~n 1935 [7]. F!ir,st, C o l e ,dem- ons t ra t ed in 1952 [8] t ha t the su r face tension of sea urchin eggs was less t han 0.1 ,dy~ne p e r cent imeter . In 1954 H a T v e y ,aI~d Sir i ~ i r,o [9] measu red t.]~e ,s~tr~aee k e~us.i,on o,f ro~il ,dr,orpl'ets ~i,us,i,de ma~ine eg~s ~ y a v e r y d i f ferent t,ech~rrique. C o 1 e irad ~marcLe iri,s m.easntrements ~ y aao~in,g tire ~oree requ i red t,o ~o,nrpres,s ,an e~g ~etwe,en tw~o thiaa glass ,s,urfaces. H ~a r v e y .and S h i p i r o me,asured tire snrf~ace ite~s,i.~n ,of ,o~il ,droplets ;by ~usi.I~g ,a cen t r i fuge ~micro,s,eo.pe me thod ~i,n ~wirich tire ,centrdf,ugal ,fgree requ i red .to b r e a k .o~il d rop le t s i~nto sin,oiler ,o~res was measured . He re a f igure o,f ,as 0.2 d y n e p,er c,enti,nmter wa,s o,bt,a,ined. I t was ~ n o w n ,that ~ o s t o,f ~h,e JoJi~s th~a:t were p resen t in ~he o,il drog,let,s ~of ~sea aa,r,clrin eg~s ,as wel l .as m a n y t a~e r l~9,id eo,rrrp,o.m~d~s wa,s in tire range ,of ~albloat 10 dyrms ~p,er centianeter. T[irese ,o~il drep$et surf.ace ~tens~on figures were, *th,e~efore, diffi,eul,t ito a,c.eo,um ~fo,r ~n ~.erms .~f a nro,d,e,1 s,u.ch a,s tirat 'pmpo,s,ed :by G ,o r t e r .aud G r ,e n d e,1. D a n ,i e 1 1 i ~a~rd H .a r v e y ~n 1935 [10], Ip,erf,ormed so,me ,experdaneuts ~nvolviaag ,t~e ,strrf, ace ~0ensi,o,n p r o p - er,ties .o,f r~acke~al ,eg~g ,oil and ,pr,o,pos~d tira~ tire 1,o~ ,strrfa,ce tens,i~n srri,ght be due to the p resence of p ro te in at the oil wa t e r in ter face . This led d i r ec t ly to t~ae ~pro,p~sal ~n 1935 [7] ,of the mode l sirown i,n F~i,g. t wki,ch ,is tire origiual
222 J .D . Robertsoa
D,a, ni,e.ll~i-D, av, s,o,n m.~d,el. Aeeo~d~i~rg ~t~o Shis, ~I~e ,cell an.en~brarre w,a,s c~rmeived 4o 'h,awe ,a ~i,pid core ~s She ~p~ol~ar ,en~d~s ,of ,fire ~i,p,i~d ~mo~ecules ~p,o~imirrg .ou,t- w a r d a'n,d ,covered ~on ~ea.ch ~s~ide 'by a ~o,nomo~ecul~ar fi~m ~of !profei.n. T h e y .4~d ~ro,t ,spec~.fy t~he .t.o~,al num'b.er ,of ~i~vid ,moleeule,s ~present because ,tlhere w.a,s n,o~hin, g 6n ,tlheir re~d.en.ee ~,hat ,al.lowed ~h~m ~t,o ~d,o s~o. The re was, ,h,owever, so,me e.-~iden,ee i,n ~t'h,e l~i4e,ra,ture .a't :t'ha:t ~t~i,me ~e~eerrr~n,g ,tih, e electr~ieoJ ~calp,a.ci,ty o,f ~s,o,~e eel4 ~m, embra~res ,a~rd flris was =f.ou.n,d '~t,o b,e a~'bo.~ ~o.n,e ,n~icrofarad. I t was ,al.so kn,own t'hat tire ele,cgrical res4stanee .o,f ~he an,em'brrane was ,abo,ut 1,000 ,t~o 100,000 O'hzn.s. In llBe .ea,se ,of ~so~re ,cell men~b,ra,rms,, i l w~s p,o,ss,i~ble
MYELIN SHEATH STRUCTURES
(RADIAL DIRECTION)
A
~CZZZS ~
~CZZZ Z 2 Z 3 ~ *-q=XZZ ~
C
il li; i i B D
Fig. 6. Diagram from $ e h m i t t, B e a r and P a 1 m e r [1Sl showing four po,s,sible arrangements of lipid and pr.otein in the repeating u~it of the myelin sheath. The
lipid non-polar chains are indieate,d by the tuning fork symbols.
t,o Lsay fI:om t'he measu red eapaci , iy Jt'ha,t ~t,t~e 't~riek~ress ~o,f ~he merrrbrane pro'ht~bly ,died .~o.t ,exceed 100 A. H~owever, .t~here were ,mea~s,ureaTrent~s ~of ere,c- tri,eal ,eap~re~ty ,of eer, tai,n ,mem~braams, n,otably ,o~f Jsk,etetial ,m~s,cle fibers i~n 'wl~i.eh ,t~h.e wal'u,es .o,f 4he ~n,ea~b.raaa.e ca,p,acity we:~e .such ,a,s ,tl~ ~su~gest ~a ,much grea~er ,fl~iek.rms.s.
In 1940 W a u g h and ,S c,h aa i t t [1 i] maAe s,orrr, e ,d41,ect ,mea~s,urement,s o,f the t'l~ick~ess :o,f red .'bl,o,od eel,1 .m, enrba, a.n~es using ,an ,i,r~s,tr~u.lrrextt call.ed t,tre "arrMyt`ical t~Ft,oseolp.e". Ttri,s inwolved e,ssent~ially a ,co~pLardsan rot i~he re- fle@ivi,ty o.f ,a igla,ss ~sur.face ,o,n wlri.e,h a ,meartbr, arm 'wa,s ,dri, ed. TGey ~rse,d the n~et,h,~dJs ,gha~t had ~been ,devel,o,p.ed Ib.y L a n , g . m ~ a : i r ,a.Ird B l o d g e l t [12] f,or ~,ald,rrg is,~ep fit.ms ,of 'b,arl~mn 'ste'a~a~e. In fllese filma,s ,bard, urn bst,earate ,is bui l t nO in st~e,p,s..on a g]Jalss ~s,~i,c[e ~it,h ,ea.eh ,s,~ep li~nvolving ~ll~e ,ad,di,t~ion o:f ,only ,(~im ,m,~Im,mo~eeul~ar filan. T,he r.etteet,iv~ty ,o.f eiaeh ~st,ep v,ar:i,ecl .in ~a ldnear fCa~s,l~i,on ~a~d the ,analytli.eal ].ep,go,seop,e was .essent4afly ~an ~i~nstru,ment v~hi.ch all~owecl ta co,rap,avis,on to 'be ~nade t) ,et~een 4he ref lect i~i ty ,o,f ,d,~i~ed re,d ,hlo,(~cl cell g~'hols~ ton a gla,ss s]$de a n d t'he r,e~ect,an.ce of ~a La.i~g, rrm.i~-Bfodg,~t't ~s,te.p- film. TJ~ey con.ef~ded f rom ~'he, ir smd.ies hhat ~he fresh ~a, sl~e,d r ed ,cell ,gh.o,st n~ean,hr,an, e ~.e,ats,ur~e,d alb.o,l~t 220 A !in ~,hiekn, ess an, d ,t'ha,t af~er ~Li.p,i,d ,extract ion it ~neas.ured .ah,o,ut 120 Y~. Tt~e ,d,iffie.u.lty wi~h tlai,s 'met,h,o,d, of .cettrse, was :the
Origin of the Unit Membrane C,oncept 223
pr,o!b.lean ,o,f removh~g ,all n,on-me,a~bra.n.o,t~s elen~ea,t~s ,t~ha~ rema~i,ned iat4.ache,d to fl:m maean~,rar~es ~,n .the ~r,ep,a~a:t~o~ .of e~he ~ed ,cell ghosts. T,h,e va lues o,bt, ali.ned w e r e hel,i.eved ~o, h,e ~dgh ]~,eca~,se ,o~f +his ,d,iffi,eufty.
E,a~}~er, S e h m i t t. B ,e a r ~ar~d P ,o n d e r i.n 1936 [13] h a d ,st.t~died ,the op~ea.1 or,o.pe~fies .of ih.e red ~l,o,o,cl .cell im.em,b~ane Sn ,polarized }igh,t ,and had dem,arts~t.r'ated .~ha t ~her.e wo~s a ,detlectaMe r a d i a l l y pos i t ive ~r,efr.i,ngence. So,me y~ear;s laCe:r, M i ~t e 'It i s o n [14] s,md~,ed '.~h.e .pr,o,p,e,r,~i,es }of l~ed Ib.lo,od ,cells ~in p,ol.arized li.ght anor:e .ex~e~s,ively a n d c(m,el~a4ed ~ha.t ~he rad,ial ~pos~i,Nvi~ty wa~s d u e t.o fo lded pr, o:tein ,eha,i~s ,arra~ge,d ma, di,atly ri,n ~a laye:r ~o.f t:he ,order o.f 0.1 t,o ~o.ne anSe.ron in thiekn.es.s. I t ~o.w algrpears, l to~ever , rthat t'his ,is no't
Table 1, Chemical Composition o/ JPuri/ied "Light" and "Heavy" Myelin.
Preparation no.
CHCla: CHaOI-I insoluble residue, % dry wt.
Proteolipid protein, % dry wt. Total lipid, % dry w~.
Cholesterol, % of total lipid Galac~olipids, % of total lipid Phospholipids, % of total lipid Plasmalogens, % of total lipid
"Light" myelin
I 2 3
1.1 1.1 0,8 21.2 21.1 21.6 77.7 77.8 77.6
25.9 26.8 28.3 28.2 29.8 29.7 42.3 43.0 43.6
- - 12.5 13.4
25.8 31.9 42.6 15.5
"Heavy" myelin
1 2 3
3.6 5.6 4,7 22.2 21.3 23.9 74,2 73.1 71.4
24.4 25.9 28.2 - - - - 29.4
41.4 -- 42.3 - - - - 13.6
5.8 23.7 70.5
25.1 29.3 44.] ' 13.8
s,o ,and t~hM :the h:ir,efr,i,n~en, ce ,de;tect.ed Iby S ,c :h an ~i t ~, 13 e a r ,a~d P o n d e r p~o~a~ly w~as [pro,dueecl .by ,,tLhe l,i~pii, d ~m, oiecuJ, e,s io.f !~h,e artei~br,a~e. Howeve r , t~ere s~ill mm.a,ins ,the 9.ossib~:~ity ~that rEhere i,s a ~ro~t~ial co~np;o,ne~t qa4t,a.ched ~,o tOae z, ed ,cell an, e~ raa , e t s :t,hM ~h.as detectable: r ad i a l l y ,po,st~ive hlir,efr,ing;e,n,ee ~ha,t s,i,m,ply ,adds 4,0 ~t,~a,t .o,f 4,he tip,~d.
I would l ike n o w .t.o ,t~urn ~LO anolther ~i.n,e ,of evi, d,ence r~lat ing to ,~Ge ~pr,o,blem of cell ,men*br,a,rm: s~truct,ure !fil~at [at first wa~s no,t quiJ~e Is,o ,dJi~,e,ct i,u its r d a - {io~sh,ilp ,~s we rmw k~n.ow ii;t &,o ,be. As a res/u}t of t,h,e ~s~tudli,es ,o,f W. J. S e h m i d t [15] .and ,of F. O . . S e h m i ~ t arrd ~a~s co,ffahorators [16, 17, 18J !i,n ,the la~te 1930's 4t was ,estahfi.s.hed tha t ,the io.W'i,e ia:vis ,of ~e rve r w.as va~d,i.al a n d ifllat tlh,ere was i,n'tr.ir~si.c .post,tire rla.dii,al birefr,~r~gen.e,e. T~hi,s po.sii,t~ve ra~c~i0al h,ir,efrir~g.en, e,e w,as fmrr~d ,to r,evemse tin s!i.gn 'up,o:n 4rea~men,t wi th li~p,id solven, ts [19]. Fr,oln ,su,eh ],ilnes ,o~f ,e:v~deace, W. J. S e,h,m i d t co,n.cl,trded .i.n 1937 [t5] .tha't ~t,he .my.e~in ;s~h!e,a~h was con:s.truot,ed o,f ,aft,ernatirrg layers ,of ~}iip/,d ,and ~0r,ot.ein .as ,i.n,d!i,eaf~ect ,in ,~he ,d~,~gram ~n Fdg. 5. I.n 1935, S .e,hm i ,t :t, B .e a r and C 1 .a r k [16] ,o~4~a~ned ~t.he fi.rs,t ,sin,all aa~gl,e X-r,ay di.ffract.i~o~a 9attterr~s frroan myel in . T h e y found ~ho:t ~t~ie ra,&i,al r e p e a t peri,od ~n f resh ,frrog ~ y e I i n ~ a s 171 A .a~d lin ~m,aan.m.al.i,an ~ay,eliat was 186 A. I~t was kn.oiwn froan var$o,us. &emi,eal st~a,d,ies ' that anye.}ixt .eo,r~tai:ned li,pid aa,d a pro'rein eall,ed .ne.u~,o.k.eratLn. T~a~s in 1942, S e h m i t r[, B ,e,a r ,and P a 1 m e r [18] we re ,ahl~e ~t,o 9ostu$a,te ,kl~a~t ~fJae r~d,i,M' .repeat~ia~g mri~t in ~lt~e ~ny, e}i,n sJaeMla mus,t con~t.ain .two t),}m,olecul,ar l~a,flets o,f ~i,pid ~i:th rass.ocialed ~n,on, o~ayers
Tab
le 2
. C
once
ntra
tion
s o]
Lip
ids
in
Gra
y M
atte
r,
Whi
te M
atte
r,
and
Mye
lin
o/
Hu
ma
n
Bra
ins.
..
..
..
1
0-m
on
th
old
6-y
r ol
d 9
-yr
old
Gra
y
Wh
ite
Gra
y
Wh
ite
Gra
y
Wh
ite
Gra
y
I M
atte
r M
atte
r M
atte
r M
atte
r M
atte
r M
atte
r M
atte
r M
yel
in
My
elin
M
yel
in
Wa
ter
To
tal
lipi
d
No
nli
pid
re
sidu
e T
ota
l g
lyce
rop
ho
sph
atid
es 1
To
tal
sph
ing
oli
pid
s ~
Un
iden
tifi
ed
Cho
lest
erol
E
than
e]am
ine
gly
cero
ph
osp
hat
ides
S
erin
e g
lyce
rop
ho
sph
atid
es
Cho
line
g
lyce
rop
ho
sph
atid
es
Sp
hin
go
my
eliu
C
ereb
rosi
de
Cer
ebro
side
su
lfat
e C
eram
ide
84.1
36.4
63
.6
20.3
5.
1
3.0
7.9
6.8
2.8
10.8
1.8
1.8
0.7
0.8
80.8
49.0
51
.0
20.3
14
.3
2.9
11.5
9.4
2.4
8.6
2.1
8.5
2,5
1.1
m
78.0
22
.0
31.7
24.7
3.0
18.6
14
.2
5.5
12.1
4.6
13.7
5.
1
1.2
83.2
35
.8
64.2
22
.5
3.8
2.9
6.6
10.6
3.6
8.3
1.3
1.0
0.6
0.8
75.5
58.4
41
.6
20.4
19
,2
5.4
13.4
8.6
3.5
8.3
2.7
12.8
2.7
0.9
80.9
19
.1
24.6
28.6
6.
1 21
.5
11.3
4.2
9.1
4.4
19.2
3.9
1.1
85.8
37.6
62
.4
21.2
5.
6
3.5
7.2
9.6
2.7
9.0
2.8
1.9
0.4
0.5
77.4
66.3
33
.7
25.9
19
.9
7.3
13.2
12.0
5,
1 8.
8 4,
9
10.5
3.9
0.5
78.0
22.0
31
.9
25.0
2.
5
18.6
14
.2
5.5
12
.2
4.6
14.0
5.
1
1.3
82.3
39.6
60
.4
21.1
5.
5
5.8
7.2
9.2
2.9
9.0
1.9
2.3
0.8
0.5
All
val
ues
, ex
cep
t w
ater
, ar
e ex
pre
ssed
as
a
per
cen
tag
e of
th
e d
ry
wei
gh
t.
1 S
um
of
EG
P,
SG
P,
and
C
GP
. 2
Ex
clu
din
g
gaug
lios
ides
.
55-y
r ol
d
Wh
ite
Mat
ter
My
elin
75.2
--
64.6
78
.0
35,4
22
.0
21.5
24
.8
21.5
24
.5
6.5
~9.
0 15
.1
19.7
9.
1 1
1.2
4.2
5.3
8.2
8.3
5.2
4.4
12.5
16
.0
3.0
3.4
0.8
0.7
O
Origin of the Unit Membrane Concept 225
of p~mteirt ,o,f the ~g,e:nera,1 ty,p,e ,s,h,own lin the i r d,ia~gra,ms ,in F~ig. 6. Th,ere were a n u m b e r ro,f p,ossli,bd,l,i.ti~es f,o.r th.e sp,e,eific ~arra,ngement ~of 4,he 1,ip,id a n d n,o,n- li,p:i,d ,eomlpo,nents ,any ~o:ne o.f wd~ieh ,wonld ihave sa,tli,stied ,l~he, X - r a y d a t a t h e n a v,aiM,bfe.
Mor.e r~e,e,ent .eheml.eal la~a,alys,es ,of ~myeld.n h a v e .es,tal~il,ishe,d anore ,f,u.}ly t:he n,atur.e ,at :the ,fi~pd, d an,d ~noa-ldo!i,d ,eo~m~p~o,nents ,t,l~at ar'e !present. I,t ,i:s, kn~own tha t t]~e l,ip,ids ,are ,m.a:i,nly ~p,hos[plho,hlpd,d,s, eh.ol,eslt~er~o,l, .~aIaeto~}i,pids an~d
z - 7 0 + 2
r- J -q
U V Fig. ? a. Electron density plot for the repea,ting unit in rat sciatic myel'in from F i n e a n and B u r g e [26J. The two deep throughs neenr in the 1,oeation of the
two unit membranes. The origin "o" is at the center of the in,traperi.od line.
,w :~:.: .:::: ..::.: :.~,~:: ; ~ Cholest ero~
P 'T~ :;? :.i.! (ii:.[?~i: ]i: :'):::i;;~! P ~ P h o s p h o l i p [d
F~g. 7 b. Idealization of the electron density ctlrves for the myel.in layer of rat sciatic nerve in which thee }ocation of the 1.ipi,d Ml.ayer.s an the central p.ortions, of
the two unit membranes are indicated very s&ematically.
pl.asm~alo~gens, s p , h i ~ o myeli,n ,aa~d ins,o,s,itol ~p,ho:sp,h,a,t,i,d.e,s. Th,e pr:in, ei'p,al p,ho,s,pho,l, iRp,i,ds a r e phos,phatirdy] .eho]~in,e, p~h,os,p,h,altd,dy] ,e~han,ol amdne, ph,os- ph,a,ti,dyl s,erli,n,e. Tah,les 1 a,n,d 2 ,summ~arize ~the ],i~p,i,d e,o~mp.osi:f:i,on ,o,f nayelda as d,eterm.ined b y two J:eee,n,t in~es~tigatdoas [20, 211.
T/he p r o t e i n eo,mpio4n,en~t .o.f ,myefi:n cal.le,d n,ear, o,k,erati~n in 4Ge o,lder 1,itera- tur'e wa,s s 'lmwn b y lP ,o 1 .c h a n d L e B a r,o n [22] to 'be ~a ,degra'd.a;tion ,pro,duct of la ]?r~o~te!in ,a~hich ~!hey were lalbl.e ~to ,&,ara,et,eriz,e ,more ~ully .anld ish,ow ~fo ,b,e a oolm;po,ne,nt ,of a ,ela,ss ,of li~p~oLpro~te'ins ref,erxed ,to ,as ,pro,~e,olirpd~d~s. Pr,o4,eo- ].i,p.ilds a r e ,o,per, atio,n.ally ,define,d a,s a ~kiitd of lipo~prote,in w}~ieh d,s .ex4r.ae,ta~ble fr.om ;tiis,s.ues ,by a ~n~ixt,ure ~o~f ehloro,f,orm and a~etihanol ,in ~,h,e r,a~io .o.f 2: i. Mo,st ]:i,pLo,pro,t,e!ias are wa'tier s,o.lu~b.~e, b,u,t ~h4s k:i~,cl of li.pop~,ote,in li,s no,t ,and for til~is r,eas~on ,the ~.erm ,pr.o,teohpd~d w~s inven ted . T~tm ehenfi,eal com~p.osi~ion of .pr,oteo,l'i~p!~ds h,a,s ~.e,en ,ex,tensiv,ely stu.d!ied. T h e ~i,p,ild .ec~mp,one~ts ar,e v e r y s imi la r t,o ~ho,s,e o,f ,my,el.i~n and ,i~d.e,ed, i~ ~he mos t ~p,ure mye]~in fra,oti,ons a.hn~o,s,t 160% o~f ~lh.e ~pro~tei~n i's pr.o~teol,i~p,i~d proteiin. Reoer~tly, a,s ~the r,es'u~ of w o r k Joy M a t s u m o t o and M a t s u m o t o I25[ and T e n n e n J o a u I n [24],
P r o t o p l a s m a , L X l I I / 1 - - 3 15
:~e6 ]. D. Roberts on
it has been found poss ib le to r e m o v e the l ip id c o m p o n e n t ].caving b e h i n d t~he ,protein .in an undenatur ,ed crate i.n ithe ,s,e:n.s,e :that i t ,is st:ill .ehlor.oform- m, et~hano:l ,s,ol,uble. I t i.s, howe.vet, v e r y ,e,asiIy de,na.mr,ed 'by hea t , is, o,me dry,ing pr, o,eedures, ebe. T,be pro,t,ein i,s ,a~so wa' ter .s,olnbie ~ar~d this .opens up n u m e r o u s kinds ,of ..phys:ieal, chemical and ~b~i.o,physl}e,al ,s.i~di.es tha,t are n.ow ju~s:t ,begin- ning. The .am~in.o ,a,eid c,o~po~ss ,o.f ,a ,s,a.m:p}e o.f s,ueh pr,o,{eol,i,p,id Ipro~t,e'in is g iven in T a b l e 3 I251. This p r o t e o l i p i d p r o t e i n m a y tu rn out to be a struct~ural ,pr,o,t,e,in o,f .s!ignifieanee ,i,n a l l ,me,m,branes.
aJpid
Pro/eln ,eea.
Fig. 8. Diagram from F i n e a n [281 showing his conception of the arrangement of molecules in the radial repeating unit of myelin.
i{.eeent X - r a y i&i.ffra,e~t~Lo,n stfudi.e,s of n e r v e Imy,elin Ih,av, e led ,t,o .a refin,e.m,ent of ,the e a r l y aal.o,cl.els pr,ogo.sed b y S e h m i t f, B e a r ,al~d P a 1 m ,e r (Fig. 6) and a plo4 ,o,f it,he ,el,eetron ,d,ensitfy ,&i,str,i'bufFi,on :in the r,epeat,ing u.ni:f 'ha,s no.w been m,ade :to ~a reso.lu,f:i,o,n ,o:f abo,u,t 39 A. This w.~s. d,o.ne i i rde.pendent ly b y F i n e a I1 ,a=~d B u r g .e [26] ~aird 'by M .o .o d y [271. T h e .electron d.en,si~y p l o t of ~t,he r.e~peat~irrg ,u~Li~t g,iv,e,n ,by F ~i n e a n ,a~nd B u r .g .e ~s ~shown in Figs. 7 a and 7 b. Fro,m ~,he k n o w n ,d.eIrs,ilty ~of 't,h.e :pr:obein arid ~i.p:i,d .emnponel~t~s p m s e m in t,he ,mpe~in ish~elalth, ~lt ,i,s :now p,o,s,si.b,~e ,to s,ay fth, a t :the ,ffene.ral ,arr ,angement of ~the $ip,i,d .a,Ird non-lig;id eo.mpo,rre.nt:s ,given ,in F.ig. 8, t a k e n fr,mn an ea r | i e r pa,per b y F :i n e,a n [27], i.s ,esserr~ially eorr,ect. I t :shoquld b.e .noted t ha t t:be d.etaS~ed arran,g,ements ,such .a;s ,the ,s.hape o.f ~{he sphi.rrgo,mye.Iin :molecule an,d po,s'ilti,o~ .of ,elro].e,s,t,erol .~iven !~n .~he ,di,agrmn r.emai:n p u r e l y spe,eulat ive.
Origin of the Unit Membrane Co.ncept 227
Desp,i:te ;i~t,s ~l,im~itations ;thee e l e c t r o n ,dens i ty 'p,lo~t n o w ~prm~i~des ~t:he stre.nges:t ,e~i~cl,ence ~c,f ,the g e n e r a l .eorre.etrre.s,s ,o,f 'the .mc~d,el ,even ~t,h,on~gh :i,t was firs,t p r o - pc~s,e,d Jby F i n e .a n :in ,the lr~id 1950's [281 b e f o r e ~t,h,e r,e,ee,rrt X - r a y Id~ffra.eti,on e~i,dene.e wa.s a,zalifable. T h e m.o,d.el r,es,ul,tecl . f rom ,a ,eorrs~i,derati,on ,of .t,he earli.e,r X - r a y finfd~ilrg,s .o,f .S e h m :i 't d ,at ,al.; n e w e r res'u.tts ,of Fli n ,e a n anicl eow, o.rkerls ,as we l l as evMen,ee wki.eh w.a~s .d,eri~eJ , exc lus ive ly froan e l e c t r o n mier'o,s,co~py. I.n f ac t , :i,t se,em,s .fa,ir !to s a y f l int , ~in ithi,s ,i.r~st.an,ce, rthe eleetr~on mi.ero~sdeo~pile ,e~i, denee pro,~ide,d ~h~e h,est 5as!i.s f o r tJhe .nro,clef w h e n ,d.t w as orig~i,nally ~adv,aneecl ar rd w.e ~thu~s th.ave ,erie :i,n,st~a~ee ~in whi.eh 4d,ireet e.lee'tr,on mieresc.oop:~e ,e~ ideaee Ih.a,s le,d 'to a imo,~eeul.ar ano,d.el whi.eh ]aa,s h.e,en ~.u]> sequ ,en t ly e,onfirlmed b y X - r a y diffr,aetl~on. T]~i~ d~s n,at a ~rri.que si.tuati,o,n, h o w e v e r , .s,i.ne,e .the .int,e~rplay :of e l e c t r o n ~mi.ero,s,eo.pde ~a'rrcl X - r a y Id:iffraet,ion st'u, cli.es ,inwo,lvelcl ,in :the ewolu.t:i,o,n o,f o,ur ,eurren,t .i,d,e'as ,of ~us .e le stru.cXqlre p r o v i d e .ano, t,ker ,/,n.stanee dn wtrieh :i:t :i,s fd.iffieul't t,o 's,ay vztri,eh l i ne ,o,f ,evid.enee w.a.s me.s,t :i~n~plo,r't, ant .
[ rsih.o~ul, d l ike ,t,o . turn no,w anor,e s p e c i f i c a l l y t~o .tlh,e .el~eetron m:i,er.o,s,e.o.Fe s~t~t~d:i.e~ o~f n e r v e m y e l i n ~an~d ,i.rrclieale h,ow ~h,ey .are relate ,d ~,o, t h e ewo.l~u,t,io.n o.f 'the: g.enera,1 ~no~eeul.ar mo, clre] a n d Ira t h e ~g, en.er'a.1 !pr:ot)~ean .of meanI ) rane stru~c:/~u:r,e. I't was a.ss.umecl ,by S e h m :i ;t t .a~d la~i.s eo.wo:rk.ers, dn rt:he ia,t,e ~ ine- te,en ~th,irties :as we l l ,a.s o the r s work~ing ~in th,e fiel,cl t.hat/3r~o~b.lems o.f n e r v e myel]'in ,s~tru.eture ,were r .e ievant 'to. 't~ho,s,e ~o,f .eel] ~neanhran.e s . trn,cture ]9.ee,aus~e repel:in ~seern~ecl t.o ]3,e ~i.n s a m e w a y ,r,e~l, aftecl to m . e m b r a n e p~he,nom,ena. I:t was c lear , fo r ~i,n.s~t~anee, , that its ~pr,e~sen,ee la.a;d ,s.o,meth,ing k,o ~d.o. wit]a eo.n~cluet,i,on velo~ei~ty .'in per~}pheral myelin.a,te,d fibbers. H o w e v e r , tlhe ~e~caclt ~ a y in w h ieh i t was~ rda. tecl ,eo,uicl n.o~t 'b,e .exp~iei:tly s~t,ate,cl u n t i l ee.rt:a'in .el,e,etr,on mi,er~),s,ea,pe smdie.s ha, el ~b,e,en eea~le . te , d. T h e firs-[ ,s.te,p .in u.n,d,er.s,tan, cl;i~ng ,tlh[e n~,orp.ho- log ica l prrobl,ems ~eaxne, n,o,t f roan e l e c t r o n micro . scope stn, cl'i~es .of n~yel:in, b u t ra: ther fr,o~m s~tu, cl~i.e.s o.f n . o n - m y e l i n a t e d n e r v e fiber,s earri , ed. ~u,t ~ y G a s ,s e r in ,t~e .ear ly 1950's. Af:ter ,a lo.n,g a n d f rn i t f ,u l c a r e e r in phys,i~ol.o,gy, G a s s e r turne,ct ,h~!s dalan,d to. e~e.etron m~iems~e,c~py a b o u t i9'50 ]o.e,eau:s,e h,e w a n t e d t.o kllo,W .s,c~me.th'ing a,bou:t , t ie , s t ruc tu re ~o,f ~ fibers. U~p to fish,at ~ti~m,e, i~t h a d b e e n 13,el~i~eved ,that '~ fi~bers, o r n, on-myeli,nate~cl pe . r Jphera l n e r v e fib,ers, eo.ns.i,s,t.ecl of ]oun, cl,les ,of .axoa~s wh ich w e r e encelo.se,cl in , syney t i a i S.ehw.ann cells. T h e S . ehwann cel ts w e r e eoaee:iv,ed ,t,o 1).e ar'r~a~a~ge, cl a,s v, e r y long syn.eyti lal tu'b.e:s. T~he ax,o~,s w e r e tlh,o.u,gh~t 4o h,e: eom,p l e t e ly ,inelu, d, ed dn 4~he s y n e y t i a l cel l anas,ses. H,o,wev.er, 'i,n 1952, ,at ~tla.e C,o.ld S~prri,r~g H,arb,or .Syanp~o~si.um [291, G a .s ,s e r gave t 'he firs't rep ,or t ~o,f ~h~i,s {in, dd~n,g~s and: showed ' tlEat ~,h~e ax, on,s were no.t 'J~s,ol.ated w~i~fahi.n ~tre Sehw.a~n~ ,cell,s, Ib,ut ra,f~h~er tlaat ea,eh .wa~s e,on- neetecl ,~ro thee ,onlter s.urfa~ee ,o,f t h e S e h w a n n ee]~l Iby a ,tenu,o,u,s anemlb,ranous s t ru .e inre wh~i.eh he eon.ee,i,~ed ,o~f ~n gro,s.s m,o.rph~o,~ogieal :t,er~'s .as t).e,i~g a kd.nd ro.f anesen~t'ery, n,o,t .u.n,]~ik,e 't~a.at ,w~a~eh eo.rtrreets tthe {,~t.es~t,i,n,e wi4,1a t~he t),o,dy w a l l dn ~er~t,ebr~age,s. H e i therefore , :in.vented it,h~e: if,eran ~ for' t~h~is s~p,eeial s:trn.et'ure. S,u'b~sequelrt stu~d;i,es b y G a s s e r ~w.~h t,h,e a;s,s!isttanee .of P a 1 a ,d e [3.0], resM,t~d ,in .t.he c l e a r ,d,e'morrs~tra~ti,on ct,h,at fla,e me , saxon eon- s.ist:ecl s i m p l y .o,f .tah,e S,ehwa,nn ce l l .s'urf,a.ee ~menab~a~e 'inv.agfin.a4,ecl ~and e x - t.en, cl,ed ,to en.&ose th.e ,axo,n ,eo~npletely. L~f.er ,on ~i,t w a s ~sh.o~vn b y .t~h~e a u t h o r [31] :th~a't S ,ehwann ,cell ~neanbranes e o ~ i , st of a ..pair ,o~f ,den,s.e 1.ines
15"
228 ]. D. Roberts.on
each ,a~b,out 20 A tkiek separ'a,t.ed ~y ,a 1}i.g~ht een,tra.1 z,o,rm about 35 5~ ,a~er~o,ss t,o wh'ieh Lke ~s,p,ee!i.al ~e.rm "m~}t membr,ane" 'was ,a.plpl,ied. Tihe rel.a~t,ion~sh~i~p,s o.f ;t,t~e un i t ,me~xbr,a,nes .i,n no~n-~nye~ina~te, d nerve fibers ~ar,e ;as; ,sh,own ~in F,ig. 9. As the d!iagra~m ,i,ndieate,s, n,o,n,my,elli~n,~te, d n~rve: fi~bem,s eons4~s:t .of .S,chwann cel.~s w~it,h w,hl!,eh .axo,n,s ar.e relatte, d ~t ~t,h,e var,~ety ,o,f way,s shown. S,o,me ,of t'he axo,ns ~slimply fie ,in ,a,pp,o,si,ti.o,n wi~t~h .the S,ehwann ,eell,s wh,ile ,oit,her~s are pushed down ,int,o ,t,l~e~ ,to v a r y i n g ,&egre,es. W~i,th itlh,e ones ly~i.ng deepe,st, the ,tw,o enve~o.p.ing slheat.h,s ,o,f S.ehw~ann eyt, o~p]~a,san ~eame t.oge~t~her s~o tam/ t,heir .membranes ~i,e in .el,~s,e ap:po,si,t,i,o~n tto m a k e :the mesaxen. In ,adul, t fiS,er~s
Fig. 9. Diagram of a xert.ebrate unmyelinated nerve fiber. See text.
ther;e ,]~s .a ~,e.marka.ble unif.orani,ty !in {~h,e spacing ]3,etween :l.hLe tw, a .S.chwann cell me~mbr:anes !in the me,saxon w~h~er:e t.hey ,are s,eparat.ed 'by .a~b.o.uf 100 t.o 15/) A. Th~s .s,epar,ati,on is als.o .seen 5.etwe,en th, e S.d~w,ann cell ,mebmbr,ane and the ax.on m,emlbranes. Ther,e i~s t~hus a dir'eot ~pa,thway be tween tile surfae.e of tihe axon ,and ,the ,onts.i,d,e 'by m,e~an=s ,of t~his ~in.t.er-,m~embra,ne g~ap. T,he gap i.s c.o,nt.i:n.uro,U;S wi.th .ex.tr,aee,ll:ul.ar ,s,ubstanee. There ,i,s a cm~dens,at:i(on ,o,[ ex t r a - eetl'u}ar ,mater,ial .a'ro,unid .~he S,dlwann eel,l tha t ,i,s J varia~bSe th,iEd~ness and var!i.ab~e dens}ty dep.e~,dling ~'p,o,n how ,t.h,e ma{e~i,al i,s tr,eate, d. T,h:i.s &, shown by .~h.e l.ig,ht s.t, ipp}ir~g ,in F,ig. 9 wh}eh ,d,e~p~iets the .app,ear,anee .o,f flh,e so-eall.ed '%asem'ent m e m b r a n e " ,o.r '~ 1,an~na". The dr.awing wa~s .eo,r~s{ru,ei, ed mailnly fr,mn ~o~b,s,er, v,at~i,on=s o,f ,per,mang,ana{.e f ixed .materli, al ~an, d 'tLhe ~,as,emen,i ]am}na was aee,ordingly .s,o,m ewha t d,e-~e;m~pha,sliz,ed. If ,i,s .much ~more pr,o,min,en{ in 'OsO4 f ixed 'mat,er~ia,1 .and w:ith lead sta.i,rdng :it,s .pr,m~i,n,enee is fur,i~er enha.need. The re .are go,o,d r,eas,o~ns ,t.o ~b,eli.eve, ~b.eoa.u,s~e ,of ,sta:i,I~ing ,ehara.ete'r- i,stics, tha,t ,th,e: ba.s,emen,t fam.lina ii.s a ,e,on.de.l~s,ati.o,n ,of ex, teae,elfuil~ar ~s,ubs,i.an,ee w~t~h a h.i.gh ,Coherent :o.f mueo!poly,saeehar:~des. In m y <~p.ini,o.n, '~/:s suSsi'anee grades ,off ,~mper,ce~pttbly ~in~o 'd~e .exir,aeellular co,nt~numn a n d lalso ~im.o the gap ~in .{ihe ,mesax,o,n and the ,s.pa,oe ]~etween {he ax.o.n .airtd ,the .Sehwann cell
Origin of the Unit Membrane Co.ncept 229
m e m b r a n e s . I thus conceive o.f the gap subs tance as b.eirtg re la ted to the bas,ement .mem,br.ane s.ub,stanee and the genera l in te rce l lu la r m a t r i x . Thus ~t i,s pro,b.aBly hi,gh in muco,p.o.lysacehar.~de and mu,eo,prot.ein. The re is also good ovid.once for a s suming tha t it ~i.s abou t 90 % h y d r a t e d as I shall ind ica te la.ter.
Fig. 10. Reprnduction of the first micrograph sh.owing an outer and inner mesaxon in a myel,inated nerve fiber. This was obtained by the author in 1954 [45].
Mag. X 176,00'0.
In 19'54 G .e r e n [52] b,e,g~an a s t u d y .of the f.orma~ion o.f ne rve m y e l i n in eh,iek periLp,heral n, er~es. At .early s tages be fo re the a p p e a r a n c e of mye l in , s,h,e ,o~b.s~erv.ed t4h,at there were numb,ors of n ,on-myel ina ted ne rve fibers h a v i n g th,e g,eneral s:tr'ue.mr,e es.ta,bli.sh,ed b y G a s s .e r ]).ut in which there was on ly o.ne .Sehwan,n cell pe r ,ax.o.n. This led .her t,o po,s,t'ulat:e t ha t m y e l i n might , in fa,ct, , s imply b,e f .ormed b y .s,p.ira,1 wingding o,f a me,saxon ar,o,u.n,d the axo,n wi th .ee.n,d,ens,atio,n .}n.t,o t~he ,eo,mpaot my.el, in ,s,trueture. Thi,s was a ~ e r y in.t~eresting thy.p,otthesi,s, b u t .o,n t,h,e ev.i,d,en.ee ava,ila,bl,e a t the t ime, if wa,s qui.te p.o,s,s,ibl,e tha.t no.n-.membrano.n,s comp,on, ents were la id d,own ,between .the mem,branes .o,f t~h.e mes,axo,n 1,o,o,ps as m y e l i n fo,rmed even ,if the s ,piral l ing o.f ~'h,e mes,ax,o~n wa,s eo,nt,inu,ed d u r i n g devel, o,pment. The first s tep in .e,s.tab- ]~is,l~i:rtg .t,his hypo thes i s e,ame wit:h ,d,em,ons.trat,i,on of an ouLer and an inner mes.ax.o,n a,~ta,ch, ed to .eomp.aet m y e l i n .i,n a l i za rd m y e l i n a t e d n e r v e fi~b,er f rom
230 J .D . Robertson
the au thor ' s [,ahox,a.tory ~a f,ew ,mo,n.~hs aft.er G e 1" e,n',s hypot~h,esi,s wa~s ~d- w n e e d . T h e .o,riginlal ~mlier,o,g.ra~.ph li,s' ,r~epmd'u,eed ,in F~i,g. 10. T~h,e r,e,la~ti,o,nshi~p,s o,b,s..er~ed ,i,n th,is ',mi.cr,o!gr,a~ph str.o.rrg,ly .s.ugg~st,ed t h a t m y e l i n wa,s f o r m e d ,by a eo,nt:inluing re.labor,at,ion of ,tl]cm ,sp,isal Lmes~axon iseen ~in interlmed~iafe fi,b,er,s. I t wa:s :alts,o i m p o r t a n t ,idn Ithi~s ,c,oknnect~i,on tha t tlhe ~o;ut,er ,m.esax,on .ertt,er,ed the myel~in in a dire.cf,io,n .op,p,o}sli,t,e ,to. t h a t ,of ~tlhe 4~nn, er ane,s,ax,o,n. [.t was ,al,s,o p,o,ssibl:e .frem ,t,he r,el.at,io,ns.hip.s ,o,b,s,erv,ed :to ,st,alte witir tea,so,nab,re ,eerta.inty th~at :no extra-m,enlbran:ous ,mat,er4a.} 'was l~a~i,d d o w n fin tire ,cyto~pla,s:m hetwe,en the m e s a x o n lo,o,ps a,s the myefi:n ,s,he~a:~h ev,olve,d. Thins w a s ;ap,par,errt fr,oun the f~aet t h a t t~he m a jor dense l~ine ,m,ela,sur, ed o,nly ,a,b,ou{ 30 A li,n tlhi&nes~s, and was 'f,ormed b y .the ,int~imat~e ~appio,siti,o~n o.f .one ~of ,tlhe: Sehwann .cell .mean- br:ane.s .e~f rtll:e m,es,ax.o~n wdtir tlhe co,mpa.ct ~myelin. I,t was noit iposLs'ib,~e at !the t ime, howeve r , ~t,o ,st.a~e w~tuh ,eerta,ilrty t ha t the myell in wa,s ~matd~e ,o~nly .o.f S.ehwann ceil rnem,brane m:aterd,al ,b,e,cau,s,e t.he S,ehwann cell imel]rrbrane:s w,e,r,e v e r y fuz.zy :in ,ap,peaxanee .and .of ,i.n, defin'ite thiekn, es~s. Tire r'el,a=t:io~s.l~ip ,of the ~membral]eS m. t~e in~tra~perioJd fine o,f the ,r,epea,t!ing myel'i,n stru.et~ur,e eo,u}d not ,b,e mad.e ,o,ut wLt,h ,cl~arlity par , f ly ,b,eeau,s,e :the S.ehwann cell m~e.mbran.e ,at the su r face ,of r Sehwan,n cell ~extern.a.lly wa,s v e r y ,fuzzy and lin~d:efin,ite ~in app,ea.ran,ee. I~t was a~ppar,ent t h a t i t measur ,ed less t h a n 100 A :in t!hiek~ess bu t t ire pro,bfem wa,s ,how nmeh fes,s. In so,me places , it seemed t~o. ~m~ea,s,ure wel l unde r 50A. T h e ~po~s,sibi}ity t,hu~s r.ema,ined t 'hat extra-m,embrano,u.s m a t e r i a l m!~ghi b,e .aidd,ed ,between ,tlhe ,outside surfaee..s ,o,f the ~mes.ax, o.n m e m - b ranes ,a,s my~elin .ev,o~v,ed.
Tih,e ,s,olu;~ion ~of ~t~he pro,blem awa.ited ~he ,d.ev.eJop~ent of perman,g~anate f ixa t ion b y L u f t [531 w h o i n d e p e n d e n t l y r ed i scove red it in 1956 "~ as a su i tab le fixati ,ze f~o.r mem,brano,us s t ruc tures . Alt,h,o~ugh ~he t y p i c a l urri~t m,em- br!an.e p a t t e r n ~ a d ,b.een seen ~n o.u:r .la,b,ora~ory in 1954 4n ,squ,i,d n e r v e fib,er~s a:fter OsO4 fix.at!i,o.n :al~d ~n.ethacry~at.e ~embed~di.n,g, ,thi,s .a, pp,ear~nee w a s n,ot u.sually s.e,en ;and !its ' import ,anee was not a,t fir,st ap,precia~ed. Itt w a s .only a~t,er it w~s ~se, en in the m e s a x o n ,in refa~io'n to my~el.i~n ~that ~it!s ,stgn,ifiean,ce b e c a m e .e~e,ar.
As ~so.on .a.s we: ,b,egan 4.o ~us,e p.er:mang.an~e 'as a fixi~ng a~ent , .an, d ar,al~d~ife for em]3e, d,d:ing, ithe uni,t me:mtbrane p ,a t tern ;~ppeareld reg'u~ar,ly in aH .our eIeetr~on ,m'icro,graphs. F'/,g. 11 ris ,a ~mi.cr,o,grap~h ,of ,a hu~m~an r,ed ,b~o,o,d ,eel.1 t h a t slmw,s tlh,e ,u~nit mean,b.ra,n,e ~t 4ts su r face . ,Fi,g. 12 s,lmws tire two, un'it ane~n- b ranes .o,f .a mes,ax,on ,in ,a rmn-~npe,l~i,n~ted ~n,erv, e fi,b,er and F,i,g. 15 ,siro,wls ,t,he r'eI~a.tiorrsihilp,s ,og the tw.o ,un,it .m.embr:an,es to .eo~mpa,et ,myeltin in ,a tmyelina~t:in~g m.o,use ,seiat~e ne rve fi,b,er. Such ,m~i,cmgra~phs .a,s ,thins ,alto,wed an~ean:irrg to ,b.e a t tached t~o the u.:ni,t anembrane pat t tern a,r~d .h~,c~iea4,ed t~h~t th,i;s ~p,a.it, e rn , of the var;i,o~ns one.s ,o,b~se,rwed ~[n ~me,mbr,altes, wa,s ,a isi,gnifica.nt ,one. T h e y ,showed eo,n.e~usively .lira4 t,Im lintr~a~per,i,od li~ne .o.f myeldn w,a,s fm~me, d ,by t~he ;in,t,imMe a~Fpo's!i:t~i.on ,o~f tire 'two ,o,u.t,s~id,e: ,d~nse ,s,tra, ta .of :~h,e ,mtit membrane ,s tuek,ed ~n a t 4ire su r f ace ~of .the ,S,ehwa,n,n ,eel1 ,to ,make tire m,esaxon. T,hey ,eo,nfirm, eid the fact , ,sa~g,g, est~ed b y O.sO4 fix.a4,i~on, t ha t t~he 'maj,oa" Id, errs,e ]~in, e ,i,s, f;or,~ed b y the :intd~mat,e ,app,ositi,o,n ,o.f t he ,two ~irrsd~d,e ,dens,e s4rat~a ,of the itrlil,a.m,inar
a P.erm,an,g'a'n;a~te was o.r~ig,~n~ally des,eribed as a fixative for nerve in t.he last century by B e t h e.
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232 J .D . Roberts on
u~i~t. ~fih,e i.m.p,ortant e.onel~u~si,o,n w.a~s then r,e.ael~e,d t h a t extr.a-memb.ran.o~us ma.ter.ial was ~ot ine}u, ded d,u.r,i.ng 't.h,e f.o.rama.tli.o~a !o,f mpeI,i,n for there was roLam ,e,n~y {,or the .otb~serve,d rnembr:an,e ~mater~al. In,deed, ,t,kere i~s, ,if ,anythintg, a ~s[iglht .eoa.tra.et,ion ~o.f the uni,t mem~brane~s a,s they ,ent,e, ,the ,eoan,paet myel,in. I t wa.s a p p a r ' e n t fro,m these el,e:etr.o.n mi.er.o.seo.pieal s tudies t ha t the r e p e a t i n g unit in the myel ' in shea th wa.s s i m p l y the mes.axon and no.thing else. i t was
Fig. 12. Portion of a nonmyelinated nerve fiber in m.ouse seiatdc nerve showing the unit membrane of the axon and of the Schwann cell. A porti.on of a mesaxon appears in the upper center. Note the gap between the two unit membranes.
Mag. X 12'8,000.
fu r the r .a,pp:ar,ent on g,ro~u~rLd.s o,f ,sym~metry tha t ,this mu,st eorr, e,s~pon.d t,o the ra.d~ial repe,a~ting un.i.t ,defected in 'myel in b y X - r a y ,diffraot'i.o,n ,s,tu,die,s. To be .sure, the ,d.imensions wer,e r,e,d.uee,d b.eeause .o,f ,shr'inka, ge d,uring the lore- para.tLo,ry proeedur,e,s. T h e exa.et ~degr.ee .of .shr inkage iilvo~v,e,d was .st ud.i,ed b y F i n e a n [34] an~d l a i e r b y F . e r n a n d e z - M o r a n and F i n . e a n [35] b y a eombd~ae, d X - r a y ,di,ffraet~i.oa an.d elee.tron mier,oseo,p,ie ,study t h a t was of va lue in ,su,p,port,ing the b,el.iJ th,a,t the reduet, i.on 4n the r ad i a l r e p e a t fro,m ab,o,u.t 170 -185 A t,o. ,a.b,ou.t 100 120 A was due t.o ,shl, ink,age du,ring pr,e- p,armi.o,n. F,itg. 14 sum,marti:z,eLs .the sJtepp~s ~i,n the format ' i~n .of n e r v e ,myelin and have been w o r k e d out m a i n l y front s tudies of mouse sciat ic ne rve fibers du r ing the first f ew d a y s a f t e r b i r th .
Fr,o,m file above, i~ was po,s,si~ble to m a k e some ,d,e, duetio~l~s ,abom th.e .under- lyialig m'oleeu~ar .ar rangeanent r,e,s.p,o~i~s,i,bfe fo,r the unlit ~membrane ~patiern.
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0.
234 j . D . Robertson
The d.i,men~sio,ns and .t,he r,ela, t.im~s:h~p t,o m y e l i n aI].o.w,ed one :t,o s,ay ,th:at t~he urrd,er]ying m~ofecul.ar ~pattern w a s f ike ly t.o Ib,e !one ,of 4he ~tlr,ee p at4errrs ,in F~i.g. 15 a - c . A.s f a r as .th,e ehelrdeal a n d ,bi.oplayls,i,eal ev idence w,errt, ,at ,that t ime role e m d d no~t .eho.o,se h,etw.een :these th ree 9o,s,siiibdfitie,s. H~ow,ewer, ,studies of mc, clel sys,tem,s o,f pur,e ]lilp~i.d,s a.rrd ~pr.o4ei~ns so,on p.o~i.ntecl 't,o .f}m .aoCrect eh.e,i,ee .in t,he la,t.e ~[950's. I'i wa.s.w.e.ll kno,wn tha,t pr,o,min m,o~l,eeul,e,s, aft,er fixa+ion, embedddng and s,eeti,o,ning a p p e a r e d as rMh,er u r d f o r m den s,i,tie,s 4n
Schwann Schwann cy'toploem
~ / / _. _ C y t O p l a s m
AXOn
�9 i ' t membrane ~ ~ u n i t g(~p/ membrane membrone gczp membrone
0 b c
Fig'. l& Diagram summarizing the steps in the formation of nerve myelin. The mesaxon is shown .at "m." Nnte that the intraperiod line originates from appositi.on of the two out~s.ide surfaces .of the unit membranes of the me.saxon and the intra-
period 1.ine by apposition of the cytoplasmic surfaces of the mes,axon loops.
~ao~
~PO~
" , /X/VX/
b c
Fig. 15. The appearance of the unit membrane in electron micrographs is .shown to the left with the approximate dimensions. Three poss{ble moleeul,ar configura- tions are shown in a, b and c. The zig-zag lines represent non-l,ipid monol,ayers; the circles represent lipid polar heads and the, bars represent the non-polar carbon
chains of the lipid molecules.
ele.etro,n m~i,crographs, as, for inst,ance, in co[l.agen fi:ber~s or mvofiLam,enf,s. Not,e, f.o,r ,ins,tance, the r a the r u n i f o r m d e n s i t y o.f tlhe cryMa] ,o.[ ,ehy,m.o- tryps~inoge:n .in Fig. 16.
T~his ~spe.eimen was fix,ed, .mr~bedded and ,sectioned dn the s a m e w a y ,that a t i ssue .s'p.ec.ian,ell w.o,uld g a v e b,een handl,ed. O f eourse, tpr,es,eal4 Ld~ay anethods m'ig~t .b.e ex,p.ee~ed t=o y ie ld ~so,m.e signs o.[ . internal ,s.trneture .in ~s~,eh a e,rys.tal, bu t tki,s pr,epara'tio.n was .daaract,eristi,e ,of the o,ne,s availal),Le ,a't the t,irne b,ef.o~e 1960. The pietur,e o.bbtained fr,mn a l ip id ~st>eeimen was , howeve r , a t t ha t d,me a.s a't the p re sen t t ime, r;ad,ieally differ,ent. Ins ' toad o~f a ~no.r,e or l,e,ss u n i f o r m ,density .or g r a n u l a r a .ppearance , t he 1,ipid .sp,ee~me.n ap,p,ea.r,e,d as a s y s t e m ,o,[ r e g u l a r r e p e a t i n g ,den.s,e ,and 1.i~ht lines as in F~i,g. 17 whi.eh sh,ow,s a ~s!p,e,ei'nle,n of egg k e p h a l i n f ixed v~ihh 'Os'O~ ,e~n.bed~ded ,in ar:aldit,e, an'd ~sec-
O
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16
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Mag
. X
l,040
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.
Origin of the Unit Membrane Co,neept 237
t4one,d. T i re ,d,en:se f ines .here rrep.eat a4 a per i ,od .obf .ab.ou,t 40 A. T,hi,s rep ,ea t per'to, el ~i,s ,a'b,o~u't tire :same a.s tthe o.n,e f~o,und ~n .such ,sp.e,e~rrrens 5 y X - r a y d,J~f- fracr T,he g.erLer'al ,m,o,le'cular arra'nlge~mel~t ,of .su.eh lip'id,s ~,s k~nown go b.e Iik,e ~hat , shown .~n F~ig. 18 a. T~a, is i,s ,~tre ,mo,s,t e o ~ m o a ,fo,zm in vzhich 's,u,eh lilp,i~d~s .e~i,st. I t i.s .ealled the :smectic .s'~ate. C l e a r l y , t h e ,den.s,e :li'nes. s een ,in tire el~e,etr,o,n ~ticr.o,gra,ph~s anu.s4 r,e,pres,ent ,ei.t,Irer ,~he ~po,lar ,end,s .o;f .the .l~i,p!i~d m,o.]~e- cures o r t h e n, oln-,p~al~ar ,end,s ,of ,~he earb.on ,eha:hrs a,s lindi,ea.t.ed ~in b a n d c res,p,eetliwely. O n .the 5.a,s,i,s o,f tire cv~d:e.nee t~h.a't w e 'ira,d ,at t h a t t 'ime, ,any
Fig. 18. Lipid molecules as. in Fig. 15. "b" and "c" respectively sh.ow ~he po.ssible location of the dense strata in mierographs ,such as that in Fdg. 17 in relat'ion to
the polar and non-polar carbon chains of the lipid mo]eeul.es.
e,ne ,of the t,hree ,d~i~agrams i,ndiea~fecl tin F:i,g. 15 a - c eo.uld ex, pla~in tire urri t men4br:ane pwt~t,ern as .o,bs,erv.ecl ~in l~'i.g. 15 a ,prowid,ing eer t ,a in a.srs.u,m,p;t,i,oa~s w e r e ma,d,e. F:ies,t, ~t~e evi.den.ee ,se,em,eld q.u~ite a,dequ,at,e to ,a l low uts 't,o ass.ua~le t h a t ,pro.~e.ia .or .o,tirer aon-l'ip,id,s i spread .in ,a t h i n f i lm wo.u$d, ,in f i xed a n d e m b e d d e d mater , ia l , a,pp,ear ,a,s a ,tiri,n d e n s e :s,trat~u,m. Fro ,m F,i,gs. 1.6 a n d 17 i,t wa~s ,c~ear :tha,t ,ei,ther ' the a~i.g,ned ,p~ol,ar ~ll.e,ads ,or t h e no~n-p,o]~ar ,earbo,n .&ai,ns .of a ,l'ip~ifd mo.n.olaper wo,u~d .al,so a~ppear a,s a ~iri'.n ~dens,e .strat.um. I f -we as ,sumed .t,hat ,the .p,o.lar Irea,d,s wo,uld a p p e a r ~dens,e, mo, d,el ",a" (F~,g. 15) .co,uld ,be c o r r e c t ~sin, c,e .the ,d.enls,e Mr'ata fro~m h.o.t,h tire ,litp:i, cl ,ancl n,on-,llio!id w.o,ul,d summa,re . I f we a.s.s,umed, ,co~nv,ersc~|y, f i rm ' the n.on-.po]Jar ,ehMns w o u l d ,ap,pear d~en.s.e, "~ ,in F.igl 15 co,u.l,d .al~s,o, 33,e ,eorr,eet. I tow,ever , width ei.t,her .as:su~np- tio,n '~ c o u l d h,e ,eft,mini,areal heea~l,se w,e sLiro,ufd ,ei:$h, er .see t~tLis ,as a ,s.in, gle ,d.e~s,e ,st.ra~tu~m .or a,s t h r e e ,eloJse,ly , spaced ,d,er~s,e ~st~ata each s e p a r a g e d 5 y 1,i,ght h,a,n, ds. ,S~i~ee th,i,s was ,never ,o,b,s,er*eed m,o,d'el % " wa~s rea ,d i ly el, i~rLinaLed. I t was e~i,d,e.nt ,M tlhi,s ,p.oJs ,tha't ~a~e 'eo.ul,d d e e i d e u n m n b i g u . o n s l y ]o,etweeal
eas j . D . Roberts.on
nm.dels "a" and "b" if we eo.ul,d ,decide `between ~h.e .tw.o alt,ernat:ive i,nt,er- p re ta l ions of the dense bands in Fig. 17 as ind ica ted in Fig. 18.
We ,set a~b,otut :the ~s.o]utli.o.n ,o.f ,thi, s ,pro`bl.em ,by ~taki.ng a~dvant.age .o[ an exl>er!iment ,d,o.n.e b y S e~.m~i t t, B,e a r ~ar~d P a lm ,e r in 1942 [18]. This is ,ill,ust.r:ate, d 'in F'i,g. 19. Tt~ey ,showed .that a l ip id .in *he ,s,meetie s ta te as fllu,str,a't,e~d ,i.n F.ig. 19 a when ~hydra~t.ed, ,e.o,u,ld be eau,sed :to spl i t off' int,o i,n,diivi, dua] s 1,e,afl,et,s ,as lind.~ea't,ed :in Fi~g. i9 b ,in a revers ible fashi.oa.
YYYYYY
rrrrrr , 0 O0 0 0 O0 0 0
H20 O 0 0 0 0 o o o o
o o o
Q b
Fig. 19. Di;agram taken frem S c h m i t t , B e a r and P a l m e r [181 showing lipid molecule:s arranged in bilayers in the smectic state. In "a" water is excluded and the bilayers are closely approximated, in "b" water has entered along the polar he,ad,s of the mol.ecules split:ring .off indivi,dual bilayer,s and increasing the repeat
period a.s detected by X-ray diffraction from 65.7 s to ~27 3..
We rea,s~oned t,hat ,if w,e e,oul, d eau,s,e the lip,i,d s.p.eeim.en in tki,s ~stat.e t,o bec,o,me hydr~ated, w,e Mrou,l~d be ab.le .to fix t'h,e ind,ivi,dual bhno,[eeular .I~.eaf~e,t.s .of 1,i~p:i,d and r,eveal them :i.n ~s,e,et~i.ons ~oy e~e.etr.o,n m,iero,s~eo~py. I~t wa,s. .dear ,t,ha.t i f we eoultd ,do t,hti:s, ~and we f.ound f, or .each blimo,l,eeu~ar leaf le t a s!ir~g,~e ,d.eils,e line~ we wou ld have to choose the a l t e rna t ive i n t e rp r e t a t i on i l lus t ra ted in Fig. 18 b. Fig. 20 s~ow,s a Ls,p,eeiLme~n which w~als hydrat ,ed ,during fix,~ti,on. Ea,ch ~,i~ay,er of 1,i'pi~d ap'pe,ar.s '.a~s ~a ,pair ,o~f ,de.rts,e ]ii,ne.s .m,ak.ing u=p ,a uni1 ~ab,o,uf 60 A ~th,iek, apla:earhLg v e r y m.ueh ]ti,ke a u,ni,t meanbr,ane, .alih.o,ugh th,inner. This a l lowed us, then, 'to .eo,ne$u,d~e tha,t l~ip~i~d !mo,leeules, .in ,in.t~eraet{ng ~w.i'i~h ,our f ixing agents, develop densi t ies at the i r po la r ends. F r o m this we were able to eli!mina,le mo,del "b" in F,ig. 18 ,and .etm.o.se the .o,ne Jn "a" a,s .the ~only o.ne w,h~i& fit,t,ed all ,of ,the fa,et~s. I t wars ,thi,s analys,i.s ,t'hat was p r i m a r i l y re,sp,on- .s.i,b~e .fo,r 'the 'unacmb,ig~o,u,s ,cho,i:e.e ,o.f ,the k,ind .o.f Im.o~eeular ,d,iagrmn for the rep,ea~irtg p,er,i,o,d in ~erve mye l in tha t i:s ,i,~d~ieat,ed i~n Fig. 8. S,u~b,sequ,ent artaly,si,s has .eonfir~med ~tri,s in'ter,pr,ei.at, i.on wi th ,the .part ial ~s,o].u'ti, on of ~he
o r r C3
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Mag
. X
280,
000.
240 J .D. Roberts.on
X-r 'ay d!iffractii.on pro,bl.em in ;morse .r,eeent year,s [26, 271. H,ow,ever, d ie ,eSeetr,on miemseo,p,ic ,evid.en,ee ~in .th,e la.t,e 1950's was quite .ad,equ,ate t.o ,allow dafi,s e.o,nehtsi.o,n ,t.o .be t,,eached wd,th .a h,i~gh Id,egr,e:e o,f ce r t a imy . I,t .should b,e p,ointed out ,thai ,tlris ,eo,n,el, uls:i,on fro,m m,od,el s,tud~i, es wa,s v e r y ,el~egantly co,nfirmed b y a s i m g a r s t u d y ,by S .t ~. e ,c k ,e n ii u ,s [351 in eorr,e]atio,n wi th ,some X - r a y ,d!if- fract,i,on stu, d'i,e8 Iby La~ z z .a 't i and H u ,s .s o n [31] ,i.n 1962 even t~hough an earl.ier ,s'tndy ,by S t ,o ,c k ,e n i n ,s ,p~,bti,s,hed ~in 1959' [381 reached .e,o.ntr:a,d,iefo,ry con eltlts;ion, s.
Fig. 21. Fig. 22.
Fig. 21. Diagram of the electron microscopic appear~ance o.f myelin at its junction with the mesaxon to the left. Tio the right is ~uperimposed the molecular diagram reduced partly by X-ray diffraction and partly from the studies of model systems
by electron microscopy. Fig. 22. Molecular diagram of the general pattern of organization of the unit membrane. The bars represent non-polar earhon cha,ins ,of lipid m,oleeules with the eircle,s representing their polar heads. The zig-zag lines represent monolayers of non-lipid of two different kinds. It is not intended t,o exclude interpenetrati,on of the lipid earbon chains. This is not shown in the diagram for simplicity, Indeed,
some degree ,of interpenetration of the lipid carbon chains is very probable.
W;i,th all ,o,f thes,e facts dn baird, it was po~s,sib~e to take ~he moh~ecular d i a g r a m f.or the rad,ia~ r,e~peat'ing uni t in 'mye]'in ,t~at was p,o,s,tulaf.e,d 'by F ,i n e a n .i~n Fig. 8, .c,o.mbine dt wi.th the electron m~eroscope ,oS,s.ervatio.ns a.s lin, d ica ted dn F!i,g. 21, ex,tr,apol, af,e ,o,nt t,o the S.chwann cell su r face and ,say tEa.t f,h,e m,o.le,6ular ~pat, fern r.e~s~po~si,ble {or .the m~if m mnbralm .i~ma,ge was tha t .ind,i.eat,ed in tim d i ag ram. Thd,s led ,di,reetly to :t'he pos+uJ~ate t.ha,t t'he unde r ly ing mole,oular c,an~g.uratio,n ~o,[ the u.ndt me/mbrane ~stru.et'ure wa,s t.hat 's~o,wn ~in Fig. 22.
Tt~s dd, ag rmn ~s lin re,any w a y s s,i~n~i~ar to ~the ,o.ne ~p'rogo.se, d ~by D a n .i e 11 i and D a v s o n dn the 1930's b~uf lit sho,uld be no,ted t:h.at :it r:esu~ted fr,o,m a eom- p le te ly , independent l ine ,of work , d r a w i n g m a i n l y ,o,n evi,d,ene,e de r ived fronl e lec t ron m, ieros.eo,pe stud4~s. I n a ~s,ens~e, i,t ~ha,d ,i:ts to,otis ,in eo,mmon wd~th ~'he D,aniei~i-Davson 'm,od.el d n ,th,e w o r k ,of L a n g m u i r. Bu,t 'it was arr,ived
Origin of the Unit Membrane Concept 241
at b y a very +d~ifferetrf p a t t t w a y and thu+s ,sh, ott~d ,be cor~si~dered ~a~s an irrde- perrdemt aaro,d,el. Tlrks ,ks not YOo ~ay tha t ik ~d,o,~ ntc~t 9~rav~t~e ~onfiranat,~on o,f the general eorreetrres,s .of fire Da:niel~i an,odel. I t fd~oes m ~in, deed td~o this, ma, d in a~d&iti~n, if !p.rovides cer ta in rrew fact,s t h a t t ake t~s a liyofle ,f, urSh, e:r ,in ,uqn,d,er- starrffi,rrg the ~noleeul,ar erffa~i,zat~i.orn ,of anexn~rarres. Most ~p ,or t :an t ly , .i~t se,~s a 1,tenet on tire mrln~ex +of t,i!p~,d aTro[ecu, les ,in t~e ~orn~rarre ,to a s,~rrgle b~,layer. Another ~mtportam a,d~d:i~i,ocra~[ .feature .thaNe i t ia,~mdu~ees ~. the noSion +at .chenrieal ,a~syamnefry ,in the an,embr~rre. To he sure, ,drean.ical asy~-nmetry eou'~d be ~km'pLied froan ~t~e ea r ly deetro-p~hy.s,iol.o~eM ,stud,~es, even 4t1~e dayoi,ng l~ack t.o B e r n ~. t e i n. However , we rrow lrave ,d3efl.ni4e ,stru.etrrral ev3d, ence of ek~rrrieal a,syxnm,etry. T.'kis 'is esyoa~blkshed s imp ly f rom She Saet t.hat the r,a,&i,al repeat ' i~g un+it ~deteet.e,d b y X-ray &i.f.fr~a,eti,o;n arrarst +i~ehrd~ ~I~ro uaaiyo aIrel~brarres i~s.tea~d of nrre. T~ris +rrea~s ,~hmat the ~o~s,~die ~srr~f, aces..nard the ~i~rs.i,d,e surfaces of :~he m,e~brane ,must be ,sl~.nifi, eaxrtly l~f~ererryo li,n ,chemi,eal t,era~s. Thks, differ,ea'ree is anal~if,esked +in ihe el, eefroaa arr~i,erogral3tr.s ~of +eetai,ons of ~lyel.ixt 13y ~he +d~iffierent a~ppear:arree ,of ~he xn, aj.o.r ,den~s,e ,line a~d yoIre i~tira- peri, od ~iaae. Thi~ differen.ee c lear ly re+.u,l.t,s f rom a ,dfiff!ererree Sn (0he . r eac t i~ ty of time eomp,o~err~s ~of yolxe ~ia~side ~an, d ,one,side ~s:urfa~ees xrf She anearrbrane '~i,tth fixilrg ag~en.~s. The ~th,er ~m~p,ortam fea,ture th~at wa,s ,a~cLd, ed i,s yoIre ]~rrrit,at~on of 'the t'tri&~tes,s, o,f ,t,he .non-l~i~p$d aa~o+rrotayer in t~e f~ua~d,a.menf~at uni t . I f t ~ Sehwann cell an~enrbra'Ire ~,s ~o bte ~taken +as ,a genera l kype ,of alr~aabr, ane, lh,en the tl~iekness ,of ,the ,~roxt-li,p.id an,on, oLayers canrrot .ex,eeecl 20-30 A, ,riot a n y t,kicker lapers ~t~han tlrks si.a~pty canon,of 'be ~p~adce,d i~tyoo ~tahe imye~in s~eat~a al:o~g w,~6h the li~pid+s Shat a re kn,own fo b,e: .present. This ~thi&nes,s, [i,n~'.'t ,d~oes, h~ow- ever, ,su+gffest 'Nat ,o,rdy :once an oaa,o,layer ,o,f n.on-li.p~i~d .~s presment a t ea,ch surface of tire ,fundam.enyoal nrrit. The exac t ,st,a:te ,of • n~on-[~pid c,o~p,orrenyos Sn terms ,of ,pr~m,ary, secondary .o.r tert:iary ,struet.ure ha~s n, ot yet. been w~o~k,ed orrt. Frrorn ~ e ,fa~t qkra~t :the: ,op:tic az~ks ,of it;he angelica she atL lies in the r.a.d~ial &ireet:ion, w.e ,ea.rr .say h.ow.e~er, ~hat ,She n.on-lip~i,d any.e~i~n ,eoanl3,onen.t,s td~ not have ,a 'preferred or,ienyoafi,on. T,ki,s Js srot t,o ~ay, ~o~ev, er, ~ghat Ithey ,cannot have o.rd,er. F,o.r exanap:l,e, hez~a,ffonat , symmetry ~s , e o ~ p , a f ~ e w~:ch ,an ~ol~t,ic axis ~n the ra+d,i,al +d.iree/~i,o,n. The ra,d.~ally p,ositive i,ntrirrsic ~birefrJrrg.enee ho,wever, str,o,ngly su,ppoet,s ,the ,[a,rrrdlar ,arrange~errt .of ithe ,~'pd, d +a~rd ~n, deecl, the X-ray ~di,ffraet,i,o,n e~iderree 4,n ~ . prasent ,state tpro,~ides quite ,go,od a d,dAyoio~ral ev, id~en,ee.
There ,are severat ,~o.od reas,on.s ,for b,eh,e,~i~r~g yohak pa r t ,of .t~e ehe~ica l a~sy,rrrmetry o.f '~e ~unit membrarre iks attri,but,~rb}e 'to a re~at, ively h igh ,content of anu.eo,po,lys+aechari,de or anu.eopro+iein ,an, d p.erhap~s .glpeoiipi,d,s i~ the .tarter sur face fff t~he ,m.ean,brane border ing ,on 'ex'tr.a.eellular ana/e.rial. Whi le there is trot ye t icLefinifive ,proo.f ' that this .is ~s,o, .there are ,several~ ~s,u~gesSive ,lines of ev~derree. In ~he first p,~a,ee, perm.ar~ganate i.s a ]~et~t,er genera l f ixat ive for p,olys,aeeha~i,des :tJhan O,sO4. It ~ks ,a.l.s+o a ~ett,er fi~ca.tive for +t~bze ~o,uter ha i f .of the urrit an,emGr.a~ne, It w.~s *his fea'~ure +flha.t fizs~ suffgest.ed rthe presence of po.ly+aeeharide eo+mp.onei~s 4n .~he .oa~t,er hal~f e.f ,flhe axnit an,ean~.rarre. O,ttter fea.t,ures tl~at ,s,trg~est t~hks, however , ~d,o. exist . T,here Its very ~oo,d ~isto.elremical e-~kd.enee ~h.at the ,g.ap sttbStranee present betw.een q3~a~irs ~f un i t memhrarr.es, in so,me :t'kss,u.es a.t lea..s~, ~has kksyooekenri.eal ,s.t~rLni.rrg ,proper~i.es ,i.udieagin,g +the
Protoplasma, LXIII/1--3 16
242 J.D. Roberts on
~pI~ese,n,ce of mu:eo,polys,aec~arides. I~1 a.dditi.o~n, tire .tw,o ,out'side anenrbrane sur- fa*c~s ~Lnit,d :i~n an~elin ~o ,malce t im ~intraperi,od ~i.ne ~ a y ~e read i ly ~soI~t apa r t b y s.oaking anye}ixt/i,n ~d~is,t~]],ed ~wat.er. T h e y anay he hro,u,girt back t.o- ~etirer a,ga'~n b y ~mmersing ~flae ~yelr in in a d i lu te salt ,s,olutioa. By co~ntrast, rite is:urfaees aza~ited ,at fire aria}or ,d,errs'e ,1,ine d,o no,t ,eoan,e ,a~p.art ,~nder iiris ~rea,tm,esrt. This l~ehaviour i,s am4 ~urrli~/,e tha t o,f a ~poly,saeeirari,cfe ,gel [39]. Ano.~irer ki.~d .o,f evidence dep,em:ls ~o~n iramun~o~o,~i.e,al ,studies ~hi, ch have su,g.~es,~ed 4ira't .s,onle .of, ~t~te dmmuno,lo,gieal ipr,ol3~t,i.es ,oFf ~eell ~s,ur faces ,ex,pos,ed t,o ~h,e ,tort.side a re ~depemdeat .oa~ 'a rpolys,aeeharide ,e~mp~o.~ent .a~rd glye~li9,ids. The Jb~].~o~cl ,grLo,u,p sttb~stanees, for !i,n.s.~avtee, ,are well ]cno~wn t~o .eon.tai~ .a ,p,oly- s,a,eeharid,e el.eanerrt. Th, ese ,are a~o,st ~roba.bly ,located :i.n ~the ,o,u~sid,e surface ~f ~he ~,nit ,m, er~brane o.f re,el ~,loo,d ~ee~l,s. M,o,s~ .a~l ~o,f :tirese v.ari, ous t~i,fls o.f evidence s,ee~ 't,o. ~b,e m~ost cornpai'ible w,ith the .assu~pii.on ~hat She ex te rna l s,urfa,ee ,o,f .fire ~trrit ,m,embra~re .eo~tM~rs ~a p,olys:ae&aride ,de~nen.t ,in ,the .f,or~n, p,erira:ps, o,f a aTm,e,o,pro,$ein a n d Iperh:ap.s a g,lye,o,}iodd ,element. This m a y very well aeeo~rrt for iire ,s,tr~e~ural d~e~i.eM asymane.try ,evs ~in She ~nye]i,n skeatir as well ,as i,n ,other m, erabranes iby s cha~aetet%i@,s.
I,t was, ,o~f eo.ars,e, neees~sary ~to es~a~b~i,sh tha't ,tire an:it ntea~br,a~e ,pati.erns observed ,in ele.etron ~n~i,er'o~g.raphs a t ~the sur face ~,f 'Lhe S,eItwann .eell.s 6n rel~t~ion to ,myelin was rro:t a ,p.eeuliar'Ry ,o'f tire Sehvca~im cell. We sat~,st]e,d ourselves tha t this was the ease in the late 1950's by conduct ing extensive ,survey,s ,of a n.Ulr~irer ,o,f ~c~ifferent 4y~.es ,o.f ,its,sues ,fr,o~n ,many ,diffe.ren.t ,organs and f.r,oan sunny ,&iger,ent a~ri~rrra}s., ewen ,of ,different ,piryl,a [40]. Tire patLern wa.s demons t r a t ed h.o,tir , ~ t h KMnO4 a~d OsO~ fix,at.fan, al.tirough ~he la~tt,er me ,hod requir,ed ~s,ev.er,al years .o,f .e~Nu~io,n ~o,f .tire ~.eehniq.ue before ,tire uni4 me~,bra'ne p a t t e r n ,beg,an fo a,pg.e,ar ~eonsishently. The pa'tte.rn ~is IrOW fa i r ly regu,l.arly seen a f te r g}u6aral~d,ehyde f ixat ion fo}lo,wed Iby OsO~ and it has b,~en ,de,moaMra~d ,in ,all anel~.r~an,o~u.s eelI ,orgaIrel,~es ian, d strewn r he reg~u- l:arly pr,esem no.t ,only in ani,m:a,fs, hwt i.n l ~ a n t ceils. I.t thus avpears i,o b,e a .u.rriv.er,sa.1 ,bi,ol, ogieal ,eo:n.sta.nt. I t isiron~fd Ire un_,&ers,to.od, Irowever, ~hat thds is ~o'.t ~n,eaat ~o i ,mply firm 4ire struetnr,e is a rli,g,id o~ae which ~ever varies. Obv.i.o,usly .mem~.rane,s h a v e .spe,eificity. I t is kno,wn tlh, a,t :the eirean,i, eaI co~n- po,si,tio,n ,o,f d i f ferent anembranes. ~s qu,i.t,e differ,eat. The ,parbi,eu,lar mole.eular s,p.eeies tIra~t ~ .ake trp ,any given anen~br.ane m a y 'vary co~nslkterat~ly. We .do n,ot ye t k n o w whe.tirer t~er.e is ,any a.rrang,emen~ .of par:t ieular ,mol,eeular species ~tha.t is cmn, m,on ,to. a l l an.e~b.ranes. Tirere ~n.ay he s,oan,e co,maTron s t ruc tura l ~protein ,or .struetural }i,po-protein tirat is .of ,general o,ceurrerme a,t least dn a :p,arti.eular s~peeie,s. Ho,w,ewer, i,t ,d,o.es ,alp~?ear ,o,rt tire basis .of the evi, d,enee t h a t Ir~s b~een .o,utl,in.ed Itira.t tire genera l ,p,attern ,o{ ,or..ga~rization o.f ee]:l m.eanhra.n,es en~b,odie, d :in tire .u,nit ,mem.br'ane .concept is ,eOLr~s.tant .and general. I t is p,assihle ' that lo,eal variatio.n,s ,in ,tire pa~tern ,o.f o.rganizat.ion m a y ,occur, such ,a,s ~phase dranges ,in the ]i:pid hil,ay,er v~iLh r,earran,g.e~ments of ,the l~ipid ~mo.leeu~es oeeurr ing '~n cer ta in r.egi,o~s, r,earrartgements o,f the k ind p,ostal'at, ed ~by S j 5 ,s t r ,a ~n ,d [41] ,on tire basis ,o~f the wo.rk ,o~f L u z z a t i and H u,s s ,o n [37] aI~d ,of S t o.e e k ,e n i u ,s [36] and this ,~s a :topic .on which m,u.eh current .activity is lee~4,ered. H.otwev.er, a't the pr,e,s,ent t,~me, there is no p,c~sit*ive evidence first s,ueh ~piras,e tra~rs,forrcrations ,ever .o.eeur in lilpo-pro.tein
Origin of the Unit Membrane Co,ncept 243
syste~rrrs .eitqher in oitro or in oioo. I t is quite cesta,in t~rat ~uch chaag,es h~ave no~ been d,e~nroa~strated !in ~rerve any,e~in. C,o,rr~el.a~ed ~riophy~i, cal s,t~d~,es b y e.l~ectron ~,cro~:eopy, p,otariz,afion .optics arr, d X-ray ,diffra.et,i,on ,hawe ,a,ls,o been carri~ed ,out .in o,ur ,labor, at,pry on intlaet .re~ina~ ~o~d Qufer ,se,gan,eats ~,here s,tacks of ~rrri~ an,embraxres o~e f , o ~ d ~ra,ldng ~p i]ae regular ,laanellae ,of the u receptors. O,ur .evi~demce fa,~s t,o snl3p,ort a gl,~bu.l~ar phase t ransfor- mation in ~ e nx.eanLbrarre ,1.ip4,d.s .~n She ,inSaet .rod l~rmdIae. How,ever, s.ome q,tresfi.on h~s been rais,ad ~y ~he wor~ ~f ,o/~her~ [42] a~s ~ ~ e ~ e r or n,~ ~l,abnlar ip~ase t.rarrsf, orma~ions ~aay occur ~n the RO S m , e a ~ a ~ e l~ipi,d~. Evidence a,~,trib~rt.a~bl.e to such changes ,h~s b ee~ {,~und ~n X-ray an,d EM st~d,~es of exterrs~ively ~legr,~d,ed ROS naem~ran~,. W~i, le .oar own s~trd.ies do a~ot sutpport t~'he prreSemee of such tr.a~forana,H~rrs ia~ !intact ROS anem- branes, we have seen evi,d.e~ree af l~i,d rearrangements o e c u r r ~ g ia ret ,~al rods afSer prolon,ged .expos.ure ~f,o the X-ray b e a ~ ,th,a.t ,could ~e ,r,daf.ed such phase transformations. We at t r ibute these changes to degradat ion and all our evidence at present is against the occurrence of such phase transitions a,s a ws or ~e~eral ~fea~fnre o,f a n y ,unit an,em~bran,es. I t wou~d be b,eyo~d ~ e ,scape ,of tke ~presen~ p a p e r ~ pro~eeed s ,~nto .~his topic and I shah not ,un, der take ,~o ,do .,so becatrs,e I ,have recerrtly ,considered it at some le~gt,h 5.n two o.t'her ar~e],es [d~3, 4,hi. Saff~ee ,i~ to say ,~hat ~at ,t,'he 9resen.t :t@ne fke ,general arrar~gement of ~,e .,l~pd,d ,an, d non-~ip:id components ~d~p.~eted s F,ig. 21 ~s~i~l.1 appe,ar, s to expresls ~dtre nros.t ~general ,pa~terrt ,of ,m.ol,eeu~ar ,orffa~r~i- zat~on ~of a:ll [4vin~g ~mexrtbra, nes.
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16"
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