The Isolation of Non-Keratin Protein Filaments from Inner Root Sheath Cells of the Hair Follicle

Vol. 56, No. 1 Printed in U.S .A.

TH E J ouRNAl.- oF lN\"J!:STIOA'l'JYE DEnl\ I A'l'OLOGY

Copyrig h t© JUiJ b y Th o Willia ms & \l" ilkins Co.

THE I SOLATION OF NON-KERATIN PROTEIN FILAMENTS FROM

INNER ROOT SHEATH CELLS OF THE HAIR FOLLICLE'" P . M . ST E I NERT, B.Sc .. P . Y . DYER

AND

G. E. ROGERS, M. c., PH.D.

ABSTRACT Protein fi la m ents obtained from th e cells of t h e inn er r oot sheath layers of the g uin ea pig hair rolli cle h ave been i olatcd a nd characteri zed . They a r e h oll ow tubes approx imately 0 A in di a m eter and a re of indete rmin ate length . The protein of t he fi lam ent is un iqu e in t hat it con tains t he a mino acid cit rullin e and its amino acid compo it.ion is ver y s imi la r to t lw t of t he tota l pro tein obtain ed from t he inner root s heath cell s as soluble polypep tides by di gestion with crysta lline t ryp sin. The int racellula r filam ents of t he inner root sheath cell s a r e chem ically a nd stru ct ura lly qui te distinct fr om t h e k e ratin mic rofibril s t h at a rc presen t in t he neig hbo rin g co r tica l cell s of th e foll icle. It is suggested t hat t h e fil aments pl a,y a rol e in t h e developm ent of cell shape in t h e h a ir fo lli cle in a manner an a logous to t hat which is accepted for mi crotubu les in many types of cells . The protein s contain ed in t he m ature inn er roo t sheath cells of h a ir fo llicles a nd the m edulla ry cells of ha ir fibr es have b een shown to contain substan tial amounts of t he a mino acid citru lli ne (1- 7). Sin ce t he proLei11 co nta in ed in t h ese t issu es is h ighly r esistan t t o dissolution by normal pro tein solvents (2), chem ical studi es h ave been p erformed on soluble polypeptides d erived from the t i sues by digest ion with cr ystalline t r ypsin or p ep sin. Limited sequence a na lyses of polypeptides d eriv ed from th e proteins of t h ese an d a related t issu e (t h e m edulla of porcupine quills) indicates that the citrullin e is chemically bound in t h e p roteins b y peptide linkages

(3, 7). Previous stud ies h ave indi ca ted t h at the p r otein of t h e inner roo t sh eath is composed of fi la men ts of t h e a -typ e (5, 6), ori ented in t he cell in t h e direction of fibre growt b, while t he protein of t he m eclu Ua is n ot franld y fi lamentous (6) . In th is paper we show t hat t he protein of t he inner roo t sh eath can b e iso la ted in t he form of morpho logically distin ct filam ents and describe t heir proper ties. MJITEHIJILS A1'
Th e hair fol licles of male alb ino guin ea 1 ig;s aged one to three weeks were exposed by t he waxRecei,·ed Mnrch 24. HJ70 ; accepted fo r pub lica tion Jul y 17. 1970. ''From the D epn rtment of Biochemistr., ·. U nive rsity of Adelaide, AdP.laid e. South Auslm lia 5001. Au tra~ i n. 49

sheet procedure (6, 8), removed with animal cliP! ers and uspen led in a buffer of 10 mM trischlorid e pH 7.4 containing 10 mM KCJ. i solation of malure inner root sheath tissue. The published method (5, 6) for Lhe preparalion of inner root sheaths was unsatisfacto ry du e to t he prese nce of hair a nd denalured insolubl e proLein. ConsequenLly, fo ll icles were dispersed by gen tl e agitation in a buffer of 8 M urea, 10 mM trischl orid e pH 7.4, 25 mM 2-mercaptoet.hanol at 4° for 5 m in. T he solu ble prekeratin a nd oth er cytoplasmic proteins were alkylated at pH 9.0 by addition of solid iodoacetic acid to 50 mM unlil -SH negative and cen trifuged at 38,000 g for 15 min. Th e pel leL containing Lhe mature inner roo t s heaths, k eratini zed hair an d cellula r debris was washed free of the so lu bilized proteins an d urea by centr ifuga tion at 500 g in 10 mM tris-chloride buffer pH 7.4, resuspended in 5 ml of 10 % sucrose in this buffe r and layered onto a 24 ml eli conLinuous sucrose gradien t in a 30 ml cellulosenil.rate lube. The o- rad ient e mployed ,,·as composed of 6 ml layers of 70, 60. 50 and 20 % sucrose in the sa me buff r a nd was centrifu ged at 22,500 r pm fo r 30 min . in a Spinco SW25.1 roto r. On lv im1 er root shenlh banded at the 50-60% su cros~ inLerr,,ce. T he denser hnir fibres bnnd ed at Lhe 60- 70 % sucro e interfa ce and Lh other cytoplasm ic deb ris remained at hi gher levels on lhe gradien t. Th e purified heat hs were exa mined by light mi croscopy u ing phase-contrast and polarized-light optics and were obser ved lo be free of olher particles. Th e sheaths we re recoYerecl a nd wa heel by centrifu gation to remo,·e. ucrose. Prepamtion of filament s. F il aments co uld be released from inner root shenths isolated by th e proced ure cle-cri bed above . T he most snlisfaclory method for release \\·as limited di gestion "·i th a 0.1 % solu lion of a proleol.,·ti c e n z~·me in Lhc Lris-

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'r HE JOURNAL OF I NVESTIGATIVE DE RMATOL OGY

chlori d -K l buffer. Four differen t en zymes were Lcsletl: crysLallinc try psin (Sigrna, T yp Ill , 2Li mes crysta ll ized ), ct-cbymotry psin C'N orLhi nglo n, 3-Limcs cr.rslallizcd) , pronase (Call iochem, B-grad e) and D ifco Lryp ·in (D ifco Labs ., 1:250 prepo.rolion ). T he enzy me solu tions were clari fi ed by centrifu gnlion aL 50,000 rpm in a Spinco 50.1 roto r for 2 !1 r im mcd ia Lely befo re use . Digestions were perform ed in a " fillro.tion ap 1 at·a Lu ·" similar Lo Lha L descri b d by Kawiak et al. (9) fo r times va rying up to 15 min a t 20 °. AL the completion of the diges tion Lhe cell suspensions were chil led to 0° and Lhe cells harvested and washed by cen trifugation in the buffer at 500 g Lo remove enzy me. F ilamenls were then released fr om Lhe cells by homogeni zation in the Lris-chl oride-K I bu ffer in a cl ose-fi LLing D ounce homogenizer (K ontes glass ; clearanc approximately 0.07 m m). The homogenates were cen trifu ged at 4,000 g for 5 min to re move cell ular debris. The resultant sup rnates contain ed Lhe inn er root sheath pro tein fila ments and were retained for furth er studies. T he same procedure could be applied to intact follicles thus avo iding the pre-isolation of the inner root sheaths. In t hese circumsta nces lh e cells of the folli cle bulbs were completely digested within about 10 min ; the surroundin g inn er root sheaths had become detached from the follicles and were disru pted in to single cells which could Lhen be readily separated in th e fil tration apparatus. T rypsin cl·io estion of inner root sheat hs to poly7Jepticles . The standard rnelhod in protein chemistry for the en zymic cleavage of pro teins to polypeptides is to use purified proteolytic enzy mes of known specificity . Accordingly, the pro cedme fo r the release of t he lo tal protein of th e inner root sheath tissue was by digestion with a pure, crystallin e proteolytic enzyme. As in earlier equ ence studi es (7), the di gestions were performed u: ing crystall in e Lry psin (Mann , minimal chy motrypsin con tent ) at 37° in 10 mM N H,H COa (pH 8.3) using an enzy me : tissue ralio of 1:100. Th e reaction was termin ated afte r 3 hr by fr eezedrying the su pem ate obta ined by centrifu gation for 5 min at 4,000 g. A m.in.o acid analy sis . Samples for a mino acid analys is were hydrolyzed for 28 hr in constan tboiling H Cl at 110°, freed of H CI by evapo ra tion and analyzed in a Technicon a mino acid analyser. Fi lament preparations were di alyzed fo r 4 hr against water and then freeze-dried I efore hydrolysis. E l.ectron microscopy. Specimens for electron micro. copy were exa min ed in a Siemens E lmiskop I cl eclron mi croscope. H omogenate preparations were exa mined after negative-staining wilh 2% uranyl acelate . F re hly depiln led guinea pig hair folli cles were fixed in 2% glu taraldehyde, postfix ed in 1% osmic acid , dehydrated in acetone and embedd ed in aralcli te by stand ard pro cedures. Sections were stain ed on t he grid for 90 min wi th 1% potassium permanganate.

RE SUL'rS

Fine struci'ure of filaments in sit u. A t ransver,;e cr oss-sec! ion t hrough t he inn er root sheath la)·cr of a gu inea p ig ha ir follicle is shown in F ig. l a at a sta ge when t he H enle lay er of t he sheath has become a hard ened r igid st ru cture. F ilam en ts appear as hollow t ube. abou t 80 A in climnet er. Fig. l b sho\\·: a t ransverse cr oss- ·eet ion t hrough t he cor t ic[il r egion of t he follicle at t he : am e level. K eratin mi crofi b ri ls app roximately 0 A in diam eter can b e seen. They a re clea rly different in t heir . taining p roper ties . Their "core " a nd t he intc rfi l::nn ent ous regions (mat ri x) are m ore densely sta ined t hnn t he inner root shent h fil::imen ts . Isolation of filaments . In ea rli er studies (2) attempts to release t he fibrous protein mate rial of t he inner root sheath by a var iety of tec hn iqu es were not succes fu l. However, it ha d b een no ted t hat durin g p roteolytic digest ion of t he inner r oot ·heat hs (to release t he p rotein as solub le p olypep t id es ) , t he sheaths were init iall y eli ru ptecl into single highly-birefrin gent cells, t he fibr ous con tent s of whi ch were removed onl y after longer di gestion. W e h:we fo und t hat hom ogeni zation of t he cells produ ced durin g t he earl y stages of t his digest ion releases t he protein filam ents . Homogeni zation o f sheath · in buff er before en zym e t reat m ent clicl not produ ce fi la ments, but inst ea d , large m embra ne-bound clumps of fibrous mat erial. Moreover, homogenates of intact hair folli cles p repared in t he t ris-chloride b uffer d id not show the presence of fi la men ts . or t he proteolytic en zy mes tested, t he m ethod employing di gestion wi t h D ifco t r ypsin, fo r approximately 15 min at 20° on inta ct fo llicles or p urified inner root sheat hs gave t he highest yields of fi la ments p er uni t weight of sta r t ing materia l. A t y pical prep nration of fi lam en t negntivelystained by u ra nyl a cetate is shown in F ig. 2a. These fil ament . were prep ared fr om whole guin ea pig hair follicle t issue by th e m ethod d escribed . The fil am ent s are ap p r ox imately 80 A in wid t h, severn! mi cron. in length and a ppenr to have a eli t inct "core" t hroughou t t heir length (Fi g. 2b ). It was d et ermin ed by electron microscopy using a vari ety of negative- ta ining t echniques, t hat such prep arat ions of fi laments were almost completely devoid of other cytoplasm ic par ticles

Fw. 1. Transverse cro. s-. ecLion t hrough a guinea pig hair follicle. (a) C ross-section through a cell of the H enl e laye r of t he inner root sheath showing Ihe holl ow Iuhu l::tr stru ct ure of the fil amen ts. The filame nts a rc app roximately SO A in cliamcle r. The inler-filnmentou.· regions are of very low electron density. (b) C ross-s -ction I hroup:h Ihe ca rl ira] region of the follicle at t he same level. Each mi crofibril is app roximnl·cl_,. SO A in d inm lcr a nd has a densely-stained core and t h microfibrils are surrounded b.v a d e n ti<' l ~·-sln inin g: matri x. Specimens prepa red as described in Materials and M ethods. X l SO,OOO. 51

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Fro. 2 . Inner root sheath fil aments prepared from whole guinea pig follicle tissue by digesLion wiLh Difco trypsin for 15 min at 20 °. The majority of the fll amcnts sho wn here arc about 80 A in diameter and are more than 1 micron long. They show a dense core throu ghout their length which appears to constitute one third of th eir diameter. NegaLively-stained with 2% uranyl acetate . (a) X 120,000. (b) X 210,000.

53

HAIR FOLLICLE FIBROUS PROTEIN

and were col1Sid cred to be sufficient ly pu re fo r fur ther studies. Amino acicl analyses . It can be seen from t he T able t hat t he amino acid com position of t he inner root sheath fi lament p repn rations and t he low sulfur k eratin p roteins (H-SCMK-A) ext racted fr om guin ea p ig hair ar e completely diffe rent (columns 1-3 cf column 4). Comparison of th e irm er roo t sheath fil ament prepa rations wit h t h e :low sulfur keratin proteins, instead of the total keratin proteins of ha ir (H-SCMK), is justified as t his fra ction is t hought to deri ve from th e microfibrillar moi ety of t he original hair fib!·e (10). Thus t he inner root sheath filam ents a re chemically as well as s tructurally unlike t hose of keratin. The ai}alyses of t he fi laments are qui te similar to the an aly es of t he t rypt ic polypeptid es deri ved from. t he total pr oteins of t he inner root sh&'lt h . There are, however, four amino acids which vary signifi cantly; in t he filam ents t he content of 'citrulline is higher and t he contents of tyrosine, phenylalanine and lysine are lower t han t hose in t he t ryptic peptides of whole inner root sheaths. This suggests th~ presence in whole inner root sheaths of other protein species that do not ·contain citrull.in e and which a re absent from t he filament p reparations. DISCUSSION

TABLE Ll m·i no aci
(A ll values a.re expressed a. mo les percent a nd repro en t Lhe average of Lwo experiments) Tryptic

iso lated

polypep tides

fro m inner

deri ved

root

from

sheaths

inll e r root

Fi laments

Amino acid

sheaths

.. SCM-cyste in e Aspar tic ac id T hreo nine Serin e Glu ta mi c ac id P roline Ci tr ull i net Glyc ine Alanine Valin e Half-cystine Methi onin e Isoleucine Leucine Ty ros ine Ph enylalanine Ornithine Lys in e H istidine Arginin e % H.ecovery of prote in material hydrolyzed as amino ac id

0 .0 10. 2 3.2 6.5

2-! .0 -1.1 4. 1 7.7 G.6 ,L S

0 .7 2.3 3.4 9.8 1.9 1.7

1. 4 4 3

0.0 9.4 3.0 5 .8 22.5 3.5 3.2 7. 1 6.2 4.8 0.7 2.1 3 2 9.1 2 .7 3 .1 1.0

:.:

~ u '1 ttl

:;a

5.3 8.7 4.2 G.4 16.7

3.5 0.0 4.4 6 .G 5.8 0.0 0. 4 3.5 10.0 1.() 1.5

3 .6

1. 4 3.7

0 .0 3. 7 0.9 7.0

74

78

03

lA

8 .8

General properties. It has been r ecognized fo r some time from electron microscope studies that the cells of t h e mature inn er root sheath mainl y *From unpublis hed s tudi es. Th e protein was contain oriented fi laments about 80 A in diame- ext.racled from guinea pig hair (H ) by red uctio n ter (5, 6). Previous studies on t he total proteins a nd then alkyl at ion wi th iodoacet ic acid to give derived fr om t he t issue have shown t he presence 1he S-carb oxy methyl kerate i ne (SCM!\:) derivative in whi ch the half-cyst ine res idues have been of t he amino acid cit rullin e ( 1-7). However, it co nver ted to SCM-cysteine res idues . Frn.cl ion A was not ]mown whether t he citrulline was lois prepared by p rec ipitation at pH 5.0. T his keracated in t he fi laments or in some ot her protein te in e fr act ion (H-SCMK-A ) is lower in SCMpresent in the inner root sheath cells. Attempts cysteine co nte nt tha n t he total hair kera l in proto isolat e th e fjb rous protein or an intact protein te ins (H-SCMK) and is beli eved lo derive from species had n ot been successful. Consequently, t·he mi crofibrils of I he origin nl hnir fib re (10). previous chemica l studies wer e perfo rm ed on t Duri ng ac id hydrolys is citrulli ne undergoes soluble polypeptides derived from t he t issue part ial decomposi ti on to orni thine. T he ci t·. rull in e which was t he only known mean · of removing valu e given is the s um of the citru llin e remainin g t he protein content from t he cells. In contrast, after 28 hr and the orni t hin e produ ced durin g 1 his Lime. t he r elease of t he filam ents from t he inner root sheath cells as described in t he present wo rk has been achi eved by empl oying a short period of in t he p rotein of t he fi laments and not p ri marily proteolytic digestion. The present isolation from in a non-filamentous p ro tein of these cells. The constitution of t he filaments in terms of t he inner root sheath cells of morph ologically distinct protein fi laments which contain citrul- the number of protein species that co ntain citline establishes t he existe nce of thi s amino acid rullin e has not been determin ed. Such a deter-

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THE JOURNAL OF INVESTIGA'l'IVE DERMATOLOGY

mination will be difficul t since recent work (H.

W. J. H arding and G. E. Rogers, unpublished)

replace microt ubules as t he determinants of cell shape and stru cture.

has shown t he presence of y-peptide (isopepWe are indebted Lo Miss C. H ayles for the tide) links between adjacent polypeptide chains. amino acid analyses. This work was supported by These cross-links prevent the separation of t he a grant from the Australian Wool Board . component chains and t heir dissolution by t he REFERENCES usual solvents for proteins. 1. Rogers, G . E.: Some observations on the proIt has not b een possible to establish a criteteins of inner root sheath cells of hair fo llicles. Biochim . Biophys . Acta, 29 : 33, rion of chemical purity for these inner root 1958. sheath filaments. Although t he possibility of 2. Rogers, G. E .: E lectron microscope studies of contamination of the filaments by other nonfihair and wool. Ann. N.Y. Acad. Sci., 83: 408, 1959. b rous protein material cannot b e overlooked, the 3. Rogers, G. E.: Occurrence of citrulline in proamino acid a na lyses suggest t hat the extent of teins. Natu re, 194: 1149, 1962. 4. Rogers, G. E.: The localisation and signifithis contamination is minimal. cance of arginine and citrulline in proteins D espite the similar diameters of inner root of the hair follicl e. J. Histochem. Cytochem., sheath fil aments and keratin microfibrils (ap11: 700, 1963. 5. Rogers, G. E.: I solation and properties of proxima tely 80 A), they differ from one another inner root sheath cells of hair follicles. Exp . in at least two properties. Keratin microfibrils Cell Res., 33: 264, 1964. show a different a ffinity for the p ermanganate 6. Rogers, G. E.: Stru ctural and biochemical features of the hair folli cle, p. 179, Th e section-stain due to t he presence of t he sulfurEpidermis. Eds ., Mon tagna, W. and Lobitz, rich m atrix within their cores and between t heir W., Academ ic Press Inc., New York, 1964. p eriph eries (11) . Secondly, t he amino acid com7. Steinert, P.M ., H arding, H. W. J. and Rogers, G. E.: I solation and characterisation of prop osit ion of the inner root sheath filaments is tein-bound citrulline. Biochim. Biophys. ent irely different from t hat of a -keratin. The Acta, 175: 1, 1969. 8. Ellis, W. J.: M ethod for obtaining wool roo ts vir tual absence of cystine and t he presence of for histochemical exa mination. N ature, 162 : citrulline are the salient features of difference. 957, 1948. Possible functional relationship with microtu9. K awiak, J ., Moskalewski , S. a nd Darzynkiewicz, Z. : Isolation of chondrocytes from calf bules. The primary function of the inner root cartilage. Exp . Cell Res., 39 : 59, 1965. sheath is considered to be a structural one (12, 10. Crewther, \V. G., Fraser, R . D. B., Lennox, F. G: and Lindl ey, H.: The chemistry of 13). T\le cells of the sheath are t hought to conkera tms. Adv. Pro tein Chern., 20: 191, 1965. strain the growin g hair in the follicle and thus 11. Filshie, B. K. and Rogers, G . E. : The fine con tribute a cooperative effect to the elongation stru cture of a-keratin. J . Malec. Biol., S: 784, 1962. of the cortical cells in tern al to them (12, 13) . 12. Auber, L.: The anatomy of follicles producing Further, t he outward movemen t of t he inner '':o?l ~bres, wi th special reference to kerattmsatJOn . Trans. Roy. Soc. Edinburgh , 62: root sheath relative to the hair is regard ed as 191 , 1952. . being t he cause of t he flattened form of t he 13. Straile,_ W .. E .: Root sheath-dermal papilla relatwnsh1ps and the con trol of hair growth, cuticle cells by establishment of a "shearing" p. 35, The Biolom; of Skin ancl If air Growth. action (12) . These morphogenic forces could be Eds. L:vne, A. G. and Sh01·t, B . F. Angus expected to b e dependent upon the fibrous eleand Rob ertson Ltd., Sydney, 1965. 14. Shelanskl, M.I:. and Taylor, E. W.: Properties m en ts of the inn er root sheath cell. of the protem subuni t of central-pa ir and The stru ctural elements t hat are prominently outer-doub let micro tubules of sea urchin flagella. J. Cell Bioi., 38 : 304, 1968. involved in t he acquisition of cell shape during 15. Renaud , F. L., H.owe, A. J. and Gibbons, I. R.: growth and development of many cells a re the Some properties of the protein forming t he outer fi bre of cilia. J. Cell Bioi., 36: 79, cytoplasm ic microtubules; t hese stru ctures are 1968 . b ecom ing well characteri zed especiall y in their 16. Marantz. R., Ventill a. M . and Shelanski. M . occurrence as outer fibres of sperm ta ils (14) L ._: Vinblastine-i nduced precipitation of n:tcrotubule 1 rotein. Science, 165 : 498 . 1969. and cilia (15) and as mi crotubules in neurones 17. We1 enberg. R. C., Borisy, G. G. and T aylor. (16-18). It is of interest t hat cytoplasmic microE. W. : The colchi cine-binding protein of mammalian brain and its relation to microtubu le are not prominent features of either de. ub_ulcs. ~ i och e mi st ry, 7: 4466, 1968. veloping or mature inner root sheath cells (un- 18. Vi/ tsmewskJ, H., Shelanski , M . L. and T erry , R . publish ed observations of this labo rator y). Thus D .: Effect. of mi totic spindle inhibi to rs on neurotubules and neurofil aments in anterior it i.· suggested t hat t he filaments o[ t hese cells horn cell . J . C II Bioi., 38: 224, 1968.