The demonstration of lysosomes by the controlled temperature freezing-sectioning method By LUCILLE BITENSKY

Transcription

1 205 The demnstratin f lyssmes by the cntrlled temperature freezing-sectining methd By LUCILLE BITESKY (Frm the Department f Pathlgy, Ryal Cllege f Surgens f England, Lincln's Inn Fields, Lndn, W.C. 2) With ne plate (fig. 1) Summary Lyssmes, as islated and defined by bichemists, are particles in which acid lytic enzymes are cntained by a lipprtein membrane; they are inert until the membrane has been rendered permeable. Previusly it has nt been pssible t demnstrate cytchemically rganelles which meet all these criteria, since all the preparatry prcedures have affected the membrane permeability. The cntrlled temperature freezingsectining methd has permitted the demnstratin f granules which are inactive fr acid phsphatase until subjected t agents that will disrupt lipid-prtein structures, such as heat, frmalin, and 'tritn X-i'. Hence such rganelles may be identified with the bichemists' lyssmes. Intrductin THE distributin f acid phsphatase in rat liver hmgenates is almst exclusively cytplasmic. Palade (1951) and Berthet and de Duve (1951) fund that 95% f the ttal activity was in the cytplasmic fractin, the nuclear activity being regarded as due t cntaminatin. Hwever, the histchemical lcalizatin f acid phsphatase activity by the lead phsphate methd (mri, 1941) cnflicted with these results and shwed, in the liver cells, a diffuse distributin with activity either in the cytplasm r in the nucleus r in bth. De Duve (e.g. 1959; see als ianett and de Duve, 1955) described a distinct grup f cytplasmic particles called 'lyssmes' which cntained a number f acid lytic enzymes including acid phsphatase. These enzymes are cntained within the lyssmes by a lipprtein membrane and are released when this membrane is damaged. Hlt (1959) examined the lead phsphate prcedure critically, and by mdifying bth the methd by which the tissue was prepared and als the cnstitutin f the incubatin medium, was able t demnstrate structures in sectins f rat liver and kidney which resembled the lyssmes f tissue fractins. Althugh his techniques did nt allw him t shw that these were inert particles which became activated nly when the membrane was mdified, yet he was able t enhance their activity and even t release the acid phsphatase cmpletely by prcedures similar t thse used by de Duve. Thus the bichemical data shw the lyssme as an inert particle which cannt be activated until sme damage is inflicted n the bunding membrane. nly after this membrane has becme altered can enzymic activity be demn- [Quarterly Jurnal f Micrscpical Science, Vl. 103, part 2, pp , June 1962.]

2 206 Bitensky Demnstratin f lyssmes strated inside the particles themselves (Hlt, 1959). The reprts f lyssmal enzymes, such as acid phsphatase, ccurring diffusely thrughut the cell, r in nuclei, wuld seem t be due either t further disruptin f the lyssmes, with subsequent diffusin f the enzyme itself, r t diffusin artifacts f the histchemical methd. Thus the cmplete identificatin f particles in sectins with lyssmes is made unreliable by varius types f diffusin artifact. Fr example, the presence f activity n small cytplasmic grains might represent nly sites f adsrptin f either the enzymes r the final dye. T prve cnclusively that particular types f granules crrespnd t the bichemists' lyssmes requires, ideally, that they shuld be shwn t be (1) inert when intact; (2) activated by treatments that will damage membranes. It might be regarded as mre satisfying if in additin it culd be shwn that the acid phsphatase f these granules can be made t diffuse thrughut the cell, and s give rise t the earlier histchemical lcalizatin f this enzyme. This might be advantageus, since it culd therwise be argued that the acid phsphatase demnstrated n the granules represented nly a very small, and perhaps even an unimprtant, fractin f the ttal cellular cntent f this enzyme. f these three criteria, nly the last tw have up till nw been demnstrated, wing t the fact that all the treatments that were required fr the prductin f suitable sectins have been damaging t membranes (Hlt, 1959). With the advent f the cntrlled temperature freezing-sectining methd, which appears t preserve lipid-prtein cmplexes (Chayen and thers, 1961), it seemed advisable t test whether a cmplete cytchemical identificatin f the particles with the bichemists' lyssmes culd be achieved. Materials and methds Albin Wistar male rats, fed n a cmplete diet (MRC rat diet B41), were killed by placing them under a funnel thrugh which nitrgen was passed frm a cylinder at a rate f ver 1 1 per min. A sample cube f side apprximately 5 mm was taken frm the liver and frzen immediately in a glass tube which had been previusly cled in slid carbn dixide ice. Sectins were cut at 8 [L n a freezing crystat micrtme at abut 25 C, with the knife cled t abut 70 0 C. Sectins were treated by the mri acid phsphatase prcedure as mdified by Hlt (1959); the incubatin time in the substrate varied frm 5 t 60 min. Serial sectins were treated fr 5 min with either a 10% slutin f neutralized frmalin (40% frmaldehyde) cntaining 0-9% sdium chlride, r an 0-25% slutin f 'tritn X-100', r an 0-25 M slutin f sucrse. The sectins were washed in running water and then incubated as described abve. The staining prcedure was cntrlled by the additin f -i M sdium fluride t the incubatin medium t inhibit the enzyme. Results The results are summarized in table 1 (see Appendix, p. 209).

3

4 Bitensky Demnstratin f lyssmes 207 Untreated sectins. stain was discernible in the sectins incubated fr 5 r 10 min (fig. 1, A), whereas when the incubatin time was extended t 20 min, small black granular structures culd be seen in sme f the liver cells (fig. 1, B). Larger stained structures distributed at the cellular periphery appeared in mst cells after incubatin fr 40 min, while prlnged incubatin (60 min) resulted in very diffuse cytplasmic and nuclear staining (fig-i.c). Sectins treated with frmalin. stain was bserved in the sectins incubated fr 5 min, but incubatin fr 10 min revealed small black granules in sme f the liver cells (fig. 1, D), similar t thse seen in the untreated sectins which had been incubated fr 20 min. Mst f the liver cells cntained blackened cytplasmic granules and there was sme nuclear reactin in the sectins treated fr 20 min (fig. 1, E). Mre prlnged incubatin resulted in diffuse cytplasmic and intense nuclear staining (fig. 1, F). Sectins treated with sucrse. Black granules appeared in sme f the liver cells after incubatin fr nly 5 min. The sectins treated fr 10 min shwed stained granules in mst cells and there was sme diffuse cytplasmic staining. Incubatin fr lnger perids resulted in diffuse cytplasmic and intense nuclear staining. Sectins treated with 'tritn X-100'. depsitin f lead sulphide was detected in these sectins even after prlnged incubatin in the substrate. Discussin It has been shwn by de Duve and his assciates (e.g. 1959) that glycerphsphate des nt penetrate the membrane f intact lyssmes which have been islated by hmgenizatin and differential centrifugatin. Hence the activity f such particles is demnstrated, bichemically, after the enzymes have been rendered sluble; t achieve this it is necessary t disrupt the bunding membrane. Hlt (1959) pstulated that frmalin fixatin may partially denature r mdify the lyssmal membrane and allw easier access f the substrate t the enzyme, withut permitting the latter t diffuse ut f the particle. This culd nt be prved by the use f tissue fixed in frmalin, but nly by the applicatin f a technique which did nt invlve chemical fixatin f the tissue. In this study, sectins prepared by the cntrlled FI. 1 (plate). Sectins f the liver f the rat (cntrlled temperature freezing-sectining technique), t which mri's methd fr acid phsphatase has been applied. A, untreated sectin; incubated 10 min; n stain is discernible. B, untreated sectin; incubated 20 min; t shw blackened granules in the liver cells. C, untreated sectin; incubated 60 min; diffuse cytplasmic and sme nuclear staining, but blackened granules are still present. D, sectin treated with frmalin; incubated 10 min; t shw blackened granules similar t thse in B. E, sectin treated with frmalin; incubated 20 min; numerus black granules with sme diffuse cytplasmic staining. F, sectin treated with frmalin; incubated 60 min; nte the diffuse cytplasmic and intense nuclear staining.

5 208 Bitensky Demnstratin f lyssmes temperature freezing-sectining methd and treated with frmalin shwed stained granules after shrt incubatin, whilst untreated sectins remained unstained. Thus the lyssmes had been activated by frmalin treatment, but remained inert in the untreated sectin. Activated lyssmes were bserved in the untreated sectins, but nly after mre prlnged incubatin. This was prbably due t the effect f heat n the lyssmal membrane and is in agreement with the finding that intact, inactive lyssmes in tissue hmgenates are activated by incubatin at 37 0 C at ph 5 (de Duve, 1959). Shrt perids f incubatin allw access f the substrate t the enzyme, while lnger perids result in diffusin f the enzyme with diffuse cytplasmic and nuclear staining. 'Tritn X-100' releases acid phsphatase frm liver lyssmes (ianett and de Duve, 1955). Similar results were btained with sectins prepared by the cntrlled temperature freezing-sectining methd. staining reactin ccurred even after prlnged incubatin in the substrate. It wuld seem that the enzyme was released frm the lyssmes and had diffused ut f the sectin. Lyssmes in tissue hmgenates are activated by repeated freezing and thawing (de Duve, 1959). Hlt (1959) fund that particles stained very rapidly in frzen-dried sectins and he suggested that sme mdificatin f the lyssmal membrane had ccurred. The cntrlled temperature freezing-sectining methd applied in this study des nt appear t have had the same effect as freezing-drying n the lyssmal membrane, since the integrity f the membrane seems t be preserved. This is shwn by the fact that the lyssmes were inactive until subjected t damaging agents like heat, frmalin, and 'tritn X-i'. With increasing severity f the treatment, activity culd be demnstrated either in the lyssmes alne, r in the lyssmes and cytplasm, r in the cytplasm and nucleus (see table 1). The mst severe treatment (tritn X 100) released the enzyme and n activity culd be demnstrated. Thus the three criteria that are required t prve that granules crrespnd t the bichemists' lyssmes have been fulfilled in this study. The granules have been shwn t be inert when intact; they have been activated by treatments that will damage membranes; and the acid phsphatase f the granules has been made t diffuse thrughut the cell and give rise t intense but diffuse cytplasmic and nuclear activity. I wish t acknwledge my gratitude t Prfessr. J. Cunningham fr his encuragement and t Dr. S. J. Hlt fr his guidance in this wrk. I am als much indebted t Dr. J. Chayen fr mst stimulating and helpful discussin. I shuld like t thank Mr. A. A. Silcx fr his skilful assistance and Mr. A. L. E. Barrn fr the phtmicrgraphy. I am indebted t the British Empire Cancer Campaign fr financial assistance and I wish t thank the Trustees f the Prphit Fund fr a research studentship.

6 The Bitensky Demnstratin f lyssmes 209 Appendix TABLE I effect f varius treatments n the intensity, lcalizatin, and speed f the acid phsphatase reactin untreated frmalin sucrse tritn X-i I + Time f incubatin (rniri) black granules; = diffuse cytplasmic staining; = nuclear staining; = strng reactin; + = mderate reactin; = n reactin. -1- References BERTHET, J., and DUVE, C. DE, Bichem. J., 50, 174. CHAYE, J., CHAYE, R., CUIHAM,. J., and BITESKY, L., Path, et Bil., 9, 925. DUVE, C. DE, 1959, in Subcellular particles, edited by T. Hayashi. Washingtn (American Physilgical Sciety). IAETT, R., and DUVE, C. DE, Bichem. J., S9, 433- MRI,., Arch. Path., 82, 189. HLT, S. J., Exp. Cell Res., Suppl. 7, 1. PALADE,. E., Arch. Bichem., 30, 144.