Restricted mobility and endocytosis of anionic sites on newborn rat jejunal brush border membranes.код для вставкиСкачать
THE ANATOMICAL RECORD 202:61-71 (1982) Restricted Mobility and Endocytosis of Anionic Sites on Newborn Rat Jejunal Brush Border Membranes RALPH A. JERSILD, JR. Department of Anatomy, Indiana University School of Medicine, Indianapolis, Indiana 4622.3 ABSTRACT The distribution and mobility of anionic sites on the microvillous surface of newborn rat jejunal absorptive cells were studied using polycationic ferritin (PCF)as a visual probe and were compared with anionic sites previously described for adult jejunum. Segments from 5- to 26-day-oldrats were incubated in PCF for 5 minutes either before or after fixation for electron microscopy. From days 5 to 20, anionic sites were distributed diffusely along the lengths of the microvilli and did not show random translational mobility. In contrast, microvilli examined from animals at weaning (21 to 26 days) resembled those from adults in which most binding sites were capable of lateral mobility and were induced by PCF to cluster into discrete patches. The diffuse pattern was altered by cortisone administration, paralleling a premature reduction in the endocytic apparatus of the cell. The difference in mobility of anionic sites with age coincides with differences in absorptive function. Evidence is presented showing that in the neonate binding of PCF to the microvilli was followed with time by endocytosis into an apical system of tubules for intracellular transport, incorporation into coated vesicles, and release through the lateral cell surface. The results suggest that endocytosis is accomplished by a mechanism that includes a directionally controlled move ment for the selective internalization of PCF binding sites from the membranes of the microvilli to those of the tubular cytoplasmic channels. It was shown previously in absorptive cells from the adult rat jejunum that receptors bearing anionic sites are distributed in a diffuse, random pattern along the length of the microvillous surface, as observed by the binding of polycationic ferritin (PCF)to fixed cells (Jersild and Crawford, 1978). PCF also can be used on living cells at physiological pH, and has been useful in determining the degree of lateral mobility of PCF receptors and their capacity for redistribution through electrostatic coupling (Danon et al., 1972). When applied to the unfixed membranes of living absorptive cells, PCF induced a redistribution of anionic sites into discrete patches separated by areas of unlabeled membrane. Other anionic sites formed a nonmobile cap of PCF binding sites over the tips of the microvilli (Jersild and Crawford, 1978). During the first 3 weeks of postnatal development in the rat, the composition of brush border enzymes and other proteins and glycoproteins differs considerably from that in 0003-276X/82/2021-0061$03.500 1982 Alan R. Liss. Inc. the adult. This reflects differences in diet and, in some species, in the ability for endocytosis of intact proteins, including antibodies, in the neonate (Brambell, 1970;Galand and Forstner, 1974; Seetharam et al., 1977). Endocytosis is accomplished by an extensive system of tubules and vesicles present throughout the apical cytoplasm (Kraehenbuhl and Campiche, 1969; Rodewald, 1973). A change from these and other features of the neonate to those of the adult absorptive cell occurs at the time of weaning about the end of the third week, and is normally completed by 24 days of age. The changes can also be induced prematurely by cortisone (Yeh and Moog, 1975, 1977). The present study was undertaken to examine the distribution and mobility of anionic sites on the brush border surface of the newborn rat jejunum through weaning. The results show considerable difference from those obtained in the adult in the distribution Received March 30, 1981. accepted June 5, 1981. 62 R.A. JERSILD, JR. of PCF binding sites and in their lateral mobility that parallel basic functional differences between the neonatal and adult jejunum. MATERIALS AND METHODS PBS for 15, 20, 30 or 60 minutes (5 animals). Care was taken not to affect the blood supply to the segment. All incubations were stopped by rinsing the tissue in PBS and then transferring the excised tissue to glutaraldehyde. In control experiments, 1 mglml native ferritin (Calbiochem,La Jolla, California) replaced PCF for incubation of unfixed or prefixed specimens on days 5, 10, and 15 after birth. Native ferritin was also injected into ligated segments for continuous in vivo exposure for 60 minutes. Other control samples of unfixed specimens were incubated in 1 mglml poly-Llysine (Miles Laboratories, Inc., Elkhart, Indiana) for 5 minutes at room temperature to block anionic sites, then rinsed and incubated in PCF as usual (Skutelsky and Danon, 1976). Some fixed specimens were treated before PCF incubation with chloroform-methanol for 20 hours according to the method of Suzuki (1965) for ganglioside extraction in aldehydefixed tissues. Methylation was performed by treatment of fixed tissues with 0.05 N HC1 in absolute methanol for 16 hours at room temperature before PCF incubation (Fisher and Lillie, 1954). Portions of these samples were saponified with 1.0% KOH in 80% ethanol (Ito, 1965). Control tissues were exposed solely to absolute methanol under the same conditions. In another series, cortisone acetate (Merck, Sharp and Dohme, West Point, PA.) was administered intraperitoneally in saline to neonates at 50 pglgm body weightlday (Yeh and Moog, 1975), or 5 mglrat on day 1 (Halliday, 1959). Data on these two groups were essentially the same and, therefore, will be presented collectively. Littermates served as saline-injected controls for each experimental condition. Animals were injected and sacrificed as presented in Table 1. Rats of Wistar strain were purchased from Harlan Industries, Indianapolis, Indiana. A total of 20 animals were examined on days 5,8, 10, 12, 15, 17, 19, 21, 22, 23, 24, and 26 after birth. Immediately after sacrifice of each animal, 5-mm segments of proximal jejunum, starting from the ligament of Treitz, were removed, opened longitudinally, and allowed to form an inverted roll. These were used for all in vitro studies following a thorough saline rinse and blotting to remove surface debris and mucus. Initial fixation was in 3% glutaraldehyde, buffered to pH 7.4 with 0.1 M sodium cacodylate, for 4 hours at room temperature. Secondary fixation in 1%cacodylate-buffered Os04for 1 hour was followed by a graded series of ethanol dehydration and embedment in Epon 812 for electron microscopy. Sections were stained briefly with lead citrate. Segments were incubated in phosphatebuffered saline (PBS) containing 1 mglml polycationic f e r r i t i n ( P C F ) (Miles Laboratories, Inc., Elkhart, Indiana) for 5 minutes at room temperature either before or after initial fixation. This concentration of PCF was used to directly compare with results obtained previously in the adult jejunum (Jersild and Crawford, 1978). It was shown in that study that mucus has a greater affinity for PCF than anionic sites on the microvillous surface of unfixed cells, and that only mucus shows binding at concentrations lower than 1 mglml. Incubations for longer than 5 minutes were performed in ligated segments of jejunum in vivo at 1or 3 mglml PCF. After an inRESULTS itial flush with PBS, the PCF was injected into the lumen for a continous exposure of 15,45,or Distribution of anionic sites from days 1 to 20 Following a 5-minute incubation of living, 60 minutes (three animals), or for a 5-minute pulse followed by a flush and chase in PCF-free unfixed intestinal segments in PCF, labeled TABLE 1. Influence of cortisone on the distribution of brush border anionic sites and endocytic tubules Anionic sites' Tubules Number of Age treatment Age at animals started sacrifice Diffuse Pat ched present ' * * Saline' 6 Cortisone 3 10 12 Cortisone 3 8 12 f.f Cortisone 3 10 15 f,f 'Littermates in each experiment were injected with saline. All retained neonatal characteristics and are combined here. or absent (-). 'PCF labeling density in diffuse or patched sites. varying from abundant (++++)to sparse (f) 'Tubules, as they typically appear in numbers in neonates before weaning (++ +).or in adults (+). *Asterisks = data is a s presented in cortisone experiments -see footnote one. Group ~ ~~ . ~~ ~~~~ ++++ ++++ + ++++ ++++ + + MOBILITY AND ENDOCYTOSIS OF ANIONIC SITES 63 anionic sites were dispersed over the lateral membranes were cut transversely. Binding of surfaces of absorptive cell microvilli, either PCF to the microvilli was greatest on the cells uniformly or interrupted by short, unlabeled of the distal (apical)villus and became generalsegments of membrane in an apparently ran- ly lighter or was lacking on cells of the proxdom manner. The tips of the microvilli usually imal villus, beginning a variable distance from were not reactive throughout this period (Fig. the villous tips. All absorptive cells in reactive I). Diffuse labeling also occurred for variable regions of the distal villus showed binding in distances along the membranes of some of the contrast to an intermixing of labeled and tubules that extend from the base of the unlabeled cells seen previously in adults (Jermicrovilli or along those of deeper profiles (Fig. sild and Crawford, 1978). A similar diffuse arrangement of anionic 1).The bound PCF appeared to be suspended among the filaments of the surface coat cover- sites was observed on microvilli of cells fixed ing the membranes of the microvilli and with glutaraldehyde before incubation in PCF. tubules. This is especially evident where the Fixed tissue treated with methanol or Fig. 1. Microvilli of an absorptive cell from the distal villus of a 10-day-old rat. Incubation was for 5 minutes in PCF before fixation. Numerous binding sites are distributed in a diffuse, random pattern over the length of the microvilli. Some gaps in labeling occur locally. Binding of PCF to the surface coat is evident where the membranes are cut transversely. The surface coat a t the microvillous tips is not labeled. One surface tubule and a vesicular profile in the terminal web zone show diffuse. membrane-bound labeling. The tubule is labeled only along the upper part of its length (arrow). Other tubules are not labeled. X 70.000. 64 R.A. JERSILD. JR. chloroform-methanol displayed the same binding characteristics as that of untreated specimens. Tissues exposed to methanol-HC1 prior to incubation showed no evidence of PCF binding. Subsequent saponification of a portion of this tissue resulted in a return to the original binding pattern. Preincubation in poly-L-lysine eliminated or greatly reduced subsequent binding with PCF. There was no retention or binding observed following incubation of fixed or living cells with native ferritin. While most cells on the distal villus continued to show abundant, diffuse binding up to day 20, some others showed a variable reduction in the number of binding sites along the microvillous length between days 15 and 20. In a few cases, cells were observed in which the PCF clustered into small patches similar to that seen in older animals (see below). Such Fig. 2. Microvilli of an absorptive cell from a postweaned rat in which the cells were fixed in glutaraldehyde before incubation in PCF for 5 minutes. PCF is distributed in a random, diffuse pattern primarily along the upper two-thirds of their length. The microvillous tips are more heavily labeled with PCF, which forms a distinct cap over each. X 57,000. cells at times were observed on the villus proximal to cells with diffuse binding sites. Distribution of anionic sites from days 21 to 26 Diffuse binding was observed only rarely in living absorptive cells incubated for 5 minutes with PCF following day 20, and not at all after day 23. Rather, the binding pattern observed throughout this 6-day period was more consistent with that found in adult animals studied previously (Jersild and Crawford, 1978). When cells were fixed before incubation in PCF, the anionic sites were distributed in a diffuse pattern along the lateral microvillous surface (Fig. 2). When incubation preceded fixation, however, a striking redistribution of anionic sites along the lateral surface occurred in which discrete patches of densely packed ferritin were separated by portions of membrane with little or no binding. The tips of the microvilli Fig. 3. Microvilli of an absorptive cell from the distal villus of a 22-day-old rat. Incubation was for 5 minutes in PCF before fixation. PCF is organized as a dense cap over each microvillous tip similar to that in prefixed specimens (Fig. 21. Along the length, the PCF is drawn into clusters of various sizes, leaving the remaming membrane relatively free of binding sites. X 57,000. MOBILITY AND ENDOCYTOSIS OF ANIONIC SITES 65 tained when the cells were continually exposed to PCF. Whereas the interior of the cell showed a progressive increase in labeling over the 60-minute test period examined, the microvilli showed a marked decrease in PCF binding, whether the PCF was presented as a pulse (Fig. 4) or continuously (Fig. 5). During this time, some PCF also could be found in multivesicular bodies in the apical cytoplasm (Fig. 6). In contrast to the diffuse pattern of PCF binding in the endocytic tubules, PCF within these bodies was largely in dense clusters with no apparent association with the membranes. Influence of hormones on anionic sites Some of the coated vesicles throughout the The results of this study are summarized in apical region also contained PCF, presumably Table 1. After injections of cortisone over a received through direct membrane continuity period of 2 days, absorptive cells showed a with the tubules (Fig. 7). By 20 minutes, small similar binding pattern to that observed in amounts of PCF were observed in the lateral neonatal absorptive cells during the first 20 intercellular spaces, often in an area where days described above. Injections over a period coated vesicles carrying PCF apparently had of 4 to 5 days, beginning on day 8 or 10 after fused to the plasma membrane (Fig. 7). The birth, respectively, resulted in a marked reduc- characteristic appearance of PCF in these tion in diffuse labeling with PCF and a con- spaces was in the form of tight clusters, often comitant decrease in number of endocytic in paracrystalline array, in which most of the tubules. A sparse, but detectable, patching of ferritin appeared detached from the membrane PCF-bound receptors was present in some surf ace. specimens, but large numbers of clustered DISCUSSION binding sites, as seen in the adult, were not achieved with any of the injection sequences. I t was shown previously that electrostatic Treatment with cortisone also did not enhance binding of PCF is specific for surface anionic the development of dense caps of PCF binding sites on absorptive cell microvilli of adult rat sites at the microvillous tips, as seen in many jejunum (Jersild and Crawford, 1978). These cells from postweaned animals (Figs. 2, 3). results were confirmed in the present study for Saline-injected littermates from all ex- jejunal absorptive cells of the neonatal rat. periments continued to show diffuse labeling Although the nature of the anionic sites was of the microvilli and the presence of numerous not determined precisely, the loss of binding apical endocytic tubules. sites upon treatment with methanol-HC1and their reappearance following saponification Pulse-labeling and long-term labeling suggest that binding of PCF chiefly reflects with PCF the distribution of exposed carboxyl groups Cells examined at the end of a 5-minute pulse (Revel, 1964). The clear association of most with PCF are the equivalent of the earlier PCF with filaments of the surface coat and the 5-minute incubation studies. I t should be em- resistance of PCF binding sites to extraction phasized here that diffusely labeled anionic with chloroform-methanol provide evidence sites only extended into some of the tubules of that the binding is primarily related to the the terminal web and, in these, labeling usually presence of surface-coat glycoproteins, did not extend the full depth of the tubule (Fig. although other membrane proteins cannot be 1).Ferritin that could be characterized as free excluded. I t seems unlikely that external in the lumen of these channels or as tight agents such as mucus, which does avidly bind clusters along the membrane was only rarely PCF, could account for the diffuse binding patobserved. No evidence was obtained for the up- terns observed on the membrane in the neonate. It is also doubtful that mucus-bound take and transport of native ferritin. Following a 15- or 20-minute chase in PCF- PCF could account for the cell-specificclusters free medium after pulsing with PCF, a much of PCF observed in postweaned rats. Morelarger number of tubular channels throughout over, binding sites in mucus have a greater afthe apical cytoplasm showed diffuse mem- finity for PCF than sites on the microvillous brane binding (Fig. 4). Similar results were ob- surface of unfixed cells. At concentrations of also frequently were covered by a dense cap of ferritin particles (Fig. 3). The number of labeled cells per villus varied considerably. As demonstrated in adult animals (Jersild and Crawford, 1978), labeled cells alternated in an apparently random fashion with cells that did not bind PCF. Surface pits and apical tubules, which are a part of the endocytic apparatus of the neonatal absorptive cell, were reduced to scattered profiles typical of adult animals. PCF labeling of these profiles following 5-minute incubation was not observed. 66 R.A. JERSILD. JR. PCF below that used in this study (e.g., 0.3 mg/ml),microvilli remain totally unlabeled and subsequent cross-linking with labeled mucus to produce the characteristic patches or caps does not occur (Jersild and Crawford, 1978). In the adult rat, it was shown previously that most anionic sites of the microvillous surface are laterally mobile and can be induced by Fig. 4. Apical portion of an absorptive cell from the upper villus of a 10-day-old rat. PCF was pulsed for 5 minutes in viva, then chased with PBS for 20 minutes. Binding to the microvilli is diffuse; however, large segments of the membrane are unlabeled. Most endocytic tubules in and below PCF to cluster into discrete patches (Jersild and Crawford, 1978). In contrast, PCF does not induce a clustering of anionic sites under similar conditions in the newborn. Rather, as shown in the present study, these sites retain a diffuse distribution. This suggests that these binding sites do not possess lateral mobility, and that subsequent patching on exposure to the terminal web zone also show diffuse or scattered labeling, most of which is membrane bound. There is no evidence for clustering of PCF including that in the endocytic pits at the base of the microvilli. X 51,000. MOBILITY AND ENDOCYTOSIS OF ANIONIC SITES 67 PCF is prevented by certain restraining mechanisms to be considered below. The diffuse pattern was seen up to the time of weaning, although there was a tendency toward a reduction in number of anionic sites in some specimens during the third postnatal week. By the 24th day, anionic sites present on the microvilli, with the exception of those at their tips, consistently formed patches in the presence of PCF, reflecting a distinct change in Fig. 5. Base of microvilli and apical cytoplasm of an a b sorptive cell from the distal villus of a 10-day-oldrat. The intestine was exposed continuously to PCF for 45 minutes in vivo. The microvilli show widely dispersed labeling with PCF. Greater concentrations of PCF still occur locally a t the base of the microvilli and in endocytic pits (arrows). Endocytic tubules show abundant, diffuse, membranebound PCF. X 61,000. 68 R.A. JERSILD. JR. their lateral mobility at this time. The possibility that patching of binding sites was induced by fixation (Burry and Wood, 1979)is negated by the diffuse binding pattern seen on cells fixed prior to PCF exposure. These features of anionic binding are comparable to those maintained through adult life (Jersild and Crawford, 1978).The change in behavior of anionic sites with time is suggested to reflect differences in absorptive cell function in the newborn and adult animals. A number of changes have been shown to occur in absorptive cell function and associated structure during the same period of time. Among these are a loss of ability to ingest intact proteins and antibodies (Halliday, 1955; Jones, 1972) concomitant with an extensive reduction in the endocytic system of tubules (Clark, 1959; Rodewald, 1973;),and changes in the composition of brush border enzymes and other proteins, including a decrease in lactase and an increase in other hydrolases (Galand and Forstner, 1974; Seetharam et al., 1977). These changes occur in a relatively short period that extends from about the 19th to the 23rd day in the rat and are under the control of the adrenal cortex. In this respect, these changes can be induced prematurely in the suckling rat by the administration of glucocorticoids (see review by Yeh and Moog, 1975).In the present study, cortisone administered over 4 to 5 days likewise produced premature changes in anionic sites, reflected primarily as a marked decrease in PCF-binding, indicating that this feature of the neonatal absorptive cell is also under hormonal control. The lack of response after only 2 days of cortisone treatment may reflect the inability of fully matured cells to respond to the hormone. A respor:se instead may require replacement with crypt cells Fig. 6. A rnultivesicular body, 20 minutes after a 5-minute pulse with PCF, contains an internal cluster of PCF. X 51,000. induced during differentiation as shown for other features controlled by hormones (Clark, 1959, 1971; Doell et al., 1965).Ths view is supported by the observation that occasional cells with patched PCF binding sites could be seen on villi in a migratory position proximal to cells that had diffuse anionic sites. The concomitant loss of detectable microvillous anionic sites and the marked reduction in endocytic tubules observed in the present study following cortisone administration suggest a relationship between these sites and endocytosis. Indeed, evidence presented here clearly demonstrates that considerable PCF is absorbed and transported through the cell by an endocytic process. In fact, the pattern of uptake and transport, the time course for traversing the cell, and the sensitivity to cortisone are very similar to that described for antibody transport in the newborn rat (Rodewald, 1973; Waldmann and Jones, 1973). For this, the proximal small intestine has been shown in important studies by Rodewald (1976, 1980)to be the site for selective absorption of antibodies by a process that involves the binding to receptors covering the microvillous membrane. Binding of PCF to the cell membrane of the microvilli meets the first requirement for selective uptake as originally suggested by Brambell (1970). While the selective uptake mechanism has been shown to be fairly specific for certain antibodies (Rodewald, 1973; Waldmann and Jones, 1973), it is clearly evident in the present study that uptake is not restricted to them but may include other molecules that are capable of binding to suitable receptors in the cell membrane, such as PCF. It cannot be stated at this time whether or not PCF is binding to the receptors that transport antibodies into the cell. The binding of PCF and its subsequent transport may indicate, however, another mechanism by which certain potential antigens enter the body of the young in addition to their uptake as immune complexes (Abrahamson et al., 1979). Binding of ligands to the cell surface is known to stimulate pinocytic activity (Silverstein et al., 1977). For some cell types, ligand binding has been reported to induce crosslinking and subsequent clustering of diffusely distributed and apparently freely diffusible receptors which are then internalized as concentrated patches (Schekman and Singer, 1976; Schreiner and Unanue, 1976; Willingham et al., 1979). This contrasts with the 69 MOBILITY AND ENDOCYTOSIS OF ANIONIC SITES results in the present study in which the anionic sites on the microvilli of neonatal jejunum were not passively mobile and were not redistributed into clusters following the binding of PCF. Yet with time after a 5-minute pulse-labeling period, there was a large in- crease in the number of endocytic tubules labeled in the apical portion of the cell accompanied by a striking decrease in labeling of the microvilli. It is of interest that labeling of the microvilli also decreased with time during continual exposure to PCF. It thus appears that . . - .. . . .- . .. . _ Fig. 7. Deep apical cytoplasm and lateral boundary b e tween two cells from a 10-day-oldrat following a continuous in vivo exposure to PCF for 45 minutes. PCF is present in small coated vesicles (short arrows). In larger coated vesicles, two of which are attached to the cell membrane, the PCF is seen to still be membrane bound (largearrows). Other PCF is in the lateral intercellular space where i t forms dense clusters. X 54,000. In the inset, diffuse, membrane-bound label is present in an endocytic tubule and in an attached coated vesicle. X 89,000. -------_ I - 70 R.A. JERSILD, JR. binding sites can be temporarily depleted during the endocytic process. The results at this time suggest that these sites interact with other membrane of submembrane structures (Edelman, 1976;Nicolson, 1976)which prevent their passive mobility. When bound to a suitable ligand, these sites then are conveyed from the microvilli to the cell interior by a directed movement that channels them into tubular components of the system in which they are further transported. A transmembrane system of directional control over binding site movement (Nicolson, 1976) is therefore proposed which would ensure their passage into subjacent tubules of considerable length. It may also be an important mechanism for the maintenance of the integrity of the structure and other functional activities of the microvilli during the event of endocytosis in a manner similar to that described for other cells (Oliver and Berlin, 1976). For this, the core actin filaments and the cross filaments connecting them to the microvillous membrane (Mukherjee and Staehelin, 1971; Mooseker and Tilney, 1975)may play some important roles as yet not recognized in neonates. Furthermore, the maintenance of diffuse binding within the endocytic tubules may indicate a regulated movement of membrane-binding PCF in these structures as well. I t has been suggested that binding of proteins to microvillous receptors not only aids in their selective uptake, but also protects them from lysosomal degradation (Brambell, 1970). In the present study, a small amount of PCF was sometimes observed within multivesicular bodies as internal clusters rather than diffusely bound to the membranes. This appearance is very similar to that previously observed in small quantities in the adult jejunum where its transport was terminated (Jersild and Crawford, 1978). It seems likely that it represents the uptake of PCF unbound or only loosely bound to membranes and ultimately destined for degradation by the lysosomal activity found in these bodies (Kraehenbuhl and Campiche, 1969). ACKNOWLEDGMENTS This project was supported by grant 5S01 RR-05371 awarded by the Division of Research Resources, NIH, DHEW. The author gratefully acknowledges the skillful technical assistance of Mrs. Vera McAdoo and the secretarial assistance of Mrs. Deborah Komlanc. LITERATURE CITED Abrahamson, D.R., A. Powers, and R. Rodewald (1979) Intestinal absorption of immune complexes by neonatal rats: A route of antigen transfer from mother to young. Science, 206: 567-569. Brambell. F.W.R. (1970) The transmission of passive immunity from mother to young. In: Frontiers of Biology, Vol. 18. A. Neuberger and E.L. Tatum, eds. NorthHolland Publishing Company, Amsterdam. Burry, R.W.. and J.G. Wood (1979) Contributions of lipids and proteins to the surface charge of membranes. An electron microscopy study with cationized and anionized ferritin. J. Cell Biol., 82 726-741. 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